Cytogenetics and

Molecular Cytogenetics
Genomic technologies provide the means of diagnosis and management of many
human diseases. Without insights from cytogenetics, correct interpretation of
modern high-throughput results is diffcult, if not impossible. This book summarizes
applications of cytogenetics and molecular cytogenetics for students, clinicians and
researchers in genetics, genomics and diagnostics. The book combines the state-of-
the-art knowledge and practical expertise from leading researchers and clinicians
and provides a comprehensive overview of current medical and research applications
of many of these technologies.

KEY FEATURES
• Provides clear summaries of fuorescence in situ hybridization technologies
and others
• Comprehensively covers established and emerging methods
• Chapters from an international team of leading researchers
• Useful for students, researchers and clinicians
 Medical Genomics and Proteomics
Series Editors
Scott O. Rogers
Science publisher
Charles R. Crumly—CRC Press/Taylor & Francis Group

Published Titles

Molecular Analyses
Edited by Scott O. Rogers

Cytogenetics and Molecular Cytogenetics

Edited by Thomas Liehr

For more information about this series, please visit: www.routledge.com/Medical-

Genomics-and-Proteomics/book-series/CRCMOLGENPRO
 Cytogenetics and
Molecular Cytogenetics

Edited by
Thomas Liehr
Legend for fgure in titlepage:
Central white Figure part consists (from left to right) of: Two schemes showing multicolor-banding
and chromosomal translocations were provided by Leon Liehr (Jena, Germany; https://leonliehr.
jimdofree.com/), the partial M-FISH karyogramm, the interphase nucleus and the chromosomal
microarray fgures are derived from chapters 14 (part of 1), 6 (part of 1D) and 21 (part of 2A),
respectively.
First Edition published 2023
by CRC Press
6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487–2742
and by CRC Press
4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN
CRC Press is an imprint of Taylor & Francis Group, LLC
© 2023 selection and editorial matter, Thomas Liehr; individual chapters, the contributors
Reasonable efforts have been made to publish reliable data and information, but the author and
publisher cannot assume responsibility for the validity of all materials or the consequences of
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Library of Congress Cataloging‑in‑Publication Data
Names: Liehr, Thomas, 1965-editor.
Title: Cytogenetics and molecular cytogenetics / edited by Thomas Liehr.
Description: First edition. | Boca Raton : CRC Press, 2023. | Series: Medical genomics and
proteomics | Includes bibliographical references and index.
Identifers: LCCN 2022026812 (print) | LCCN 2022026813 (ebook) | ISBN 9781032121628
(hardback) | ISBN 9781032122212 (paperback) | ISBN 9781003223658 (ebook)
Subjects: LCSH: Cytogenetics. | Molecular genetics.
Classifcation: LCC QH441.5 .C98 2023 (print) | LCC QH441.5 (ebook) | DDC 572.8—dc23/
eng/20220713
LC record available at https://lccn.loc.gov/2022026812
LC ebook record available at https://lccn.loc.gov/2022026813
ISBN: 978-1-032-12162-8 (hbk)
ISBN: 978-1-032-12221-2 (pbk)
ISBN: 978-1-003-22365-8 (ebk)
DOI: 10.1201/9781003223658
Typeset in Times
by Apex CoVantage, LLC
To my two teachers in Human Genetics and Chromosomics:
Prof. Erich Gebhart (Erlangen, Germany), who showed
to me the importance of publishing results, and
Prof. Uwe Claussen (Jena, Germany), who was
a role model for unconventional thinking.
Contents
Series Preface............................................................................................................xi
Scott O. Rogers
Preface.................................................................................................................... xiii
Acknowledgements .................................................................................................. xv
Editor .....................................................................................................................xvii
List of Contributors.................................................................................................xix

Chapter 1 Cytogenetics and Chromosomics......................................................... 1

Thomas Liehr

Chapter 2 Banding Cytogenetics .......................................................................... 7

Hongyan Chai and Peining Li

Chapter 3 Generation of Microdissection-Derived Painting Probes from

Single Copy Chromosomes ................................................................ 27
Svetlana Romanenko and Vladimir Trifonov

Chapter 4 FISH—An Overview.......................................................................... 35

Thomas Liehr

Chapter 5 FISH—Microscopy and Evaluation ................................................... 43

Ivan Y. Iourov

Chapter 6 FISH—in Routine Diagnostic Settings .............................................. 49

Chenghua Cui and Peining Li

Chapter 7 FISH—in Leukemia Diagnostics....................................................... 71

Roberto Matos, Mariana de Souza, Gerson Ferreira,
Amanda Figueiredo, Marcelo Land and Maria Luiza Silva

Chapter 8 FISH—in Tissues ............................................................................. 105

Thomas Liehr

Chapter 9 FISH—in Human Sperm and Infertility .......................................... 111

Martina Rincic and Thomas Liehr

vii
viii Contents

Chapter 10 FISH—in Spontaneously Aborted Products of Conception ............ 117

Thomas Liehr

Chapter 11 FISH—Characterization of Chromosomal Alterations,

Recombination, and Outcomes after Segregation............................ 121
Ilda Patrícia Ribeiro, Eunice Matoso, Joana Barbosa Melo,
Ana Jardim, Thomas Liehr and Isabel Marques Carreira

Chapter 12 Multicolor-FISH—Methods and Applications................................. 151

Thomas Liehr

Chapter 13 FISH—Centromere- and Heterochromatin-Specifc

Multicolor Probe Sets....................................................................... 157
Thomas Liehr

Chapter 14 FISH—Detection of Individual Radio Sensitivity ........................... 163

Thomas Liehr

Chapter 15 FISH—Detection of CNVs .............................................................. 171

Tigran Harutyunyan, Anzhela Sargsyan
and Rouben Aroutiounian

Chapter 16 FISH—Interphase Applications Including Detection of

Chromosome Instability (CIN) ........................................................ 181
Ivan Y. Iourov, Svetlana G. Vorsanova and Yuri B. Yurov

Chapter 17 FISH—Determination of Telomere Length

(Q-FISH/CO-FISH).......................................................................... 189
Gordana Joksić, Jelena Filipović Tričković and Ivana Joksić

Chapter 18 FISH—in Three Dimensions—3D-FISH ........................................ 201

Thomas Liehr

Chapter 19 FISH—On Fibers .............................................................................207

Thomas Liehr

Chapter 20 FISH—and Single-Cell Gel Electrophoresis Assay

(Comet Assay) .................................................................................. 211
Galina Hovhannisyan, Tigran Harutyunyan
and Rouben Aroutiounian
Contents ix

Chapter 21 Molecular Karyotyping .................................................................... 225

Ilda Patrícia Ribeiro, Luís Miguel Pires, Susana Isabel Ferreira,
Mariana Val, Joana Barbosa Melo and Isabel Marques Carreira

Chapter 22 FISH—Mitochondrial DNA ............................................................ 251

Tigran Harutyunyan

Chapter 23 FISH—in Birds ................................................................................ 263

Rafael Kretschmer, Michelly da Silva dos Santos, Ivanete de
Oliveira Furo, Edivaldo Herculano Correa de Oliveira
and Marcelo de Bello Cioff

Chapter 24 FISH—in Fish Chromosomes .......................................................... 281

Francisco de M. C. Sassi, Gustavo A. Toma
and Marcelo de Bello Cioff

Chapter 25 FISH—and the Characterization of Synaptonemal Complex.......... 297

Victor Spangenberg

Chapter 26 RNA-FISH—on Lampbrush Chromosomes: Visualization of

Individual Transcription Units .........................................................307
Tatiana Kulikova and Alla Krasikova

Chapter 27 FISH—in Insect Chromosomes ....................................................... 319

Vladimir E. Gokhman and Valentina G. Kuznetsova

Chapter 28 FISH—in Plant Chromosomes......................................................... 339

Susan Liedtke, Sarah Breitenbach and Tony Heitkam

Chapter 29 FISH—and CRISPR/CAS9.............................................................. 353

Thomas Liehr

Index...................................................................................................................... 357
Series Preface
In 2005, the Encyclopedia of Medical Genomics and Proteomics was published.
It consisted of two large volumes of analytical methods and equipment that were
applicable to medical and other studies of genomics and proteomics. While it was
a comprehensive collection at the time, many variations, improvements, and new
technological developments have occurred during the past two decades. Also, the
alphabetical arrangements of the chapters (by title) made searching by topic some-
what cumbersome. Finally, the Encyclopedia was originally available in print ver-
sion only. All of these factors necessitated an extensive revision in content, format,
and organization. The new collection is entitled Series in Medical Genomics and
Proteomics. The revised version will be offered as volumes, organized by topic,
and will be published in print and electronic formats. Currently, at least one dozen
volumes are planned.
This is the second volume in the Series, entitled Cytogenetics and Molecular
Cytogenetics. Because of their importance in several felds, the methods described
in this volume have been developed and used increasingly over the past several
decades to study chromosomal loci, genetic alterations associated with diseases,
chromosomal translocations, congenital abnormalities, tissue-specifc changes,
development, gene expression, molecular taxonomy, evolution, epigenetic changes
associated with diseases (including cancer), and others. The chapters included in this
volume provide a detailed comprehensive collection of descriptions of cytogenetic
and molecular cytogenetic methods, with special emphasis on FISH (fuorescence in
situ hybridization) procedures and applications. This includes an overview of FISH
methods, as well as descriptions of techniques used to identify specifc components
of cells (e.g., centromeres, portions of synaptonemal complexes), disease diagnoses
(e.g., leukemia, chromosome rearrangements), and protocols to characterize cellular
components in various organisms (e.g., birds, fshes, plants). Additionally, chapters
are included on basic cytogenetics, chromosome banding techniques, molecular
karyotyping, and microdissection. This volume is poised to be a valuable resource
for those in cell, molecular, and medical sciences.

Scott O. Rogers
Medical Genomics and Proteomics Book Series Editor

xi
Preface
With this book, a contribution to the highly actual feld of cytogenetics, cytogenom-
ics and chromosomics is provided. The book presents applications and protocols
in clinical and research applications. It is not restricted to human genetics, but also
includes animal and plant cytogenetics and provides some insights in actual develop-
ments. This book is divided in following sections:

I. Basics on (molecular) cytogenetics, cytogenomics and chromosomics

(Chapters 1 to 5);
II. (Molecular) cytogenetics in human genetic diagnostics (Chapters 6 to
13);
III. (Molecular) cytogenetics in clinical genetic research settings (Chapters 14
to 20);
IV. Molecular karyotyping (Chapter 21);
V. Molecular cytogenetics to detect mitochondrial DNA (Chapter 22);
VI. (Molecular) cytogenetics in animal cytogenomics and chromosomics
(Chapters 23 to 27);
VII. (Molecular) cytogenetics in plant cytogenomics and chromosomics
(Chapter 28); and
VIII. CRISPR/Cas9 and its role in cytogenomic and chromosomic research
(Chapter 29).

Overall, the reader gets an up-to-date overview on possibilities of fuorescence in

situ hybridization and related approaches, how they work and why they cannot be
thought away from clinical genetic routine diagnostics and basic research.

xiii
Acknowledgements
The Editor thanks to all authors investing time and work to provide chapters to this
book on cytogenetics and molecular cytogenetics. Only together could we provide
this important contribution to the feld of cytogenomics and chromosomics.

xv
Editor
Thomas Liehr has been working in human genetics since 1991. He is a biologist
by education and head of the molecular cytogenetic group in Jena, Germany.
Research felds include clinical genetics, leukemia cytogenetics, interphase structure
of human chromosomes, and research on chromosomal evolution. The results of
his research are published in >10 books, ~100 book chapters, >800 referred papers,
and ~1000 abstracts. His particular expertise includes small supernumerary marker
chromosomes (sSMC), chromosomal heteromorphisms and uniparental disomy—
see ChromosOmics databases (http://cs-tl.de/DB.html). He is active member of the
European Board of Medical Genetics and received multiple prizes, two invited pro-
fessorships and a Dr.h.c. as summarized at http://cs-tl.de/.

xvii
Contributors
Rouben Aroutiounian Gerson Ferreira
Yerevan State University Instituto Nacional de Câncer José
Yerevan, Armenia de Alencar Gomes da Silva
(INCA-RJ)
Sarah Breitenbach Rio de Janeiro, Brazil
Technische Universität Dresden
Dresden, Germany Susana Isabel Ferreira
Faculty of Medicine of the University
Isabel Marques Carreira of Coimbra
Faculty of Medicine of the University Coimbra, Portugal
of Coimbra
Coimbra, Portugal Amanda Figueiredo
Federal University of Rio de Janeiro
Hongyan Chai Rio de Janeiro, Brazil
Yale School of Medicine
New Haven, CT, USA Jelena Filipović Tričković
University of Belgrade
Chenghua Cui Belgrade, Serbia
Chinese Academy of Medical Sciences
& Peking Union Medical College Vladimir E. Gokhman
Tianjin, China Moscow State University
Moscow, Russia
Marcelo de Bello Cioff
Universidade Federal de São Carlos Tigran Harutyunyan
São Carlos, SP, Brazil Yerevan State University
Yerevan, Armenia
Edivaldo Herculano Correa de Oliveira
Universidade Federal do Pará Tony Heitkam
Belém, Brazil Technische Universität Dresden
Dresden, Germany
Ivanete de Oliveira Furo
Universidade Federal Rural da Galina Hovhannisyan
Amazônia (UFRA) Yerevan State University
Parauapebas, Brazil Yerevan, Armenia

Mariana de Souza Ivan Y. Iourov

Instituto Nacional de Câncer José de Mental Health Research Center
Alencar Gomes da Silva (INCA-RJ) Moscow, Russia
Rio de Janeiro, Brazil
Ana Jardim
Michelly da Silva dos Santos Faculty of Medicine of the University
Universidade Federal do Pará of Coimbra
Belém, Brazil Coimbra, Portugal

xix
xx Contributors

Gordana Joksić Eunice Matoso

University of Belgrade Faculty of Medicine of the University
Belgrade, Serbia of Coimbra
Coimbra, Portugal
Ivana Joksić
University of Belgrade Joana Barbosa Melo
Belgrade, Serbia Faculty of Medicine of the University
of Coimbra
Alla Krasikova Coimbra, Portugal
Saint Petersburg State University
Saint Petersburg, Russia Luís Miguel Pires
University of Coimbra
Rafael Kretschmer Coimbra, Portugal
1. Departamento de Genética e
Evolução, Universidade Federal de Ilda Patrícia Ribeiro
São Carlos Faculty of Medicine of the University
São Carlos, SP, Brazil of Coimbra
2. Departamento de Ecologia, Zoologia Coimbra, Portugal
e Genética, Instituto de Biologia,
Universidade Federal de Pelotas, Martina Rincic
Pelotas, RS, Brazil School of Medicine, University
of Zagreb
Tatiana Kulikova Zagreb, Croatia
Saint Petersburg State University
Saint Petersburg, Russia Svetlana Romanenko
Russian Academy of Sciences
Valentina G. Kuznetsova Novosibirsk, Russia
Russian Academy of Sciences
St. Petersburg, Russia Anzhela Sargsyan
Yerevan State University
Marcelo Land Yerevan, Armenia
Federal University of Rio de Janeiro
Rio de Janeiro, Brazil Francisco de M. C. Sassi
Universidade Federal de São Carlos
Peining Li São Carlos, SP, Brazil
Yale School of Medicine
New Haven, CT, USA Maria Luiza Silva
Instituto Nacional de Câncer José de
Susan Liedtke Alencar Gomes da Silva
Technische Universität Dresden (INCA-RJ)
Dresden, Germany Rio de Janeiro, Brazil
Roberto Matos Victor Spangenberg
Instituto Nacional de Câncer José de Vavilov Institute of General Genetics
Alencar Gomes da Silva (INCA-RJ) RAS
Rio de Janeiro, Brazil Moscow, Russia
Contributors xxi

Gustavo A. Toma Svetlana G. Vorsanova

Universidade Federal de São Carlos Mental Health Research Center
São Carlos, SP, Brazil Moscow, Russia

Vladimir Trifonov Yuri B. Yurov

Russian Academy of Sciences Mental Health Research Center
Novosibirsk, Russia Moscow, Russia

Mariana Val
Faculty of Medicine of the University of
Coimbra
Coimbra, Portugal
 1 Cytogenetics and
Chromosomics
Thomas Liehr

CONTENTS
Introduction................................................................................................................ 1
Chromosomics and Cytogenomics ............................................................................ 1
Application Fields of (Molecular) Cytogenetics........................................................ 2
Classical/Solid Staining .................................................................................... 3
C-Banding and NOR Silver Staining................................................................ 3
Banding Cytogenetics....................................................................................... 3
Fluorescence In Situ Hybridization (FISH)...................................................... 3
Primed In Situ Hybridization (PRINS)............................................................. 4
Comparative Genomic Hybridization (CGH)................................................... 4
Molecular Combing .......................................................................................... 4
Conclusions................................................................................................................ 4
References.................................................................................................................. 4

INTRODUCTION
This chapter is a general introduction to the book Cytogenetics and Molecular
Cytogenetics published in this new “Medical Genomics and Proteomics” book series.
The specifc topic of this book on (molecular) cytogenetics is embedded in the feld
of chromosomics, which can be only realized based on cytogenomic approaches.[1, 2]

CHROMOSOMICS AND CYTOGENOMICS

The idea of chromosomics, as an overarching designation to integrate all research
directions on the (human) genome, was introduced by Prof. Uwe Claussen (Jena,
Germany) in 2005.[3] Chromosomic research is about all genetics-related presenta-
tions of life, including DNA-basepair to chromosome- and interphase-nucleus level,
but also epigenetic aspects (including three-dimensional morphologically of nuclei,
micro-RNAs, imprinting, etc.), breakpoint characteristics and interspecies genomic
studies. Overall, chromosomics has the goal of introducing novel ideas and concepts
in biology and medicine.[1–3]
To get closer to this noble goal, cytogenomic approaches are needed. In
Table 1.1, a list of the most commonly used cytogenomic techniques is provided.[4]
Cytogenetics and molecular cytogenetics were, together with classical approaches of

DOI: 10.1201/9781003223658-1 1
2 Cytogenetics and Molecular Cytogenetics

TABLE 1.1
Cytogenomic Approaches Adapted acc. to[4] Are Listed Here
Cytogenomic Field Cytogenomic Technique
Cytogenetics – Classical/solid staining
– C-banding
– NOR silver staining
– Banding cytogenetics
Molecular Cytogenetics – Fluorescence in situ hybridization
– Primed in situ hybridization
– Comparative genomic hybridization (CGH)
– Molecular combing
Molecular Genetics – Restriction fragment polymorphism analyses
(classical approaches) – DNA-cloning in vectors
– Blotting approaches like Southern blotting
– DNA-fngerprinting
– PCR approaches incl. MLPA
– Sanger sequencing
Molecular Genetics – Array-CGH/chromosomal microarray analyses (CMA)
(modern approaches) – Second generation sequencing
– Third generation sequencing
– Gene editing (CRISP/CAS9)
Others – Electron microscopy–based approaches
– Laser scanning–based approaches
– Optical mapping approaches
– Uniparental disomy/Epigenetic changes oriented approaches—incl.
studies on long non-coding RNAs, etc.
– Bioinformatics

molecular genetics, the frst possibilities of chromosomics. Groundbreaking insights

into the secrets of inheritance were and are still provided by these basic cytoge-
nomic tools.[1,  4] Without chromosome numbers being known, including informa-
tion about sex-determination systems, modern (high-throughput) molecular genetic
approaches are (if at all) only partly informative.[5, 6] It is a truism, which cannot be
repeated enough especially in the human genetics feld, that no classical cytoge-
nomic technique is ever outdated.[7] Each approach has advantages and limitations,
which can be substituted by each other in the optimal case. Thus, to answer questions
in chromosomic research and/or diagnostics, a sensible combination of cytogenomic
approaches to be applied is always necessary.[7]

APPLICATION FIELDS OF (MOLECULAR) CYTOGENETICS

Here the techniques listed in Table 1.1 for the cytogenomic felds (i) cytogenetics and
(ii) molecular cytogenetics are treated, and it is shown in some examples where these
approaches are necessary in routine diagnostics and chromosomic research.
Cytogenetics and Chromosomics 3

CLASSICAL/SOLID STAINING
Classical solid Giemsa or Orcein staining—also called classical cytogenetics[8]—is
the basic approach used to determine constitutional chromosome numbers in spe-
cies previously not studied by cytogenetics.[9] Also, there are species in which chro-
mosome banding is not applicable[10, 11]; here classical cytogenetics is necessary in
research.
Solid chromosome stains are also applied in research settings of radiation or
mutagenesis—and here also diagnostic applications are reported to determine num-
ber of chromosomal breaks per metaphases after irradiation and/or exposure to a
mutagen.[12]

C-BANDING AND NOR SILVER STAINING

C-banding is applied to visualize the heterochromatic regions of genomes in cytoge-
netic preparations, including centromeres; the latter is eponymous for this approach.
NOR silver staining highlights the location of active nucleolus organizing region(s)
in a genome.[13]
Both techniques are done in initial, research-oriented cytogenetic studies to char-
acterize the karyotype of new plant or animal species.[9] Also they are applied in
many routine settings of cytogenetic pre- and postnatal diagnostics.[13] The latter
helps defning if an aberrant chromosome banding pattern (see next) may be just due
to a heteromorphism of heterochromatic DNA in pericentromeric, acrocentric-p-arm
or Yq12 regions of the human genome.[14]

BANDING CYTOGENETICS
Banding cytogenetics was introduced by Lore Zech in the 1970s.[15] Nowadays most
countries apply GTG-banding = G-bands by trypsin using Giemsa for routine band-
ing cytogenetics in prenatal, postnatal and tumor cytogenetic diagnostics. Besides,
banding cytogenetics is used in animal cytogenetics, in case the corresponding chro-
mosomes allow for introduction of a protein-based banding pattern.[16]

FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Fluorescence in situ hybridization (FISH) is one of the major topics of this book; so
here are just some general statements. FISH is an approach that enables the in situ
localization and mapping of specifcally defned DNA-sequences.[1, 2] It is indispens-
able in mapping of a genome, as sequencing alone is yet insuffcient to reconstruct
a karyotype.[5] The latter is due to the fact, that highly repetitive regions of genomes
cannot be accessed by routine approaches, even though frst tools are available also
to solve that problem.[17] Still, in most cases based on NGS data, an end of chromo-
some cannot be distinguished from a high-repeat copy number region being typical
for a centromere. Overall, FISH is a highly fexible approach, which can be adapted
to many research and diagnostic questions, e.g. by multicolor-approaches that enable
targeting many different DNA-sequences at a time.[1, 2, 18]
4 Cytogenetics and Molecular Cytogenetics

PRIMED IN SITU HYBRIDIZATION (PRINS)

Primed in situ hybridization (PRINS) is the second technique, besides FISH, origi-
nally included in the feld of molecular cytogenetics. As this approach exclusively
worked for repetitive sequences, it is nowadays practically no longer applied, even
though it has been used both in research and diagnostics.[19]

COMPARATIVE GENOMIC HYBRIDIZATION (CGH)

Comparative genomic hybridization (CGH) is a variant of FISH.[20] However, origi-
nally here two differently labelled while human genomes are hybridized to a normal
human metaphase plate. Thus, gains or losses in e.g. a tumor probe can be detected.
In diagnostics, this approach is nowadays replaced by array-CGH/chromosomal
microarray analyses (CMA), providing higher resolution and accuracy.[21]
In evolution research, CGH is still a great tool to, e.g. get insights into more stable
compared to regions undergoing more rapid evolution in two closely related species.[6]

MOLECULAR COMBING
So-called fber-FISH is a several decades–old variant of FISH, where DNA-probes
are hybridized instead of on interphases or metaphases on to extended DNA-fbers,
providing thus a higher in situ resolution. Recently, fber-FISH on extremely stretched
DNA-fbers became available as a standardized protocol, and is commercially avail-
able as “molecular combing”. This enables applications in research and in diagnos-
tics for studying repetitive DNA-stretches yet not reliably accessible by any other
cytogenomic approach.[22]

CONCLUSIONS
Chromosomic research and diagnostics, in general are enabled and in parts moti-
vated by new technical developments.[4] New cytogenomic approaches are always
welcome; however, due to specifc advantages of each technique, older ones should
not be considered too hastily as outdated.[7] As shown exemplarily here and with
no claim to completeness, cytogenetics and molecular cytogenetics are still, and
also will be in future, the most relevant participants in the concert of actual cytoge-
nomic approaches being necessary to get as comprehensive as possible chromosomic
insights.[1, 2]

REFERENCES
1. Liehr, T. Molecular cytogenetics in the era of chromosomics and cytogenomic
approaches. Front. Genet. 2021, 12, 720507.
2. Liehr, T. From human cytogenetics to human chromosomics. Int J Mol Sci. 2019, 20,
E826.
3. Claussen, U. Chromosomics. Cytogenet. Genome Res. 2005, 111, 101–106.
4. Liehr, T. A Defnition for cytogenomics: Also may be called chromosomics. In:
Cytogenomics; Liehr, T., Ed. Academic Press, London, 2021, pp. 1–7.
Cytogenetics and Chromosomics 5

5. Reichwald, K.; Petzold, A.; Koch, P.; Downie, B.R.; Hartmann, N.; Pietsch, S.; Baumgart,
M.; Chalopin, D.; Felder, M.; Bens, M.; Sahm, A.; Szafranski, K.; Taudien, S.; Groth, M.;
Arisi, I.; Weise, A.; Bhatt, S.S.; Sharma, V.; Kraus, J.M.; Schmid, F.; Priebe, S.; Liehr,
T.; Görlach, M.; Than, M.E.; Hiller, M.; Kestler, H.A.; Volff, J.N.; Schartl, M.; Cellerino,
A.; Englert, C.; Platzer, M. Insights into sex chromosome evolution and aging from the
genome of a short-lived fsh. Cell. 2015, 163, 1527–1538.
6. Yano, C.F.; Sember, A.; Kretschmer, R.; Bertollo, L.A.C.; Ezaz, T.; Hatanaka, T.; Liehr,
T.; Ráb, P.; Al-Rikabi, A.; Ferreira Viana, P.; Feldberg, E.; de Oliveira, E.A.; Toma,
G.A.; Cioff, M.d.B. Against the mainstream: Exceptional evolutionary stability of l ZW
sex chromosomes across fsh families Triportheidae and Gasteropelecidae (Teleostei:
Characiformes). Chromosome Res. 2021, 29, 391–416.
7. Liehr, T.; Mrasek, K.; Klein, E.; Weise, A. Modern high throughput approaches are not
meant to replace ‘old fashioned’ but robust techniques. J. Genet. Genomes. 2017, 1, e101.
8. Liehr, T. “Classical cytogenetics” is not equal to “banding cytogenetics”. Mol Cytogenet.
2017, 10, 3.
9. Chaiyasan, P.; Mingkwan, B.; Jantarat, S.; Suwannapoom, C.; Cioff, M.d.B.; Liehr, T.;
Talumphai, S.; Tanomtong, A.; Supiwong, W. Classical and molecular cytogenetics of
Belontia hasselti (Perciformes: Osphronemidae): Insights into the ZZ/ZW sex chromo-
some system. Biodiversitas. 2021, 22, 546–554.
10. D’Amato, G.; Bianchi, G.; Capineri, R.; Marchi, P. N-band staining in plant chromo-
somes with a HCl-Giemsa technique. Caryologia. 1979, 32, 455–459.
11. Martínez-Lage, A.; González-Tizón, A.; Méndez, J. Characterization of different chro-
matin types in Mytilus galloprovincialis L. after C-banding, fuorochrome and restric-
tion endonuclease treatments. Heredity. 1994, 72, 242–249.
12. Natarajan, A.T. Chromosome aberrations: Past, present and future. Mutat. Res. 2002,
504, 3–16.
13. Weise, A.; Liehr, T. Cytogenetics. In: Cytogenomics; Liehr, T., Ed. Academic Press,
London, 2021, pp. 25–34.
14. Liehr, T. Cases with heteromorphisms. http://cs-tl.de/DB/CA/HCM/0-Start.html
[accessed on 01/12/2022].
15. Schlegelberger, B. In memoriam: Prof. Dr. rer. nat. Dr. med. h.c. Lore Zech; 24.9.1923–
13.3.2013: Honorary member of the European Society of Human Genetics, Honorary
member of the German Society of Human Genetics, Doctor laureate, the University of
Kiel, Germany. Mol. Cytogenet. 2013, 6, 20.
16. Claussen, U.; Michel, S.; Mühlig, P.; Westermann, M.; Grummt, U.W.; Kromeyer-
Hauschild, K.; Liehr, T. Demystifying chromosome preparation and the implications for
the concept of chromosome condensation during mitosis. Cytogenet. Genome Res. 2002,
98, 136–146.
17. Nurk, S.; Koren, S.; Rhie, A.; Rautiainen, M.; Bzikadze, A.V.; Mikheenko, A.; Vollger,
M.R.; Altemose, N.; Uralsky, L.; Gershman, A.; Aganezov, S.; Hoyt, S.J.; Diekhans, M.;
Logsdon, G.A.; Alonge, M.; Antonarakis, S.E.; Borchers, M.; Bouffard, G.G.; Brooks,
S.Y.; Caldas, G.V.; Cheng, H.; Chin, C.-S.; Chow, W.; de Lima, L.G.; Dishuck, P.C.;
Durbin, R.; Dvorkina, T.; Fiddes, I.T.; Formenti, G.; Fulton, R.S.; Fungtammasan, A.;
Garrison, E.; Grady, P.G.S.; Graves-Lindsay, T.-A.; Hall, I.M.; Hansen, N.F.; Hartley,
G.A.; Haukness, M.; Howe, K.; Hunkapiller, M.W.; Jain, C.; Jain, M.; Jarvis, E.D.;
Kerpedjiev, P.; Kirsche, M.; Kolmogorov, M.; Korlach, J.; Kremitzki, M.; Li, H.; Maduro,
V.V.; Marschall, T.; McCartney, A.M.; McDaniel, J.; Miller, D.E.; Mullikin, J.C.; Myers,
E.W.; Olson, N.D.; Paten, B.; Peluso, P.; Pevzner, P.A.; Porubsky, D.; Potapova, T.; Rogaev,
E.I.; Rosenfeld, J.A.; Salzberg, S.L.; Schneider, V.A.; Sedlazeck, F.J.; Shafn, K.; Shew,
C.J.; Shumate, A.; Sims, Y.; Smit, A.F.A.; Soto, D.C.; Sović, I.; Storer, J.M.; Streets, A.;
6 Cytogenetics and Molecular Cytogenetics

Sullivan, B.A.; Thibaud-Nissen, F.; Torrance, J.; Wagner, J.; Walenz, P.P.; Wenger, A.;
Wood, J.M.D.; Xiao, C.; Yan, S.M.; Young, A.C.; Zarate, S.; Surti, U.; McCoy, R.C.;
Dennis, M.Y.; Alexandrov, I.A.; Gerton, J.L.; O’Neill, R.J.; Timp, W.; Zook, J.M.;
Schatz, M.C.; Eichler, E.E.; Miga, K.H.; Phillippy, A.M.The complete sequence of a
human genome. bioRxiv. 2021. preprint doi: https://doi.org/10.1101/2021.05.26.445798.
18. Liehr, T. Basics and literature on multicolor fuorescence in situ hybridization applica-
tion. http://cs-tl.de/DB/TC/mFISH/0-Start.html [accessed on 01/12/2022].
19. Pellestor, F. Development and adaptation of the PRINS technology: An overview.
Methods Mol. Biol. 2006, 334, 211–220.
20. Kallioniemi, A.; Kallioniemi, O.P.; Sudar, D.; Rutovitz, D.; Gray, J.W.; Waldman, F.;
Pinkel, D. Comparative genomic hybridization for molecular cytogenetic analysis of
solid tumors. Science. 1992, 258, 818–821.
21. Weise, A.; Liehr, T. Molecular karyotyping. In: Cytogenomics; Liehr, T., Ed. Academic
Press, London, 2021, pp. 72–85.
22. Bisht, P.; Avarello, M.D.M. Molecular combing solutions to characterize replication
genetics and genome rearrangements. In: Cytogenomics; Liehr, T., Ed. Academic Press,
London, 2021, pp. 47–72.
 2 Banding Cytogenetics
Hongyan Chai and Peining Li

CONTENTS
Introduction................................................................................................................ 7
Chromosome Structure and Various Banding Techniques................................ 8
Human Chromosome Structure in Interphases and Metaphases .......... 8
Various Banding Techniques ................................................................ 9
Molecular Basis of Banding in Human Chromosomes ...................... 10
G-Banding in Normal Population................................................................... 12
International System for Human Cytogenetic/Cytogenomic
Nomenclature (ISCN).......................................................... 12
Chromosome Heteromorphisms in Humans....................................... 13
Inducible Fragile Sites in Human Chromosomes ............................... 15
Clinical Application of Banding Cytogenetics ............................................... 16
Detection of Constitutional Chromosome Abnormalities................... 16
Detection of Somatic Chromosome Abnormalities ............................ 18
Report and Interpretation of Diagnostic Karyotype ........................... 20
Conclusions.............................................................................................................. 20
Web Resources......................................................................................................... 21
Acknowledgement ................................................................................................... 21
References................................................................................................................ 21

INTRODUCTION
Cytogenetics is an important feld of genetics focusing on the study of chromosome
structure and related functions in heredity and evolution. In 1902, W. Sutton and
T. Boveri independently developed the chromosome theory of inheritance that identi-
fes chromosomes as the carrier of genetic material.[1, 2] Earlier studies of human chro-
mosomes struggled with the correct number of chromosomes.[3] In 1923, T.S. Painter
analyzed chromosomes in human spermatogonial cells at mitotic prophase and made
three important fndings: i) the size and shape of the chromosomes were described,
and the diploid chromosome number was determined to be in the range of 46–48 with
an emphasis on 48; ii) human sex was found to be determined by an X/Y heteromor-
phic chromosomal pair mechanism; and iii) Caucasian and African-American males
had indistinguishable chromosome sets.[4, 5] In 1952, T.C. Hsu introduced a hypotonic
shock method to improve the quality of chromosome preparation from in vitro cul-
tured cells.[6] In 1956, J.H. Tjio and A. Levan used this improved technique to rec-
ognize the correct number of 46 chromosomes in a human genome.[7] This certainty
of chromosome number in normal individuals facilitated cytogenetic investigations

DOI: 10.1201/9781003223658-2 7
8 Cytogenetics and Molecular Cytogenetics

of numerical chromosomal abnormalities on different patients. In 1959, J. Lejeune

and his coworkers discovered that trisomy 21 causes Down syndrome, C.E. Ford
reported that monosomy X causes Turner syndrome, and P.A. Jacobs and J.A. Strong
found out that presence of an extra sex chromosome XXY causes Klinefelter syn-
drome.[8–10] In 1960, P. Nowell and D. Hungerford identifed a tiny chromosome
termed ‘Philadelphia chromosome’ in patients with chronic myelogenous leuke-
mia.[11] All these discoveries prompted great efforts from cytogenetic investigators to
improve the cell culture techniques for various types of human tissues and to develop
different staining methods for identifying individual chromosomes with recogniz-
able banding patterns.[12] At the same time, the necessity of standardized documenta-
tion of human chromosomes was recognized and proposed. An International System
for Human Cytogenetic Nomenclature (ISCN) was developed through a series of
conferences and continuously updated to incorporate new techniques for extended
clinical applications.[13] Banding cytogenetics has enabled the characterization of
constitutional and somatic numerical and structural chromosomal abnormalities
through different stages of human development and enriched the medical knowledge
of these abnormalities on spontaneous abortion, developmental delay, intellectual
disability, multiple congenital anomalies, infertility, tumorigeneses, and metastasis.

CHROMOSOME STRUCTURE AND VARIOUS BANDING TECHNIQUES

Human Chromosome Structure in Interphases and Metaphases
The double-stranded DNA of the human genome consisting of three billion nucleo-
tides is packed into interphase chromatins and metaphase chromosomes through
a multi-stage process during cell cycles. The frst stage of compaction from naked
double-stranded DNA with a width of 2 nm to a nucleosome fber with a width of
11 nm is achieved by winding approximately 200-basepair (bp) of DNA around a
core of eight histone molecules, two each for H2A, H2B, H3, and H4. In the next
stage, the nucleosome fber is twisted in a regular helix with about six nucleosomes
per turn to form a 30 nm–width solenoid. The solenoid involves a loop arrangement
radiating from a scaffold to form interphase chromatins. Chromatins form numer-
ous compact dynamic domains such as topologically associating domain, contact
domain, and loop domain to govern various functions. Further packing of chroma-
tins to metaphase chromosomes with a width of 1,400 nm can be visualized by a
light microscope.[14]
From earlier cytologic observation on interphase nuclei, condensed and darkly
stained chromatins were termed heterochromatin, and decondensed and diffused
chromatins were termed euchromatin.[15] In 1957, M.L. Barr discovered a dense mass
by Giemsa staining of interphase nuclei in female cells from oral mucosal smear,
skin biopsy, and blood specimen.[16] Later this dense stained mass termed ‘Barr
body’ was found to be facultative heterochromatin from an inactivated X chromo-
some; this simple nuclear staining is a useful diagnostic tool for detecting X chromo-
some inactivation.[17, 18] Giemsa staining of a normal human metaphase showed solid
stained 46 chromosomes of 23 pairs. Based on the size of chromosomes from large
to small, the 22 pairs of autosomes were numbered chromosomes 1 to 22, and one
Banding Cytogenetics 9

pair of sex chromosomes was named XY for male and XX for female. Each chromo-
some has a primary constriction designated as centromere. Based on the position
of the centromeres, these numbered autosomes were further divided into: group A
of metacentric chromosomes 1, 2, and 3, group B of submetacentric chromosomes
4 and 5, group C of submetacentric chromosomes 6 to 12, group D of acrocentric
chromosomes 13, 14, and 15, group E of submetacentric chromosomes 16, 17, and 18,
group F of metacentric chromosomes 19 and 20, and group G of acrocentric chromo-
somes 21 and 22. Chromosomes were ordered according to their size, and fnally it
was determined that indeed chromosome 21 is the smallest human chromosome and
not 22; still the designations were not changed. The constricted or narrow regions
outside the centromere in some chromosomes are termed secondary constrictions.
This original solid stained karyotype has been the reference for detecting numeri-
cal chromosomal abnormalities in patients, but the diffculty in distinguishing indi-
vidual chromosomes within a group severely affects its accuracy.

Various Banding Techniques

Banding techniques for human chromosomes were pursued to identify individual
chromosomes by recognizable and reproducible patterns. A band is defned as a spe-
cifc locus or a part of a chromosome with clearly distinguishable darker or lighter
staining from its adjacent segments. When metaphase chromosome preparations are
treated successively with HCl, RNase, DNA denatured by NaOH, renatured by saline
incubation, and then stained by Giemsa, a dark staining pattern in the centromere
of each chromosome is shown. This specifc C-band pattern for centromeres and the
distal long arm of chromosome Y reveals constitutive heterochromatin in the human
genome.[17] Hybridization of radioactive nucleic acids to mouse cytological prepara-
tions identifed the location of satellite DNA in the centromeric heterochromatin
regions.[19] Historically, satellite DNA was defned as genomic DNA fractions with
different buoyant densities on cesium chloride or cesium sulfate gradients from the
main fraction of genomic DNA.[20, 21] Inside nuclei, the nucleolus is the largest organ-
elle with functions in producing and assembling cellular ribosomes. The nucleolus
organizer regions (NORs) contain repeat units of ribosomal genes, which are located
in the short arms of D and G group acrocentric chromosomes. Active NORs can be
stained by silver nitrate solution to reveal the proteins in secondary constrictions on
the short arms of chromosomes 13, 14, 15, 21, and 22.[22]
In 1968, L. Zech, coworker of T. Caspersson, discovered that a banding pat-
tern on individual chromosomes by a fuorescent staining with quinacrine can be
visualized under a fuorescent microscope.[23] This Q-banding technique, named
after quinacrine staining, required a fuorescent microscope, and the fuorescent
intensity quenched quickly; therefore, it was less optimal for routine diagnostic
use. However, the Q-band pattern prompted the development of more banding
techniques. By treating metaphase chromosome preparations with dilute trypsin
and then staining with Giemsa, one can see G-band patterns along each individ-
ual chromosome under a light microscope.[24] G-banding is easy to perform, and
the staining patterns on euchromatin of each chromosome are reliable and repro-
ducible. Accordingly, G-banding was quickly accepted in clinical cytogenetics
10 Cytogenetics and Molecular Cytogenetics

laboratories and has been the most widely used banding method in chromosome
analysis. When incubating chromosome preparations in phosphate buffer at a high
temperature followed by Giemsa staining, an R-band pattern with dark and light
staining reverse of the G-band pattern was noted. Extending this treatment to a lon-
ger time or a higher temperature could erase most of the R bands but retain terminal
bands, so called T-banding at the end of each chromosome. Further improvement
in the banding technique involved the synchronization of cell cycle to capture late
prophase and prometaphase and the integration of DNA intercalating agents such
as ethidium bromide to elongate chromosomes for a high-resolution G-banding
pattern.[25, 26] Standardized protocols for these traditional banding of chromosomes
for cytogenetic analysis have been published.[27–29] A three-letter code with the frst
letter denoting the type of banding, the second letter for method of chromosome
treatment, and the third letter for the staining dye is used to describe different
banding techniques.[13] For example, GTG refers to G-Banding by Trypsin treat-
ment and Giemsa staining, and CBG refers to C-banding by Barium hydroxide
treatment and Giemsa staining. All these banding patterns refect the organization
of heterochromatin and euchromatin in human chromosomes and thus have differ-
ent applications.

Molecular Basis of Banding in Human Chromosomes

Q-banding involves staining with quinacrine, which intercalates into chromosomal
DNA and shows brighter fuorescence in regions of AT-rich DNA. G-banding in
protease-treated chromosomes is resulted from interactions of both DNA and pro-
tein with the thiazine and eosin components of the Giemsa dye. The bright and
dull Q-band patterns are consistent with the dark and light stained G-band pat-
terns. R-banding involves denaturing of chromosomes in hot acidic saline fol-
lowed by Giemsa staining. This treatment preferentially denatures AT-rich DNA
and leaves under-denatured GC-rich regions for staining; therefore, the R-banding
shows a reversed pattern of the Q/G-banding. T-banding is the staining of a subset of
GC-richest R-banding resistant to more severe heat treatment and refects the repeat
sequences at the subtelomeric regions and telomeres.
Banding techniques reveal an inherent pattern of chromosome organization in
the context of DNA sequences.[30, 31] Approximately one-third of the human genome
is composed of short- and long-interspersed repeat elements abbreviated as SINEs
and LINEs, respectively. The most abundant SINEs are Alu elements, and the
predominant LINE is retrotransposon L1. Further molecular studies revealed that
G-band positive regions are AT-rich, Alu poor, LINE rich, gene poor, and repli-
cate late, while R-band positive regions are GC-rich, Alu rich, LINE poor, gene
rich, and replicate early.[31] The structure of human metaphase chromosomes was
also analyzed under atomic force microscopy; the G-band treated chromosomes
showed a characteristic spiral pattern of chromatid fbers with ridges and grooves
corresponding to the dark and light bands.[32] Using cryo-electron tomography
and small-angle X-ray scattering analyses on metaphase chromosomes revealed
an interdigitated multilayer structure where compact chromosomes are composed
of many chromatin layers stacked along the chromosome axis.[33] Table  2.1 lists
Banding Cytogenetics 11

TABLE 2.1
Various Banding Techniques and Related Staining Patterns and Genomic
Sequences
Banding Staining Pattern Genomic Sequences Replication
Technique
Q-banding Brighter Q band AT-rich
G-banding G-band positive (dark stain) AT-rich, Alu poor, LINE rich, late
gene poor
  G-band negative (light stain) GC-rich, Alu rich, LINE poor, early
gene rich
R-banding R-band positive (dark stain) (similar to G-band negative)
  R-band negative (light stain) (similar to G-band positive)
C-banding C-band positive (dark stain) centromeric satellite sequences and
Yq12
Silver staining Positive on short arm of proteins on 5S/18S/28S ribosomal
D/G group chromosomes DNA at active NORs
T-banding Positive on both ends of subtelomeric and telomeric repeats
each chromosome

the various banding techniques and their related staining patterns and genomic
sequence organizations.
Studies from genome sequencing further defned the organization of heterochro-
matin and euchromatin in human chromosomes.[34, 35] The primary constriction or
centromere in each chromosome contains heterochromatin consisting largely of
high-order arrays of α-satellite DNA and other satellite repeats (beta, gamma, I,
II, and III). The α-satellite DNA monomers, consisting of an approximately 171 bp
repeat unit with 40% sequence divergence in different chromosomes, are tandemly
organized into distinct high-order repeat units at the centromere. A 9 bp degenerate
motif in the α-satellite serves as the binding site for centromere protein B. There are
centromeric transition regions with euchromatic gene-containing segments dupli-
catively transposed toward the pericentric regions.[35] The secondary constrictions
in the fve acrocentric chromosome arms 13p, 14p, 15p, 21p, and 22p encode the
5S, 18S, and 28S ribosomal DNA genes, which lie on a 43-kb sequence present in
approximately 50 tandem copies on each arm and are fanked by additional repeats
arranged in a complex structure.[34] Silver staining reveals the protein components
associated with these ribosomal gene transcriptions. Finally, there is a single large
region on distal Yq composed primarily of thousands of copies of several repeat
families. The heterochromatic regions all tend to be highly polymorphic in the
human population. The euchromatin of the human genome are bounded proximally
by heterochromatin and distally by a telomere consisting of several kilobases of the
hexamer repeat TTAGGG.[34]
12 Cytogenetics and Molecular Cytogenetics

G-BANDING IN NORMAL POPULATION

International System for Human Cytogenetic/Cytogenomic
Nomenclature (ISCN)
In 1971, at the fourth International Congress of Human Genetics in Paris, a group
of human cytogeneticists agreed upon a uniform system of human chromosome
identifcation. The report from this Paris Conference proposed the basic ISCN for
designating not only the short arm (p arm) and long arm (q arm) of individual chro-
mosomes but also chromosome landmarks, regions, and bands. Landmarks include
the ends of chromosome arms, the centromere, and certain bands in each chromo-
some. A region is defned as an area of a chromosome lying between two adjacent
landmarks. Regions and bands are numbered consecutively from the centromere
outward along each chromosome arm. When an existing band is subdivided in
a high-resolution banding, sub-bands are numbered and placed after a decimal
point.[13] For example, 1q31.1 stands for chromosome 1, long arm, region 3, band 1,
and sub-band 1.
Current idiograms of G-banding for normal human chromosomes are pre-
sented in fve different levels of resolution based on the number of bands. Given
the size of three billion bp or 3,000 megabase (Mb) of human genome, karyotypes
at approximately 300, 400, 550, 700, and 850 band levels have an average analytic
resolution of 10-Mb, 7.5-Mb, 5.5-Mb, 4-Mb, and 3.5-Mb per band, respectively. A
multicenter study on the subjectivity in chromosome band-level estimation recom-
mended the use of representative bands from two to seven selected chromosomes to
assess the banding level.[36] Symbols and formats for the description of numerical
and structural chromosomal abnormalities have been standardized and continu-
ously updated in the ISCN.[13] For examples, symbols +, -, t, del, dup, and der refer
to gain of a chromosome, loss of a chromosome, translocation, deletion, duplica-
tion, and derivative chromosome, respectively. Therefore, 47,XY,+21 denotes a
numerical chromosomal abnormality in a male with trisomy 21; 46,XX,t(9;22)
(q34;q11.2) denotes a structural chromosomal abnormality in a female with a
reciprocal translocation of the segment of chromosome 9 distal to band 9q34
and the segment of chromosome 22 distal to band 22q11.2; and 45,X,del(5)(p14)
denotes compound numerical and structural abnormalities in a female with a loss
of one copy of sex chromosome X and a deletion of the segment of chromosome
5 distal to band 5p14. With the integration of chromosome microarray analy-
sis, region-specifc assays, and genomic sequencing techniques, the ISCN was
changed to an International System for Human Cytogenomic Nomenclature in
the ISCN 2016 edition. The current ISCN 2020 provides not only a standardized
nomenclature but also general principles applicable to multiple techniques for the
documentation of numerical and structural chromosome abnormalities, genomic
copy number variants, and gene rearrangements.[13] Furthermore, ISCN desig-
nation of chromosomes and bands has been a reference for the human genome
sequence in the genome browser (https://genome.ucsc.edu/). Figure 2.1 shows a
normal male karyotype, the designation of chromosomes, and common chromo-
some heteromorphisms.
Banding Cytogenetics 13

FIGURE 2.1 G-banded karyotype and chromosomal heteromorphisms.

A. A normal male karyotype.
B. Designation of short arm (p), long arm (q), and centromere in a metacentric chromo-
some 1, and short arm stalk (stk) and satellite (s) of an acrocentric chromosome 14.
C. Common chromosome heteromorphisms of 1qh+, inv(9)(p12q13), 13ps+, 16qh+, 21pstk+,
and Yqh+.

Chromosome Heteromorphisms in Humans

Chromosome heteromorphisms refer to the observed variations of different size
or staining on the pericentric regions of autosomes, the short arms of acrocentric
chromosomes, and the distal long arm of Y chromosome.[37, 38] The biological and
clinical implications of these variations in normal populations were frst analyzed in
a New Haven study of 4,482 consecutively born infants. This study by solid stain-
ing of metaphase chromosomes noted heterochromatin-associated polymorphisms
14 Cytogenetics and Molecular Cytogenetics

in chromosomes 1, 9, 16, 18, and Y and estimated the relative frequency in 3,476
Caucasian and 807 African-American infants.[39] Further studies of chromosome
variations in Q-band and C-band recognized increase (+) or decrease (-) in length of
heterochromatin in the long arm (q) of chromosomes 1, 9, 16, and Y, and in the short
arm (p) or centromere (cen) of chromosomes of 13, 14, 15, 21, and 22 and X.[40] The
molecular basis of these variations related to the large arrays of tandemly repeated
DNA sequences.
Another type of human chromosome heteromorphism is pericentric inversions
likely resulting from the shuffing of satellite DNA in the heterochromatic regions.
An early study on heterochromatin size and pericentric inversion by C-banding on
80 normal Caucasians noted that the frequency of size heteromorphisms for chro-
mosomes 1, 9, and 16 were 11.3%, 47.5%, and 7.5%, respectively, and pericentric
inversions of chromosomes 1 and 9 were 20% and 23%, respectively. These was
no signifcant difference between males and females for size and position of these
heteromorphisms.[41] A more recent large case series of 1,276 individuals from India
detected pericentric inversion of chromosome 9, inv(9)(p11q13), and Robertsonian
translocation of chromosomes 13 and 14, rob(13;14)(q10;q10), in 3.68% and 1.25%
of individuals, respectively; while other chromosomal heteromorphisms of chromo-
somes 1, 9, 13, 14, 15, 21, 22, and Y were seen in 0.15%–1.95% of individuals.[42] A
molecular cytogenetic study of 334 carriers of heterochromatic variants of chromo-
some 9 recognized the presence of different type of variants and absence of evidence
linking these variants to infertility.[43] The pericentromeric region of chromosome 9
is the most obvious heteromorphism in human chromosomes; the proximal 5-Mb on
9p11 to distal 4-Mb on 9q12 comprise a mere 0.3% of the genome. These two regions
have a high density of segmental duplications and a high degree of intrachromosomal
sequence identity (98.7%). This high sequence similarity between the two regions is
likely to be the reason for a polymorphic inv(9) present in approximately 1% of
the human population.[34] A study of 2,970 prenatal cases in China showed an inci-
dence of approximately 8.8% for chromosomal heteromorphisms. The most frequent
was found to be in chromosome Yqh (5.28% males), followed by chromosome 1qh
(1.65%), inv(9) (0.94%), 22p/cen (0.77%), 15p/cen (0.64 %), 9qh (0.58%), and 16qh
(0.34%). The frequency of common chromosomal heteromorphisms and pericentric
inversions from three large case series of different populations are reviewed and
summarized in Table 2.2. The earlier New Haven study showed a higher frequency
of heteromorphisms likely due to the solid staining and related size estimation for
variant patterns.[39] Recent studies showed comparable frequencies of heteromor-
phisms between Indian and Chinese populations; furthermore, there was no evi-
dence for an association between chromosome heteromorphisms and infertility or
recurrent spontaneous abortions.[42, 44]
Chromosome heteromorphisms are inheritable traits within a family, so it is a
useful biomarker to track parental origin of chromosomal abnormalities such as the
maternal or paternal origin of trisomy 21, derivative chromosomes, and uniparental
disomy of chromosome 15.[45–47] The reporting practice on chromosome heteromor-
phisms varies among clinical cytogenetics laboratories. Survey results from more
than 200 cytogeneticists indicated that 61% of them would include selected hetero-
morphism data in a clinical report. More than 90% considered prominent short arms,
Banding Cytogenetics 15

TABLE 2.2
The Estimated Frequency of Common Chromosomal Heteromorphisms from
Different Populations
  Caucasian African-American[39] Indians[42] Chinese[44]
Infants[39] (n=807, 411 males) (n=1276, (n=2970,
(n=3476, 638 males) 1533 males)
1741 males)

Heteromorphisms Frequency Frequency (%) Frequency (%) Frequency (%)

(%)
1qh 0.23 0.37 0.54 1.65
9qh 0.15 0.58
16qh 2.59 4.33 0.34
Yqh 15.27 (males) 15.31 (males) 5.28 (males)
13cenh/ps/pstk 0.31 0.17
14cenh/ps/pstk 16.47* 26.63* 0.15 0.24
15cenh/ps/pstk 1.95 0.64
21cenh/ps/pstk 0.54 0.49
22cenh/ps/pstk 5.16* 10.04* 1.17 0.77
inv(9) 0.06 1.24 3.68 0.94
inv(Y) 0.11 (males) 1.72 (males) 0.39 (males)

qh: long arm heterochromatin, cenh: centromeric heterochromatin, ps: short arm satellite, pstk: short arm
stalk, inv: inversion
* frequency given by D or G groups

large or double satellites, or increased stalk length on acrocentric chromosomes to

be heteromorphisms; 24% to 36% stated that they would include these in a clinical
report. Heterochromatic regions on chromosomes 1, 9, 16, and Y were considered
heteromorphisms by 97% of participants, and 24% indicated they would report these
fndings. Pericentric inversions of chromosomes 1, 2, 3, 5, 9, 10, 16, and Y were
considered heteromorphisms, with more than 75% of respondents indicating they
would report these fndings.[48] More information on specifc heteromorphisms can
be found online at http://cs-tl.de/DB/CA/HCM/0-Start.html.

Inducible Fragile Sites in Human Chromosomes

Under microscopy, human chromosomes have apparent constrictions. The primary
constriction at the centromere can be seen by C-banding, and the secondary con-
striction on a site of NORs can be seen by silver staining. Other inheritable non-
random ‘constrictions’ of breaks or gaps induced by specifc cell culture conditions
are termed fragile sites.[49, 50] Fragile sites are classifed as common or rare depend-
ing on their frequency within the population and are further subdivided into dif-
ferent groups based on their specifc induction chemistry. The induction chemicals
such as folate acid, aphidicolin, bromodeoxyuridine, or 5-azatidine function as
16 Cytogenetics and Molecular Cytogenetics

partial DNA replication inhibitors at constituent sites of chromatin and make

them fail to compact during mitosis.[49] Examples of common fragile sites include
aphidicolin-inducible FRA3B (3p14.2), FRA16D (16q23.2), and FRAXD (Xq27.2),
while rare fragile sites include folate-sensitive FRA11B (11q23.3), FRA16A
(16p13.11), FRAXA (Xq27.3), FRAXE (Xq28), and FRAXF (Xq28). Most fragile
sites occur as normal variants. Some fragile sites such as FRA6E (6q26), FRA11B
(11q23.3), FRA16D (16q23.2) have been suggested as ‘hotspots’ for constitutional
and somatic terminal deletions in cancer.[49, 51] Sequencing analysis of several frag-
ile sites revealed a CCG/CGG trinucleotide repeat sequence adjacent to a CpG
island. FRAXA at Xq27.3 with an unstable CGG repeat within the fragile X men-
tal retardation 1 gene (FMR1) causes fragile X syndrome (FXS, OMIM#300624).
The CGG repeat at FRAXA is polymorphic with six to 52 repeats in normal peo-
ple and is considered as a ‘premutation’ with 48 to 200 repeats in parental carriers.
The affected males and females exhibit a massive expansion resulting in so-called
‘full mutation’ of 230 to more than 1,000 repeats. This dynamic expansion to full
mutation is associated with aberrant methylation of the FMR1 gene leading to
transcriptional suppression. FXS is an X-linked dominant disorder with an esti-
mated incidence of one in 1,500 males and one in 2,500 females. Patients with the
full mutation show reduced penetrance of 80% in males and 30% in females. The
understanding of unstable CGG repeats in the FMR1 gene has led to the switch of
its diagnosis from cytogenetic analysis of FRAXA to DNA testing of the trinucleo-
tide expansion.[52]

CLINICAL APPLICATION OF BANDING CYTOGENETICS

Banding cytogenetics has been used to detect numerical and structural chromosomal
abnormalities for spontaneous abortions, for prenatal and postnatal patients of vari-
ous clinical indications, and for various types of cancers. Technical standards and
guidelines for cytogenetic studies and reporting of constitutional (germline) and
acquired (somatic) chromosomal abnormalities have been developed.[53, 54] Routine
chromosome analysis is performed on 15–20 metaphase cells; increased cell count
is required to detect constitutional mosaicism and somatic clones in a portion of
cells. It is recommended that the analysis of 20, 50, and 100 metaphases could detect
a mosaic pattern or a clonal abnormality in 14%, 6%, and 3% of cells with a 95%
confdence, respectively.[55]

Detection of Constitutional Chromosome Abnormalities

Chromosomal abnormalities have been detected in different human development
stages. Studies on products of conception from spontaneous abortions recognized
that approximately 50% of the spontaneous abortions are resulted from aneuploi-
dies, polyploidies, and structural abnormalities with a relative frequency of 77%,
18%, and 5%, respectively. The most frequently seen aneuploidies are monosomy X,
trisomy 16, trisomy 21, trisomy 22, trisomy 15, and trisomy 18.[56] Loss of one copy
of any autosome is rarely observed and incompatible to life. It is estimated that 94%
of fetuses carrying chromosomal abnormalities will end in a spontaneous abortion,
Banding Cytogenetics 17

indicating a strong natural selection against major chromosomal abnormalities in

human biology.
Clinical indications for prenatal cytogenetics testing include advanced mater-
nal age, abnormal ultrasound fnding, serum screening, and non-invasive prenatal
screening (NIPS—also called non-invasive prenatal testing or NIPT) on cell-free
fetal DNA from maternal serum. NIPS/NIPT has a high positive predictive value
for aneuploidy, which have reduced invasive procedures by approximately 60% and
almost doubled the diagnostic yield of chromosomal abnormalities in current pre-
natal diagnosis.[57, 58] The detection rate of chromosomal abnormalities is approxi-
mately 15%; the diagnostic effcacy measured by detected cases vs expected cases by
newborn incidence reaches 92% for trisomy 21 (Down syndrome, OMIM#190685)
and monosomy X (Turner syndrome), but only 0.1%–0.4% for other sex chromo-
some aneuploidies (XXY/XXX/XYY).[59] The low detection rate for sex chromo-
some aneuploidies is likely due to the variable positive predictive value from NIPS/
NIPT, reduced follow-up rate for cytogenetic diagnosis, and less severe clinical
implications. Since there is an estimated 3% to 20% chance of a mosaic pattern
for sex chromosome aneuploidies and 3% chance for structural rearrangements of
sex chromosomes, follow-up cytogenetic studies could provide accurate diagnosis
for karyotype-phenotype correlation and for genetic counseling on parental studies
for future pregnancies.[60] Prenatal diagnosis of common syndromic chromosomal
abnormalities contributed signifcantly to reduce birth defects and improve clinical
management.
For pediatric patients with intellectual disability, developmental delay, multiple
congenital anomalies, and dysmorphic features, the detection rate for numerical and
structural chromosomal abnormalities were approximately 5% and 3%, respectively.
The diagnostic effcacy for Down syndrome, Turner syndrome, Klinefelter syndrome
(47,XXY), 47,XXX, 47,XYY, and balanced Robertsonian translocations were 63%,
48%, 14%, 4%, 3%, and 2% respectively.[59] The low detection rate for the latter three
abnormalities likely related to the mild to normal phenotypes at childhood and may
be noted in adulthood due to reproductive problems. Aneuploidy has been observed
in approximately 1–4% of sperms, 10–35% of eggs and polar bodies, 20–40% of
preimplantation embryos, 50% of spontaneous abortions, and 0.3% of newborns.[61]
Inter-chromosomal heterogeneity of centromeric features infuences chromosome
segregation fdelity and results in non-random aneuploidy.[62]
Various types of balanced and unbalanced structural chromosomal abnormalities
have been detected in approximately 2% of prenatal and 3% of pediatric patients.[59]
The detection of balanced translocations could bring attention to the risk of unbal-
anced derivative chromosomes for fetuses. A well-known example is the carriers of
a Robertsonian translocation between the short arms of D/G group acrocentric chro-
mosomes. With an incidence of 1 in 1,000 in the general population, Robertsonian
translocations are likely resulted from fusions of the repeat sequences at NOR sites.[34]
The risk of Down syndrome for carriers of a Robertsonian translocation involving a
chromosome 21 and another D/G group chromosome was estimated to be 10%–15%
for a maternal carrier and <1% for a paternal carrier. However, for a carrier of a
Robertsonian translocation of 21q, rob(21;21), the risk for Down syndrome is 100%.
18 Cytogenetics and Molecular Cytogenetics

Unbalanced chromosomal structural abnormalities include chromosomally vis-

ible deletions, duplications, inversions, ring chromosomes, and complex rearrange-
ments of several chromosomes. Chromosome microarray analysis provides further
characterization of genomic imbalances and involved gene content in these struc-
tural abnormalities. For example, chromosomally observed deletions of 4q could be
further defned as containing distal duplications and deletions.[63] Prenatal diagnosis
and postnatal fndings of a fetus with partial trisomy of 13q21.33-qter and partial
monosomy of 10p15.3-pter led to the detection of a balanced translocation, t(10;13)
(p15.3;q21.33) in the mother and explained a history of two neonatal deaths and one
spontaneous abortion.[64] Ring chromosomes 21 showed unique genomic structure
with a terminal deletion and interstitial duplication or deletion and distinct mitotic
behaviors.[65] Prenatal diagnosis on recurrence of a duplication of Xq26.1-q26.3 in
two male fetuses indicated that the normal mother could have gonadal mosaicism.[66]
Further clinical, bioinformatic and functional analyses could provide precise geno-
type-phenotype correlations for dosage sensitive genes in the human genome.[51,  67]
Online databases for interpreting gene dosage effects (ClinVar) and pathogenic copy
number variants (ClinGen, DECIPHER) are available. Figure  2.2 shows various
types of autosomal and sex chromosome abnormalities by G-banding.

Detection of Somatic Chromosome Abnormalities

Banding cytogenetics played an important role on detecting recurrent chromosomal
rearrangements of diagnostic signifcance, prognostic risk stratifcation, and targeted
therapy or treatments for various types of tumors.[68] The classical example is the so-
called ‘Philadelphia chromosome’ observed initially by solid staining of metaphases
from patients with chronic myelogenous leukemia (CML). Further banding analysis
indicated that the Philadelphia chromosome is the derivative chromosome 22 from
a reciprocal translocation t(9;22)(q34.1;q11.2). Molecular characterization of this
translocation defned underlying BCR‑ABL1 gene fusion with increased expression
of tyrosine kinase, which led to the development of tyrosine kinase inhibitor for the
treatment of this leukemia.[69, 70]
Following World Health Organization (WHO) classifcation of tumors of hemato-
poietic and lymphoid tissues, many recurrent somatic numerical and structural chro-
mosomal rearrangements are of diagnostic, prognostic, and therapeutic values.[71]
For example, in pediatric patients with B-cell precursor acute lymphoblastic leuke-
mia (B-ALL), a hyperdiploid clone with trisomies of chromosomes 4, 10, and 17, or
a translocation (12;21)(p13;q22) with ETV6‑RUNX1 gene rearrangement predicts a
good prognosis, while a translocation t(9;22)(q34.1;q11.2), rearrangements involving
the KMT2A gene at 11q23, and intra-chromosomal amplifcation of chromosome 21
(iAMP21) are associated with an unfavorable prognosis.[72] Other specifc rearrange-
ments such as jumping translocations of 1q likely related to the heterochromatic
region have been reported in myelodysplastic syndrome (MDS) and acute myeloid
leukemia (AML).[73]
Specifc recurrent chromosomal abnormalities have been seen in various type
of solid tumors. For example, reciprocal and three-way translocations involv-
ing the ETV6 gene at 12p13.2 and the NTRK3 gene at 15q25.3 have been associ-
ated with congenital fbrosarcoma.[74] Complex rearrangements involving multiple
Banding Cytogenetics 19

FIGURE 2.2 Various types of autosomal and sex chromosome abnormalities by G-banding.

A. A pedigree showing a mother (I.1) carrying a balanced translocation t(10;13) with a his-
tory of two neonatal deaths (II.1, II.2), a spontaneous abortion (SAB, II.3), and a daughter
(II.4) carrying a derivative chromosome 10.
B. Cases of ring chromosomes 6, 9, 18 and 21 and derived variant ring broken chromosome,
dicentric (dic) and tricentric (trc) ring chromosomes.
C. A female with an isochromosome of Xq.
D. A male with a derivative Y chromosome from a distal duplication and interstitial deletion
in the short arm and deletion of the long arm as defned by a microarray analysis.
E. A pedigree showing mother to daughter transmission of a derivative chromosome 14 with
enlarged short arm satellite region containing translocated Yq material as shown in the
insert of a positive hybridization signal of DYZ1 probe to the derivative der(14).

chromosomes such as a giant ring or marker chromosome have been seen in lipo-
sarcoma.[75] A review of profciency test results from over 200 cytogenetics labo-
ratories for a 20-year period showed that more than 96% of laboratories provided
correct interpretation of chromosomal abnormalities of hematological neoplasms.[76]
Online databases such as the ‘Atlas of Genetics and Cytogenetics in Oncology and
20 Cytogenetics and Molecular Cytogenetics

Haematology’ http://atlasgeneticsoncology.org/ are available for interpreting somatic

recurrent chromosomal abnormalities.

Report and Interpretation of Diagnostic Karyotype

Reporting and interpretation of constitutional or acquired chromosomal abnor-
malities follows the general principles of ISCN 2020 and standardized statements
from diagnostic practice.[13, 77] For constitutional abnormalities, clinical impact on
physical and intellectual development, congenital anomalies on organs and systems,
reproductive ftness, cancer predisposition risk, and family history should be taken
into consideration. Extended work up on additional metaphases should be pursued
to differentiate true mosaicism from pseudomosaicism and to resolve mosaicism
confned to the placenta in chorionic villus sampling.[55] Occasional metaphases
with random gain or loss of one chromosome, inversion inv(14)(q11.2q32), inversion
inv(7)(p14q35), or translocation t(7;14) in phytohemagglutinin-stimulated T cells
could represent a culture artifact.[77] However, these inversions and translocations
between chromosomes 7 and 14 in 10–45% of stimulated T cells is indicative for
Nijmegen breakage syndrome (OMIM#251260), and further sequencing analysis on
the NBS1 gene should be recommended.
For acquired abnormalities, tumor classifcation and stratifcation, prognostic
risk, and targeted therapy related to the clonal abnormalities should be presented in
the report. Chromosomal abnormalities with impact on therapy such as transloca-
tion t(15;17)(q24;q21) with PML‑RARA gene fusion in acute promyelocytic leukemia
should be reported and communicated to referring physicians in a timely manner.
Clonal abnormalities with a complex karyotype defned by the presence of three or
more unrelated structural and numerical aberrations have been seen in a signifcant
portion of cancers. The interpretation of complex karyotypes requires clinical evi-
dence, expert consensus, and molecular and genomic fndings. For example, a scor-
ing practice counting the number of chromosome aberrations in complex karyotype
has been proposed to correlate with the international prognosis scoring system for
patients with myelodysplastic syndrome (MDS).[78] Furthermore, the poor prognosis
associated with complex karyotypes in MDS could be driven by adverse TP53 gene
mutations.[79] Refnement of cytogenetic classifcation in acute myeloid leukemia
revised the prognostic classifcation for rare recurring chromosomal abnormalities
and complex karyotypes.[80] A retrospective analysis on a large case series of chronic
lymphocytic leukemia defned cytogenetic complexity at levels of low or interme-
diate (3 to 4 aberrations) and high (≥5 aberrations) and presented their associated
prognostic risk in combination with molecular fndings.[81] For many patients, diag-
nostic karyotype provides the framework for further molecular analysis and clinical
investigation.

CONCLUSIONS
Banding cytogenetics has provided structural basis for the understanding of
sequence organization in human chromosomes and cytological landmarks for draft-
ing the human genome. Technical standards and guidelines of G-band analysis have
Banding Cytogenetics 21

been developed and implemented for clinical cytogenetics. Current ISCN 2020 pro-
vides general principles applicable to multiple molecular and genomic techniques
and standardized documentation of chromosomal abnormalities, pathogenic copy
number variants, and gene rearrangements. Banding cytogenetics has been and con-
tinues to be an effective and highly profcient cell-based approach to detect constitu-
tional numerical and structural chromosomal abnormalities in prenatal and pediatric
patients and somatic clonal chromosomal abnormalities in various types of cancers.

WEB RESOURCES
Atlas of Genetics and Cytogenetics in Oncology and Haematology (http://
atlasgeneticsoncology.org/)
Cases with heteromorphisms (http://cs-tl.de/DB/CA/HCM/0-Start.html)
ClinVar (www.ncbi.nlm.nih.gov/clinvar/)
ClinGen (https://clinicalgenome.org/)
DECIPHER (www.deciphergenomics.org/)
Human Genome Browser (https://genome.ucsc.edu/)

ACKNOWLEDGEMENT
Special thanks to Autumn DiAdamo and Jiadi Wen of Yale Clinical Cytogenetics
Laboratory for helpful discussion and careful editing on the content of this chapter.

REFERENCES
1. Boveri, O.G.M. Über Mitosen bei einseitiger Chromosomenbindung. Jenaische
Zeitschrift für Naturwissenschaft. 1903, 37, 401–443.
2. Sutton, S.W. The chromosomes in heredity. Biol. Bull. 1903, 4, 231–251.
3. Gartler, S.M. The chromosome number in humans: A brief history. Nat. Rev. Genet.
2006, 7, 655–660.
4. Painter, S.T. Studies in mammalian spermatogenesis. II: The spermatogenesis of man.
J. Exp. Zool. 1923, 37, 291–338.
5. Ruddle, F.H. Theophilus painter: First steps toward an understanding of the human
genome. J. Exp. Zool. A. Comp. Exp. Biol. 2004, 301, 375–377.
6. Hsu, T.C. Mammalian chromosomes in vitro, I. Karyotype of man. J. Heredity. 1952, 43,
167–172.
7. Tjio, J.H.; Levan, A. The chromosome number of man. Hereditas. 1956, 42, U1–6.
8. Ford, C.E.; Jones, K.W.; Polani, P.E.; De Almeida, J.C.; Briggs, J.H. A sex-chromosome
anomaly in a case of gonadal dysgenesis (Turner’s syndrome). Lancet. 1959, 1, 711–713.
9. Jacobs, P.A.; Strong, J.A. Case of human intersexuality having a possible XXY sex-
determining mechanism. Nature. 1959, 183, 302–303.
10. Lejeune, J.; Turpin, R.; Gautier, M. Chromosomic diagnosis of mongolism. Arch. Fr.
Pediatr. 1959, 16, 962–963.
11. Nowell, P.C.; Hungerford, D.A. A minute chromosome in human chronic granulocytic
leukemia. Science. 1960, 132, 1497.
12. Priest, J.H. Medical Cytogenetics and Cell Culture. Henry Kimpton Publisher, London;
1977.
22 Cytogenetics and Molecular Cytogenetics

13. McGowan-Jordan, J.; Hastings, R.J.; Moore, S. ISCN 2020: An International System for
Human Cytogenomic Nomenclature. Karger, Basel; 2020.
14. Maeshima, K.; Iida, S.; Tamura, S. Physical nature of chromatin in the nucleus. Cold
Spring Harb. Perspect. Biol. 2021, 13, a040675.
15. Brown, S.W. Heterochromatin. Science. 1966, 151, 417–425.
16. Barr, M.L. Dysgenesis of the seminiferous tubules. Br. J. Urol. 1957, 29, 251–257.
17. Arrighi, F.E.; Hsu, T.C. Localization of heterochromatin in human chromosomes.
Cytogenetics. 1971, 10, 81–86.
18. Barr, M.L. Sex chromatin and phenotype in man. Science. 1959, 130, 679–685.
19. Pardue, M.L.; Gall, J.G. Chromosomal localization of mouse satellite DNA. Science.
1970, 168, 1356–1358.
20. Corneo, G.; Ginelli, E.; Polli, E. Repeated sequences in human DNA. J. Mol. Biol. 1970,
48, 319–327.
21. Ginelli, E.; Corneo, G. The organization of repeated DNA sequences in the human
genome. Chromosoma. 1976, 56, 55–68.
22. Tantravahi, R.; Miller, D.A.; Dev, V.G.; Miller, O.J. Detection of nucleolus orga-
nizer regions in chromosomes of human, chimpanzee, gorilla, orangutan and gibbon.
Chromosoma. 1976, 56, 15–27.
23. Caspersson, T.; Farber, S.; Foley, G.E.; Kudynowski, J.; Modest, E.J.; Simonsson, E.;
Wagh, U.; Zech, L. Chemical differentiation along metaphase chromosomes. Exp. Cell.
Res. 1968, 49, 219–222.
24. Seabright, M. A rapid banding technique for human chromosomes. Lancet. 1971, 2,
971–972.
25. Hoo, J.J.; Jamro, H.; Schmutz, S.; Lin, C.C. Preparation of high resolution chromosomes
from amniotic fuid cells. Prenat. Diagn. 1983, 3, 265–267.
26. Yunis, J.J.; Sawyer, J.R.; Ball, D.W. The characterization of high-resolution G-banded
chromosomes of man. Chromosoma. 1978, 67, 293–307.
27. Arsham, M.S.; Barch, M.J.; Lawce, H.J. The AGT Laboratory Manual. 4th Edition.
Wiley-Blackwell, Hoboken, NJ; 2017.
28. Rooney, D.E.; Czepulkowski, B.H. Human Cytogenetics: A Practical Approach Volume
I: Constitutional Analysis. 2nd Edition. Oxford University Press, Oxford; 1992.
29. Rooney, D.E.; Czepulkowski, B.H. Human Cytogenetics: A Practical Approach Volume
II. Malignancy and Acquired Abnormalities. 2nd Edition. Oxford University Press,
Oxford; 1992.
30. Bickmore, W.A. Patterns in the genome. Heredity (Edinb). 2019, 123, 50–57.
31. Gardiner, K. Human genome organization. Curr. Opin. Genet. Dev. 1995, 5, 315–322.
32. Ushiki, T.; Hoshi, O.; Iwai, K.; Kimura, E.; Shigeno, M. The structure of human meta-
phase chromosomes: Its histological perspective and new horizons by atomic force
microscopy. Arch. Histol. Cytol. 2002, 65, 377–390.
33. Chicano, A.; Crosas, E.; Oton, J.; Melero, R.; Engel, B.D.; Daban, J.R. Frozen-hydrated
chromatin from metaphase chromosomes has an interdigitated multilayer structure.
EMBO J. 2019, 38, e99769.
34. Collins, F.S.; Lander, E.S.; Rogers, J.; Waterston, R.H.; Conso, I.H.G.S. Finishing the
euchromatic sequence of the human genome. Nature. 2004, 431, 931–945.
35. She, X.W.; Horvath, J.E.; Jiang, Z.S.; Liu, G.; Furey, T.S.; Christ, L.; Clark, R.; Graves,
T.; Gulden, C.L.; Alkan, C.; Bailey, J.A.; Sahinalp, C.; Rocchi, M.; Haussler, D.; Wilson,
R.K.; Miller, W.; Schwartz, S.; Eichler, E.E. The structure and evolution of centromeric
transition regions within the human genome. Nature. 2004, 430, 857–864.
Banding Cytogenetics 23

36. Geiersbach, K.B.; Gardiner, A.E.; Wilson, A.; Shetty, S.; Bruyère, H.; Zabawski, J.; Saxe,
D.F.; Gaulin, R.; Williamson, C.; Van Dyke, D.L. Subjectivity in chromosome band-level
estimation: A multicenter study. Genet. Med. 2014, 16, 170–175.
37. Bishop, A.; Blank, C.E.; Hunter, H. Heritable variation in the length of the Y chromo-
some. The Lancet. 1962, 280, 18–20.
38. Starkman, M.N.; Shaw, M.W. Atypical acrocentric chromosomes in Negro and Caucasian
Mongols. Am. J. Hum. Genet. 1967, 19, 162–173.
39. Lubs, H.A.; Ruddle, F.H. Chromosome polymorphism in American Negro and White
populations. Nature. 1971, 233, 134–136.
40. McKenzie, W.H.; Lubs, H.A. Human Q and C chromosomal variations: Distribution and
incidence. Cytogenet. Cell Genet. 1975, 14, 97–115.
41. Verma, R.S.; Dosik, H.; Lubs, H.A. Size and pericentric inversion heteromorphisms of
secondary constriction regions (h) of chromosomes 1, 9, and 16 as detected by CBG
technique in Caucasians: Classifcation, frequencies, and incidence. Am. J. Med. Genet.
1978, 2, 331–339.
42. Rawal, L.; Kumar, S.; Mishra, S.R.; Lal, V.; Bhattacharya, S.K. Clinical manifestations
of chromosomal anomalies and polymorphic variations in patients suffering from repro-
ductive failure. J. Hum. Reprod. Sci. 2020, 13, 209–215.
43. Kosyakova, N.; Grigorian, A.; Liehr, T.; Manvelyan, M.; Simonyan, I.; Mkrtchyan,
H.; Aroutiounian, R.; Polityko, A.D.; Kulpanovich, A.I.; Egorova, T.; Jaroshevich, E.;
Frolova, A.; Shorokh, N.; Naumchik, I.V.; Volleth, M.; Schreyer, I.; Nelle, H.; Stumm,
M.; Wegner, R.D.; Reising-Ackermann, G.; Merkas, M.; Brecevic, L.; Martin, T.;
Rodríguez, L.; Bhatt, S.; Ziegler, M.; Kreskowski, K.; Weise, A.; Sazci, A.; Vorsanova,
S.; Cioff, M.deB.; Ergul, E. Heteromorphic variants of chromosome 9. Mol. Cytogenet.
2013, 6, 14.
44. Zhu, J.J.; Qi, H.; Cai, L.; Wen, X.H.; Zeng, W.; Tang, G.D.; Luo, Y.; Meng, R.; Mao,
X.Q.; Zhang, S.Q. C-banding and AgNOR-staining were still effective complementary
methods to indentify chromosomal heteromorphisms and some structural abnormalities
in prenatal diagnosis. Mol. Cytogenet. 2019, 12, 41.
45. Ceylaner, G.; Ceylaner, S.; Danişman, N.; Ergün, A.; Ekici, E.; Schinzel, A.; Baumer,
A. Chromosomal heteromorphisms may help for the diagnosis of uniparental disomy
(UPD): A case report. Prenat. Diag. 2007, 27, 1072–1074.
46. Magenis, R.E.; Overton, K.M.; Chamberlin, J.; Brady, T.; Lovrien, E. Parental origin of
the extra chromosome in Down’s syndrome. Hum. Genet. 1977, 37, 7–16.
47. Magenis, R.E.; Sheehy, R.R.; Brown, M.G.; McDermid, H.E.; White, B.N.; Zonana, J.;
Weleber, R. Parental origin of the extra chromosome in the cat eye syndrome: Evidence
from heteromorphism and in situ hybridization analysis. Am. J. Med. Genet. 1988, 29,
9–19.
48. Brothman, A.R.; Schneider, N.R.; Saikevych, I.; Cooley, L.D.; Butler, M.G.; Patil, S.;
Mascarello, J.T.; Rao, K.W.; Dewald, G.W.; Park, J.P.; Persons, D.L.; Wolff, D.J.; Vance,
G.H.; Cytogenetics Resource Committee.; College of American Pathologists/American
College of Medical Genetics. Cytogenetic heteromorphisms: Survey results and report-
ing practices of Giemsa-band regions that we have pondered for years. Arch. Pathol.
Lab. Med. 2006, 130, 947–949.
49. Lukusa, T.; Fryns, J.P. Human chromosome fragility. Biochim. Biophys. Acta. 2008,
1779, 3–16.
50. Sutherland, G.R.; Richards, R.I. The molecular basis of fragile sites in human chromo-
somes. Curr. Opin. Genet. Dev. 1995, 5, 323–327.
24 Cytogenetics and Molecular Cytogenetics

51. Xie, X.; Chai, H.; DiAdamo, A.; Grommisch, B.; Wen, J.; Zhang, H.; Li, P. Genotype-
phenotype correlations for putative haploinsuffcient genes in deletions of 6q26-q27:
Report of eight patients and review of literature. Glob. Med. Genet. 2022, 9, 166–174.
52. Warren, S.T.; Nelson, D.L. Advances in molecular analysis of fragile X syndrome.
JAMA. 1994, 271, 536–542.
53. Mikhail, F.M.; Heerema, N.A.; Rao, K.W.; Burnside, R.D.; Cherry, A.M.; Cooley, L.D.
Section E6.1–6.4 of the ACMG technical standards and guidelines: Chromosome stud-
ies of neoplastic blood and bone marrow-acquired chromosomal abnormalities. Genet.
Med. 2016, 18. 635–642.
54. Silva, M.; de Leeuw, N.; Mann, K.; Schuring-Blom, H.; Morgan, S.; Giardino, D.; Rack,
K.; Hastings, R. European guidelines for constitutional cytogenomic analysis. Eur. J.
Hum. Genet. 2019, 27, 1–16.
55. Hook, E.B. Exclusion of chromosomal mosaicism: Tables of 90%.; 95% and 99% conf-
dence limits and comments on use. Am. J. Hum. Genet. 1977, 29, 94–97.
56. Zhou, Q.H.; Wu, S.Y.; Amato, K.; DiAdamo, A.; Li, P.N. Spectrum of cytogenomic
abnormalities revealed by array comparative genomic hybridization on products of con-
ception culture failure and normal karyotype samples. J. Genet Genomics. 2016, 43,
121–131.
57. Chai, H.; DiAdamo, A.; Grommisch, B.; Boyle, J.; Amato, K.; Wang, D.; Wen, J.; Li,
P. Integrated FISH, karyotyping and aCGH analyses for effective prenatal diagnosis of
common aneuploidies and other cytogenomic abnormalities. Med. Sci. (Basel). 2019,
7, 16.
58. Meng, J.; Matarese, C.; Crivello, J.; Wilcox, K.; Wang, D.; DiAdamo, A.; Xu, F.; Li,
P. Changes in and effcacies of indications for invasive prenatal diagnosis of cytoge-
nomic abnormalities: 13 years of experience in a single center. Med. Sci. Monit. 2015,
21, 1942–1948.
59. Chai, H.; DiAdamo, A.; Grommisch, B.; Xu, F.; Zhou, Q.; Wen, J.; Mahoney, M.; Bale,
A.; McGrath, J.; Spencer-Manzon, M.; Li, P.; Zhang, H. A retrospective analysis of
10-year data assessed the diagnostic accuracy and effcacy of cytogenomic abnormali-
ties in current prenatal and pediatric settings. Front. Genet. 2019, 10, 1162.
60. Xie, X.; Tan, W.; Li, F.; Ramirez, P.; DiAdamo, A.; Grommisch, B.; Amato, K.; Chai,
H.; Wen, J.; Li, P. Diagnostic cytogenetic testing following positive noninvasive prenatal
screening results of sex chromosome abnormalities: Report of fve cases and systematic
review of evidence. Mol. Genet. Genomic Med. 2020, 8, e1297.
61. Nagaoka, S.I.; Hassold, T.J.; Hunt, P.A. Human aneuploidy: Mechanisms and new
insights into an age-old problem. Nat. Rev. Genet. 2012, 13, 493–504.
62. Dumont, M.; Gamba, R.; Gestraud, P.; Klaasen, S.; Worrall, J.T.; De Vries, S.G.;
Boudreau, V.; Salinas-Luypaert, C.; Maddox, P.S.; Lens, S.M.; Kops, G.J.; McClelland,
S.E.; Miga, K.H.; Fachinetti, D. Human chromosome-specifc aneuploidy is infuenced
by DNA-dependent centromeric features. EMBO J. 2020, 39, e102924.
63. Rossi, M.R.; DiMaio, M.S.; Xiang, B.; Lu, K.; Kaymakcalan, H.; Seashore, M.; Mahoney,
M.J.; Li, P. Clinical and genomic characterization of distal duplications and deletions of
chromosome 4q: Study of two cases and review of the literature. Am. J. Med. Genet. A.
2009, 149A, 2788–2794.
64. Wei, Y.; Gao, X.; Yan, L.; Xu, F.; Li, P.; Zhao, Y. Prenatal diagnosis and postnatal fol-
lowup of partial trisomy 13q and partial monosomy 10p: A case report and review of the
literature. Case Rep. Genet. 2012, 2012, 821347.
65. Zhang, H.Z.; Xu, F.; Seashore, M.; Li, P. Unique genomic structure and distinct mitotic
behavior of ring chromosome 21 in two unrelated cases. Cytogenet. Genome Res. 2012,
136, 180–187.
Banding Cytogenetics 25

66. Cook, S.; Wilcox, K.; Grommisch, B.; Li, P.; Xu, F. Prenatal diagnosis of Xq26.1-q26.3
duplication in two fetuses of a woman with gonadal mosaicism. North Am. J. Med. Sci.
2014, 7, 176–179.
67. Xu, F.; Li, L.; Schulz, V.P.; Xiang, B.; Zhao, H.; Li, P. Cytogenomic mapping and bioin-
formatic mining reveal interacting brain expressed genes for intellectual disability. Mol.
Cytogenet. 2014, 7, 4.
68. Qumsiyeh, M.B.; Li, P. Chapter 5: Molecular Biology of Cancer: Cytogenetics.
Philadelphia: Lippincott Williams & Wilkins Publishers; 2001.
69. Nowell, P.C.; Hungerford, D.A. Chromosome studies on normal and leukemic human
leukocytes. J. Natl. Cancer. Inst. 1960, 25, 85–109.
70. Rowley, J.D. Genetics: A story of swapped ends. Science. 2013, 340, 1412–1413.
71. Swerdlow, S.H.; Campo, E.; Pileri, S.A.; Harris, N.L.; Stein, H.; Siebert, R.; Advani, R.;
Ghielmini, M.; Salles, G.A.; Zelenetz, A.D.; Jaffe, E.S. The 2016 revision of the World
Health Organization classifcation of lymphoid neoplasms. Blood. 2016, 127, 2375–2390.
72. Moorman, A.V. New and emerging prognostic and predictive genetic biomarkers in
B-cell precursor acute lymphoblastic leukemia. Haematologica. 2016, 101, 407–416.
73. Couture, T.; Amato, K.; DiAdamo, A.; Li, P. Jumping translocations of 1q in myelo-
dysplastic syndrome and acute myeloid leukemia: Report of three cases and review of
literature. Case Rep. Genet. 2018, 2018, 8296478.
74. Marino-Enriquez, A.; Li, P.; Samuelson, J.; Rossi, M.R.; Reyes-Mugica, M. Congenital
fbrosarcoma with a novel complex 3-way translocation t(12;15;19) and unusual histo-
logic features. Hum. Pathol. 2008, 39, 1844–1848.
75. Chai, H.; Xu, F.; DiAdamo, A.; Grommisch, B.; Mao, H.; Li, P. Cytogenomic character-
ization of giant ring or rod marker chromosomes on four cases of well-differentiated and
dedifferentiated liposarcomas. Case Rep. Genet. 2022, 2022, 6341207 .
76. Larson, D.P.; Akkari, Y.M.; Van Dyke, D.L.; Raca, G.; Gardner, J.A.; Rehder, C.W.;
Kaiser-Rogers, K.A.; Eagle, P.; Yuhas, J.A.; Gu, J.; Toydemir, R.M.; Kearney, H.; Conlin,
L.K.; Tang, G.; Dolan, M.M.; Ketterling, R.P.; Peterson, J.F. Conventional cytogenetic
analysis of hematologic neoplasms: A 20-year review of profciency test results from the
college of American pathologists/American college of medical genetics and genomics
cytogenetics committee. Arch. Pathol. Lab. Med. 2021, 145, 176–190.
77. Giersch, A.B.S.; Bieber, F.R.; Dubuc, A.M.; Fletcher, J.A.; Ligon, A.H.; Mason-Suares,
H.; Morton, C.C.; Weremowicz, S.; Xiao, S.; Cin, P.D. Reporting of diagnostic cytoge-
netic results. Curr. Protoc. Hum. Genet. 2016, 89, A 1D 1-A 1D 23.
78. Chun, K.; Hagemeijer, A.; Iqbal, A.; Slovak, M.L. Implementation of standardized inter-
national karyotype scoring practices is needed to provide uniform and systematic evalu-
ation for patients with myelodysplastic syndrome using IPSS criteria: An International
Working Group on MDS cytogenetics study. Leuk. Res. 2010, 34, 160–165.
79. Grimwade, D.; Hills, R.K.; Moorman, A.V.; Walker, H.; Chatters, S.; Goldstone, A.H.;
Wheatley, K.; Harrison, C.J.; Burnett, A.K; National Cancer Research Institute Adult
Leukaemia Working Group. Refnement of cytogenetic classifcation in acute myeloid
leukemia: Determination of prognostic signifcance of rare recurring chromosomal
abnormalities among 5876 younger adult patients treated in the United Kingdom Medical
Research Council trials. Blood. 2010, 116, 354–365.
80. Baliakas, P.; Jeromin, S.; Iskas, M.; Puiggros, A.; Plevova, K.; Nguyen-Khac, F.; Davis,
Z.; Rigolin, G.M.; Visentin, A.; Xochelli, A.; Delgado, J.; Baran-Marszak, F.; Stalika,
E.; Abrisqueta, P.; Durechova, K.; Papaioannou, G.; Eclache, V.; Dimou, M.; Iliakis,
T.; Collado, R.; Doubek, M.; Calasanz, M.J.; Ruiz-Xiville, N.; Moreno, C.; Jarosova,
M.; Leeksma, A.C.; Panayiotidis, P.; Podgornik, H.; Cymbalista, F.; Anagnostopoulos,
A.; Trentin, L.; Stavroyianni, N.; Davi, F.; Ghia, P.; Kater, A.P.; Cuneo, A.; Pospisilova,
26 Cytogenetics and Molecular Cytogenetics

S.; Espinet, B.; Athanasiadou, A.; Oscier, D.; Haferlach, C.; Stamatopoulos, K.; ERIC.;
the European Research Initiative on CLL. Cytogenetic complexity in chronic lympho-
cytic leukemia: Defnitions, associations, and clinical impact. Blood. 2019, 133(11),
1205–1216.
81. Haase, D.; Stevenson, K.E.; Neuberg, D.; Maciejewski, J.P.; Nazha, A.; Sekeres,
M.A.; Ebert, B.L.; Garcia-Manero, G.; Haferlach, C.; Haferlach, T.; Kern, W.; Ogawa,
S.; Nagata, Y.; Yoshida, K.; Graubert, T.A.; Walter, M.J.; List, A.F.; Komrokji, R.S.;
Padron, E.; Sallman, D.; Papaemmanuil, E.; Campbell, P.J.; Savona, M.R.; Seegmiller,
A.; Adès, L.; Fenaux, P.; Shih, L.Y.; Bowen, D.; Groves, M.J.; Tauro, S.; Fontenay, M.;
Kosmider, O.; Bar-Natan, M.; Steensma, D.; Stone, R.; Heuser, M.; Thol, F.; Cazzola,
M.; Malcovati, L.; Karsan, A.; Ganster, C.; Hellström-Lindberg, E.; Boultwood, J.;
Pellagatti, A.; Santini, V.; Quek, L.; Vyas, P.; Tüchler, H.; Greenberg, P.L.; Bejar, R.;
International Working Group for MDS Molecular Prognostic Committee. TP53 muta-
tion status divides myelodysplastic syndromes with complex karyotypes into distinct
prognostic subgroups. Leukemia. 2019, 33, 1747–1758.
 3 Generation of
Microdissection-
Derived Painting
Probes from Single
Copy Chromosomes
Svetlana Romanenko and Vladimir Trifonov

CONTENTS
Introduction.............................................................................................................. 27
Materials .................................................................................................................. 28
Instruments ..................................................................................................... 28
Reagents.......................................................................................................... 28
Solutions ......................................................................................................... 29
Description of Methods............................................................................................ 29
Micro-Needle and Micropipette Preparation .................................................. 29
Slide Preparation............................................................................................. 29
Chromosome Staining .................................................................................... 29
Microdissection............................................................................................... 30
Generation of Paint Probe by Whole Genome Amplifcation (WGA) ........... 31
Expected Results...................................................................................................... 31
Acknowledgements.................................................................................................. 32
References................................................................................................................ 32

INTRODUCTION
The method of chromosome microdissection with a subsequent DNA isolation was
developed over 40 years ago. It allows physically isolating either a whole or a large
fragment of a chromosome. After early works on Drosophila polytene,[1] murine,[2]
and human[3] metaphase chromosomes, the method was considerably improved by
Senger and colleagues,[4] who frst employed a combination of an inverted micro-
scope equipped with a rotating plate, including a deproteinization step, followed
by DNA amplifcation. From 1992 onwards, most paints for fuorescence in situ
hybridization (FISH) have been made by direct polymerase chain reaction (PCR)
amplifcation, using a degenerate universal primer (DOP-PCR).[5] Subsequent major

DOI: 10.1201/9781003223658-3 27
28 Cytogenetics and Molecular Cytogenetics

technological improvements included the addition of a second left-handed micro-

manipulator equipped with a pipette, containing a collection drop, which enabled a
more convenient handling of dissected chromosomal parts.[6] Chromosome-specifc
libraries for multicolor FISH and multicolor banding constructed by microdissection
have found wide and successful application in clinical genetics, comparative inter-
species studies, and interphase cytogenetics.[7, 8] Semi-archived material can also be
used for chromosome microdissection.[9]
However, one of the signifcant limitations of microdissection method was the
need to obtain multiple copies of the chromosome or its fragment of interest in order
to create high-quality paints. The development of multiple displacement amplif-
cation (MDA) whole-genome amplifcation methods[10] makes it possible yet to
reduce the amount of starting material and work with a single chromosome copy.
The approach showed high effciency in obtaining samples for FISH in compara-
tive genomic studies[11, 12] and in medical genomics.[13] Moreover, it was shown that
sequencing of DNA from single isolated chromosomes (ChromSeq) can be done and
is suitable to determine the chromosome content and assign genomic scaffolds to
chromosomes, thus bridging the gap between cytogenetics and genomics.[14]
Here we provide an updated protocol for generating paint probes by an MDA-
based system from microdissected chromosomes or chromosomal segments.

MATERIALS
INSTRUMENTS
• Inverted microscope the IX Series (Olympus Shinjuku, Tokyo, Japan) or
Axiovert 10 or 135 (Zeiss, Jena, Germany)
• Right-handed and left-handed micromanipulators (Narishige, Tokyo, Japan
or Eppendorf, Germany)
• Pipette puller (Narishige, Japan)
• Glass rods, 2mm diameter (Schott Glas, Mainz, Germany)
• Pasteur pipettes, 230mm (Deltalab, Barcelona, Spain, or Assistent,
Sondheim, Germany)
• Aspirator with trap fask (“Grant”, UK).

REAGENTS
• Glycerol (Sigma-Aldrich)
• Proteinase K from Tritirachium album (Sigma-Aldrich, USA)
• Trypsin (Sigma-Aldrich, USA)
• 2×SSC
• 1×PBS
• Giemsa (Merck)
• Carbon tetrachloride (Sigma-Aldrich, USA)
• Dimethyldichlorosilane (Sigma-Aldrich, USA)
• GenomePlex® Whole Genome Amplifcation (WGA) Kit, WGA1 (Sigma-
Aldrich, USA)
Generation of Microdissection-Derived Painting Probes 29

• GenomePlex® WGA Reamplifcation Kit, WGA3 (Sigma-Aldrich, USA)

• QIAquick PCR Purifcation Kit (Quiagen, USA)

SOLUTIONS
• Collection drop solution: 30% Glycerol, 10mM Tris-HCl pH 7.5, 10mM
NaCl, 0.1% SDS, 1mM EDTA, 0.1% Trition X100, 1.44mg/ml Proteinase K.

DESCRIPTION OF METHODS
MICRO-NEEDLE AND MICROPIPETTE PREPARATION
1. Prepare micro-needles from 2mm glass rods on a vertical two-step puller,
to get very sharp but not very long edges.
2. Expose the needles to UV light for at least 30min.
3. Prepare micropipettes on the same puller from Pasteur pipettes.
4. Break the micropipette tips carefully to obtain a small round opening
(~30–70µm).
5. Siliconize micropipettes by immersing into 1% dimethyldichlorosilane in
carbon tetrachloride using an aspirator with a trap fask (“Grant”).
6. Air dry and wash twice in 1mM EDTA (pH 7.5).
7. Incubate micropipettes for 30min at 100°C.
8. Keep micropipettes and micro-needles in closed boxes prior to use.
Caution: Dimethyldichlorosilane and carbon tetrachloride are highly
toxic—all manipulations should be done under the draft hood. Two ml of
siliconization solution is enough to siliconize more than 30 micropipettes.

SLIDE PREPARATION
Traditionally, preparations for microdissection were dropped on coverslips.[15]
However, in our experience, the use of microscope slides is more convenient. To
achieve a good spreading of chromosomes, the conditions for slides preparation
should be optimized. In some cases, for better spreading, it is recommended to keep
the slide over a water bath, or add more fxative over the freshly dropped preparation.

1. Put clear 76×26 mm microscope slides (Menzel-Glaeser, Braunschweig,

Germany) in cold DNA-free distilled water (i.e. PCR water).
2. Take a wet slide and drop 15µl of metaphase preparation in 3:1 methanol/
acetic acid fxative onto the slide.
3. Let slide air dry at room temperature.

CHROMOSOME STAINING
Traditionally, chromosome staining was made using trypsin and phosphate buf-
fer incubation.[15] However, conventional GTG-banding[16] allows for better stain-
ing of chromosomes and does not affect the quality of probes. In cases where the
30 Cytogenetics and Molecular Cytogenetics

desired chromosome is easily identifed (the smallest or the biggest chromosome in

karyotype, the only acrocentric, and etc.), the trypsin treatment step can be skipped.
Obtaining a high-quality GTG-banding is required for an accurate identifcation of
chromosomes or their regions.

1. Incubate the slide in trypsin solution (0.12% or 0.25% trypsin in 1×PBS)

for 30 to 90s (the required time will vary depending on chromosome
preparation).
2. Rinse in 2×SSC.
3. Incubate in Giemsa solution (35ml phosphate buffer or sterile water and
3.5ml Giemsa) for 0.5–2min (the time will vary depending on chromosome
preparation).
4. Rinse in sterile water.
5. Air-dry the slide.
6. Assess the quality of the metaphase spreading and banding achieved under
the microscope.
7. Keep the slides refrigerated until the microdissection step.

MICRODISSECTION
1. Prepare 10–20µl of collection drop solution.
2. Use a 10× or 20× objective.
3. Move the objective down to adjust the needle.
4. Load a needle into the holder, and then load onto the micromanipulator, so
that the tip of the needle is close to the center of visual feld of the objective.
5. Find the needle in the visual feld and center it; then switch to 40× objec-
tive and ensure that the tip of the needle can be seen with the 40× objective.
6. Put the slide on the specimen stage and fnd a suitable metaphase spread.
By rotating the table, place the chromosome to be cut at a right angle to
the needle.
7. Bring the needle down carefully until it is just above the chromosome.
Forward movement of the tip will lead to the excision of the chromosome
material.
8. Touch the excised fragment with the tip of the needle several times until it
is attached to the needle. Carefully elevate the needle.
9. Switch to a 10× or 20× objective.
10. Remove the microscope slides.
11. Take a siliconized pipette; gently touch the surface of the collection drop
solution using the siliconized pipette, so that a tiny amount of collection
drop solution will be sucked up by capillary force.
12. Suspend the pipette above the objective.
13. Place the needle to the same plane as the pipette tip using micromanipu-
lator. Transfer the chromosome fragment by touching the solution inside
of the pipette with the needle, and then withdraw the needle. Large frag-
ments will remain visible in the collection drop for several seconds.
14. Put the pipette in a humidifed tray at 60°C for 1–2h.
Generation of Microdissection-Derived Painting Probes 31

GENERATION OF PAINT PROBE BY WHOLE GENOME AMPLIFICATION (WGA)

Contamination is one of the major problems when performing a sequence-indepen-
dent DNA amplifcation starting from low amounts of DNA. We recommend using
separate micropipettes for pre- and post-primary DOP-PCR procedures. UV treat-
ment of the microdissection room and instruments as well as DNA-EX (Genaxis)
cleaning of working surfaces is highly recommended.

1. Aliquot 5µl DNA-free water into 0.2ml tubes.

2. Transfer the collection drop with the microdissected material from the
Pasteur pipette into the tube containing the water by breaking off the
pipette tip into the tube.
3. Briefy spin tube. The solution can be kept on ice for several hours before
DNA amplifcation (for longer periods store at −20°C).
4. Use Whole Genome Amplifcation Kit (WGA1) for DNA amplifcation.
Follow manufacturer’s protocol.
5. Purify the amplifcation product using any available PCR purifcation kit
(e.g. QIAquick PCR Purifcation Kit). Determine the concentration of the
purifed amplifcation product using NanoDrop spectrophotometry.
6. Run 2µl of PCR products on a 1.5% agarose gel. The products should look
as a smear with an average size of 0.2 to 0.8kb.
7. Use WGA Reamplifcation Kit (WGA3) with 0.1µM of added modifed
nucleotides (bio-dUTP or dig-dUTP) for DNA labeling. Follow manufac-
turer’s protocol.
8. Purify the amplifcation product using a PCR purifcation kit (e.g.
QIAquick PCR Purifcation Kit).
9. Run 2µl of PCR products on a 1% agarose gel. The products should appear
as a smear with an average size of 0.2 to 0.8kb.
10. For FISH use, precipitate the PCR product in ethanol and resuspend in
30–40µl hybridization buffer.

According to the manufacturer’s protocol, starting material for WGA1 is 10ng of

DNA in 10µl volume of the reaction mixture. In our protocol, the starting mate-
rial is well below 1pg; therefore, we recommend transferring the collection drop
content into 5µl of sterile water and proportionally halving all other reagents when
working with WGA1. The number of amplifcation cycles should be increased up
to 30.

EXPECTED RESULTS
The resulted libraries may demonstrate different qualities depending on the repetitive
DNA content in studied genomes and chromosomes. Therefore, we recommend using
COT DNA for repetitive DNA suppression during FISH.[17] Figure 3.1 demonstrates
expected results of FISH with painting probes obtained from single-copies of chro-
mosomes 15 and 16 of the Nile crocodile (Crocodylus niloticus). Although both
chromosomes are specifcally painted by the respective libraries, heterochromatic
32 Cytogenetics and Molecular Cytogenetics

FIGURE  3.1 FISH-result on a metaphase spread from Nile crocodile CNO (Crocodylus
niloticus) hybridized with painting probes obtained from single-copies of chromosomes 15
(red) and 16 (green) of this species. Painting probe CNO15 additionally paints heterochro-
matic regions of most CNO chromosomes, despite suppression with sonicated total genomic
CNO-DNA.

regions on other chromosomes are also highlighted with the CNI15 probe, despite
suppression with sonicated total genomic DNA of Crocodylus niloticus.

ACKNOWLEDGEMENTS
While this protocol was written, SR and VT were supported by RFBR grant
# 19-54-26017.

REFERENCES
1. Scalenghe, F.; Turco, E.; Edstrom, J.E.; Pirrotta, V.; Melli, M. Microdissection and clon-
ing of DNA from a specifc region of Drosophila melanogaster polytene chromosomes.
Chromosoma. 1981, 82, 205–216.
2. Röhme, D.; Fox, H.; Herrmann, B.; Frischauf, A.M.; Edström, J.E.; Mains, P.; Silver,
L.M.; Lehrach, H. Molecular clones of the mouse t complex derived from microdissected
metaphase chromosomes. Cell. 1984, 36, 783–788.
3. Bates, G.P.; Wainwright, B.J.; Williamson, R.; Brown, S.D. Microdissection and
microcloning from the short arm of human chromosome 2. Mol. Cell. Biol. 1986, 6,
3826–3830.
Generation of Microdissection-Derived Painting Probes 33

4. Senger, G.; Lüdecke, H.J.; Horsthemke, B.; Claussen, U. Microdissection of banded

human chromosomes. Hum. Genet. 1990, 84, 507–511.
5. Telenius, H.; Carter, N.P.; Bebb, C.E.; Nordenskjold, M.; Ponder, B.A.; Tunnacliffe, A.
Degenerate oligonucleotide-primed PCR: General amplifcation of target DNA by a sin-
gle degenerate primer. Genomics. 1992, 13, 718–725.
6. Weimer, J.; Kiechle, M.; Senger, G.; Wiedemann, U.; Ovens-Raeder, A.; Schuierer, S.;
Kautza, M.; Siebert, R.; Arnold, N. An easy and reliable procedure of microdissec-
tion technique for the analysis of chromosomal breakpoints and marker chromosomes.
Chromosome. Res. 1999, 7, 355–362.
7. Liehr, T.; Heller, A.; Starke, H.; Rubtsov, N.; Trifonov, V.; Mrasek, K.; Weise, A.;
Kuechler, A.; Claussen, U. Microdissection based high resolution multicolor banding for
all 24 human chromosomes. Int. J. Mol. Med. 2002, 9, 335–339.
8. Liehr, T.; Starke, H.; Heller, A.; Kosyakova, N.; Mrasek, K.; Gross, M.; Karst, C.;
Steinhaeuser, U.; Hunstig, F.; Fickelscher, I.; Kuechler, A.; Trifonov, V.; Romanenko,
S.A.; Weise, A. Multicolor fuorescence in situ hybridization (FISH) applied to FISH-
banding. Cytogenet. Genome Res. 2006, 114, 240–244.
9. Al-Rikabi, A.B.H.; Cioff, M.B.; Liehr, T. Chromosome microdissection on semi-
archived material. Cytometry A. 2019, 95, 1285–1288.
10. Höckner, M.; Erdel, M.; Spreiz, A.; Utermann, G.; Kotzot, D. Whole genome amplifca-
tion from microdissected chromosomes. Cytogenet. Genome Res. 2009, 125, 98–102.
11. Andreyushkova, D.A.; Makunin, A.I.; Beklemisheva, V.R.; Romanenko, S.A.;
Druzhkova, A.S.; Biltueva, L.B.; Serdyukova, N.A.; Graphodatsky, A.S.; Trifonov V.A.
Next generation sequencing of chromosome-specifc libraries sheds light on genome
evolution in paleotetraploid sterlet (Acipenser ruthenus). Genes. 2017, 8, 318.
12. Rajičić, M.; Romanenko, S.A.; Karamysheva, T.V.; Blagojević, J.; Adnađević, T.;
Budinski, I.; Bogdanov, A.S.; Trifonov, V.A.; Rubtsov, N.B.; Vujošević, M. The origin
of B chromosomes in yellow-necked mice (Apodemus favicollis): Break rules but keep
playing the game. PLoS ONE. 2017, 12, e0172704.
13. Lemskaya, N.A.; Romanenko, S.A.; Rezakova, M.A.; Filimonova, E.A.; Prokopov, D.Y.;
Dolskiy, A.A.; Perelman, P.L.; Maksimova, Y.V.; Shorina, A.R.; Yudkin, D.V. A rare
familial rearrangement of chromosomes 9 and 15 associated with intellectual disability:
A clinical and molecular study. Mol. Cytogenet. 2021, 14, 47.
14. Iannucci, A.; Makunin, A.I.; Lisachov, A.P.; Ciof, C.; Stanyon, R.; Svartman, M.;
Trifonov, V.A. Bridging the gap between vertebrate cytogenetics and genomics with
single-chromosome sequencing (ChromSeq). Genes. 2021, 12, 124.
15. Yang, F.; Trifonov, V.; Ng, B.L.; Kosyakova, N.; Carter, N.P. Generation of paint
probes from fow-sorted and microdissected chromosomes. In: Fluorescence In Situ
Hybridization (FISH); Liehr, T., Ed. Springer Protocols Handbooks, Berlin, 2016,
pp. 63–79.
16. Seabright, M. A rapid banding technique for human chromosomes. The Lancet. 1971,
298(7731), 971–972.
17. Trifonov, V.A.; Vorobieva, N.V.; Serdyukova, N.A.; Rens, W. FISH with and without
COT1 DNA. In: Fluorescence In Situ Hybridization (FISH); Liehr, T., Ed. Springer
Protocols Handbooks, Berlin, 2016, pp. 123–132.
 4 FISH—An Overview
Thomas Liehr

CONTENTS
Introduction.............................................................................................................. 35
FISH Probes ............................................................................................................. 36
Repetitive Probes ............................................................................................ 36
Locus-Specifc Probes .................................................................................... 36
Partial Chromosome Paints............................................................................. 37
Whole Chromosome Paints ............................................................................ 37
Whole Genome ............................................................................................... 37
FISH Probe Sets.............................................................................................. 37
Basic FISH-Protocol ................................................................................................ 38
Yield = FISH Applications....................................................................................... 38
Conclusions.............................................................................................................. 39
References................................................................................................................ 39

INTRODUCTION
FISH = fuorescence in situ hybridization is one of two basic approaches originally
being considered to constitute the feld of molecular cytogenetics.[1] FISH is the
approach that at present is widely applied in chromosomic research, while its sister
approach PRINS = primed in situ hybridization (also sometimes called primed in
situ amplifcation, or primed in situ labeling) is practically no more in use.[1, 2] FISH
can be used to visualize DNA or RNA in tissues, interphase cells and metaphases.[1, 3]
Here only DNA-oriented FISH is considered.
FISH is in general single cell–oriented; single to multiple loci in a genome can
be accessed by single to multicolor-FISH approaches. The possibilities of FISH are
only limited by ideas whose questions can be solved by this approach.[1] Thus, since
the frst radioactive ISH experiments in the 1970s[4] and FISH-experiments in the
1980s,[5] practically each year new applications in human genetic, animal- and plant-
genetics and even in single-cell eukaryotic and prokaryotic research are reported.[1, 6]
The FISH-principle is depicted in Figure 4.1. Necessary for FISH are only a target-
DNA and a probe-DNA to hybridize.[3]
In the following, basics on DNA-probes being used in FISH and some examples
for application of FISH—mainly in human genetics—are provided.

DOI: 10.1201/9781003223658-4 35
36 Cytogenetics and Molecular Cytogenetics

FIGURE 4.1 FISH-principle in a scheme: A chromosome is depicted from which schemati-

cally and at greater magnitude the DNA-double strand is emanating. After denaturation, it
is single-stranded and brought together with single-stranded, labeled probe-DNA, which
hybridizes at homologous regions in the target region.

FISH PROBES
FISH probes = probe-DNA in human genetics are nowadays mainly commercially
available ones, being ready to use and labeled already with fuorophores or other
haptens like biotin or digoxigenin.[1, 3] Besides, there also can be used homemade/
homebrewed probes being labeled either by PCR-methods[7] or Nick-translation.[8]
Here fve basic probe DNA types suitable and used for FISH are listed:

REPETITIVE PROBES
Repetitive and/or heterochromatic DNA stretches can be easily visualized by FISH.[9]
Basic units of the repeats are 1 to 200 base pairs; however, they become detectable
by FISH as soon as they span 10s to 1000s of kilo base pairs.[9] In FISH, randomly
selected 2n to 6n repeats, telomeric repeats (6n), centromeric repeats (~170n) and
repeats being present in nucleolus organizing regions (NOR) are applied as FISH-
probes in general.[10] As in human chromosomes most centromeres have a private
satellite-DNA sequence, these centromeric/alphoid besides telomeric probes are
popular, commercially available probes.[3]

LOCUS-SPECIFIC PROBES
Locus-specifc probes (LSPs) are normally euchromatic DNA-stretches cloned into
genetic vectors; nowadays in most cases bacterial artifcial chromosome (BAC)
clones are used as basis for LSPs.[3] To produce good and reproducible FISH-signals,
FISH—An Overview 37

a vector needs to carry inserts of at least 12 kb,[11] while for mapping purposes excep-
tionally 0.44 kb–sized cDNA probes can be useful.[12]

PARTIAL CHROMOSOME PAINTS

A “painting probe” stains at least one or two euchromatic chromosomal subbands
and not only a single locus or a heterochromatic block.[13] To establish a partial chro-
mosome paint = pcp, only glass needle–based chromosome microdissection (midi) is
suited.[14] The maximum size of a pcp is spanning the arm of a meta- or submetacen-
tric chromosome,[15] and they can be as small as the diameter of the extended glass
needle, i.e. a part of a chromosomal subband,[16] or, in case of doing midi lampbruch
chromosome, even in LSP size.[17] Just for completeness, it must be mentioned that
also radiation hybrid cells[18] or ZOO-FISH probes like applied for RX-FISH[19] have
been used to create kinds of pcps.

WHOLE CHROMOSOME PAINTS

A whole chromosome paint = wcp can be established in two ways: either, as pcps, by
midi,[14] or by chromosome fow sorting.[20] Also, interspecies hybrids (e.g. mouse/
human somatic cell hybrid) have been used to set up wcp probes.[21]

WHOLE GENOME
FISH is normally used to access single cells. However, when doing compara-
tive genomic hybridization (CGH),[22] the single cell–derived metaphase chromo-
somes of a normal individual is just used as a kind of matrix, or low-resolution
DNA-chip. Towards this matrix, DNA extracted from millions of cells derived
from two different body tissues or even two closely related species is hybrid-
ized. Certainly tested DNAs are labeled in two different colors. Thus, it is pos-
sible to detect copy number gains and losses between the two probes. This CGH
approach has been used in human genetic research for solid tumors and has
been further evolved as an important tool in diagnostics nowadays as chromo-
some microarray (CMA) technique.[23, 24] In addition, CGH is currently applied
in evolution-research.[10]

FISH PROBE SETS

All different kinds of FISH-probes can be used for one- to multicolor-FISH (mFISH)
experiments.[3, 6] Important mFISH probe sets in diagnostics are all 24 human wcp
probes taken into one probe set,[25, 26] LSP/centromeric probe sets being tumor-type
specifc[27] or for prenatal testing.[28] LSPs and/or repetitive probes are the probes
normally being applied in molecular combing, where FISH is possible in almost base
pair resolution on maximally stretched DNA-fbers.[29]
38 Cytogenetics and Molecular Cytogenetics

BASIC FISH-PROTOCOL
As each laboratory uses a specifc protocol to do FISH, here just basic steps to be
included in each metaphase FISH-protocol are listed—see also ref[3]:

• Slides with metaphase spreads (e.g. obtained from a cytogenetic laboratory)

have to air-dry at room temperature (RT) overnight.
• To obtain optimal results later, a pretreatment of the slides is recommended;
here a three step procedure led to best plasma-free metaphases with strong
FISH-signals and optimal inverted 4′,6-diamidino-2-phenylindole (DAPI)
banding pattern:
• Step 1: incubate slides in a 3% paraformaldehyde solution for ~5 min.
• Step 2: incubate slides in a 0.5% pepsin/1% 1N HCl solution for ~2–3 min.
• Step 3: repeat step 1.
• Dry slides in a 70%/90%/100% ethanol series at RT.
• Do washing in between steps 1–3 with 2×SSC or 1×PBS; also consider envi-
ronment protection when discarding formaldehyde-contaminated solutions.
• Denature slides in a 70% formamide solution in 2×SSC—formamide
reduces the melting point of DNA; SSC-salts stabilize DNA structure—at
75°C for 3–5 min.
• Dry slides in a 70%/90%/100% ethanol series at RT—only 70% ethanol
should be at −20°C.
• Denature FISH-probe (here a commercial probe is suggested, which has a
ready to use) at 95°C for 5 min. Then either do prehybridization at 37°C for
10–30 min or place directly on ice.
• Pipette denatured FISH-probe on slide with denatured target-DNA, cover
with a coverslip, and seal with rubber cement.
• Incubate in a humid chamber over night at 37°C.
• Remove coverslip and do post-hybridization washings acc. to probe used.
• If necessary, a hapten detection must be done in case of a probe labeled e.g.
by biotin.
• Also, consider environment protection when discarding formamide-
contaminated solutions.
• Add antifade with DAPI and a coverslip.
• Evaluate slide under a fuorescence microscope with suited flter sets.

YIELD = FISH APPLICATIONS

Here a subjective list of possible FISH-application without claim of completeness is
provided:

(i) mosaic characterization[30, 31]

(ii) diagnostics in clinical genetics and acquired diseases[1, 3]
(iii) microdissection, reverse FISH and sequencing can be combined[32]
(iv) detection of radio- or mutagen-sensitivity[33]
FISH—An Overview 39

(v) chromothripsis detection[34–36]

(vi) CGH in ZOO-FISH experiments[10, 37]
(vii) gene mapping in metaphase[12] or by fber-FISH Nguyen[29]
(viii) nucleomics = interphase architecture studies[38–40]
(ix) in situ visualization of uniparental disomy[41]

CONCLUSIONS
As a conclusion, still best suited is a statement of Serakinci and Koelvraa from 2009:

FISH techniques were originally developed as extra tools in attempts to map genes and
a number of advances were achieved with this new technique. However, it soon became
apparent that the FISH concept offered promising possibilities also in a number of
other areas in biology and its use spread into new areas of research and also into the
area of clinical diagnosis. In very general terms the virtues of FISH are in two areas
of biology, namely genome characterization and cellular organization, function and
diversity. . . . To what extend FISH technology will be further developed and applied
in new areas of research in the future remains to be seen.[42]

REFERENCES
1. Liehr, T. Molecular cytogenetics in the era of chromosomics and cytogenomic
approaches. Front. Genet. 2021, 12, 720507.
2. Pellestor, F. Development and adaptation of the PRINS technology: An overview.
Methods Mol. Biol. 2006, 334, 211–220.
3. Liehr, T. (Ed.). Fluorescence in situ Hybridization (FISH): Application Guide. 2nd
Edition. Springer, Berlin; 2017.
4. Gall, J.G.; Pardue, M.L. Formation and detection of RNA-DNA hybrid molecules in
cytological preparations. Proc. Natl. Acad. Sci. U. S. A. 1969, 63, 378–383.
5. Langer, P.; Waldrop, A.; Ward, D. Enzymatic synthesis of biotin-labeled olynucleo-
tides: Novel nucleic acid affnity probes. Proc. Natl. Acad. Sci. U. S. A. 1981, 78,
6633–6637.
6. Liehr, T. Basics and literature on multicolor fuorescence in situ hybridization applica-
tion. http://cs-tl.de/DB/TC/mFISH/0-Start.html [accessed on 01/12/2022].
7. Telenius, H.; Carter, N.P.; Bebb, C.E.; Nordenskjöld, M.; Ponder, B.A.J.; Tunnacliffe,
A. Degenerate oligonucleotide-primed PCR: General amplifcation of target DNA by a
single degenerate primer. Genomics. 1992, 13, 718–725.
8. Rigby, P.W.J.; Dieckmann, M.; Rhodes, C.; Berg, P. Labeling deoxyribonucleic acid to
high specifc activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol.
1977, 113, 237–251.
9. Liehr, T. Repetitive elements in humans. Int. J. Mol. Sci. 2021, 22, 2072.
10. Xu, D.; Sember, A.; Zhu, Q.; de Oliveira, E.A.; Liehr, T.; Al-Rikabi, A.B.H.; Xiao, Z.;
Song, H.; de Bello Cioff, M. Deciphering the origin and evolution of the X1X2Y system
in two closely-related oplegnathus species (Oplegnathidae and Centrarchiformes). Int. J.
Mol. Sci. 2019, 20, 3571.
11. Liehr, T.; Thoma, K.; Kammler, K.; Gehring, C.; Ekici, A.; Bathke, K.; Grehl, H.;
Rautenstrauss, B. Direct preparation of uncultured EDTA-treated or heparinized blood
for interphase FISH analysis. Appl. Cytogenet. 1995, 21, 185–188.
40 Cytogenetics and Molecular Cytogenetics

12. Pröls, F.; Liehr, T.; Rinke, R.; Rautenstrauss, B. Assignment of the microvascular endo-
thelial differentiation gene 1 (MDG1) to human chromosome band 14q24.2→q24.3 by
fuorescence in situ hybridization. Cytogenet. Genome Res. 1997, 79, 149–150.
13. Liehr, T. Human Genetics: Edition 2020: A Basic Training Package. Epubli, Berlin;
2020.
14. Al-Rikabi, A.B.H.; Cioff, M.d.B.; Liehr, T. Chromosome microdissection on semi-
archived material. Cytometry Part A. 2019, 95, 1285–1288.
15. Kytölä, S.; Rummukainen, J.; Nordgren, A.; Karhu, R.; Farnebo, F.; Isola, J.; Larsson, C.
Chromosomal alterations in 15 breast cancer cell lines by comparative genomic hybrid-
ization and spectral karyotyping. Genes Chromosomes Cancer. 2000, 28, 308–317.
16. Senger, G.; Lüdecke, H.J.; Horsthemke, B.; Claussen, U. Microdissection of banded
human chromosomes. Hum. Genet. 1990, 84, 507–511.
17. Zlotina, A.; Kulikova, T.; Kosyakova, N.; Liehr, T.; Krasikova, A. Microdissection of
lampbrush chromosomes as an approach for generation of locus-specifc FISH-probes
and samples for high-throughput sequencing. BMC Genomics. 2016, 17, 126.
18. Guyon, R.; Rakotomanga, M.; Azzouzi, N.; Coutanceau, J.; Bonillo, C.; D’Cotta, H.;
Pepey, E.; Soler, L.; Rodier-Goud, M.; D’Hont, A.; Conte, M.; van Bers, N.; Penman,
D.; Hitte, C.; Crooijmans, R.; Kocher, T.; Ozouf-Costaz, C.; Baroiller, J.; Galibert, F.
A high-resolution map of the Nile tilapia genome: A resource for studying cichlids and
other percomorphs. BMC Genomics. 2012, 13, 222.
19. Müller, S.; Wienberg, J. “Bar-coding” primate chromosomes: Molecular cytogenetic
screening for the ancestral hominoid karyotype. Hum. Genet. 2001, 109, 85–94.
20. Ferguson-Smith, M.; Yang, F.; Rens, W.; O’Brien, P. The impact of chromosome sorting
and painting on the comparative analysis of primate genomes. Cytogenet. Genome Res.
2005, 108, 112–121.
21. Sabile, A.; Poras, I.; Cherif, D.; Goodfellow, P.; Avner, P. Isolation of monochromosomal
hybrids for mouse chromosomes 3, 6, 10, 12, 14, and 18. Mamm. Genome. 1997, 8,
81–85.
22. Kallioniemi, A.; Kallioniemi, O.P.; Sudar, D.; Rutovitz, D.; Gray, J.W.; Waldman, F.;
Pinkel, D. Comparative genomic hybridization for molecular cytogenetic analysis of
solid tumors. Science. 1992, 258, 818–821.
23. Pinkel, D.; Segraves, R.; Sudar, D.; Clark, S.; Poole, I.; Kowbel, D.; Collins, C.; Kuo,
W.L.; Chen, C.; Zhai, Y.; Dairkee, S.H.; Ljung, B.M.; Gray, J.W.; Albertson, D.G. High
resolution analysis of DNA copy number variation using comparative genomic hybrid-
ization to microarrays. Nat. Genet. 1998, 20, 207–211.
24. Brady, P.D.; Vermeesch, J.R. Genomic microarrays: A technology overview. Prenat.
Diagn. 2012, 32, 336–343.
25. Schröck, E.; du Manoir, S.; Veldman, T.; Schoell, B.; Wienberg, J.; Ferguson-Smith, M.;
Ning, Y.; Ledbetter, D.; Bar-Am, I.; Soenksen, D.; Garini, Y.; Ried, T. Multicolor spec-
tral karyotyping of human chromosomes. Science. 1996, 273, 494–497.
26. Speicher, M.; Gwyn Ballard, S.; Ward, D. Karyotyping human chromosomes by combi-
natorial multi-fuor FISH. Nat. Genet. 1996, 12, 368–375.
27. Nagai, T.; Okamura, T.; Yanase, T.; Chaya, R.; Moritoki, Y.; Kobayashi, D.; Akita, H.;
Yasui, T. Examination of diagnostic accuracy of UroVysion fuorescence in situ hybrid-
ization for bladder cancer in a single community of Japanese hospital patients. Asian
Pac. J. Cancer. Prev. 2019, 20, 1271–1273.
28. Eiben, B.; Trawicki, W.; Hammans, W.; Goebel, R.; Pruggmayer, M.; Epplen, J. Rapid
prenatal diagnosis of aneuploidies in uncultured amniocytes by fuorescence in situ
hybridization: Evaluation of >3,000 cases. Fetal. Diagn. Ther. 1999, 14, 193–197.
FISH—An Overview 41

29. Nguyen, K.; Broucqsault, N.; Chaix, C.; Roche, S.; Robin, J.; Vovan, C.; Gerard, L.;
Mégarbané, A.; Urtizberea, J.; Bellance, R.; Barnérias, C.; David, A.; Eymard, B.; Fradin,
M.; Manel, V.; Sacconi, S.; Tiffreau, V.; Zagnoli, F.; Cuisset, J.M.; Salort-Campana, E.;
Attarian, S.; Bernard, R.; Lévy, N.; Magdinier, F. Deciphering the complexity of the 4q
and 10q subtelomeres by molecular combing in healthy individuals and patients with
facioscapulohumeral dystrophy. J. Med. Genet. 2019, 56, 590–601.
30. Vorsanova, S.G.; Zelenova, M.A.; Yurov, Y.B.; Iourov, I.Y. Behavioral variability and
somatic mosaicism: A cytogenomic hypothesis. Curr. Genomics. 2018, 19, 158–162.
31. Mkrtchyan, H.; Gross, M.; Hinreiner, S.; Polytiko, A.; Manvelyan, M.; Mrasek, K.;
Kosyakova, N.; Ewers, E.; Nelle, H.; Liehr, T.; Bhatt, S.; Thoma, K.; Gebhart, E.;
Wilhelm, S.; Fahsold, R.; Volleth, M.; Weise, A. The human genome puzzle: The role of
copy number variation in somatic mosaicism. Curr. Genomics. 2010, 11, 426–431.
32. Jancuskova, T.; Plachy, R.; Stika, J.; Zemankova, L.; Hardekopf, D.W.; Liehr, T.;
Kosyakova, N.; Cmejla, R.; Zejskova, L.; Kozak, T.; Zak, P.; Zavrelova, A.; Havlikova,
P.; Karas, M.; Junge, A.; Ramel, C.; Pekova, S. A method to identify new molecular
markers for assessing minimal residual disease in acute leukemia patients. Leuk. Res.
2013, 37, 1363–1373.
33. Hovhannisyan, G.G. Fluorescence in situ hybridization in combination with the comet
assay and micronucleus test in genetic toxicology. Mol. Cytogenet. 2010, 3, 17.
34. Shimizu, N. Extrachromosomal double minutes and chromosomal homogeneously
staining regions as probes for chromosome research. Cytogenet. Genome Res. 2009,
124, 312–326.
35. Koltsova, A.; Pendina, A.; Efmova, O.; Chiryaeva, O.; Kuznetzova, T.; Baranov, V.S. On
the complexity of mechanisms and consequences of chromothripsis: An update. Front.
Genet. 2019, 10, 393.
36. Nazaryan-Petersen, L.; Bjerregaard, V.; Nielsen, F.; Tommerup, N.; Tümer, Z.
Chromothripsis and DNA repair disorders. J. Clin. Med. 2020, 9, 613.
37. Spangenberg, V.; Arakelyan, M.; Cioff, M.; Liehr, T.; Al-Rikabi, A.; Martynova, E.;
Danielyan, F.; Stepanyan, I.; Galoyan, E.; Kolomiets, O. Cytogenetic mechanisms of
unisexuality in rock lizards. Sci. Rep. 2020, 10, 8697.
38. Lemke, J.; Claussen, J.; Michel, S.; Chudoba, I.; Mühlig, P.; Westermann, M.; Sperling,
K.; Rubtsov, N.; Grummt, U.; Ullmann, P.; Kromeyer-Hauschild, K.; Liehr, T.; Claussen,
U. The DNA-based structure of human chromosome 5 in interphase. Am. J. Hum. Genet.
2002, 71, 1051–1059.
39. Cremer, T.; Cremer, M.; Hübner, B.; Silahtaroglu, A.; Hendzel, M.; Lanctôt, C.;
Strickfaden, H.; Cremer, C. The interchromatin compartment participates in the struc-
tural and functional organization of the cell nucleus. Bioessays. 2020, 42, e1900132.
40. Daban, J. Supramolecular multilayer organization of chromosomes: Possible functional
roles of planar chromatin in gene expression and DNA. FEBS Lett. 2020, 594, 395–411.
41. Weise, A.; Othman, M.A.K.; Bhatt, S.; Löhmer, S.; Liehr, T. Application of BAC-probes
to visualize copy number variants (CNVs). Meth. Mol. Biol. (Clifton, N.J.). 2015, 1227,
299–307.
42. Serakinci, N.; Koelvraa, S. Molecular cytogenetic applications in diagnostics and
research: An overview. In In Fluorescence In Situ Hybridization (FISH): Application
Guide; Liehr, T., Ed. 1st Edition. Springer, Berlin, 2009.
 5 FISH—Microscopy
and Evaluation
Ivan Y. Iourov

CONTENTS
Introduction .............................................................................................................. 43
Fluorescence Microscopy ........................................................................................ 44
Imaging and Image Analysis.................................................................................... 45
Human Eye versus Electric Eye ............................................................................... 46
Conclusions .............................................................................................................. 46
Acknowledgments.................................................................................................... 47
References ................................................................................................................ 47

INTRODUCTION
In the postgenomic era, microscopic (visual) analysis of human chromosomes is
often regarded as an optional procedure in genomic research. However, the avail-
ability of fuorescence-based microscopic (genomic) techniques (like fuorescence
in situ hybridization = FISH) gradually expands the area of genetic and genomic
research.[1–3] Moreover, practical issues in medical genetics cannot be properly
addressed without FISH, the essence of which lies in the fuorescence microscopy,
visualization approaches, and DNA-based chromosome research.[1, 3, 4] Accordingly,
to understand the role of FISH in current bio- and medical science,[5–7] fuorescence
microscopy is an area to start with. Currently, FISH-based assays are required for
analyzing structure and numbers of human chromosomes in interphase (the longest
part of the cell cycle or the most usual state of a cell of an eukaryotic organism),[7–9]
genome organization at the chromosomal level,[10, 11] developing single-cell genomic
approaches,[12] and cytogenetic genotoxicological studies.[13] In total, there is a huge
and increasing amount of FISH-based assays applicable for different biomedical
tasks.[5, 7, 8, 14] Fluorescence microscopy is the basis for evaluation of FISH results,
which determines the resolution and effciency of the molecular cytogenetic analysis.
Therefore, fuorescence microscopy and evaluation are of prime importance for suc-
cessful application of FISH-based molecular cytogenetic technologies. Here, a brief
overview of fuorescence microscopy and FISH results evaluation is provided.

DOI: 10.1201/9781003223658-5 43
44 Cytogenetics and Molecular Cytogenetics

FLUORESCENCE MICROSCOPY
The frst key concept in fuorescence microscopy is the resolution. The smallest vis-
ible molecular target in the feld of the vision determines the microscopic resolution
of FISH. Usually, FISH analysis is referred to as the evaluation of small fuorescing
objects of roundish, globular, or spherical shape (i.e. FISH signals or fuorescence
signals). The diffraction may cause problems in distinguishing the closest fuores-
cence signals. Accordingly, fuorescence microscopy resolution is related to the
determination of the closest distinguishable visible objects. The resolution depends
on the wavelength (λ), the numerical aperture (NA), the magnifcation, and detection
devices. The smallest diameter of a visible FISH signal is determined by the follow-
ing ratio: d=1.22 λ/NA. Visible FISH signals are still distinguished when the dis-
tance between fuorescing objects is more than half a diameter, determined by the
aforementioned ratio.[5]
The second key concept in fuorescence microscopy is the background autofuo-
rescence. The related phenomena affect actually all the visualization-based fores-
cence assays. The decrease of the effective contrast of the adjacent FISH signals
impeding distinguishing between the signals may help to autofuorescence-related
problems in visualization of FISH results. However, reducing the image contrast
would result in resolution reduction. This dependence determines interplay between
autofuorescence and resolution (i.e. the frst and the second key concept of fuores-
cence microscopy). Actually, the background may be removed by imaging software
(imaging techniques). A number of procedures for pre-treatment of microscopic
slides before or after FISH is able to increase the signal removing background auto-
fuorescence are available, as well.[5]
The third key concept in fuorescence microscopy is the detection. The use of
specifc detection systems or algorithms is the way to increase the microscopic res-
olution of FISH-based techniques. Generally, a digital camera is used as a device
for FISH detection. The resolution may be modifed by an increase in the quantum
effciency of the detection system. Automatically or manually, the increase of the
quantum effciency reduces the noise and optical NA of the microscope and cam-
era. The dependence between NA and d is as follows: increasing NA decreases
d. In the detection context, FISH resolution may also be affected by the maxi-
mum number of distinguishable intensity levels. This number is defned by the
parameters of the detection system; exposure time may be adjusted manually or
automatically. Alternatively, detection using digitalization for quantitative analy-
sis allows differentiation between FISH signals and background autofuorescence.
The digitalization and quantitative analysis underlie quantitative FISH (QFISH),
which is discussed hereafter. For details about fuorescence microscopy concepts
considered in the FISH context, readers are recommended to address following
sources.[5–7]
The essential device for FISH-based microscopic analysis is the fuorescence
microscope. The device is required to include a fuorescence light source and a set
of fuorescence flters, which are composed of dichroic mirror and excitation and
emission flters. For FISH-based molecular cytogenetic analyses, a high-pressure
Hg (mercury) lamp is generally the most preferable to be used as a light source. The
FISH—Microscopy and Evaluation 45

latter provides suitable high-energy excitations at wavelengths specifc for the major-
ity of FISH-based techniques. The lamp produces heat. Accordingly, lamp cooling is
systematically requited. The hot lamp should not be turned on. Usually, the overall
working time of these lamps is less than 200 burning hours. Since recently, light-
emitting diodes (LED) are an Hg-free alternative as light source, which also have
longer ‘burning times’ in the range of 2000 hours or more.[15] FISH specimens are
not to be exposed to the light. To avoid fading, light shutter is used to block the expo-
sure. Multicolor nature of FISH is realized through the application of fuorescence
flters. The combination of flters has to provide proper contrast between fuoro-
chromes used for probe labeling.[5]

IMAGING AND IMAGE ANALYSIS

Imaging and image analysis are aimed at enhancing the effciency of FISH.
Technologically, FISH imaging is available when three steps are performed: (i) acqui-
sition of a microscopic (FISH) image, (ii) image pre-processing, and (iii)  digital
analysis. To perform the analysis, a number of devices are to be used. These are a
fuorescence microscope, image acquisition devise (the image sensor, e.g., a charge
coupled device (CCD) camera, a device of choice for FISH), and hardware/software
for digitizing and processing acquired microscopic images.[4–7]
The key element of FISH image acquisition is focusing. Numerous opportuni-
ties of manual and automatic focusing are provided by modern imaging systems.
Specifc FISH approaches require autofocusing (e.g. FISH analysis merging colors
obtained by processing intensities of signals; volumetric FISH analyses for 3D stud-
ies of chromosomal organization in interphase nuclei[7, 8, 10]), which may cause a prob-
lem referred to differing between FISH signals and autofuorescence particles. An
adequate solution for different FISH image acquisition is to apply both manual focus
and autofocusing.
FISH image analysis is not generally associated with extreme diffculties either
due to availability of specifc digital imaging systems or to DNA probe specifcity
and high hybridization effciency. However, raw FISH images may cause some inter-
pretational problems.[8, 14] To solve these, interactive or manual removal of fuores-
cence background by thresholding and contrast normalization is performed.
Canonical FISH protocols (i.e. FISH using site-specifc and chromosome-
enumerating DNA probes) are usually designed for visual/express analysis. More
sophisticated FISH techniques are impossible to perform without digital analysis.
The latter is specifcally designed for each corresponding FISH assay.[5, 14] Regardless
of being secondary to basic FISH procedures (sample preparation, hybridization,
and detection), imaging digital analysis may underlie sophisticated molecular cyto-
genetic analyses, which are focused on detection of multicolor FISH results.[5] An
important example of digital imaging, which opens new opportunities for FISH-
based chromosomal analysis, is QFISH (quantitative FISH).
QFISH has been demonstrated to be useful for a variety of molecular cytogenetics
studies of chromosomes. The approach is applicable for analyses of somatic chromo-
somal mosaicism and chromosome instability in interphase nuclei. It allows detect-
ing chromosomal DNA variations (e.g. chromosomal variants or pericentromeric
46 Cytogenetics and Molecular Cytogenetics

DNA variations) between homologous human chromosomes at the single cell

level.[16] Through the last two decades, QFISH has been demonstrated to be valu-
able for different basic research and diagnostic purposes in medical genetic and
cancer research.[16–18] Thus, it allows specifc chromosomal organization in inter-
phase nuclei to be depicted.[19] More importantly, QFISH is applicable for the defni-
tion of the parental origin of homologous chromosomes.[20, 21] The ratio of relative
intensities obtained by integrating intensity profles of FISH signals are compared
to differ between chromosomal associations and chromosome loss in interphase.
These measurements of FISH-painted hypervariable chromosomal loci are used for
defning homologous chromosome parental origins. In total, FISH result evaluations
using QFISH may be highly helpful as an additional tool for a molecular cytogenetic
study.[22]

HUMAN EYE VERSUS ELECTRIC EYE

The aforementioned advantages offered by FISH result evaluations using digital
imaging may be used for promoting ideas suggesting that microscopic analysis per-
formed by researcher’s eyes is outdated. Accordingly, one can propose to diminish
the signifcance of visual analysis as a contributor to successful molecular cytoge-
netic research. This might be further supported by a number of multicolor FISH
techniques, which are useless without digitalization of FISH signals. Probably, the
most important FISH-based approach to be mentioned in this context is multicolor
banding (MCB). This is an important technique for basic and applied chromosomal
analysis at molecular resolution.[23] MCB is based almost exclusively on microscopic
evaluation using digital imaging and is systematically proven effective in molecular
cytogenetic studies.[24] Moreover, the approach may be used for developing methods
of interphase chromosomal analysis, providing an unprecedented resolution for visu-
alization of an interphase chromosome at any cell cycle stage and at molecular reso-
lutions using digital image analysis.[25, 26] However, one can argue that microscopic
analyses performed by researcher’s eyes are outdated. The confrontation between
digital and visual (researcher’s eye) analyses is to be recognized as insignifcant.
Human eye microscopic evolution ensures effciency through researcher’s experi-
ence, ability of immediate decision considering the signifcance of FISH results,
and the selection of FISH results for further digital analysis. Digital microscopic
evolution (= ‘electric eye’ microscopic evolution) provides for specifc FISH image
analysis, which undoubtedly increases the resolution and/or gives an opportunity
of DNA-based chromosomal analysis unavailable to researcher eye chromosomal
evaluations. Thus, the combination of both is the most effective way to succeed (as
usual).

CONCLUSIONS
Microscopy is the basis of FISH analysis. Actually, a number of biomedical areas
require FISH to have an important place in the methodology. Somatic human
genetics/genomics, cytogenomics, and cancer research are certainly among these
areas.[27–29] Therefore, knowledge of basic principles underlying FISH may be useful
FISH—Microscopy and Evaluation 47

for researchers undertaking related biomedical studies. Since FISH and microscopy
are intimately linked to each other, it is to acknowledge the importance of knowing
opportunities and limitations of fuorescence microscopy and evaluations in the con-
text of this molecular cytogenetic platform.

ACKNOWLEDGMENTS
The chapter is dedicated to professors Svetlana G. Vorsanova and Yuri B. Yurov
and to Dr. Ilia V. Soloviev. The author’s work is supported by the Government
Assignment of the Russian Ministry of Science and Higher Education, Assignment
no. AAAA-A19-119040490101-6 and by the Government Assignment of the Russian
Ministry of Health, Assignment no. 121031000238-1.

REFERENCES
1. Liehr, T. From human cytogenetics to human chromosomics. Int J Mol Sci. 2019, 20,
826.
2. Ye, C.J.; Stilgenbauer, L.; Moy, A.; Liu, G.; Heng, H.H. What is karyotype coding and
why is genomic topology important for cancer and evolution? Front Genet. 2019, 10,
1082.
3. Iourov, I.Y.; Yurov, Y.B.; Vorsanova, S.G. Chromosome-centric look at the genome. In:
Human Interphase Chromosomes: Biomedical Aspects; Iourov, I.Y.; Vorsanova, S.G.;
Yurov, Y.B., Eds. Springer, Berlin, 2020, pp. 157–170.
4. Gersen, S.L.; Keagle, M.B. The Principles of Clinical Cytogenetics. 2nd Edition.
Humana, Totowa, NJ; 2005.
5. Liehr, T. (Ed.). Fluorescence In Situ Hybridization (FISH): Application Guide. Springer,
Berlin, Heidelberg, Germany; 2017.
6. Garini, Y.; Young, I.T.; McNamara, G. Spectral imaging: Principles and applications.
Cytometry A. 2006, 69, 735–747.
7. Yurov, Y.B.; Vorsanova, S.G.; Iourov, I.Y. Human Interphase Chromosomes: Biomedical
Aspects. Springer, Berlin; 2013.
8. Vorsanova, S.G.; Yurov, Y.B.; Iourov, I.Y. Human interphase chromosomes: A review of
available molecular cytogenetic technologies. Mol Cytogenet. 2010, 3, 1.
9. Bakker, B.; van den Bos, H.; Lansdorp, P.M.; Foijer, F. How to count chromosomes in a
cell: An overview of current and novel technologies. Bioessays. 2015, 37, 570–577.
10. Manvelyan, M.; Hunstig, F.; Bhatt, S.; Mrasek, K.; Pellestor, F.; Weise, A.; Simonyan, I.;
Aroutiounian, R.; Liehr, T. Chromosome distribution in human sperm: A 3D multicolor
banding-study. Mol Cytogenet. 2008, 1, 25.
11. McClelland, S.E. Single-cell approaches to understand genome organisation throughout
the cell cycle. Essays Biochem. 2019, 63, 209–216.
12. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Single cell genomics of the brain: Focus on
neuronal diversity and neuropsychiatric diseases. Curr Genomics. 2012, 13, 477–488.
13. Hovhannisyan, G.G. Fluorescence in situ hybridization in combination with the comet
assay and micronucleus test in genetic toxicology. Mol Cytogenet. 2010, 3, 17.
14. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Recent patents on molecular cytogenetics.
Recent Pat DNA Gene Seq. 2008, 2, 6–15.
15. Lang, D.S.; Zeiser, T.; Schultz, H.; Stellmacher, F.; Vollmer, E.; Zabel, P.; Goldmann, T.
LED-FISH: Fluorescence microscopy based on light emitting diodes for the molecular
analysis of Her-2/neu oncogene amplifcation. Diagn Pathol. 2008, 3, 49.
48 Cytogenetics and Molecular Cytogenetics

16. Iourov, I.Y.; Soloviev, I.V.; Vorsanova, S.G.; Monakhov, V.V.; Yurov, Y.B. An approach
for quantitative assessment of fuorescence in situ hybridization (FISH) signals for
applied human molecular cytogenetics. J Histochem Cytochem. 2005, 53, 401–408.
17. Konsti, J.; Lundin, J.; Jumppanen, M.; Lundin, M.; Viitanen, A.; Isola, J. A public-domain
image processing tool for automated quantifcation of fuorescence in situ hybridisation
signals. J Clin Pathol. 2008, 61, 278–282.
18. Amakawa, G.; Ikemoto, K.; Ito, H.; Furuya, T.; Sasaki, K. Quantitative analysis of cen-
tromeric FISH spots during the cell cycle by image cytometry. J Histochem Cytochem.
2013, 61, 699–705.
19. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Fluorescence intensity profles of in situ
hybridization signals depict genome architecture within human interphase nuclei. Tsitol
Genet. 2008, 42, 3–8.
20. Vorsanova, S.G.; Iourov, I.Y.; Beresheva, A.K.; Demidova, I.A.; Monakhov, V.V.; Kravets,
V.S.; Bartseva, O.B.; Goyko, E.A.; Soloviev, I.V.; Yurov, Y.B. Non-disjunction of chro-
mosome 21, alphoid DNA variation, and sociogenetic features of Down syndrome. Tsitol
Genet. 2005, 39, 30–36.
21. Weise, A.; Gross, M.; Mrasek, K.; Mkrtchyan, H.; Horsthemke, B.; Jonsrud, C.; Von
Eggeling, F.; Hinreiner, S.; Witthuhn, V.; Claussen, U.; Liehr, T. Parental-origin-
determination fuorescence in situ hybridization distinguishes homologous human chro-
mosomes on a single-cell level. Int J Mol Med. 2008, 21, 189–200.
22. Iourov, I.Y. Quantitative fuorescence in situ hybridization (QFISH). Methods Mol Biol.
2017, 1541, 143–149.
23. Liehr, T.; Heller, A.; Starke, H.; Rubtsov, N.; Trifonov, V.; Mrasek, K.; Weise, A.;
Kuechler, A.; Claussen, U. Microdissection based high resolution multicolor banding for
all 24 human chromosomes. Int J Mol Med. 2002, 9, 335–339.
24. Alhourani, E.; Aroutiounian, R.; Harutyunyan, T.; Glaser, A.; Schlie, C.; Pohle, B.;
Liehr, T. Interphase molecular cytogenetic detection rates of chronic lymphocytic
leukemia-specifc aberrations are higher in cultivated cells than in blood or bone mar-
row smears. J Histochem Cytochem. 2016, 64, 495–501.
25. Iourov, I.Y.; Liehr, T.; Vorsanova, S.G.; Kolotii, A.D.; Yurov, Y.B. Visualization of inter-
phase chromosomes in postmitotic cells of the human brain by multicolour banding
(MCB). Chromosome Res. 2006, 14, 223–229.
26. Iourov, I.Y.; Liehr, T.; Vorsanova, S.G.; Yurov, Y.B. Interphase chromosome-specifc
multicolor banding (ICS-MCB): A new tool for analysis of interphase chromosomes in
their integrity. Biomol Eng. 2007, 24, 415–417.
27. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B.; Kutsev, S.I. Ontogenetic and pathogenetic
views on somatic chromosomal mosaicism. Genes. 2019, 10, 379.
28. Ye, C.J.; Sharpe, Z.; Heng, H.H. Origins and consequences of chromosomal instability:
From cellular adaptation to genome chaos-mediated system survival. Genes. 2020, 11,
1162.
29. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Systems cytogenomics: Are we ready yet?
Curr Genomics. 2021, 22, 75–78.
 6 FISH—in Routine
Diagnostic Settings
Chenghua Cui and Peining Li

CONTENTS
Introduction.............................................................................................................. 49
Analytic and Clinical Validity of FISH Assays........................................................ 50
Design of Locus-Specifc, Gene-Targeted, Regional or Whole
Chromosome Painting Probes............................................................. 50
Validation and Verifcation of FISH Assays for Clinical Testing.................... 50
Detection of Constitutional Cytogenomic Abnormalities........................................ 52
Rapid Prenatal Screening of Common Aneuploidy........................................ 53
Detection of Microdeletion and Microduplication Syndromes ...................... 53
Detection of Numerical and Structural Chromosomal Abnormalities............ 55
Detection of Somatic Numerical and Structural Chromosomal
Abnormalities ..................................................................................... 57
Detection of Recurrent Rearrangements in Hematopoietic
and Lymphoid Tissues ........................................................................ 57
Detection of Recurrent Rearrangements in Solid Tumors.............................. 60
Conclusions.............................................................................................................. 64
Acknowledgements.................................................................................................. 65
References................................................................................................................ 65

INTRODUCTION
Fluorescence in situ hybridization (FISH) is a cell-based macromolecule rec-
ognition technique based on the complementary nature of DNA double strands.
Fluorophore-coupled nucleotides can be enzymatically incorporated into cloned or
amplifed DNA fragments. These fuorescence-labeled DNA fragments can be used
as probes to hybridize onto the complementary sequences in interphase nuclei and
metaphase chromosomes, and then visualized through a fuorescence microscope.
This technique was initially developed to replace radioactive probes for physical
mapping of genes to chromosomes.[1–4] Human genomic DNA fragments of 100 to
300 kilobases (kb) cloned in bacterial artifcial chromosomes (BAC) were com-
piled in a database and mapped to chromosomes by FISH.[5] These mapped BAC
clones provided frstly cytogenetic landmarks for the Human Genome Project and
later a resource for developing locus-specifc and gene-targeted FISH probes. FISH
assays have higher analytical resolution than karyotyping and high sensitivity and

DOI: 10.1201/9781003223658-6 49
50 Cytogenetics and Molecular Cytogenetics

specifcity on detecting targeted DNA. The design of multicolor probes enabled

simultaneous detection of chromosomes 13, 18, 21, X, and Y in uncultured amni-
otic fuid cells; this FISH assay was immediately accepted as the frst clinical
application for rapid prenatal screening of common aneuploidies.[6, 7] The intro-
duction of FISH into clinical cytogenetics excited the black-white banding pat-
tern with fuorescent colors and evolved the feld into molecular cytogenetics with
expanded diagnostic applications.[8, 9]

ANALYTIC AND CLINICAL VALIDITY OF FISH ASSAYS

DESIGN OF LOCUS-SPECIFIC, GENE-TARGETED, REGIONAL
OR WHOLE CHROMOSOME PAINTING PROBES

BAC clones mapped across the human genome in the genome browser (https://
genome.ucsc.edu/) are a reliable resource for developing FISH probes target-
ing to specifc loci and genes.[10] A wide variety of fuorophores are available,
and several enzymatic reactions such as nick translation, random priming, and
degenerative oligonucleotide primed polymerase chain reaction have been used
to incorporate fuorophore-coupled nucleotides.[11] Locus-specifc, gene-targeted,
regional or whole chromosome painting probes have been developed for diagnos-
tic uses. Enumerative locus-specifc probes containing highly repetitive α-satellite
sequences at centromere and unique DNA sequences at pericentric or specifc
chromosome loci are used to detect numerical chromosome abnormalities.[6, 10]
Probes containing repetitive sequences at telomeres and subtelomeric sequences
are used to detect telomeric integrity and subtelomeric rearrangements.[12] Gene-
targeted design of dual-color double fusion probes for two genes are used to detect
conjugated signals for a reciprocal translocation involving the two genes, while
dual-color break apart probes juxtaposing the 5′ and 3′ regions of a gene are used
to detect separated 5′ and 3′ signals for rearrangements of the gene.[13, 14] Contig
probes for a chromosome band or collection of probes for a whole chromosome
can be labeled in a multicolor pattern to generate a painting effect for an individual
chromosome or a whole metaphase.[15–17] Due to the standardized probe design and
widespread diagnostic usage, all these probes are available commercially or can be
made custom. Figure 6.1 shows various designs of FISH probes and typical signal
patterns on interphase nuclei and metaphase cells.

VALIDATION AND VERIFICATION OF FISH ASSAYS FOR CLINICAL TESTING

The analytical resolution of FISH is defned as the ability to distinguish two points
along the length of interphase chromatins or metaphase chromosomes. The limit
of this resolution is determined as 200–500 nm by the light microscope. The width
of naked DNA double strands, packed chromatins, and metaphase chromosomes is
2 nm, 30 nm, and 1,400 nm, respectively, which explains the resolvable visibility
only for chromosomes but not for chromatins or naked DNA under a light micro-
scope. The length of 1 kb DNA double helix is 340 nm. Considering the labeling eff-
ciency of fuorophore-couple nucleotide incorporation and hybridization effciency
FISH—in Routine Diagnostic Settings 51

FIGURE  6.1 FISH probes designs and typical signal patterns on interphase nuclei and
metaphase chromosomes. A. FISH using locus-specifc probes targeted to chromosomes 13
and 21 shows an abnormal pattern of three signals for the KCNJ6 probe indicating trisomy
21 in a patient and a normal two signals for both RB1 and KCNJ6 probes in a normal control
(NL). B. FISH using whole chromosome painting (WCP) probes for chromosomes 2 and 16
shows translocated segment of 2q onto a 16p. C. FISH using locus-specifc probes for 5p and
5q detects a deletion of 5q in an abnormal cell and a disomic pattern in a normal cell. D. FISH
using gene-specifc dual-color double fusion probes for the ABL1 and BCR genes shows one
independent signal and two conjugated signals for BCR‑ABL1 rearrangement (pointed by
arrow).

of FISH probes onto nuclei or chromosomes, the targeted region of unique sequence
FISH probes for clinical use is in the range of 100–200 kb. This has been generally
accepted as the analytic resolution for FISH assays using a fuorescence microscope.
The size of G-band in the 550-band high resolution level is in an average of 5–6
megabases (Mb) per band. The analytical resolution of FISH is approximately 50
times higher than karyotyping.
To comply with clinical laboratory standards and guidelines, FISH assays need to
be verifed and validated before their usage on clinical specimens.[18, 19] Verifcation
refers to analytical accuracy by ensuring the location of hybridized probes on the
expected chromosome loci and analytical precision by measuring quantitative agree-
ment between repeated assessments of the same sample.[18] Accuracy of FISH probes
can be evaluated by a hybridization to normal control metaphases for a correct signal
pattern onto the expected loci in chromosomes and to abnormal metaphases for the
52 Cytogenetics and Molecular Cytogenetics

intended abnormal signal pattern. The latter approach has the advantage of con-
frming both chromosomal location and numerical or structural rearrangements.
Accuracy can be evaluated by scoring a minimum of fve metaphase cells to verify
the presence of hybridized probes to the appropriate chromosome loci and absence
of cross-hybridization to other chromosomes. Precision of FISH assays measures the
reproducibility by performing consecutive three to ten repeat FISH assays over three
to ten days on a selected specimen with a known proportional of normal and abnor-
mal cells. The precision is calculated as the mean, standard deviation, and range of
the results; intra-assay and inter-assay concordance for signal pattern scoring should
be ≥ 95%.[20, 21]
Validation refers to the assessment of analytical sensitivity, specifcity, reportable
range, and other performance characteristics required to ensure assay performance.
For the examination of a FISH signal at the targeted location, probe sensitivity and
specifcity are equivalent to analytical sensitivity and specifcity.[18] Probe sensi-
tivity is the percentage of scorable metaphase chromosomes or interphase nuclei
with the expected probe signal pattern. Probe specifcity is the percentage of all
scored signals that occur at the expected location at metaphase chromosomes or
at the expected signal pattern at interphase nuclei. Probe sensitivity and specifcity
should be established by analyzing signal patterns of probe hybridization to at least
40 chromosomes in 20 metaphases or 100 interphase nuclei. Reportable range is
defned by a cut-off value for normal signal patterns.[20, 21] This cut-off value for the
upper limit at the 95% and 99% confdence intervals can be easily ascertained using
the β inversion function (BETAINV) in a Microsoft excel spreadsheet.[22] In addition
to the pre-clinical verifcation and validation, quality control, quality assurance, and
quality management procedures should be implemented in the pre-analytic, ana-
lytic, and post-analytic phases for each FISH assay. Interpretation and reporting of
FISH results should follow the technical standards and guidelines.[18, 19] The results
of FISH assays should be described following the current International System for
Human Cytogenomic Nomenclature (ISCN 2020).[23] Profciency testing for labo-
ratories performing FISH assays showed a consensus of 91.7% of participants with
the same diagnostic conclusion, indicating the reliability of current FISH assays in
detecting constitutional and somatic chromosomal abnormalities.[24]

DETECTION OF CONSTITUTIONAL CYTOGENOMIC

ABNORMALITIES
The newborn incidence for numerical and structural chromosomal abnormalities is
1/154 and for recurrent microdeletions and microduplications and other pathogenic
copy number variants is estimated as 1/291; together these cytogenomic abnormali-
ties occur in approximately 1% of newborns.[25] Karyotyping, FISH, and chromo-
some microarray analysis (CMA) have been routinely used in clinical cytogenetics
laboratories to detect these abnormalities. FISH has the advantages of being directly
performed on uncultured interphase nuclei for a short turn-around time, accurately
detecting cryptic chromosomal imbalances and rearrangements, and reliably track-
ing mosaic patterns at a single cell level. It plays an integral role in the detection of
constitutional cytogenomic abnormalities.
FISH—in Routine Diagnostic Settings 53

RAPID PRENATAL SCREENING OF COMMON ANEUPLOIDY

Numerical chromosomal abnormalities involving autosomes 13, 18, and 21 and sex
chromosomes X and Y are considered common aneuploidies with syndromic pheno-
types. For example, trisomy 21 causes Down syndrome (OMIM #190685), trisomy 18
causes Edwards syndrome, and monosomy X causes Turner syndrome. In current pre-
natal clinics, advanced maternal age and results from ultrasound examination, maternal
serum screening, and non-invasive prenatal testing (NIPT) of cell-free fetal DNA in
maternal serum plasma are used to recognize fetuses highly suspected for common
aneuploidies and other cytogenomic abnormalities. Invasive procedures will be per-
formed on high-risk pregnancies to collect amniotic fuid or chorionic villus for diag-
nostic testing. AneuVysion assay, using tri-color centromeric probes for chromosomes
X, Y, and 18 and dual-color locus-specifc probes for chromosomes 13 and 21, was the
frst FISH assay approved by the US Food and Drug Administration (FDA) for rapid
prenatal aneuploidy screening in uncultured amniocytes.[6, 7] A cut-off value from con-
secutive normal cases should be calculated to differentiate a normal disomic pattern
from abnormal trisomic or monosomic patterns.[26] A multi-center retrospective study
showed an extremely high concordance rate of 99.8% between FISH and karyotyp-
ing results.[27] This highly effective FISH assay was further extended to the analysis of
uncultured cells from chorionic villi. AneuVysion FISH assay on uncultured cells can
provide results within 24 hours, while chromosome analyses on cultured amniocytes or
villi cells take seven to twelve days. This rapid screening can release the psychological
distress from pregnant women. However, the risk of misdiagnosis for as low as 0.2–
0.4% by AneuVysion could have signifcant impact in a prenatal setting.[26–28] False neg-
ative or positive results could be caused by technical errors in processing and scoring,
sampling-related issues of twin or triplet pregnancies and maternal cell contamination,
as well as occasionally inherent factors of low-level mosaicism, mosaicism confned to
the placenta, structural chromosome abnormalities, and cryptic rearrangements. It is
recommended that the results from AneuVysion should be used exclusively as a pre-
amble to full chromosome analysis, and irreversible clinical actions should not be per-
formed solely based on FISH result.[26, 28] Approximately 10% of cases with common
aneuploidy showed a mosaic pattern, and mosaicism for sex chromosome aneuploidy
varied from 3%–20%; structural rearrangements affecting the FISH interpretation, such
as isochromosome of Xq, isodicentric Y, and derivative chromosome from a familial
translocation t(Y;14)(q12;p13), were reported.[29, 30] An integrated FISH, karyotyping,
and CMA approach should be applied for safe and accurate diagnosis of common aneu-
ploidies and other cytogenomic abnormalities in current prenatal cytogenetic testing.[29]

DETECTION OF MICRODELETION AND MICRODUPLICATION SYNDROMES

High-resolution prometaphase chromosome banding has allowed the detection of
subtle interstitial or terminal deletions of 8q23.3/24.1 for Langer-Giedion syndrome,
11p13 for WAGR (Wilms tumor aniridia gonadoblastoma retardation) syndrome
(OMIM #612469), 15q11/12 for Prader-Willi syndrome (PWS, OMIM #176270) or
Angelman syndrome (AS, OMIM #105830), 17p13.3 for Miller-Dieker lissencephaly
syndrome (MDLS, OMIM #247200), and 22q11.2 for DiGeorge syndrome (DGS,
54 Cytogenetics and Molecular Cytogenetics

OMIM #188400).[31] These microdeletions could be missed by routine karyotyping

but are reliably detected by targeted FISH probes.[32, 33] FISH analysis enabled the
detection of a microduplication at 22q11.2 as an emerging syndrome, and further
confrmed that low copy repeats (LCRs) on 22q mediated nonallelic homologous
recombination (NAHR) for recurrent microdeletions and microduplications.[34, 35]
Microdeletion and microduplication syndromes are a group of genomic disorders
caused by LCR-mediated NAHR in the human genome. The estimated newborn
incidence for the most frequently seen DiGeorge syndrome and all microdeletions
and microduplications were approximately 1/4000 and 1/400, respectively. The diag-
nostic effcacy in current prenatal and pediatric settings was estimated as 8% and
92% for DiGeorge syndrome and 3% and 46% for all microdeletions and micro-
duplications, respectively.[25] To improve the diagnostic effcacy in current prenatal
setting, enhanced NIPT to screen for microdeletions and microduplications followed
by diagnostic FISH testing and CMA on amniocytes or villi cells should be imple-
mented. Table 6.1 lists commonly used FISH probes for detecting aneuploidy, micro-
deletions, and microduplications in current prenatal and pediatric settings.

TABLE 6.1
FISH Probes for Common Aneuploidies and Microdeletion/Duplication
Syndromes
Diseases Chromosomal FISH Probes
Abnormalities
Aneuploidy
Sex chromosome aneuploidies 45,X, 47,XXX, DXZ1, DYZ3
47,XXY, 47,XYY
Pätau syndrome trisomy 13 RB1, KCNJ6
Down syndrome trisomy 21
Edwards syndrome trisomy 18 D18Z1
Microdeletion/microduplication syndromes
Kallman syndrome del(X)(p22.31p22.31) KAL1
X-linked ichthyosis del(X)(p22.31p22.31) STS
Cri-du-Chat syndrome del(5)(p15.2p15.2) CTNND2
Sotos syndrome del(5)(q35q35) NSD1
Saethre-Chotzen syndrome del(7)(p21.1p21.1) TWIST1
Williams-Beuren syndrome del(7)(q11.23q11.23) ELN
Williams-Beuren region duplication syndrome dup(7)(q11.23q11.23) ELN
Langer-Giedion Syndrome del(8)(q23.3q23.3) TRPS1
Multiple hereditary exotoses del(8)(q24.1q24.1) EXT1
Prader-Willi/Angelman syndromes del(15)(q11q13) SNRPN, UBE3A
15q11-q13 duplication syndrome dup(15)(q11q13) SNRPN, UBE3A
Smith-Magenis syndrome del(17)(p11.2p11.2) RAI1
Miller-Dieker syndrome del(17)(p13.3p13.3) LIS1
DiGeorge syndrome del(22)(q11.21q11.21) TUPLE1
22q11.2 duplication syndrome dup(22)(q11.21q11.21) TUPLE1
Phelan-McDermid syndrome del(22)(q13q13) SHANK3

Abbreviations: del: deletion, dup: duplication

FISH—in Routine Diagnostic Settings 55

DETECTION OF NUMERICAL AND STRUCTURAL CHROMOSOMAL ABNORMALITIES

Unique numerical chromosomal abnormalities such as small supernumerary marker
chromosomes (sSMC) posed an interpretation challenge because the nature of these
markers cannot be unambiguously identifed by their morphology and banding pat-
tern. The newborn incidence of sSMC was estimated as 0.043%, and approximately
34% of the sSMC cases are correlated with known clinical syndromes. The isochro-
mosome of 12p, i(12p), detected preferentially in skin fbroblasts and less likely in
peripheral blood lymphocytes, is diagnostic for Pallister-Killian syndrome (PKS;
OMIM #601803). The isochromosome of 18p, i(18p), causes i(18p) syndrome (OMIM
#614290). The derivative chromosome 22 from malsegregation of parental carriers
with constitutional balanced translocation t(8;22)(q24.13;q11.2) or t(11;22)(q23;q11.2)
caused supernumerary der(22)t(8;22) syndrome (OMIM #613700) or Emanuel syn-
drome (OMIM #609029) with distinct phenotype of severe intellectual and physi-
cal disabilities. An inverted duplication of 22q, inv dup(22), is diagnostic for cat
eye syndrome (CES; OMIM #115470). The i(12p), der(22)t(11;22), inv dup(22), and
i(18p) accounted for approximately 11%, 10%, 7%, and 6% of sSMC, respectively.[36]
Multiplex centromeric and pericentromeric probes and multicolor probes have been
used to characterize sSMC.[10, 37, 38] A study of 28,000 prenatal cases from four diag-
nostic laboratories detected 23 cases with sSMC (0.082%); 10 pregnancies were ter-
minated due to ultrasound abnormalities and syndromic associated sSMC by FISH
assays, and 13 pregnancies resulted in normal healthy neonates.[39] Approximately
50% of sSMC cases showed different levels of somatic mosaicism in different tis-
sues; the vast majority of these somatic mosaicism had no direct clinical effect, but
a de novo sSMC in a somatic state may be a hint of uniparental disomy (UPD).[40]
UPD of chromosomes 6, 7, 14, 15, 16, and 20 have been reported in cases with sSMC
in those chromosomes.[41] Collected data from literature also demonstrated that the
presence of sSMC raises clinical concern of infertility, including spermatogenesis
impairment, amenorrhea, premature ovarian failure, implantation diffculties, and
recurrent pregnancy loss.[42]
Unbalanced and balanced structural chromosomal abnormalities could be further
characterized using FISH assays. Subtelomeric rearrangements, including familial
cases with carriers of balanced cryptic subtelomeric translocations, may be missed
by routine banding analysis but can be detected by multiplex FISH using a panel of
subtelomeric probes.[12] Of 11,688 individuals with developmental disabilities ana-
lyzed by a panel of subtelomeric FISH probes, the detection rate for clinically signif-
cant subtelomeric abnormalities was approximately 2.5% with an additional 0.5%
for presumed familial variants.[43]
Constitutional ring chromosomes are a rare type of structural abnormalities with
an estimated newborn incidence of 1 in 50,000. Ring chromosomes present pos-
sible genomic imbalances from the ring formation, variable levels of dynamic mosa-
icism through mitosis, and selective karyotype evolution in different tissues. This
cytogenomic heterogeneity is likely correlated with clinical heterogeneity resulting
from compound effects of generalized features of ‘ring syndrome’ and chromosome-
specifc and dosage-sensitive gene-related phenotypes, risks of infertility, and can-
cer predisposition.[44] FISH assays are a valuable tool to visualize the deletions and
duplications in the ring chromosomes and to track the dynamic mosaic patterns in
56 Cytogenetics and Molecular Cytogenetics

interphase cells. For example, FISH mapping identifed the presence of terminal
deletions in a ring chromosome 6 and the association of a deletion of the FOXC1
gene with eye anomalies in the patient.[45] Cases of ring chromosomes 13 and 21
with terminal deletions and interstitial duplication detected by CMA were further
confrmed by FISH.[46, 47] Figure 6.2A shows the FISH assays performed on a case
with a ring chromosome 13 and the tracking of a variant ring and ring chromosome
loss in interphase cells.
CMA has been the method of choice to defne genomic imbalances, but karyotyp-
ing and FISH are needed to construct these imbalances into the rearranged chromo-
somes.[48, 49] For example, in a case with a reciprocal translocation t(5;8)(p15.3;p23.1)
and derivative chromosomes 11 and 16 from likely an 11q/16q rearrangement by band-
ing analysis and deletions in an 11q and a 16q defned by CMA, FISH assays using
whole chromosome painting probes, subtelomeric probes, and locus-specifc probes
on metaphase chromosomes revealed multiple-breakpoint inter-/intra-chromosome
rearrangements during the formation of these derivative chromosomes and identifed
FZD4 haploinsuffciency for exudative vitreoretinopathy in this patient.[50] In a case of
spontaneous abortion with a 1.5 Mb deletion at 5p15.33 detected by CMA, chromo-
some analysis and FISH assay revealed inverted duplication, triplication, and quin-
tuplication resulted from sequential breakage–fusion–bridge events (Figure 5.2B).[51]
For balanced translocations with clinical symptoms, physical mapping of breakpoints
by FISH probes could lead to the identifcation of candidate genes. For example, in a
unique case with a balanced translocation t(9;13), the delineation of the breakpoint and

FIGURE  6.2 FISH analysis for chromosomal structural abnormalities. A. FISH using
probes for the RB1 gene at 13q14.2 and the D13S1825 locus at 13q34 on a metaphase shows a
ring chromosome 13 with an interstitial duplication and a terminal deletion. Further examina-
tion of signal pattern on interphase nuclei noted this ring 13, a dicentric ring 13, and loss of
this ring chromosome 13 for monosomy 13 in 90%, 1%, and 9% of cells. B. FISH using dual-
color probes for distal 5p detects three types of rearrangements. A diagram shows a sequen-
tial breakage–fusion–bridge process through mitosis in the formation of this mosaic pattern.
FISH—in Routine Diagnostic Settings 57

further expression analysis on the gene at the junction identifed that increased expres-
sion of the α‑Kloth gene results in hypophosphatemic rickets and hyperparathyroid-
ism.[52] These cases demonstrated that FISH is an integral part of cytogenomic analysis
to facilitate accurate reporting and interpretation.

DETECTION OF SOMATIC NUMERICAL AND STRUCTURAL

CHROMOSOMAL ABNORMALITIES
The identifcation of Philadelphia chromosome in patients with chronic myelogenous
leukemia (CML) and further characterization of this reciprocal translocation t(9;22)
(q34.1;q11.2) with underlying BCR‑ABL1 gene fusion marked the start of cancer
cytogenetics.[53, 54] A FISH assay using dual-color probes detected one conjugated
signal for BCR‑ABL1 gene fusion in metaphase derivative chromosome 22 and inter-
phase nuclei.[55] Follow up FISH assays detected trisomy 12 in chronic lymphocytic
leukemia (CLL) and trisomy 8 in myeloid leukemia and thus initiated diagnostic
application of FISH on various types of tumors.[56–58] A multicenter investigation
validated the dual-color FISH assay using BCR/ABL1 probes with a clinical sensitiv-
ity of 94% and high concordance for well-trained FISH readers.[59] With the develop-
ment of professional guidance for FISH testing and specifc panels of FISH probes
for recurrent clonal abnormalities of various types of cancers, FISH assays have been
an indispensable tool with expanding applications in cancer diagnostics.[60–61]

DETECTION OF RECURRENT REARRANGEMENTS IN HEMATOPOIETIC

AND LYMPHOID TISSUES

The World Health Organization (WHO) continuously updated the classifcation of

tumors of hematopoietic and lymphoid tissues and correlated recurrent numerical
and structural chromosomal rearrangements.[62] Most of these chromosome rear-
rangements have distinctive diagnostic and prognostic signifcance, and some of
them can guide targeted therapy.[63] Conventional karyotyping and FISH assays are
used to establish the cytogenetic profle at the initial diagnosis and to monitor the
clonal evolution, disease progression, and therapeutic effect in follow-up studies.
CML is classifed under myeloproliferative neoplasms. The translocation t(9;22)
(q34.1;q11.2) with BCR‑ABL1 gene fusion is seen in 90–95% of CML, and the
remaining 5%–10% have variant translocations involving three or four chromo-
somes and/or cryptic translocation involving BCR‑ABL1 gene fusion. The cryptic
translocations are hard to identify in the banding analysis but can be detected by
FISH. Approximately 43% of variant translocations were observed with deletions in
the formation of three-way translocations and worse response to treatment.[64] Even
FISH may miss some rare cases carrying insertional translocation in BCR‑ABL1
positive CML. The chimeric BCR‑ABL1 protein with more potent tyrosine kinase
activity are subjected to FDA-approved targeted therapy by small molecule tyrosine
kinase inhibitors such as imatinib, dasatinib, nilotib, and ponatinib.[63]
Myeloid and lymphoid neoplasms with eosinophilia are often a result of rear-
rangements of the PDGFRA, PDGFRB, and FGFR1 genes fusing with multiple
58 Cytogenetics and Molecular Cytogenetics

partner genes; these abnormal gene fusions encode constitutively activated tyrosine
kinases. Routine karyotype can detect most of these rearrangements but escape
cryptic rearrangements such as a submicroscopic deletion del(4)(q12q12) for FIP1L1‑
PDGFRA gene fusion. FISH assays using dual-color break apart probes for these
genes can detect cryptic rearrangements and infer the partner genes in chromosom-
ally observed or unobserved rearrangements. Imatinib is the frst line therapy for
patients with rearrangements of the PDGFRA and PDGFRB genes, where patients
with FGFR1 gene rearrangements are resistant to this therapy and carry a poor
prognosis.[65]
A panel of 20 probes for 10 FISH assays was utilized to detect recurrent numeri-
cal and structural rearrangements for myelodysplastic syndromes (MDS) and acute
myeloid leukemia (AML); the concordance rate between karyotyping and FISH
was more than 98%.[66] Dual-color two loci enumeration probes for 5q, 7q, and
8q/20q detect −5/5q−, −7/7q−, +8, and −20/20q−, respectively. Dual-color double
fusion probes for the RUNX1‑RUNX1T1, BCR‑ABL1, PML‑RARA, and CBFB‑
MYH11 genes can detect translocations t(8;21)(q22;q22), t(9;22)(q34.1;q11.2), t(15;17)
(q24;q21), and inversion inv(16)(p13q22), respectively. Dual-color break apart probes
for the MECOM gene at 3q26.2, the KMT2A gene at 11q23, and the NUP98 gene at
11p15.4 can detect translocations t(3q;var), t(11q;var), and t(11p;var) for rearrange-
ments of these genes with multiple partner genes. Patients with translocations t(8;21),
t(15;17), and inversion inv(16) have a favorable prognosis, trisomy 8 for an intermedi-
ate prognosis, and 5q-, 7q-, translocation t(11;var), and translocation t(9;22) for unfa-
vorable prognosis. Rapid FISH can give a result within a few hours, which could be
vital for specifc leukemias, such as acute promyelocytic leukemia (APL). APL has
characteristic reciprocal translocation t(15;17) and variant rearrangements involving
the PML‑RARA gene fusion. APL is prone to intra-vascular coagulation and causes
a high risk of mortality due to cerebral hemorrhage. All-trans retinoic acid therapy
is essential for complete remission in these patients. Rapid FISH test can provide
important evidence for the correct diagnosis and treatment of APL, which leads to
more possibilities for patients to survive.
B-cell precursor acute lymphoblastic leukemia (pre B-ALL) is a heterogeneous
disease with associated recurrent chromosomal abnormalities for diagnostic sub-
typing, prognostic stratifcation, and treatment strategy.[67] Current FISH testing for
B-ALL uses two panels of FISH probes and additional FISH probes for specifc rear-
rangements. The primary panel has four sets of probes including the ETV6‑RUNX1
genes for translocation t(12;21)(p13;q22) and an intrachromosomal amplifcation of
21q (iAMP21), the CEP4/CEP10/CEP17 centromeric probes for hyperdiploidy and
low hypodiploidy, the KMT2A gene for an 11q23 translocation t(11q;var) with vari-
ous partners, and the BCR‑ABL1 genes for translocation t(9;22). The detection of
iAMP21 by FISH is defned by fve or more signals for the RUNX1 probe in inter-
phases or three or more signals in a single chromosome 21. Additionally, FISH using
probes for the TCF3‑PBX1 genes for translocation t(1;19)(q23;p13.3) and the TCF3‑
HLF genes for translocation t(17;19)(q22;p13.3) can be performed. The secondary
panel uses dual-color break apart probes for the ABL2 gene at 1q25, the PDGFRB
gene at 5q32, the JAK2 gene at 9p24.1, the ABL1 gene at 9q34, and the CRLF2 gene
at Xp22.3 or Yp11.2 to detect rearrangements for BCR‑ABL1-like B-ALL (Ph-like
FISH—in Routine Diagnostic Settings 59

B-ALL). Patients detected with iAMP21, hypodiploidy, translocations t(17;19),

t(9;22), and t(11q,var) have a high risk stratifcation. Patients with t(1;19) have an
intermediate risk. Patients with hyperdiploidy and translocation t(12;21) have a good
prognosis. T-cell acute lymphoblastic leukemia (T-ALL) showed clonal rearrange-
ments of the T-cell receptor genes. FISH assays using dual-color break apart probes
for the TCRA/D gene at 14q11.2, the TCRB gene at 7q34, the TCRG gene at 7p14, and
the TCL gene at 14q32 were introduced as a sensitive and accurate approach for rapid
detection of rearrangements involving the T-cell receptor genes.[68]
Mature B-cell neoplasms include CLL/small lymphocytic lymphoma (SLL),
plasma cell neoplasms, and various types of B-cell lymphomas. FISH assays on
interphase nuclei are an essential part of the clinical evaluation for CLL patients.
A CLL research consortium tested a panel of FISH probes for CLL and noted a 2%
false negative rate and 3% false positive rate.[69] Current FISH panel include probes
of the ATM and TP53 genes for deletions of 11q and 17p, the D12Z3/DLEU1/CDC16
loci for trisomy 12 and deletions of 13q or monosomy 13, and the IGH gene for
translocation t(14q;var). An additional FISH application using probes of the MYB/
D6Z3 loci for a deletion of 6q can be performed. CLL patients with deletions of 13q
have a favorable prognosis, with trisomy 12 for an intermediate prognosis, and with
deletions of 11q and 17p for a poor prognosis.[70]
Plasma cell neoplasms include monoclonal gammopathy of undetermined sig-
nifcance (MGUS) and plasma cell myeloma (PCM). Because of the low percentage
of plasma cells in bone marrow specimen and low mitotic index of abnormal cells
under in vitro cell culture, chromosomal abnormalities could escape routine karyo-
typing or present in a very low percentage of cells by interphase FISH. Enrichment of
plasma cells by CD138 magnetic micro bead sorting has been introduced to improve
the diagnostic yield.[71] Current FISH arrays for MGUS/PCM is ideally performed
on enriched plasma cells (CD138+) using a panel of probes of the CDKN2C/CKS1B
genes for deletion of 1p and duplication of 1q, the D9Z3/PML/TP53 loci for hyper-
diploidy and a deletion of 17p, the DLEU1/CDC16 loci for deletions of 13q or mono-
somy 13, the IGH gene for translocation t(14q;var) with various partner genes, and the
MYB gene for a deletion of 6q. Further characterization of partner genes for positive
IGH break apart can be performed by using dual-color double fusion probes of the
IGH‑FGFR3, IGH‑CCND3, IGH‑CCND1, IGH‑MAF, and IGH‑MAFB genes for
translocations t(4;14)(p16;q32), t(6;14)(p21;q32), t(11;14)(q13;q32), t(14;16)(q32;q23),
and t(14;20)(q32;q12), respectively. Patients with deletions of 13q and 17p, hypodip-
loidy, translocations t(4;14), t(14;16), or t(14;20) have unfavorable risk. Patients with
absence of unfavorable abnormalities and presence of hyperdiploidy and transloca-
tions t(11;14) or t(6;14) have favorable risk.[71]
A panel of FISH probes for various types of lymphomas include dual-color break-
apart probes for the BCL6 gene at 3q27, the MYC gene at 8q24, the IGH gene at
14q32, and the BCL2 and MALT1 genes at 18q21. More specifcally, extranodal mar-
ginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma)
associated with translocations t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21),
and t(3;14)(p14.1;q32) can be indicated by FISH assays using the IGH and MALT1
probes. Follicular lymphoma (FL) caused by translocation t(14;18)(q32;q21) can be
detected using dual-color double fusion IGH‑BCL2 probes. Diffuse large B-cell
60 Cytogenetics and Molecular Cytogenetics

lymphoma (DLBCL) with BCL6 gene rearrangement and Burkitt lymphoma (BL)
with MYC gene rearrangement can also be detected by this FISH panel for lymphoma.
For cutaneous T-cell lymphoma (CTCL), a panel of 11 FISH probes was developed
and validated to capture gene copy number alterations. This panel includes enumera-
tion probes for genes ARID1A at 1p35.3, DNMT3A at 2p23, CARD11 at 7p22, MYC at
8q24.2, CDKN2A at 9p21.3, ZEB1 at 10p11.2, FAS at 10q24.1, ATM at 11q22.3, RB1
at 13q14.2, TP53 at 17p13.1, and STAT3/5B at 17q21.3.[72]
In general, FISH is an effective way to detect recurrent rearrangements with sig-
nifcant clinical value on diagnosis, prognosis, and treatment. FISH is mandatory
when the karyotype is normal or analyzable metaphases are not present. Different
kinds of specimens such as bone marrow, blood, and lymph node are available for
FISH testing in hematology and lymphoma. Selecting a suitable specimen is criti-
cal for a precise result. It is better to use bone marrow for myeloid diseases, bone
marrow or blood for CLL, CD138+ sorted plasma cells for MGUS/PCM, and lymph
nodes for lymphoma. FISH assays offer a rapid detection of recurrent clonal rear-
rangements, many of which are cryptic and escape the detection by banding analysis,
such as translocation t(4;14)(p16.3;q32) with IGH‑FGFR3 gene fusions, translocation
t(12;21)(p13;q22) with ETV6‑RUNX1 gene fusions, gene deletions of 9p21(CDKN2A),
12p13(ETV6), 13q14.2(RB1), and 17p13.1(TP53), and rearrangements leading to
Ph-like or ETV6‑RUNX1-like ALL. Some chromosome rearrangements involve
genes with many partner genes, such as KMT2A, MYC, IGH, and ETV6. FISH is
recommended to detect rearrangements for these genes. For example, KMT2A rear-
rangement is one of the major drivers and generally regarded as a poor prognostic
factor in acute leukemias. There are more than 120 partner genes of KMT2A rear-
rangement identifed currently, many of which are cryptic and unidentifed by chro-
mosome analysis. FISH is a good approach to detect KMT2A rearrangement using
the break-apart probe. Additionally, FISH assay using probes for sex chromosomes
X and Y has been used to check engraftment of donor cells from sex-mismatched
bone marrow and blood stem cell transplantation. Online resources such as the ‘Atlas
of Genetics and Cytogenetics in Oncology and Haematology’ (http://atlasgeneticson-
cology.org/) are very helpful in interpreting chromosome and FISH results. Table 6.2
lists the routinely used FISH panels for tumors of hematopoietic and lymphoid tis-
sues in clinical cytogenetics.

DETECTION OF RECURRENT REARRANGEMENTS IN SOLID TUMORS

Conventional cytogenetics has detected recurrent chromosomal abnormalities from
various types of solid tumors. However, this cell culture–dependent karyotyping
has experienced reduced success rate in many solid tumor specimens. Interphase
FISH assays performed directly on tumor cells have been a supplement to overcome
this diffculty and applied to different types of tumors such as brain tumors, malig-
nant melanoma, and head and neck squamous cell carcinoma.[73–75] The feasibility
of FISH assays on sections of paraffn-embedded tissues was evaluated and then
quickly applied to detect numerical aberrations from renal cortical neoplasms and
gonadal yolk sac tumors.[76–78] This development extended FISH assays onto fxed
tumor tissues.
 FISH—in Routine Diagnostic Settings
TABLE 6.2
FISH Probes for Various Tumors of Hematopoietic and Lymphoid Tissues
Disease Recurrent Rearrangements Gene Rearrangement FISH Probes Prognosis Targeted Therapy
Myeloproliferative neoplasms (chronic myelogenous leukemia, CML)
t(9;22)(q34.1;q11.2) BCR‑ABL1 BCR, ABL1 good following therapy BCR-ABL1 tyrosine
kinase inhibitor
Myeloid/lymphoid neoplasms with eosinophilia
del(4q12) or t(4q12;var) FIP1L1‑PDGFRA FIL1L1, CHIC2, PDGFRA favorable prognosis tyrosine kinase inhibitor
t(5q32;var) PDGFRB PDGFRB good following therapy tyrosine kinase inhibitor
t(8p11;var) FGFR1 FGFR1 poor
t(8;9)(p22;p24.1) PCM1‑JAK2 JAK2 survival highly variable
Myelodysplastic syndrome (MDS)
del(5q)/-5 TAS2R1, EGR1 good
del(7q)/-7 RELN, TES intermediate or poor
trisomy 8, del(20q) MYC, PTPRT intermediate, good
del(17p) TP53 poor
Acute myeloid leukemia (AML)
t(8;21)(q22;q22) RUNX1‑RUNX1T1 RUNX1, RUNX1T1 good
t(9;22)(q34.1;q11.2) BCR‑ABL1 BCR, ABL1 poor
t(11p15.4;var) NUP98 NUP98
t(11q23;var) KMT2A KMT2A intermediate
t(15;17)(q24;q21) PML‑RARA PML, RARA good tretinoin and arsenic
trioxide
inv(16)(p13q22), t(16;16) CBFB‑MYH11 CBFB, MYH11 good
inv(3)(q21.3q26.2), t(3;3) MECOM MECOM poor

(Continued)

61
 62
TABLE 6.2
(Continued)
Disease Recurrent Rearrangements Gene Rearrangement FISH Probes Prognosis Targeted Therapy
B-cell precursor acute lymphoblastic leukemia (B-ALL)
t(9;22)(q34.1;q11.2) BCR‑ABL1 BCR, ABL1 poor tyrosine kinase inhibitors
t(11q23;var) KMT2A KMT2A poor
t(12;21)(p13.2;q22.1), iAMP21 ETV6‑RUNX1 ETV6, RUNX1 very favorable
Hyperdiploid 4/10/17 D4Z1, D10Z1, D17Z1 very favorable
t(1;19)(q23;p13.3) TCF3‑PBX1 TCF3, PBX1 intermediate risk intensive therapy
t(17;19)(q22;p13.3) TCF3‑HLB TCF3, HLB
Ph-like B-cell ALL
t(1q25;var) ABL2 ABL2 high risk ABL-class tyrosine
kinase inhibitors

Cytogenetics and Molecular Cytogenetics

t(5q32;var) PDGFRB PDGFRB high risk tyrosine kinase inhibitors
t(9p24.1;var) JAK2 JAK2 high risk tyrosine kinase inhibitors
t(9q34;var) ABL1 ABL1 tyrosine kinase inhibitors
t(Xp22.3/Yp11.2;var) CRLF2‑P2RY8 CRLF2, P2RY8 high risk tyrosine kinase inhibitors
T-lymphoblastic leukemia/lymphoma
t(14q11.2;var) TCRA/D TCRA/D
t(7q35;var) TCRB TCRB
t(7p14;var) TCRG TCRG
t(14q32;var) TCL TCL
Mature B-cell neoplasms (chronic lymphocytic leukemia, CLL)
del(11q), del(17p) ATM, TP53 poor
trisomy 12, del(13q) D12Z6, DELU1, CDC16 intermediate, favorable
t(14q32;var) IGH IGH
del(6q) MYB, D6Z3
 FISH—in Routine Diagnostic Settings
Plasma cell neoplasms/multiple myeloma
del(1p), dup(1q) CDKN2C, CKS1B
hyperdiploid, 9/15/17 D9Z3, PML, TP53
del(13q) DLEU1, CDC16
t(14q32;var) IGH IGH
t(4;14)(p16.3;q32) IGH‑FGFR3 IGH, FGFR3 intermediate risk
t(6;14)(p21;q32) IGH‑CCND3 IGH, CCND3 standard risk
t(11;14)(q13;q32) IGH‑CCND1 IGH, CCND1 standard risk
t(14;16)(q32;q23) IGH‑MAF IGH, MAF high risk
t(14;18)(q32;q21) IGH‑BCL2 IGH, BCL2 BCL2 inhibitors
t(14;19)(q32;q13) IGH‑BCL3 IGH, BCL3
t(14;20)(q32;q11.2) IGH‑MAFB IGH, MAFB high risk
t(8;14)(q24.2;q32) IGH‑MYC IGH, MYC
Lymphomas
t(3q27;var) BCL6 BCL6
t(8q24;var) MYC MYC
t(14q32;var) IGH IGH
t(18q21;var) BCL2 BCL2
t(18q21;var) MALT1 MALT1
Follicular lymphoma
t(14;18)(q32;q21) IGH‑BCL2 IGH, BCL2
Mantle cell lymphoma
t(11;14)(q13;q32) IGH‑CCND1 IGH, CCND1 median survival 3–5 years
ALK-positive large B-cell lymphoma
t(2;17)(p23;q23) CLTC‑ALK ALK
t(2;5)(p23;q35) ALK‑NPM1 ALK inhibitors of ALK kinase

Abbreviations: var: variant partner genes

63
64 Cytogenetics and Molecular Cytogenetics

Recurrent chromosomal rearrangements involving tumorigenesis gene fusions

have been detected in various types of solid tumors.[79] For example, rearrangements
involving the EWSR gene at 22q12 with various partner genes, translocation t(22q;var),
have been diagnostic for Ewing sarcoma, angiomatoid fbrous histocytoma, clear
cell sarcoma, desmoplastic round cell tumor, myxoid round cell liposarcoma, and
extraskeletal myxoid chondrosarcoma. Rearrangements involving the ALK gene at
2p23, translocation t(2p;var), are diagnostic for infammatory myofbroblastic tumor.
Rearrangements involving the FOXO1A, PAX3, NCOA1 and PAX7 genes are diag-
nostic for alveolar rhabdomyosarcoma. Translocations of t(7;16)(q33;p11.2) for FUS‑
CREB3L2 gene fusion and t(11;16)(p11;p11.2) for FUS‑CREB3L1 gene fusion are
diagnostic for low-grade fbromyxoid sarcoma. Translocations of t(X;18)(p11.2;q11.2)
for SS18‑SSX1/2/4 gene fusions and t(X;20)(p11.2;q13) for SS18L1‑SSX1 gene fusion
are diagnostic for synovial sarcoma. Specifc translocation t(X;17)(p11.2;q25) with
ASPSCR1‑TFE3 gene fusion is diagnostic for alveolar soft-part sarcoma and rear-
rangements of TFE3 with other genes are diagnostic for renal cell carcinoma. The
rearrangements involving the TMPRSS2 gene at 22q22.3 are indicative of early stage
of prostate cancer. The translocation t(17;22) with COL1A1‑PDGFRB gene fusion
and its variants is diagnostic for dermatofbrosarcoma protuberans.[80] FISH probes
are available for the detection of these rearrangements on metaphase chromosomes
and interphases nuclei from tumor specimens.
Recurrent numerical chromosome abnormalities with histologic and clinical asso-
ciation to various solid tumors have been characterized.[79] Genomic approaches like
CMA can detect genome-wide somatic copy number alterations, but FISH is still
the gold standard to determine losses and gains of specifc genes and chromosome
regions at a single-cell level.[81] For renal cell carcinoma, deletions of 3p, gains of
chromosomes 7 and 17, losses of chromosomes 1 and 17, and losses of chromosomes
1 and 14 are for differential diagnosis of clear cell carcinoma, papillary carcinoma,
chromophobe carcinoma, and oncocytoma, respectively. Amplifcation of the EGFR
gene distinguishes glioblastoma with small cell phenotype. For bladder cancer, an
UroVysion FISH panel, including centromeric probes for chromosomes 3, 7, and 17
and a locus-specifc probe for the CDKN2A gene at 9p21, was approved by the FDA
for surveillance and determining therapy effectiveness.[82] Additional deletions of
the RB1 gene at 13q14.2, the PTEN gene at 10q23, and the TP53 gene at 17p13.1, and
amplifcation of the ERBB2 gene at 17q21 predict poor outcome for bladder cancer.
Amplifcation of ERBB2 gene (HER2) and EGFR gene at 17p12 have been seen in
breast carcinoma. Guidelines for HER2 FISH testing in breast cancer have been
updated for accurate scoring of HER2-postive breast cancer for anti-HER2 targeted
therapy.[83]

CONCLUSIONS
In summary, FISH assays are a clinically validated and cell-based diagnostic
tool capable of detecting numerical and structural chromosomal abnormalities
and genomic copy number alterations. FISH assays have been routinely used in
rapid prenatal screening of common aneuploidies, detection of microdeletion and
microduplication syndromes, and further characterization of sSMC, subtelomeric
FISH—in Routine Diagnostic Settings 65

rearrangements, ring chromosome structure, and complex chromosome rearrange-

ments. FISH assays play an essential role in cancer cytogenetics by detecting recur-
rent numerical aberrations and gene rearrangements of clinical signifcance in
diagnosis, prognosis, and targeted therapy for various types of tumors.

ACKNOWLEDGEMENTS
Special thanks to Autumn DiAdamo and Jiadi Wen of Yale Clinical Cytogenetics
Laboratory (USA) for helpful discussion and careful editing on the content of this
chapter.

REFERENCES
1. Gall, J.G.; Pardue, M.L. Formation and detection of RNA-DNA hybrid molecules in
cytological preparations. Proc. Natl. Acad. Sci. U. S. A. 1969, 63, 378–383.
2. Rudkin, G.T.; Stollar, B.D. High resolution detection of DNA-RNA hybrids in situ by
indirect immunofuorescence. Nature. 1977, 265, 472–473.
3. Lichter, P.; Cremer, T.; Borden, J.; Manuelidis, L.; Ward, D.C. Delineation of individual
human chromosomes in metaphase and interphase cells by in situ suppression hybridiza-
tion using recombinant DNA libraries. Hum. Genet. 1988, 80, 224–234.
4. Lichter, P.; Tang, C.J.; Call, K.; Hermanson, G.; Evans, G.A.; Housman, D.; Ward, D.C.
High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid
clones. Science. 1990, 247, 64–69.
5. Cheung, V.G.; Nowak, N.; Jang, W.; Kirsch, I.R.; Zhao, S.; Chen, X.N.; Furey, T.S.; Kim,
U.J.; Kuo, W.L.; Olivier, M.; Conroy, J.; Kasprzyk, A.; Massa, H.; Yonescu, R.; Sait, S.;
Thoreen, C.; Snijders, A.; Lemyre, E.; Bailey, J.A.; Bruzel, A.; Burrill, W.D.; Clegg,
S.M.; Collins, S.; Dhami, P.; Friedman, C.; Han, C.S.; Herrick, S.; Lee, J.; Ligon, A.H.;
Lowry, S.; Morley, M.; Narasimhan, S.; Osoegawa, K.; Peng, Z.; Plajzer-Frick, I.; Quade,
B.J.; Scott, D.; Sirotkin, K.; Thorpe, A.A.; Gray, J.W.; Hudson, J.; Pinkel, D.; Ried, T.;
Rowen, L.; Shen-Ong, G.L.; Strausberg, R.L.; Birney, E.; Callen, D.F.; Cheng, J.F.; Cox,
D.R.; Doggett, N.A.; Carter, N.P.; Eichler, E.E.; Haussler, D.; Korenberg, J.R.; Morto,
C.C.; Albertson, D.; Schuler, G.; de Jong, P.J.; Trask, B.J.; BAC Resource Consortium.
Integration of cytogenetic landmarks into the draft sequence of the human genome.
Nature. 2001, 409, 953–958.
6. Ried, T.; Landes, G.; Dackowski, W.; Klinger, K.; Ward, D.C. Multicolor fuorescence in
situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18,
21, X and Y in uncultured amniotic fuid cells. Hum. Mol. Genet. 1992, 1, 307–313.
7. Weise, A.; Liehr, T. Rapid prenatal aneuploidy screening by fuorescence in situ hybrid-
ization (FISH). Methods Mol. Biol. 2019, 1885, 129–137.
8. Speicher, M.R.; Ward, D.C. The coloring of cytogenetics. Nat. Med. 1996, 2, 1046–1048.
9. Cui, C.; Shu, W.; Li, P. Fluorescence in situ hybridization: Cell-based genetic diagnostic
and research applications. Front. Cell. Dev. Biol. 2016, 4, 89.
10. Castronovo, C.; Valtorta, E.; Crippa, M.; Tedoldi, S.; Romitti, L.; Amione, M.C.;
Guerneri, S.; Rusconi, D.; Ballarati, L.; Milani, D.; Grosso, E.; Cavalli, P.; Giardino, D.;
Bonati, M.T.; Larizza, L.; Finelli, P. Design and validation of a pericentromeric BAC
clone set aimed at improving diagnosis and phenotype prediction of supernumerary
marker chromosomes. Mol. Cytogenet. 2013, 6, 45.
11. Morrison, L.E.; Ramakrishnan, R.; Ruffalo, T.M.; Wilber, K.A. Labeling fuorescence in
situ hybridization probes for genomic targets. Methods Mol. Biol. 2002, 204, 21–40.
66 Cytogenetics and Molecular Cytogenetics

12. Ning, Y.A.R.; Smith, A.C.M.; Macha, M.; Precht, K.; Riethman, H.; Ledbetter, D.H.; Flint,
J.; Horsley, S.; Regan, R.; Kearney, L.; Knight, S.; Kvaloy, K.; Brown, W.R.A. A complete
set of human telomeric probes and their clinical application: National Institutes of Health
and Institute of Molecular Medicine collaboration. Nat. Genet. 1996, 14, 86–89.
13. Bentz, M.; Cabot, G.; Moos, M.; Speicher, M.R.; Ganser, A.; Lichter, P.; Döhner, H.
Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in
chronic myeloid and acute lymphoblastic leukemia by in situ hybridization. Blood. 1994,
83, 1922–1928.
14. van der Burg, M.; Beverloo, H.B.; Langerak, A.W.; Wijsman, J.; van Drunen, E.; Slater,
R.; van Dongen, J.J. Rapid and sensitive detection of all types of MLL gene transloca-
tions with a single FISH probe set. Leukemia. 1999, 13, 2107–2113.
15. Pinkel, D.; Landegent, J.; Collins, C.; Langerak, A.W.; Wijsman, J.; van Drunen, E.;
Slater, R.; van Dongen, J.J. Fluorescence in situ hybridization with human chromosome-
specifc libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc.
Natl. Acad. Sci. U. S A. 1988, 85, 9138–9142.
16. Meltzer, P.S.; Guan, X.Y.; Burgess, A.; Trent, J.M. Rapid generation of region specifc
probes by chromosome microdissection and their application. Nat. Genet. 1992, 1,
24–28.
17. Speicher, M.R.; Gwyn Ballard, S.; Ward, D.C. Karyotyping human chromosomes by
combinatorial multi-fuor FISH. Nat. Genet. 1996, 12, 368–375.
18. Mascarello, J.T.; Hirsch, B.; Kearney, H.M.; Ketterling, R.P.; Olson, S.B.; Quigley,
D.I.; Rao, K.W.; Tepperberg, J.H.; Tsuchiya, K.D.; Wiktor, A.E; Working Group of the
American College of Medical Genetics Laboratory Quality Assurance Committee.
Section E9 of the American College of Medical Genetics technical standards and guide-
lines: Fluorescence in situ hybridization. Genet. Med. 2011, 13, 667–675.
19. (CLSI) CaLSI. MM07-A2. Fluorescence In Situ Hybridization Methods for Clinical
Laboratories Approved. Guideline. 2nd Edition. Vol. 33. Clinical and Laboratory
Standards Institute, Wayne, PA; Aug. 2013.
20. Wiktor, A.E.; Van Dyke, D.L.; Stupca, P.J.; Ketterling, R.P.; Thorland, E.C.; Shearer,
B.M.; Fink, S.R.; Stockero, K.J.; Majorowicz, J.R.; Dewald, G.W. Preclinical validation
of fuorescence in situ hybridization assays for clinical practice. Genet. Med. 2006, 8,
16–23.
21. Zneimer, S.M. Validation of fuorescence in situ hybridization (FISH) for chromosome 5
monosomy and deletion. Curr. Prot. Hum. Genet. 2020, 105, e96.
22. Ciolino, A.L.; Tang, M.E.; Bryant, R. Statistical treatment of fuorescence in situ hybrid-
ization validation data to generate normal reference ranges using Excel functions. J. Mol.
Diagn. 2009, 11, 330–333.
23. McGowan-Jordan, J.; Hastings, A.; Moore, S. ISCN 2020 an International System for
Human Cytogenomic Nomenclature (2020). Karger, Basel/Freiburg; 2020.
24. Mascarello, J.T.; Brothman, A.R.; Davison, K.; Dewald, G.W.; Herrman, M.; McCandless,
D.; Park, J.P.; Persons, D.L.; Rao, K.W.; Schneider, N.R.; Vance, G.H.; Cooley, L.D;
Cytogenetics Resource Committee of the College of American Pathologists and
American College of Medical Genetics. Profciency testing for laboratories performing
fuorescence in situ hybridization with chromosome-specifc DNA probes. Arch. Pathol.
Lab. Med. 2002, 126, 1458–1462.
25. Chai, H.; DiAdamo, A.; Grommisch, B.; Xu, F.; Zhou, Q.; Wen, J.; Mahoney, M.; Bale,
A.; McGrath, J.; Spencer-Manzon, M.; Li, P.; Zhang, H. A retrospective analysis of
10-year data assessed the diagnostic accuracy and effcacy of cytogenomic abnormali-
ties in current prenatal and pediatric settings. Front. Genet. 2019, 10, 1162.
FISH—in Routine Diagnostic Settings 67

26. Liehr, T.; Ziegler, M. Rapid prenatal diagnostics in the interphase nucleus: Procedure
and cut-off rates. J. Histochem. Cytochem. 2005, 53, 289–291.
27. Tepperberg, J.; Pettenati, M.J.; Rao, P.N.; Lese, C.M.; Rita, D.; Wyandt, H.; Gersen, S.;
White, B.; Schoonmaker, M.M. Prenatal diagnosis using interphase fuorescence in situ
hybridization (FISH): 2-year multi-center retrospective study and review of the litera-
ture. Prenat. Diagn. 2001, 21, 293–301.
28. Lall, M.; Mahajan, S.; Saviour, P.; Paliwal, P.; Joshi, A.; Setai, N.; Verma, J.C. FISH is
not suitable as a standalone test for detecting fetal chromosomal abnormalities. J. Fet.
Med. 2015, 2, 53–59.
29. Chai, H.; DiAdamo, A.; Grommisch, B.; Boyle, J.; Amato, K.; Wang, D.; Wen, J.; Li,
P. Integrated FISH, karyotyping and aCGH analyses for effective prenatal diagnosis of
common aneuploidies and other cytogenomic abnormalities. Med. Sci. (Basel). 2019,
7, 16.
30. Xie, X.; Tan, W.; Li, F.; Carrano, E.; Ramirez, P.; DiAdamo, A.; Grommisch, B.; Amato,
K.; Chai, H.; Wen, J.; Li, P. Diagnostic cytogenetic testing following positive noninvasive
prenatal screening results of sex chromosome abnormalities: Report of fve cases and
systematic review of evidence. Mol. Genet. Genomic Med. 2020, 8, e1297.
31. Schinzel, A. Microdeletion syndromes, balanced translocations, and gene mapping. J.
Med. Genet. 1988, 25, 454–462.
32. Kuwano, A.; Ledbetter, S.A.; Dobyns, W.B.; Emanuel, B.S.; Ledbetter, D.H. Detection
of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridiza-
tion. Am. J. Hum. Genet. 1991, 49, 707–714.
33. Desmaze, C.; Prieur, M.; Amblard, F.; Aikem, M.; LeDeist, F.; Demczuk, S.; Zucman,
J.; Plougastel, B.; Delattre, O.; Croquette, M.F.; Brevière, G.-M.; Huon, C.; Le Merrer,
M.; Mathieu, M.; Sidi, D.; Stephan, J.-L.; Aurias, A. Physical mapping by FISH of the
DiGeorge critical region (DGCR): Involvement of the region in familial cases. Am. J.
Hum. Genet. 1993, 53, 1239–1249.
34. Stankiewicz, P.; Lupski, J.R. Genome architecture, rearrangements and genomic disor-
ders. Trends Genet. 2002, 18, 74–82.
35. Ensenauer, R.E.; Adeyinka, A.; Flynn, H.C.; Michels, V.V.; Lindor, N.M.; Dawson, D.B.;
Thorland, E.C.; Lorentz, C.P.; Goldstein, J.L.; McDonald, M.T.; Smith, W.E.; Simon-
Fayard, E.; Alexander, A.A.; Kulharya, A.S.; Ketterling, R.P.; Clark, R.D.; Jalal, S.M.
Microduplication 22q11.2, an emerging syndrome: Clinical, cytogenetic, and molecular
analysis of thirteen patients. Am. J. Hum. Genet. 2003, 73, 1027–1040.
36. Liehr, T.; Claussen, U.; Starke, H. Small supernumerary marker chromosomes (sSMC)
in humans. Cytogenet. Genome Res. 2004, 107, 55–67.
37. Henegariu, O.; Bray-Ward, P.; Artan, S.; Vance, G.H.; Qumsyieh, M.; Ward, D.C. Small
marker chromosome identifcation in metaphase and interphase using centromeric mul-
tiplex fsh (CM-FISH). Lab. Invest. 2001, 81, 475–481.
38. Brecevic, L.; Michel, S.; Starke, H.; Müller, K.; Kosyakova, N.; Mrasek, K.; Weise, A.;
Liehr, T. Multicolor FISH used for the characterization of small supernumerary marker
chromosomes (sSMC) in commercially available immortalized cell lines. Cytogenet,
Genome Res. 2006, 114, 319–324.
39. Manolakos, E.; Kefalas, K.; Neroutsou, R.; Lagou, M.; Kosyakova, N.; Ewers, E.;
Ziegler, M.; Weise, A.; Tsoplou, P.; Rapti, S.M.; Papoulidis, I.; Anastasakis, E.; Garas,
A.; Sotiriou, S.; Eleftheriades, M.; Peitsidis, P.; Malathrakis, D.; Thomaidis, L.; Kitsos,
G.; Orru, S.; Liehr, T.; Petersen, M.B.; Kitsiou-Tzeli, S. Characterization of 23 small
supernumerary marker chromosomes detected at pre-natal diagnosis: The value of fuo-
rescence in situ hybridization. Mol. Med. Rep. 2010, 3, 1015–1022.
68 Cytogenetics and Molecular Cytogenetics

40. Liehr, T.; Ewers, E.; Hamid, A.B.; Kosyakova, N.; Voigt, M.; Weise, A.; Manvelyan, M.
Small supernumerary marker chromosomes and uniparental disomy have a story to tell.
J. Histochem. Cytochem. 2011, 59, 842–848.
41. Liehr, T.; Klein, E.; Mrasek, K.; Kosyakova, N.; Guilherme, R.S.; Aust, N.; Venner, C.;
Weise, A.; Hamid, A.B. Clinical impact of somatic mosaicism in cases with small super-
numerary marker chromosomes. Cytogenet. Genome Res. 2013, 139, 158–163.
42. Armanet, N.; Tosca, L.; Brisset, S.; Liehr, T.; Tachdjian, G. Small supernumerary marker
chromosomes in human infertility. Cytogenet Genome Res. 2015, 146, 100–108.
43. Ravnan, J.B.; Tepperberg, J.H.; Papenhausen, P.; Lamb, A.N.; Hedrick, J.; Eash, D.;
Ledbetter, D.H.; Martin, C.L. Subtelomere FISH analysis of 11 688 cases: An evaluation
of the frequency and pattern of subtelomere rearrangements in individuals with develop-
mental disabilities. J. Med. Genet. 2006, 43, 478–489.
44. Hu, Q.; Chai, H.; Shu, W.; Li, P. Human ring chromosome registry for cases in the
Chinese population: Re-emphasizing cytogenomic and clinical heterogeneity and
reviewing diagnostic and treatment strategies. Mol. Cytogenet. 2018, 11, 19.
45. Zhang, H.Z.; Li, P.; Wang, D.; Huff, S.; Nimmakayalu, M.; Qumsiyeh, M.; Pober, B.R.
FOXC1 gene deletion is associated with eye anomalies in ring chromosome 6. Am. J.
Med. Genet. 2004, 124A, 280–287.
46. Zhang, H.Z.; Xu, F.; Seashore, M.; Li, P. Unique genomic structure and distinct mitotic
behavior of ring chromosome 21 in two unrelated cases. Cytogenet. Genome Res. 2012,
136, 180–187.
47. Xu, F.; DiAdamo, A.J.; Grommisch, B.; Li, P. Interstitial duplication and distal deletion
in a ring chromosome 13 with pulmonary atresia and ventricular septal defect: A case
report and review of literature. N. Am. J. Med. Sci. 2013, 6, 208–212.
48. Rossi, M.R.; DiMaio, M.S.; Xiang, B.; Lu, K.; Kaymakcalan, H.; Seashore, M.; Mahoney,
M.J.; Li, P. Clinical and genomic characterization of distal duplications and deletions of
chromosome 4q: Study of two cases and review of the literature. Am. J. Med. Genet.
2009, 149A, 2788–2794.
49. Khattab, M.; Xu, F.; Li, P.; Bhandari, V. A de novo 3.54 Mb deletion of 17q22-q23.1 asso-
ciated with hydrocephalus: A case report and review of literature. Am. J. Med. Genet.
2011, 155A, 3082–3086.
50. Li, P.; Zhang, H.Z.; Huff, S.; Nimmakayalu, M.; Qumsiyeh, M.; Yu, J.; Szekely, A.; Xu,
T.; Pober, B.R. Karyotype-phenotype insights from 11q14.1-q23.2 interstitial deletions:
FZD4 haploinsuffciency and exudative vitreoretinopathy in a patient with a complex
chromosome rearrangement. Am. J. Med. Genet 2006, 140A, 2721–2729.
51. Chai, H.; Grommisch, B.; DiAdamo, A.; Wen, J.; Hui, P.; Li, P. Inverted duplication:
Triplication and quintuplication through sequential breakage-fusion-bridge events
induced by a terminal deletion at 5p in a case of spontaneous abortion. Mol. Genet.
Genomic. Med. 2019, 7, e00965.
52. Brownstein, C.A.; Adler, F.; Nelson-Williams, C.; Iijima, J.; Li, P.; Imura, A.; Nabeshima,
Y.; Reyes-Mugica, M.; Carpenter, T.O.; Lifton, R.P. A translocation causing increased
alpha-klotho level results in hypophosphatemic rickets and hyperparathyroidism. Proc.
Natl. Acad. Sci. U. S. A. 2008, 105, 3455–3460.
53. Nowell, P.C.; Hungerford, D.A. Chromosome studies on normal and leukemic human
leukocytes. J. Natl. Cancer Inst. 1960, 25, 85–109.
54. Rowley, J.D. Genetics: A story of swapped ends. Science. 2013, 340, 1412–1413.
55. Tkachuk, D.C.; Westbrook, C.A.; Andreeff, M.; Donlon, T.A.; Cleary, M.L.;
Suryanarayan, K.; Homge, M.; Redner, A.; Gray, J.; Pinkel, D. Detection of bcr-abl
fusion in chronic myelogeneous leukemia by in situ hybridization. Science. 1990, 250,
559–562.
FISH—in Routine Diagnostic Settings 69

56. Losada, A.P.; Wessman, M.; Tiainen, M.; Hopman, A.H.; Willard, H.F.; Solé, F.;
Caballín, M.R.; Woessner, S.; Knuutila, S. Trisomy 12 in chronic lymphocytic leukemia:
An interphase cytogenetic study. Blood. 1991, 78, 775–779.
57. Anastasi, J.; Le Beau, M.M.; Vardiman, J.W.; Fernald, A.A.; Larson, R.A.; Rowley,
J.D. Detection of trisomy 12 in chronic lymphocytic leukemia by fuorescence in situ
hybridization to interphase cells: A simple and sensitive method. Blood. 1992, 79,
1796–1801.
58. Jenkins, R.B.; Le Beau, M.M.; Kraker, W.J.; Borell, T.J.; Stalboerger, P.G.; Davis,
E.M.; Penland, L.; Fernald, A.; Espinosa, R 3rd.; Schaid, D.J.; Noel, P.; Dewald, G.W.
Fluorescence in situ hybridization: A sensitive method for trisomy 8 detection in bone
marrow specimens. Blood. 1992, 79, 3307–3315.
59. Dewald, G.; Stallard, R.; Alsaadi, A.; Arnold, S.; Blough, R.; Ceperich, T.M.; Rafael
Elejalde, B.; Fink, J.; Higgins, J.V.; Higgins, R.R.; Hoeltge, G.A.; Hsu, W.T.; Johnson,
E.B.; Kronberger, D.; McCorquodale, D.J.; Meisner, L.F.; Micale, M.A.; Oseth, L.;
Payne, J.S.; Schwartz, S.; Sheldon, S.; Sophian, A.; Storto, P.; Van Tuinen, P.; Wenger,
G.D.; Wiktor, A.; Willis, L.A.; Yung, J.F.; Zenger-Hain, J. A multicenter investigation
with D-FISH BCR/ABL1 probes. Cancer Genet. Cytogenet. 2000, 116, 97–104.
60. Wolff, D.J.; Bagg, A.; Cooley, L.D.; Dewald, G.W.; Hirsch, B.A.; Jacky, P.B.; Rao, K.W.;
Rao, P.N; Association for Molecular Pathology Clinical Practice Committee; American
College of Medical Genetics Laboratory Quality Assurance Committee. Guidance for
fuorescence in situ hybridization testing in hematologic disorders. J. Mol. Diagn. 2007,
9, 134–143.
61. Liehr, T.; Othman, M.A.; Rittscher, K.; Alhourani, E. The current state of molecular
cytogenetics in cancer diagnosis. Expert Rev. Mol. Diagn. 2015, 15, 517–526.
62. Swerdlow, S.H.; Campo, E.; Pileri, S.A.; Harris, N.L.; Stein, H.; Siebert, R.; Advani,
R.; Ghielmini, M.; Salles, G.A.; Zelenetz, A.D.; Jaffe, E.S. The 2016 revision of the
World Health Organization classifcation of lymphoid neoplasms. Blood. 2016, 127,
2375–2390.
63. Li, M.M.; Ewton, A.A.; Smith, J.L. Using cytogenetic rearrangements for cancer progno-
sis and treatment (Pharmacogenetics). Curr. Genet. Med. Rep. 2013, 1, 99–112.
64. Gorusu, M.; Benn, P.; Li, Z.; Fang, M. On the genesis and prognosis of variant transloca-
tions in chronic myeloid leukemia. Cancer Genet. Cytogenet. 2007, 173, 97–106.
65. Savage, N.; George, T.I.; Gotlib, J. Myeloid neoplasms associated with eosinophilia and
rearrangement of PDGFRA, PDGFRB, and FGFR1: A review. Int. J. Lab. Hematol.
2013, 35, 491–500.
66. Vance, G.H.; Kim, H.; Hicks, G.A.; Cherry, A.M.; Higgins, R.; Hulshizer, R.L.; Tallman,
M.S.; Fernandez, H.F.; Dewald, G.W. Utility of interphase FISH to stratify patients into
cytogenetic risk categories at diagnosis of AML in an Eastern Cooperative Oncology
Group (ECOG) clinical trial (E1900). Leuk. Res. 2007, 31, 605–609.
67. Moorman, A.V. New and emerging prognostic and predictive genetic biomarkers in
B-cell precursor acute lymphoblastic leukemia. Haematologica. 2016, 101, 407–416.
68. Gesk, S.; Martin-Subero, J.I.; Harder, L.; Luhmann, B.; Schlegelberger, B.; Calasanz,
M.J.; Grote, W.; Siebert, R. Molecular cytogenetic detection of chromosomal break-
points in T-cell receptor gene loci. Leukemia. 2003, 17, 738–745.
69. Smoley, S.A.; Van Dyke, D.L.; Kay, N.E.; Heerema, N.A.; Dell’ Aquila, M.L.; Dal Cin,
P.; Koduru, P.; Aviram, A.; Rassenti, L.; Byrd, J.C.; Rai, K.R.; Brown, J.R.; Greaves,
A.W.; Eckel-Passow, J.; Neuberg, D.; Kipps, T.J.; Dewald, G.W. Standardization of fuo-
rescence in situ hybridization studies on chronic lymphocytic leukemia (CLL) blood and
marrow cells by the CLL Research Consortium. Cancer Genet. Cytogenet. 2010, 203,
141–148.
70 Cytogenetics and Molecular Cytogenetics

70. Döhner, H.; Stilgenbauer, S.; Benner, A.; Leupolt, E.; Kröber, A.; Bullinger, L.; Döhner,
K.; Bentz, M.; Lichter, P. Genomic aberrations and survival in chronic lymphocytic leu-
kemia. N. Engl. J. Med. 2000, 343, 1910–1916.
71. Chen, Z.; Issa, B.; Huang, S.; Aston, E.; Xu, J.; Yu, M.; Brothman, A.R.; Glenn, M. A
practical approach to the detection of prognostically signifcant genomic aberrations in
multiple myeloma. J. Mol. Diagn. 2005, 7, 560–565.
72. Weed, J.; Gibson, J.; Lewis, J.; Carlson, K.; Foss, F.; Choi, J.; Li, P.; Girardi, M. FISH
panel for leukemic CTCL. J. Invest. Dermatol. 2017, 137, 751–753.
73. Van Dekken, H.; Pizzolo, J.G.; Reuter, V.E.; Melamed, M.R. Cytogenetic analysis of
human solid tumors by in situ hybridization with a set of 12 chromosome-specifc DNA
probes. Cytogenet. Cell Genet. 1990, 54, 103–107.
74. Arnoldus, E.P.; Noordermeer, I.A.; Peters, A.C.; Voormolen, J.H.; Bots, G.T.; Raap,
A.K.; van der Ploeg, M. Interphase cytogenetics of brain tumors. Genes Chromosomes
Cancer. 1991, 3, 101–107.
75. Poetsch, M.; Dittberner, T.; Woenckhaus, C.; Kleist, B. Use of interphase cytogenet-
ics in demonstrating specifc chromosomal aberrations in solid tumors: New insights in
the pathogenesis of malignant melanoma and head and neck squamous cell carcinoma.
Histol. Histopathol. 2000, 15, 1225–1231.
76. Arnoldus, E.P.; Dreef, E.J.; Noordermeer, I.A.; Verheggen, M.M.; Thierry, R.F.; Peters,
A.C.; Cornelisse, C.J.; Van der Ploeg, M.; Raap, A.K. Feasibility of in situ hybridisa-
tion with chromosome specifc DNA probes on paraffn wax embedded tissue. J. Clin.
Pathol. 1991, 44, 900–904.
77. El-Naggar, A.K.; van Dekken, H.D.; Ensign, L.G.; Pathak, S. Interphase cytogenetics in
paraffn-embedded sections from renal cortical neoplasms: Correlation with cytogenetic
and fow cytometric DNA ploidy analyses. Cancer Genet. Cytogenet. 1994, 73, 134–141.
78. Jenderny, J.; Koster, E.; Meyer, A.; Borchers, O.; Grote, W.; Harms, D.; Jänig, U.
Detection of chromosome aberrations in paraffn sections of seven gonadal yolk sac
tumors of childhood. Hum. Genet. 1995, 96, 644–650.
79. Nanjangud, G.; Amarillo, I.; Rao, P.N. Solid tumor cytogenetics: Current perspectives.
Clin. Lab. Med. 2011, 31, 785–811.
80. Marino-Enriquez, A.; Li, P.; Samuelson, J.; Rossi, M.R.; Reyes-Mugica, M. Congenital
fbrosarcoma with a novel complex 3-way translocation t(12;15;19) and unusual histo-
logic features. Hum. Pathol. 2008, 39, 1844–1848.
81. Parisi, F.; Ariyan, S.; Narayan, D.; Bacchiocchi, A.; Hoyt, K.; Cheng, E.; Xu, F.; Li, P.;
Halaban, R.; Kluger, Y. Detecting copy number status and uncovering subclonal markers
in heterogeneous tumor biopsies. BMC Genomics. 2011, 12, 230.
82. Nagai, T.; Naiki, T.; Etani, T.; Iida, K.; Noda, Y.; Shimizu, N.; Isobe, T.; Nozaki, S.;
Okamura, T.; Ando, R.; Kawai, N.; Yasui, T. UroVysion fuorescence in situ hybridiza-
tion in urothelial carcinoma: A narrative review and future perspectives. Transl. Androl.
Urol. 2021, 10, 1908–1917.
83. Geiersbach, K.B.; Sill, D.R.; Meyer, R.G.; Yuhas, J.A.; Sukov, W.R.; Mounajjed, T.;
Carter, J.M.; Jenkins, R.B.; Chen, B. HER2 testing for breast cancer in the genomics
laboratory: A sea change for fuorescence in situ hybridization. Arch. Pathol. Lab. Med.
2021, 145, 883–886.
 7 FISH—in Leukemia
Diagnostics
Roberto Matos, Mariana de Souza,
Gerson Ferreira, Amanda Figueiredo,
Marcelo Land and Maria Luiza Silva

CONTENTS
Introduction.............................................................................................................. 72
Cytogenetic Analysis ............................................................................................... 73
G-Banding Karyotyping ................................................................................. 73
Molecular Cytogenetics .................................................................................. 74
Chronic Leukemia.................................................................................................... 75
Chronic Myeloid Leukemia............................................................................ 75
Chronic Lymphocytic Leukemia .................................................................... 75
Chronic Myelomonocytic Leukemia (CMML) .................................. 76
Acute Leukemia ....................................................................................................... 76
Acute Myeloid Leukemia ............................................................................... 76
Favorable Prognosis AML .................................................................. 77
Unfavorable Prognosis AML .............................................................. 78
Acute Lymphoblastic Leukemia ..................................................................... 79
Favorable/Intermediate Prognosis ALL.............................................. 80
Unfavorable Prognosis ALL ............................................................... 81
Uncertain Prognosis ALL ................................................................... 82
Recurrent Numerical Abnormalities in Acute Leukemia................................ 82
Complex Karyotypes ...................................................................................... 83
Flexibility in the Application of FISH Probes for Leukemia Diagnosis......... 83
Samples/Tissues ....................................................................................................... 83
Description of Methods............................................................................................ 84
Cell Culture for Chromosome Preparation ..................................................... 84
Cell Extraction Protocol ................................................................................. 85
Slides Preparation Protocol............................................................................. 85
G-Banding Protocol ........................................................................................ 86
Fluorescence In Situ Hybridization (FISH) Protocol ..................................... 87
Pepsin Treatment ................................................................................ 87
FISH Slides Denaturation and Hybridization ..................................... 88
FISH Slides Post Hybridization.......................................................... 89

DOI: 10.1201/9781003223658-7 71
72 Cytogenetics and Molecular Cytogenetics

Yields ....................................................................................................................... 89
FISH Panel and Probes in Leukemia Diagnostics .......................................... 89
FISH in Favorable Prognosis Leukemia ......................................................... 91
RUNX1(21q22)-RUNX1T1(8q22) Probe........................................... 91
CBFb(16q22)-MYH11(16p13) Probe ................................................ 91
PML(15q24)-RARA(17q21) Probe .................................................... 91
KMT2A(11q23)-MLLT3(9p22) Probe ............................................... 92
cMYC(8q24)-IGH(14q32) Probe ....................................................... 92
TCRA/D(14q11) Probe....................................................................... 94
ETV6(12p13)-RUNX1(21q22) Probe ................................................ 94
FISH in Unfavorable Prognosis Leukemia ..................................................... 94
del(5q)/del(7q) Probe.......................................................................... 94
BCR(22q11)-ABL1(9q34) Probe ....................................................... 94
KMT2A(11q23)(MLL) Probe ............................................................ 94
TP53 del(17p13) Probe....................................................................... 94
Conclusions.............................................................................................................. 95
References................................................................................................................ 96

INTRODUCTION
Leukemia comprises a heterogeneous group of hematological neoplasias character-
ized by clonal proliferation of hematopoietic cell lineages that underwent genetic
alterations, leading to be expressed in a specifc differentiation phase. These altera-
tions affect genes with pivotal roles in the control of natural mechanisms of prolif-
eration, differentiation, and apoptosis.[1, 2]
The main clinical symptoms result in accumulation of immature precursors in the
bone marrow (BM). This can harm or even prevent the production of normal blood
cells, such as erythrocytes (causing anemia), or neutrophils (resulting in a suscepti-
bility to infections and platelets, causing hemorrhages). With the excess proliferation
of BM-blast cells, infltration of tissues such as tonsils, lymph nodes, skin, spleen,
kidneys, central nervous system (CNS), and others may occur.[3]
Leukemia cells may be immunophenotypically similar to mature (as observed in
Chronic Lymphocytic Leukemia (CLL)), or precursor cells (originating from several
lineages, as in Acute Leukemia (AL)), or still cells similar to precursor and mature
ones (as in Chronic Myeloid Leukemia (CML)). Leukemia can affect people of any
age. However, there are particular distributions depending on leukemia cell types and
patients’ ages. In this sense, Acute Lymphoblastic Leukemia (ALL) is more common
in early childhood compared with Acute Myeloid Leukemia (AML), which is more
frequently observed in older adults. CML is extremely rare in younger children, and
CLL is almost exclusive to patients over 40 years old, with a median age of 70 years.[4]
Leukemia are a very heterogeneous and vast group of diseases, covering a wide
range of ages.[5] Thus, in this chapter, we will focus on a particular group of leuke-
mia, more specifcally, childhood ALs, to demonstrate the benefts of fuorescence
in situ hybridization (FISH)–based technologies in the daily care of these patients.
Nonetheless, we reiterate that the entire study fowchart detailed here can be applied
to all patients, regardless of age or leukemia type.
FISH—in Leukemia Diagnostics 73

Herein we aim to address the importance of FISH-based technologies to charac-

terize the most prevalent cytogenetic abnormalities in the main forms of leukemia.
We also will discuss its usefulness in disease diagnosis, classifcation and prognosti-
cation, and therapeutic implications.

CYTOGENETIC ANALYSIS
The possibility to undertake (banding) cytogenetic analysis of malignant cells repre-
sented the beginning of one of the greatest advances in understanding the biological
nature of neoplasms. This improvement was particularly important in the leukemogen-
esis feld, shedding light on the prognostic implications of chromosomal abnormalities.
Regarding the study of genes involved in disease-specifc translocations, cytogenetics
is considered one of the most powerful tools, leading to a better understanding of chro-
mosomal rearrangements and the mechanisms underlying leukemic transformation.
The precise identifcation of chromosomal breakpoints enabled the cloning of many
genes whose interaction with other genes contributes to the neoplastic process.[6]
For more than 30 years, cytogenetic analysis of patients with leukemia, mainly
AL, led to the discovery of several recurrent chromosome abnormalities, guiding
more effcient treatment regimens. Nonetheless, the prognostic value of rare abnor-
malities is still unknown. In this regard, further cytogenetic studies gathering data
on patients harboring such rare abnormalities are necessary to defne prognosis.[7]
Therefore, an adequate experimental design, individualized for each patient, con-
sidering the knowledge on FISH-based technical possibilities, can help characterize
chromosomal aberrations that in the past would be characterized only as complex/
undefned, thus helping with patient’s risk stratifcation.

G-BANDING K ARYOTYPING
Refnements of chromosome banding techniques over the last 40 years allowed
for improved detection of chromosomal abnormalities in malignant hemopathies.
The advent of these techniques brought the frst revolution in cytogenetic analysis.
Chromosome banding techniques enabled the clear identifcation of individual chro-
mosomes and the description of chromosomic abnormalities.[7]
Pioneers in this feld, Drs. Nowell and Hungerford (1960), described a minute
chromosome in BM samples from patients with CML, which was a game-changer
for hematology. This observation later proved to be the translocation t(9;22)(q34;q11),
generating the breakpoint cluster region protein (BCR)—Abelson tyrosine‑protein
kinase 1 (ABL1) chimeric fusion gene, the product of which was a constitutively
activated tyrosine kinase.[8] Another important step in the feld of tumor cytogenetics
was the description of the reciprocal translocation t(8;21)(q22;q22) by Dr.  Rowley
(1973). This translocation is a frequent non-random cytogenetic abnormality in
AML. Dr. Rowley described the translocation t(8;21) from her observation of repeat-
ing pattern of karyotypes analyzed, using banding techniques.[9, 10] Since then, cyto-
genetic analysis has provided remarkable knowledge about the heterogeneity of a
cell population. This approach enabled the detection of several genetic and chromo-
somal abnormalities in different malignancies, making it possible to classify the dis-
ease based on the alteration observed in the patient’s karyotype. For such neoplasms,
74 Cytogenetics and Molecular Cytogenetics

the presence or absence of specifc chromosomal aberrations can be a fundamental

prognostic indicator.[11–13]
G-banding cytogenetics can reveal chromosomal markers and complex karyo-
types. However, it cannot point to the origin of these rearrangements. On the other
hand, FISH-based techniques can pinpoint the genes/gene fusions involved in the
abnormalities present in the karyotype.[14] In this context, FISH has expanded the
capabilities of cytogenetic analysis not only through its higher sensitivity but also,
especially because of its quick detection of alterations. A FISH experiment can be
processed in 4–24h, and the analysis of 100–200 cells accomplished in 15–45min.
This agility/effciency enables the information about the panorama of karyotype
alterations to be achieved within an appropriate period for treatment strategies.[15]

MOLECULAR CYTOGENETICS
In situ hybridization (ISH) is a cytogenetic method used for high-resolution detection
and localization of DNA sequences in tissues and cells.[16] The frst description of
nucleic acid hybridization was made by Gall and Pardue in 1969[17] when DNA dena-
turation was performed in an integrated cytological sample from the ovary of the
frog Xenopus and hybridized with specifc tritium-labeled radioactive RNA. John
and colleagues (1969)[18] also reported in situ hybridization of DNA from the Hela
cell line and Xenopus oocytes with labeled RNA. In 1970, Buongiorno-Nardelli and
Amaldi[19] created a hybridization method using paraffn-embedded hamster brain
and liver tissues.
Immunofuorescence-based protein detection was described in 1941.[20] Until then,
only radioactive-based techniques, which took long exposure times and low resolv-
ing power, were used. One of the frst attempts was the indirect immunofuorescence
procedure using an antiserum against a poly(rA)-poly(dT). This approach was used
to detect RNA-DNA hybrids of Drosophila chromosomes with a rhodamine-conju-
gated secondary antibody.[21] In the early 1980s, Bauman and colleagues[22] devel-
oped the direct detections of Trypanosoma nucleic acids, adenovirus in human tissue
cells, and Polytene chromosomes from the salivary gland of Drosophila. The latter
became possible by using the covalent binding of the fuorochrome rhodamine mol-
ecule to the 3′-terminus of the RNA. The year 1981 was a landmark for molecular
cytogenetics due to the introduction of nonradioactive labeling methods. The use of
fuorescently labeled probes, such as biotin-labeled nucleotides with avidin-based
detection methods, was crucial to improving in situ hybridization technologies.[23]
The advent of FISH-based techniques boosted the diagnosis and study of leuke-
mia. The application of new multicolor karyotyping approaches allowed the com-
plete characterization of complex chromosome rearrangements and the discovery of
new abnormalities and putative genes involved in leukemic transformation.[16]
Therefore, the advent of FISH in the late ’80s paved the way to the second revo-
lution in cytogenetic analysis. Initially, the use of specifc chromosome probes led
to the exact identifcation of chromosomes involved in complex rearrangements.
Moreover, the Human Genome Project prompted the development of several locus-
specifc probes. This technology improved gene mapping strategies and breakpoints
FISH—in Leukemia Diagnostics 75

identifcation, besides facilitating the delineation of deleted regions associated with

specifc disease subtypes. Subsequently, the cloning of these translocation break-
points made possible the generation of probes specifc for the detection of these
alterations.[16]
Regarding leukemia diagnostics, the applicability of FISH in clinical and labora-
tory practices includes the hybridization of a fuorochrome-labeled DNA probe on
in situ chromosomal targets for the confrmation/characterization of one or more
cytogenetic abnormalities.[24] Routinely, FISH is performed on metaphase spreads,
but unlike the G-banding technique, it does not rely exclusively on metaphases. FISH
can be performed in interphase nuclei, monitoring specifc chromosomal and gene
rearrangements and numerical abnormalities, maintaining reliability in the experi-
ment.[24] On the other hand, FISH requires a prior conception or knowledge of the
abnormality to be screened for selecting the most appropriate probe.[16] Therefore,
FISH is a powerful tool combined with cytogenetic and molecular studies in the
evaluation of chromosomal abnormalities associated with hematological malignan-
cies and other types of cancers.

CHRONIC LEUKEMIA
CHRONIC MYELOID LEUKEMIA
CML is an extensively described malignant disorder of hematopoietic stem cells
(HSCs) that accounts for 15–20% of all adult leukemia cases and about 3% in chil-
dren.[25, 26] This disease is a chronic myeloproliferative neoplasm originating in a
defective pluripotent BM stem cell. The hallmark of this disease is the presence of
the Philadelphia (Ph) chromosome, translocation t(9;22)(q34;q11.2), generating the
BCR‑ABL1 fusion.[8, 27] Pediatric and adult CML share the same molecular features.
However, better outcomes have been observed in pediatric transplant patients due to
the lack of other chronic diseases or organ dysfunction associated with increased age
and lower incidence of graft versus host disease.[26]

CHRONIC LYMPHOCYTIC LEUKEMIA

CLL is characterized by the clonal proliferation and accumulation of mature and
typically CD5-positive B-cells in the peripheral blood (PB), BM, lymph nodes, and
spleen. The frst hit leading to this disease may involve multipotent and self-renew-
ing HSCs.[28, 29] Genomic studies revealed a model of progression of acquired cyto-
genetic abnormalities in the course of this disease, making the prognosis worse,
including resistance to treatments. In early stages, we can observe deletion of the
long arm of chromosome 13 in approximately 55% of cases, in addition to trisomy
12 being present in 10–20% of CLL patients. In later stages, we can observe long
arm deletions in chromosome 11 in about 10% of the cases, besides the deletion of
the short arm of chromosome 17 (5–8%). The deletion del(17p) has been described
as a poor prognostic factor since the TP53 gene is located in this chromosome
region.[30, 31]
76 Cytogenetics and Molecular Cytogenetics

Chronic Myelomonocytic Leukemia (CMML)

CMML is a rare clonal disorder originating from HSCs. This disease encompasses
features of both myelodysplastic syndromes and myeloproliferative neoplasms.
CMML affects mostly (90%) male patients over 60 years of age.[5] According to
the WHO, the main diagnostic criteria for CMML are PB monocytosis (≥1 × 109/l),
comprising at least 10% of the white blood cell (WBC) count. Besides, (i) dyspla-
sia involving one or more myeloid lineages or (ii) the presence of acquired clonal
genetic/cytogenetic abnormalities in hematopoietic cells are present.[5] About
20–30% of patients with CMML present cytogenetic abnormalities at diagnosis.
Risk stratifcation can be categorized according to the abnormalities observed: (i)
low risk when presenting a normal karyotype, or Y chromosome loss as an isolated
abnormality; (ii) high risk when presenting trisomy 8, chromosome 7 abnormali-
ties, or complex karyotype; (iii) intermediate-risk when low and high-risk fea-
tures are not observed.[32, 33] FISH is important to exclude typical (BCR‑ABL1)
and atypical CML (PDGFRA, PDGFRB, FGFR1) rearrangements, or PCM1‑JAK2
fusions.[5]

ACUTE LEUKEMIA
ALs are a heterogeneous group of diseases that may originate from the interaction
between genetic and epigenetic alterations in hematopoietic progenitors, leading to
dysregulation of multiple important signal transduction pathways, resulting in hema-
topoietic insuffciency due to a clonal proliferation of immature precursors.[34] These
neoplastic precursor cells originate in the BM and can infltrate PB and tissues.[35]
ALs are divided into two main groups, myeloid leukemia, when the myeloblast
lineage is affected, or lymphoid leukemia when the lymphoblastic lineage is affected.
AL groups differ from each other in specifc characteristics regarding cytogenetic
and genomic abnormalities, morphological and immunophenotypic profle, response
to therapy, and the clinical course of the disease. In general, ALs present a rapid fast
clinical course and if not properly treated can lead to death in a few months or even
weeks.[36, 37] AL is the most common form of childhood cancer, and its incidence is
increasing.[38]

ACUTE MYELOID LEUKEMIA

AML is a molecularly heterogeneous group of diseases affecting individuals of all
ages. Despite constituting only 20% of pediatric ALs, AML is overtaking ALL as
the leading cause of childhood leukemic mortality.[38] Approximately 80% of chil-
dren with AML present chromosomal alterations in BM and PB blast cells at the
time of diagnosis.[39, 40] This frequency is considerably higher compared to adult
patients affected by the disease, in which these alterations are detected in approxi-
mately 60% of the cases.[41]
About 80% of children with AML present genomic structural alterations associ-
ated with the disease and approximately 76% of patients presenting normal karyo-
type have at least one known mutation (FLT3, WT1, NPM1). Thus, currently, about
FISH—in Leukemia Diagnostics 77

90% of pediatric patients with AML have at least one recurrent type I and type II
mutation.[42]
In this sense, molecular cytogenetic analyses have conferred substantial evidence
with regards to the chromosomal architectures in AML and other cancers. Most
importantly, the FISH technique that plays a leading role in diagnostic pathology for
its single-cell analysis has provided crucial information regarding the heterogeneity
of AML malignant cells.[14]
In AML, cytogenetic alterations of numerical and structural types can be found.
Numerical alterations include monosomies and nullisomies, which result in a karyo-
type with less than 46 chromosomes. Trisomies and tetrasomies, among others,
will generate a hyperdiploid karyotype with more than 46 chromosomes. Structural
abnormalities are mainly translocations, insertions, and inversions.[40]
The presence of specifc cytogenetic abnormalities has important prognostic
implications for the disease. Thus, karyotypic alterations in leukemic blasts are con-
sidered one of the most signifcant markers that defne the biology of AML. In addi-
tion to karyotypic changes, gene mutations with clinical relevance associated with
the disease are also part of the classifcation scheme for AML.[5, 39, 40, 43]

Favorable Prognosis AML

Cytogenetic markers with favorable prognoses in AML are listed in the following.

• t(8;21)(q22;q22): The reciprocal translocation t(8;21)(q22;q22) results in the

fusion of the RUNX1 gene on 21q22 and RUNX1T1 on 8q22. This trans-
location presents the highest incidence in childhood AML, occurring in
approximately 12% of the cases.[44 – 46] In adults, the translocation t(8;21) can
be found in about 5% of patients with AML.[41, 46] As this abnormality is
associated with a favorable prognosis, its identifcation is highly signifcant
for diagnosis and therapy.[40, 47]
• inv(16)(p13q22): Inversion inv(16)(p13q22) encompasses 3–8% of pediatric
cases, and about 4% of adults with AML, resulting in the fusion of the core
binding factor b (CBFb) gene at region 16q22 with the smooth muscle myo-
sin heavy chain (MYH11) at 16p13.[40, 46] Additional chromosomal abnor-
malities such as +22, +8, and 7q alterations are seen in association with this
AML subtype.[40, 46, 48]
• t(15;17)(q22;q21): Acute promyelocytic leukemia (APL) accounting for
about 2–10% of pediatric AMLs (7–8% in adults) is usually related with
a good prognosis. However, in about 20% of patients whose outcome is
not favorable, ~10% pass away due to early complications, and the other
10% experiences disease relapse or drug resistance. These include the
APL variants translocation t(11;17)(q23;q21)/PLZF‑RAR, translocation
t(5;17)(q35;q21)/NPM‑RAR, t(11;17)(q13;q21)/NUMA‑RAR, and derivative
der(17) that generates the STAT5b‑RAR. Prompt diagnosis, including FISH
for a fast initiation of treatment, is critical to lowering the risk of early
mortality.[40, 41, 46] Although the hallmark of APL is the PML‑RARA fusion,
generated from translocation t(15;17)(q24;q21), it is common to observe
78 Cytogenetics and Molecular Cytogenetics

additional chromosomal abnormalities, with a clinical impact not yet estab-

lished.[49, 50]
• t(9;11)(p22;q23): The translocation t(9;11)(p22;q23) has been reported in
approximately 2% of adult cases and about 6% of pediatric AML.[41, 46] This
abnormality is the most commonly observed in infants younger than 12
months old. At the molecular level, the translocation t(9;11) is defned by
the involvement of the KMT2A gene at 11q23 with the MLLT3 at 9p22.
This aberration presents controversial prognostic implications. However,
children and infants with t(9;11) tend to have a more favorable prognosis.[11]
Still, karyotypes carrying KMT2A gene rearrangements (KMT2A-r) are
more frequent in infants, presenting an intermediate prognosis or being
associated with an adverse risk in AML.[51, 52]

Unfavorable Prognosis AML

Cytogenetic markers with unfavorable prognoses in AML are listed in the following.

• t(9;22)(q34;q11): The reciprocal translocation t(9;22)(q34;q11)/BCR‑ABL1

is a rare event in AML, accounting for 1–2% of total cases.[8, 53] The most
common fusion protein in acute leukemia is BCR‑ABL1 p190, but BCR‑
ABL1 p210 can also occur, conferring an adverse prognosis. Due to this
rarity, more data is still necessary on the characteristics of this AML
subtype.[8, 54]
• t(6;9)(p23;q34): Translocation t(6;9)(p23;q34) is often associated with an
intermediate or poor outcome. Besides, due to its rarity (1–2%) and asso-
ciation with type I mutations (i.e. FLT3/ITD), the prognostication of trans-
location t(6;9) has not been fully defned in children.[41, 55] In adults, it is
even rarer, accounting for <1%.[41, 46] The translocation t(6;9) results in the
DEK‑NUP214 chimeric fusion gene, making it possible to be monitored by
FISH.[56]
• t(6;11)(q27;q23): Translocation t(6;11)(q27;q23) is observed in about 5% of
ALs with KMT2A‑r and can be found in ALL and AML. It is more frequent
in AML (7.8% of AML; 1.8% of ALL). The translocation t(6;11) is rare in
infants (0.3%) and more frequent in children (6.6%) and adults (6%).[51] This
abnormality can be very subtle, escaping detection by G-banding cytoge-
netics, and has been associated with a poor prognosis. Additional chromo-
some abnormalities have been reported in 10% of the cases. Molecularly,
the translocation t(6;11)(q27;q23) is characterized by the fusion between the
KMT2A and the AFDN gene.[57, 58]
• t(11;19)(q23; p13.3): The subtle translocation t(11;19)(q23; p13.3), which
results in the KMT2A‑MLLT1 gene fusion, is a rare abnormality in AML,
accounting for less than 1% of cases.[51, 59] It is important to highlight that
this abnormality has a frequency of 4% in infants with AML. The translo-
cation t(11;19) is related to an unfavorable prognosis in AML.[11]
• t(10;11)(p12;q23): A KMT2A gene fusion with MLLT10 at 10p12 is frequently
observed in childhood AML as translocation t(10;11)(p12;q23).[58] The trans-
location t(10;11) is diffcult to detect by G-banding since rearrangements
FISH—in Leukemia Diagnostics 79

involving KMT2A and MLLT10 often result in cryptic/complex rearrange-

ments.[60, 61] This abnormality is defned as an independent prognostic pre-
dictor, so patients with translocation t(10;11)/KMT2A‑MLLT10 should be
referred for a high-risk AML treatment protocol.[58]
• t(1;22)(p13;q13): The translocation t(1;22)(p12;q13) juxtaposes the RBM15
gene located at 1p13 to the MLK1 at 22q13, generating the RBM15‑MLK1
fusion on derivative chromosome 22.[40, 62] It is a rare translocation in child-
hood AML (~3%), being practically restricted to acute megakaryoblastic
leukemia (AMKL). In this regard, literature data suggest that prenatal
genetic factors are involved in the leukemogenesis of this disease. The
prognostication is generally poor.[40, 63]
• Chromosome 3 abnormalities: Abnormalities involving the long arm of
chromosome 3, including inversion inv(3)(q21q26), translocation t(3;3)
(q21;q26), translocation t(3;21), 3q+ and 3q- are considered rare (<1%)
and of high risk in AML.[46, 55] Alterations in these chromosomal regions
are usually associated with AMKL.[40] Identifcation of rearrangements in
the long arm of chromosome 3 is highly important, especially concerning
3q26.2–26.31, as they can result in rearrangement and overexpression of the
MECOM gene, conferring a poor prognosis for the patient.[64–66]

ACUTE LYMPHOBLASTIC LEUKEMIA

ALL accounts for 75% of ALs and 25% of all cancers in this age group, with a peak
of incidence at 2 to 5 years of age. ALL present a signifcant heterogeneity at the
genetic level but homogeneous morphologic and immunophenotypic aspects. These
genetic lesions defne disease subsets biologically distinct as well as are used for risk
stratifcation once they lead to different responses to therapy.[66, 67]
In addition, ALL presents distinct subtypes with therapeutic implications, which
can be accessed by fow cytometry that enables distinguishing ALL in B-cell precur-
sor ALL, mature (Burkitt) ALL, and T-cell ALL. Cytogenetics, as well as molecular
genetic fndings at ALL diagnosis represent important prognostic factors in ALL.
Therefore, these alterations are mandatory for analyzing outcomes in different clini-
cal trials and detecting specifc chromosomal aberrations and their equivalents.[68, 69]
Current risk-stratifed regiments in childhood ALL have led to long-term, event-
free survival rates of more than 80% and approximately 90% of ultimately cured
patients.[69]
ALL cytogenetic diagnostics can be impaired by suboptimal cytogenetic prepa-
rations most of the time. Poor chromosome morphology and indistinct banding can
make the interpretation of karyotypes challenging. In this matter, molecular cyto-
genetic analysis is an important tool to classify and thus rescue patients in which
G-banding is not informative.[70]
Both B and T-cell ALL are associated with characteristic and recurrent cytoge-
netic alterations. B-cell lineage comprises the majority of ALL cases (80–85%), with
well-established cytogenetic subgroups. Hyperdiploidy and translocation t(12;21)
confer a favorable prognosis, whereas translocation t(9;22), KMT2A-r, intrachro-
mosomal amplifcation of chromosome 21 (iAMP21), and translocation t(9;22) or
80 Cytogenetics and Molecular Cytogenetics

Philadelphia chromosome-like ALL (Ph-like ALL) are known predictors of poor

outcomes.[70]
Fifteen percent of childhood ALLs are T-cell lineage, where most commonly
T-cell receptor genes (TCR) either alpha/delta (14q11.2) or beta (7q34) are involved
in translocations with transcription factors that are upregulated by the TCR enhancer
regions. Risk stratifcation for T-ALL treatment is not currently related to chromo-
somal abnormalities. However, there are prognostic implications and potential thera-
peutic options for some of the recurrent abnormalities.[70]

Favorable/Intermediate Prognosis ALL

Cytogenetic markers with favorable/intermediate prognoses in ALL are listed in the
following.

• t(8;14)(q24;q32) and variants: Translocation t(8;14)(q24;q32) juxtaposes

MYC (8q24) with heavy chain locus (14q32) or light chain loci (2p12 and
22q11), leading to MYC overexpression, which is believed to play a central
role in Burkitt Lymphoma (BL) pathogenesis.[71] Because of the high simi-
larity among mature B cell ALL (FAB L3 subtype) (less than 2% of ALL
cases) and BL cells, the World Health Organization (WHO) classifcation
has included this subtype as equivalent to BL in the leukemic phase, under
the common denomination of Burkitt lymphoma/leukemia (BL/L).[72, 73]
Mature B-cell ALL fare poorly with conventional ALL therapy, once
it is a high-grade disease, with a duplicating time of 24 hours. Although,
it presents an 80% event-free survival rate when treated with three to six
months of rapid-sequence, intensive chemotherapy.[74, 75]
• Hyperdiploidy: Hyperdiploidy is the most common recurrent abnormality
in childhood B-ALL, accounting for 30% of the patients. This group pre-
dominantly has a chromosome count of 51–65, with a nonrandom pattern
of gain, leading to one additional copy of chromosomes X, 4, 6, 10, 14, 17,
18, and two additional copies of chromosome 21 as the most frequently
observed alterations in hyperdiploid clones. High hyperdiploidy with spe-
cifc gains of chromosomes 4, 10, 17, and 18 has been associated with an
excellent prognosis in pediatric B-ALL. Chromosome counts of 57–60
chromosomes typically include trisomies of chromosomes 5, 8, 11, and 12,
while karyotypes with 63–67 chromosomes frequently also have gains of
chromosomes 2, 3, 9, 16, and 22.[70, 76] Structural abnormalities associated
with hyperdiploid karyotypes generally do not alter favorable prognosis.[70]
• t(1;19)(q23;p13): Translocation t(1;19)(q23;p13) fuses the TCF3 (E2A) gene
on band 1q23 to PBX1 on 19p13.3. It is reported in 5% to 6% pediatric ALL
and 1% to 3% adult ALL, and can be either balanced or unbalanced, as
der(19)t(1;19) with two normal chromosomes 1. Most patients have pseu-
dodiploid karyotypes, and almost all are diagnosed with pre-B-ALL.[41]
Despite being previously considered high risk because of central nervous
system involvement and relapse, current intensive protocols lead this rear-
rangement to be used as a favorable/intermediate-risk mark nowadays.[76]
FISH—in Leukemia Diagnostics 81

Unfavorable Prognosis ALL

Cytogenetic markers with unfavorable prognoses in ALL are listed in the following.

• t(9;22)(q34;q11): Rarely seen in children (1–3%), translocation t(9;22)

(q34;q11.2) is more common in adults B and T-ALL (11–29%) and leads to
BCR‑ABL1 fusion or Ph-chromosome. With rare exceptions, Ph-patients
are diagnosed with B-ALL. These patients generally present a poor progno-
sis, with the only curative therapy strategy being allogeneic hematopoietic
stem-cell transplantation (HSCT), although this procedure is associated
with increased treatment-related mortality.[70]
• t(v;11q23): Rearrangements involving band 11q23 (KMT2A gene) are
detected in two-thirds of infants with ALL, representing 80% of this age
group. In older children, the incidence is much lower (1–2%),[69, 77] and in
adults it is up to 7%.[41] It has already been demonstrated that KMT2A may
be partner of more than 80 different genes, which distinguish distinct sub-
types of leukemia with both lymphoid or myeloid features and poor out-
comes.[76] Among 11q23 rearrangements, the most expressively found in
ALL are translocation t(4;11)(q21;q23)/MLL‑AFF1(AF4), detected in more
than 50% of patients, followed by translocation t(11;19)(q23;p13.3)/MLL‑
MLLT1(ENL) and, less commonly, translocation t(9;11)(p22;q23)/MLL‑
MLLT3(AF9), translocation t(10;11)(p13–15;q14–21)/MLL‑MLLT10(AF10),
and others.[77]
• t(4;11)(q21;q23): Most common among ALL KMT2A-r is translocation t(4;11)
(q21;q23), where KMT2A (11q23) gene partners AF4 (4q21). KMT2A‑AF4
fusion is particularly common in infant ALL and represents 2% of pediatric
and 3–7% of adult ALL. The translocation t(4;11) is of general interest once
it predicts a poor prognosis.[41]
• Hypodiploidy: Hypodiploid karyotypes present an overall frequency of
5% in childhood ALL. These cases are characterized by a chromosome
count of <44 (43 or fewer) chromosomes.[69] There are three well-defned
subgroups of hypodiploid to note: near haploidy (24–31 chromosomes),
low hypodiploid (32–39 chromosomes), which are associated with an
adverse prognosis, and high hypodiploid (40–43 chromosomes). In the
near-haploid cases, the chromosomes that usually are kept in two copies
are chromosomes 14, 18, 21, and the sex chromosomes. In the low hypo-
diploid karyotypes, the most frequent monosomy chromosomes are 3, 4,
7, 13, 15, 16, and 17.[70]
• iAMP21: Intrachromosomal amplifcation of chromosome 21 (iAMP21)
was frst discovered during a routine screening for ETV6‑RUNX1. Using
the FISH approach, multiple copies of the RUNX1 gene were observed.[67]
This abnormality is characterized by the gain of three or more extra
RUNX1 (21q22) copies and is mostly seen in older children. iAMP21 is
usually seen as a sole cytogenetic alteration, occurring in 2% of child-
hood ALL and associated with older ages at diagnosis,[69, 78] Patients with
iAMP21 show a poor outcome with a high rate of relapse when treated
82 Cytogenetics and Molecular Cytogenetics

under standard risk protocols. However, intensive therapy can greatly

improve the outcome.[76, 78]

Uncertain Prognosis ALL

• t(12;21)(p12;q22): Translocation t(12;21)(p12;q22) fuses the ETV6 gene
(12p13) with RUNX1 gene (21q22) and is observed in up to 25% of child-
hood pre-B ALL and less than 3% in adult ALL.[79] The translocation
t(12;21) is cryptic, highlighting the importance of ISH tools for its detec-
tion. Approximately 75% of translocation t(12;21) patients harbor addition-
al genetic changes, most often deletions of 12p with loss of the second copy
of ETV6 gene (55–70% of patients), +21 (15–20%) and an extra der(21)
t(12;21) (10–15%).[80] Due to excellent molecular response to treatment and
benefcial clinical outcome, it was believed that t(12;21) conferred favor-
able prognostic, but it was subsequently disrupted as some series found pre-
dominantly late relapses occurring in up to 20% of patients.[76, 81]

RECURRENT NUMERICAL ABNORMALITIES IN ACUTE LEUKEMIA

Chromosome 5/del(5q) monosomy is present in <1–2.5% of children with AML,
while in adults the percentage is higher, reaching 7% of cases. In ALL, del(5q) is
rarer, being observed in 1% of pediatric patients and <2% of adults. This abnormal-
ity is associated with a poor prognosis.[41, 55, 82]
Abnormalities of the long arm of chromosome 6 (6q) are seen in about 11%
of children with ALL and 3–16% of adults.[41, 83, 84] This subtle alteration has been
related to a poor prognosis, since a deletion in this region may lead to the loss of
tumor suppressor genes, whose absence may contribute to the genesis of both B-ALL
and T-ALL.[11, 84]
Aberrations involving chromosome 7 can occur as a sole abnormality, such as
−7/del(7q), or in balanced translocations 7q22/7q32-q35 in AML.[85, 86] Such aberra-
tions are present in 2–7% of pediatric cases and 2.5–8.5% in adults with AML. In
ALL, abnormalities involving chromosome 7 have a frequency of 4% in children
and 6–11% in adults. For both groups, these changes are more often associated with
a poor outcome.[41, 82, 87]
Trisomy of chromosome 8 is seen in 5–10% of pediatric patients with AML and
about 9% of adults. It is a frequent alteration in patients with AML and Down syn-
drome, affecting 27% of cases.[41, 83, 87, 88] In ALL, a higher frequency is reported in
adults compared to children, with 10–12% vs. 2%, respectively. This alteration can
be observed (i) as an isolated abnormality and thus is commonly associated with a
favorable prognosis, and (ii) as a secondary cytogenetic alteration, which may confer
a more aggressive prognosis.[83, 89, 90]
Cytogenetic abnormalities in the short arm of chromosome 9 (9p) are present
in about 7–9% of cases of childhood ALL.[83] Most of these abnormalities involve
the deletion of cell cycle regulatory genes and tumor suppressor genes (CDKN2,
CDKN2B).[11, 91] Del(9q) is a recurrent alteration in AML being present in about
2–4% of children and 2% of adults. They are also usually associated with a lower
clinical outcome.[88, 92, 93]
FISH—in Leukemia Diagnostics 83

COMPLEX K ARYOTYPES
A complex karyotype (CC) is defned as any karyotype with at least three chromosomal
aberrations, regardless of the type of alterations and the chromosomes involved.[94–96]
The frequency of CCs increases with the age of the patients.[41, 55, 60, 97] Presence of CCs
in the BM blast cells represents a possible indicator of a poor prognosis, which may
negatively infuence the patient’s clinical evolution. The literature has also shown that,
in most cases, the disease has been resistant to the initial treatment, with early relapse
of patients.[97–99] CCs have been associated with an unfavorable clinical evolution, and
it is important to emphasize that these karyotypes can carry, among their alterations,
in a cryptic way, chromosomal abnormalities that can be responsible for an adverse
prognosis.[61, 100] Since the literature suggests a possible correlation between the com-
bination of aberrations detected in a CC and the clinical response, the importance of
refnement in the characterization of this karyotypic profle becomes clear.

FLEXIBILITY IN THE APPLICATION OF FISH PROBES FOR LEUKEMIA DIAGNOSIS

FISH-based techniques revealed the involvement of several genes and allowed the
description of specifc abnormalities, uncovering several therapeutic targets related
to a specifc type of disease.[101, 102] Consequently, treatment has improved with higher
cure levels for leukemia patients. Having prior knowledge about the disease and the
probes available for testing can provide a vast hall of possibilities to properly and
quickly evaluate strategies for risk-adapted therapy.[101, 102]
In this context, the ETV6‑RUNX1 FISH probe, designed for the detection of
the cryptic translocation t(12;21)(p13;q22), can be used to detect other cytogenetic
abnormalities such as trisomies typical of hyperdiploidy. This probe can also aid in
the detection of other molecular alterations involving chromosomes derived from
variants of translocation t(12;21)(p13;q22) and ETV6‑RUNX1 gene fusion itself,
which have been reported as indicators of different clinical evolutions within this
cytogenetic group.[103] Furthermore, in cases where mitoses cannot be obtained and
G-banding analysis cannot be performed, this probe allows detection of the iAMP21,
a rare alteration of poor prognosis.[104]
Likewise, as the EVT6‑RUNX1 probe can also be used to help detect and char-
acterize other alterations involving chromosomes 12 and 21, the RUNX1‑RUNX1T1
probe can also be used to help uncover abnormalities in chromosome 21, in addition
to trisomy 8, a recurrent aberration of important prognosis in leukemia.[83, 90] Hence,
the use of gene/gene fusion probes specifc to certain diseases and abnormalities can
be made fexible to assist in the elucidation of other disease-related alterations. In
this context, the application of this knowledge in daily practice can widely contribute
to research and diagnostic laboratories.

SAMPLES/TISSUES
For (molecular) cytogenetic analysis, PB and BM samples are processed. The sam-
ples are collected in test tubes with anticoagulant heparin and referred to the labora-
tory by the responsible physicians.
84 Cytogenetics and Molecular Cytogenetics

To assist in the risk stratifcation of patients and in the experimental design based
on FISH panels, clinical diagnoses of cytochemistry, immunophenotyping, and mor-
phology are obtained through the study of medical records, slide review, and patient
fles provided by the responsible physicians in the respective institutions of origin.
The clinical-laboratory data of the diagnosis, as well as the clinical information of
the follow-up of the patients, are also obtained through the study of the medical
records in the institutions of origin.

DESCRIPTION OF METHODS
CELL CULTURE FOR CHROMOSOME PREPARATION
Equipment/Materials
• Class II biological safety cabinet
• Sterile 15ml conic tubes
• Centrifuge
• Sterile transfer pipettes
• Micropipette 50µl (p20–200–1000)
• Pipette tips 50µl (p20–200–1000)
• Neubauer chamber
• Coverslip 24×24mm
• Optic microscope
• Cell culture fasks
• Humidifed 37°C, 5% CO2 incubator

Reagents
• Ready to use growth medium: RPMI 1640 medium at 37ºC
• Fetal bovine serum, previously inactivated in a water bath at 56ºC for
40 minutes, 12ml of penicillin & streptomycin
• L-glutamine (add in RPMI 1640 medium—1:100 proportion)
• Thomas buffer solution: Dilute 10ml of acetic acid and 10 drops of Giemsa
stain in 1l of distilled water. Store in a dark bottle.[105]

Workfow
1. Into the biological safety cabin, transfer the sample (BM/PB) for a 15ml
conic tube;
2. Dilute 50µl of the sample in 1ml Thomas buffer solution using a micropipette;
3. Apply this solution to the surface of the Neubauer chamber and place the
coverslip;
4. Use the optic microscope (20×) to count the cells in the four chamber’s
quadrants;
5. The number of cells is obtained by adding the number of counted cells,
multiplying the result for 21 and afterwards for 2,500;
6. Culture is established using 80% growth medium, 20% fetal bovine serum,
and 5×106 cells of the sample in each cell culture fask;
FISH—in Leukemia Diagnostics 85

7. Cell culture fasks should stay in the humidifed 37°C incubator for 24
hours.

CELL EXTRACTION PROTOCOL

Equipment/Materials
• Syringe and needle
• Disposable Pasteur pipettes
• Benchtop centrifuge
• Laboratory water bath 37ºC

Reagents
• Colcemid solution 10µg/ml
• KCl (potassium chloride): Add 0.6g of KCl to 100ml of distilled water. Mix
by swirling until dissolved.
• Carnoy’s solution/fxative solution (acetic acid and methanol solution—1:3
proportion); combine three parts of methanol to one part of glacial acetic
acid (v/v). Use freshly. Store at room temperature (RT).[106]

Workfow
1. After 23 hours of incubation, 50µl of Colcemid solution should be added
to each of the cell culture fasks;
2. Return the fasks to the humidifed 37°C incubator;
3. One hour later (after completing 24 hours total), transfer the cell culture
in the fasks to 15ml conic tubes. The contents in each fask shall be trans-
ferred to a different conic tube;
4. Centrifuge the material for 6 minutes at 1,500 rpm;
5. Remove the supernatant from the tubes and homogenize the pellet. To
perform the hypotonic shock, add, with the aid of a disposable Pasteur
pipette, the KCl solution until completing 8 ml;
6. Accommodate the tubes in a water bath at 37°C for 20 minutes;
7. Centrifuge for 6 minutes at 1,500rpm;
8. Remove the supernatant from the tubes, homogenize the pellet, and slowly
add the Carnoy’s solution until completing 8ml;
9. Leave the tubes for 20 minutes at RT;
10. Centrifuge for 6 minutes at 1,500rpm;
11. Remove the supernatant, homogenize the pellet, unite the contents of all
the tubes in a single one and add Carnoy’s solution until completing 8ml;
12. Repeat steps 7 and 8 until the material is clear;
13. Store the material at 4°C.

SLIDES PREPARATION PROTOCOL

Equipment/Materials
• Microscope slides
86 Cytogenetics and Molecular Cytogenetics

• Disposable Pasteur pipettes

• Glass Coplin jar
• Bunsen burner

Reagents
• Ethanol 70%
• Giemsa solution (Merck): Dilute 2.5ml of Giemsa stain and 40ml of phos-
phate buffer.[107, 108]
• Phosphate buffer: Dissolve 6.808g of KH2PO4 and 0.882g of NaOH in 1l of
distilled water. Adjust pH to 6.8.

Workfow
1. Centrifuge the tubes containing the chromosome preparation for 6minutes
at 1,500 rpm;
2. Remove the supernatant (until it is twice as large as the pellet) and homog-
enize the content left in the tube;
3. Clean the slides using ethanol 70%;
4. Puff on the cleaned slides;
5. Add three drops from the material onto the slides. Before adding mate-
rial, keep the pipette at arm’s length from the slides for better chromosome
dispersion;
6. Quickly fame the opposite part of the slides over medium heat in a Bunsen
burner;
7. Add the slides in a Coplin jar with Giemsa solution for 12 minutes;
8. Observe the slides under an optical microscope (in phase contrast) to check
for the presence of metaphases;
9. After confrming the presence of metaphases, follow steps 4–6 again to each
new slide, and then store the slides in a microscope slide case for aging.

G-BANDING PROTOCOL
Equipment/Materials
• Spatula
• Precision balance
• Beaker 100ml
• Laboratory water bath
• Disposable Pasteur pipettes
• Glass Coplin jar

Reagents
• Dulbecco’s Phosphate Buffered Saline (PBS): Dilute 8.0 g of NaCl, 0.2 g of
KCl, 0.2 g of KH2PO4, and 1.44g of Na2HPO4H20 in 1l of distilled water.
Adjust pH to 7.8.
• Trypsin solution: Dilute 0.1g of Trypsin from porcine pancreas in 100ml of
Dulbecco’s PBS
FISH—in Leukemia Diagnostics 87

• Phosphate buffer: Dissolve 6.808g of KH2PO4 and 0.882g of NaOH in 1l of

distilled water. Adjust pH to 6.8.
• Giemsa solution (Merck): Dilute approximately 2.5ml of Giemsa stain and
40ml of phosphate buffer.[107, 108]

Workfow
1. Heat the trypsin solution (0.1g of trypsin and 100ml of Dulbecco PBS) in
the water bath for 30 minutes;
2. Dip the slide in the trypsin solution for 1–10 seconds;
3. Wash the slide in saline solution;
4. Stain the slide in Giemsa solution for 15 minutes;
5. Wash the slide in saline solution and let it dry at RT;
6. Under the microscope, observe whether the time of exposure to trypsin
was suffcient to generate the chromosomal banding pattern; if the chro-
mosomes are digested too much, decrease the exposure time. If the time is
not enough for the chromosomes to be banded, increase the time until the
desired result;
7. Analyze at least 20 metaphases to confrm the cytogenetic diagnosis.

FLUORESCENCE IN SITU HYBRIDIZATION (FISH) PROTOCOL

Pepsin Treatment
Equipment/Materials
• Glass Coplin jar
• Polypropylene Coplin jar
• Glass microscope slides
• Sterile Pasteur pipettes
• Laboratory water bath
• Thermometer

Reagents
• Ethanol 70%, 85%, and 100%; prepare v/v dilutions of 100% ethanol with
distilled water to make stock solutions of 70% and 85% ethanol. Between
uses, store covered at ambient temperature. Discard stock solutions after
six months.
• 2×SSC (pH 7.0): Dilute stock solution (20×SSC) in distilled water in the
proportion 1:10. Adjust pH for 7. 20×SSC (stock solution): Dilute 175.34g
of NaCl and 88.24g of C6H5Na3O7.2H2O in 500ml of distilled water on
shaker. Add distilled water to bring fnal volume of solution to 1l. Adjust
pH for 5.3.
• Pepsin 0.005%, HCl 0.001M: Dilute 400µl pepsin 0.5% in 39.6ml HCl
0.01M; pepsin 0.5% HCl 0.001M (Stock solution): Dilute 0.5 g pepsin in
100ml HCl 0.01M. Keep in refrigerator.
• 1×PBS: Dilute stock solution (10×PBS) in distilled water the proportion
1:10. Adjust pH for 7.2. 10×PBS (stock solution): Dilute 80g of NaCl, 2g
88 Cytogenetics and Molecular Cytogenetics

KCl, 14.4 g of Na2HPO4, and 2.4g of KH2PO4 in 800ml of distilled water.

Add distilled water to bring fnal volume of solution to 1l.
• Formaldehyde 1%/1×PBS: Dilute 1.1ml formaldehyde P.A. (37%) in 38.9ml
of 1×PBS. Mix well.

Workfow
1. Drop the cell suspension in slides washed in ethanol 70% and leave for dry-
ing in a wet plate for 2–3 minutes. It is recommended to observe the dropped
slides in the optic microscope to pre-select target areas to apply the probes;
2. Incubate the slides in a 2×SSC solution (pH 7.0) at 37°C for 20 minutes;
3. Incubate the slides in pepsin solution (0.005%, 0.001M HCl) at 37ºC for 10
minutes;
4. Wash the slides for 3 minutes in 1×PBS at RT;
5. Incubate the slides in a formaldehyde solution (1% 1×PBS) at RT for 10
minutes;
6. Wash the slides in 1×PBS at RT for 3 minutes;
7. Dehydrate slides in a series of ethanol (70%, 85%, and 100%; 2 minutes
each);
8. Leave the slides for drying at RT.

FISH Slides Denaturation and Hybridization

Equipment/Materials
• Micropipette 10µl
• Pipette tips
• Eppendorf tube
• Coverslip 20×20mm
• Rubber cement glue
• Laboratory dry bath/dry block
• Thermometer
• Microscope slides case
• CO2 incubator

Reagents
• Pre-selected FISH probe
• Hybridization buffer
• MiliQ (purifed) water

Workfow
Slides Denaturation
1. Add 8µl of the probe hybridization mix (according to the manufactur-
er’s instructions) in the target slides’ areas and cover with the coverslip
20×20mm;
2. Seal the edges of the coverslips with the rubber cement glue;
3. Accommodate the slides in the dry bath at 75ºC for 7 minutes.
FISH—in Leukemia Diagnostics 89

Hybridization
1. Incubate the slides into a humidifed microscope slides case (keep the case
closed to simulate a dark chamber environment). Leave the case in a CO2
incubator at 37ºC overnight (12–16 hours).[107, 109]

FISH Slides Post Hybridization

Equipment/Materials
• Clamp
• Micropipette 20µl
• Pipette tips
• Polypropylene Coplin jar
• Laboratory water bath
• Thermometer
• Coverslips 24×32mm
• Dark chamber

Reagents
• 0.4×SSC/3%Tween20 solution (pH=7.0); Dilute 10ml of 20×SSC solution in
distilled water in the proportion 1:10. Adjust pH for 7; keep in refrigerator;
fnally dilute 120µl Tween20 in 40ml 0.4×SSC.
• 2×SSC/0.1%Tween20 solution (pH=7.0): Dilute 40µl Tween20 in 40ml
2×SSC.[109]
• DAPI antifade staining

Workfow
1. Remove the coverslips and wash the slides for 2 minutes in 0.4×SSC, 3%
Tween20 solution (pH=7.0) at 72ºC;
2. Transfer the slides to a 2×SSC 0.1% Tween20 solution (pH=7.0) for 1 minute
at RT;
3. Let the slides dry at RT;
4. Apply 15µl antifade staining e.g. DAPI (Vysis) over the hybridization area
and cover with a coverslip 24×32mm;
5. Incubate the slides into a humidifed microscope slides case (keep the case
closed to simulate a dark chamber environment) for at least 10 minutes;
6. Observe the slides in a fuorescent microscope to detect the FISH signals.

YIELDS
FISH PANEL AND PROBES IN LEUKEMIA DIAGNOSTICS
The WHO recommends an integrated approach for ALs cytogenetic diagnosis.
Chromosomal banding cytogenetic analysis and FISH-based techniques should be
used in the initial diagnosis and monitoring of clonal abnormalities.[5, 101, 102]
The application of a FISH panel enables a quick assessment of the karyotype and
the pattern of abnormalities present in it, including its percentage in BM blast cells.
90 Cytogenetics and Molecular Cytogenetics

In this sense, for hematological emergencies such as APL, a rapid FISH result is
mandatory for the administration of all-trans retinoic acid (ATRA).[101, 110] For cryptic
rearrangements that escape detection by chromosome banding, such as translocation
t(12;21)(p13;q22)/ETV6‑RUNX1, translocation t(10;11)(p12;q23)/KMT2A‑MLLT10,
translocation t(6;11)(q27;q23)/KMT2A‑AFDN, and abnormalities in the 17p13 (TP53)
region, FISH can be considered as a stand-alone diagnostic assay.[42, 101, 103] The FISH
probes and disease-specifc panels most commonly used in clinical cytogenetic labo-
ratories are listed in Table 7.1.

TABLE 7.1
FISH Panel and Probes in Leukemia Diagnostics
Cytogenetic Gene Fusion Prognosis Disease Types Probe Types
Abnormality
t(8;21)(q22;q22) RUNX1‑RUNX1T1 favorable AML DCDF
inv(16)(p13;q22) CBFb‑MYH11 favorable AML DCDF
t(15;17) PML‑RARA favorable AML DCDF, DCSF
(q22;q21)
t(9;11)(p22;q23) KMT2A‑MLLT3 favorable in AML ALL, AML DCDF
patients without
additional
abnormalities or
FAB M5 subtype)
11q23 KMT2A-r intermediate ALL, AML DCBAP
or poor in AML,
Unfavorable in
ALL
del(5q) EGR1(5q31) unfavorable AML DCE
t(6;9)(p23;q34) DEK‑NUP214 often unfavorable AML DCDF
t(6;11)(q27;q23) KMT2A‑AFDN unfavorable in ALL, AML DCDF
AML
t(11;19) KMT2A‑MLLT1 unfavorable in ALL, AML DCDF
(q23;p13.3) AML
t(10;11)(p12;q23) KMT2A‑MLLT10 unfavorable in AML ALL, AML DCDF
t(1;22)(p13;q13) RBM15‑MKL1 unfavorable AMKL DCDF
inv(3) MECOM (3q26) unfavorable AML DCBAP
7q (RELN/ORC5) unfavorable AML, T-ALL DCE, DCBAP
7q22.1-q22.2/
TES(7q31.2)/TCRB(7q34)
t(9;22)(q43;q11) BCR‑ABL1 unfavorable B-ALL, T-ALL, DCDF
AML, CML
t(12;21) ETV6‑RUNX1 favorable B-ALL DCDF
(p13;q22)
+4/+10/+17 D4Z1(4cen),D10Z1 favorable B-ALL TCE
(10cen),D17Z1(17cen)

(Continued)
FISH—in Leukemia Diagnostics 91

TABLE 7.1 (Continued)
FISH Panel and Probes in Leukemia Diagnostics
Cytogenetic Gene Fusion Prognosis Disease Types Probe Types
Abnormality
t(11;14) TCRA/D(14q11) favorable T-ALL DCBAP
(p13;q11)
t(1;19)(q23;p13) PBX1‑E2A controversial, B-ALL DCDF
improved with
therapy
t(4;11)(q21;q23) KMT2A‑AFF1 unfavorable ALL DCDF
iAMP21 RUNX1(21q22) unfavorable B-ALL DCDF, DCE
6q MYB(6q23),D6Z1(6cen) unfavorable ALL DCE
del(9p) CDKN2A(9p21), D9Z3 unfavorable Pre-B-ALL, DCE, DCBAP
(9 cen), PAX5(9p13.2) T-ALL
t(8;14)(q24;q32) cMYC(8q24), favorable Burkitt DCBAP,
IGH(14q32) lymphoma/ DCDF, DCE
leukemia
(BL/L)
del(13q)/+12 DLEU1(13q14), favorable CLL TCE
D13S25 (13q34),
D12Z3 (12 cen)
del(11q)/ ATM(11q22), unfavorable CLL DCE
del(17p) TP53(17p13)

ALL—acute lymphoblastic leukemia; AML—acute myeloid leukemia; CLL—chronic lymphocytic leu-

kemia; CML—chronic myeloid leukemia; DCDF—dual-color, dual fusion; DCSF—dual-color, single
fusion; DCBAP—dual-color, break-apart; DCE—dual-color enumerate; TCE—tricolor enumerate;
r—rearrangement; FAB—French-American-British Classifcation; cen—centromere; del—deletion;
inv—inversion; t—translocation; p—short chromosome arm; q—long chromosome arm; (+)—trisomy.

FISH IN FAVORABLE PROGNOSIS LEUKEMIA

RUNX1(21q22)-RUNX1T1(8q22) Probe
The test shown in Figure 7.1A is important to reveal the presence of the RUNX1‑
RUNX1T1 cryptic fusion on the derivative chromosome 8. In addition, it is possible
to observe the presence of a RUNX1T1 split signal on the derivative chromosome 13,
which is a rare variant with only a few cases described in the literature.[47, 111]

CBFb(16q22)-MYH11(16p13) Probe
Application of FISH-probe as shown in Figure  7.1B is important to stratify the
patient’s risk since inversion inv(16) is related to a good prognosis, with well-defned
treatment protocol guidelines.[5, 40, 68]

PML(15q24)-RARA(17q21) Probe
FISH for rapid detection of PML‑RARA fusion (Figure 7.1C) is mandatory in APL
characterization since a prompt diagnosis and initiation of treatment are critical to
lowering the risk of early mortality.[110]
92 Cytogenetics and Molecular Cytogenetics

FIGURE 7.1 A) FISH result for the translocation t(8;21)(q22;q22)—RUNX1 (green signal),

RUNX1T1 (red signal), and RUNX1‑RUNX1T1 fusion gene (yellow signal)—dual-color dual
fusion probe (DCDF).
B) FISH dual-color dual fusion assay characterizing a chromosome 16 inversion—CBFb
(red), MYH11 (green), CBFb‑MYH11 gene fusion (yellow).
C) FISH test with (1) dual-color single fusion probe on a metaphase spread and (2) dual-color
dual fusion in interphase nuclei; PML (red), RARA (green), PML‑RARA fusion (yellow).

KMT2A(11q23)-MLLT3(9p22) Probe
With a combination of FISH probes, it can be possible to delineate the patient’s
karyotypic alterations, confrming, in this case (Figure 7.2A), the recurrent translo-
cation t(9;11) and a trisomy of chromosome 9 with an extra KMT2A‑MLLT3 fusion
gene.[11, 77, 112]

cMYC(8q24)-IGH(14q32) Probe
The combination of IGH‑MYC with a probe for centromere 8 is essential to dis-
close the recurrent translocation t(8;14), enabling rapid detection and diagnosis of
BL (Figure 7.2B). BL is an aggressive disease with a fast clinical course. However,
FISH—in Leukemia Diagnostics 93

FIGURE  7.2 A) Experiment with FISH in interphase nuclei for the translocation t(9;11)
(p22;q23)— 1) Two normal KMT2A gene signals (yellow),
2) One standard KMT2A (yellow) and KMT2A (red and green) split signals with an extra
KMT2A (red) signal—KMT2A dual-color break-apart probe (DCBAP).
3) Standard KMT2A (red) and MLLT3 (green) signals, besides three copies of KMT2A‑
MLLT3 fusion (yellow)—KMT2A-MLLT3 dual color dual fusion.
B) MYC-directed FISH using
1) assay with dual color dual fusion plus centromeric probe (CEP) characterizing the translo-
cation t(8;14)(q24;q32)—IGH (green) cMYC (red) CEP8 (blue);
2) MYC DCBAP showing a cMYC gene rearrangement.
C) FISH results for translocation t(11;14)(p13;q11)/TCRA/D—dual-color break-apart probe in
interphase nuclei showing a TCRA/D gene rearrangement.
D) FISH test with
1) dual color dual fusion on metaphase spread for translocation t(12;21) (p13;q22)—ETV6
(red) RUNX1 (green) and ETV6‑RUNX1 fusion gene (yellow);
2) Flexibility of ETV6-RUNX1 DCDF in interphase nuclei for iAMP21 monitoring—
two ETV6 (red) normal signals and RUNX1 (green) multiple signals revealing the cryptic
iAMP21; G-banding showed normal karyotype (result not shown).
94 Cytogenetics and Molecular Cytogenetics

excellent clinical outcomes can be achieved provided that it is early diagnosed and
properly treated.[71, 74]

TCRA/D(14q11) Probe
Translocation t(11;14)(p13;q11) is associated with an excellent prognosis, so its pre-
cise characterization is fundamental for diagnosis and therapy (Figure  7.2C). The
translocation t(11;14)(p13;q11) and translocation t(7;11)(q35;p13)/TCRB are variant
translocations of each other.[41, 113]

ETV6(12p13)-RUNX1(21q22) Probe
The translocation t(12;21) is subtle to the G-banding technique and is associated
with a favorable prognosis once adequate treatment is administered.[78, 114] On the
other hand, iAMP21 is considered to confer a poor prognosis, including a high risk
of relapse. In this example (Figure 7.2D), the ETV6‑RUNX1 probe can be used for a
prompt diagnosis of this hematological urgency. Besides, the same probe could help
detect the cryptic iAMP21 even with the G-banding karyotype being apparently
normal.[104]

FISH IN UNFAVORABLE PROGNOSIS LEUKEMIA

del(5q)/del(7q) Probe
Monosomy 5, 7, or del(5q/7q) as karyotypic changes are associated with a poor out-
come in AML. Multiple tumor suppressor genes on the long arms of chromosomes 5
and 7 may play an important role in leukemogenesis.[115–117] They can be detected by
FISH as shown in Figure 7.3A.

BCR(22q11)-ABL1(9q34) Probe
The application of a BCR‑ABL1 probe is important to stratify the patient’s risk indi-
cating targeted therapy against BCR‑ABL1 fusion (Figure 7.3B) by treatment with
tyrosine kinase inhibitors.[5, 101]

KMT2A(11q23)(MLL) Probe
KMT2A—specifc probe (11q23) is essential to disclose KMT2A-r, a group of aber-
rations mainly associated with infant leukemia and related to intermediate/adverse
prognoses.[51] In this example (Figure 7.3C), the probe helped identify a cryptic inser-
tion in a translocation t(10;11), with chromosomal material between the green and
red signals.[61]

TP53 del(17p13) Probe

Loss of the TP53 gene is an aberration of high prognostic impact (Figure 7.3D), con-
sidered to confer the poorest clinical outcome in CLL and an unfavorable prognosis
in ALs.[118–120]
FISH—in Leukemia Diagnostics 95

FIGURE 7.3 A) Experiment with FISH in

1) metaphase and interphase nuclei for deletion del(5q)—EGR1 (5q31) (red), control (5p15)
(green) dual-color enumerate probe (DCE);
2) interphase nuclei for del(7q)—(RELN/ORC5) 7q22.1-q22.2 (red)/TES (7q31.2) (green)
DCE.
B) FISH dual color dual fusion result for the translocation t(9;22)(q34;q11)—BCR (green),
ABL1 (red), and BCR‑ABL1 fusion genes (yellow signals).
C) FISH assay in metaphase and interphase nuclei with MLL dual-color break-apart probe
for KMT2A-r monitoring—Normal KMT2A (yellow) signal; KMT2A split signals (red and
green).
D) Experiment with TP53 deletion dual-color enumerate probe (DCE) in interphase nuclei of
CLL BM cells—TP53 (red) and CEP17 (green), showing the del(17p).

CONCLUSIONS
Hematological neoplasms are classifed according to their differences in clinical and
biological characteristics. Thus, acknowledging the association between specifc
cytogenetic abnormalities and clinical-morphological features is fundamental.[121]
In this sense, FISH-based techniques can be applied to metaphase chromosomes
96 Cytogenetics and Molecular Cytogenetics

and interphase nuclei, providing an accurate cell-based diagnosis complementary to

genomic tests.[122]
In leukemia, the application of FISH has brought remarkable knowledge in the
feld of cancer cytogenetics. The characterization of recurrent chromosomal abnor-
malities and clonal evolution is crucial for classifying the different disease subtypes,
selecting treatment strategies, and monitoring minimal residual disease.[123] Besides,
at the molecular level, FISH can be the basis for the indication of putative genes with
an impact on leukemogenesis.[101] In this context, FISH-based technologies play a
major role in the diagnosis, prognosis, and therapeutic choice, facilitating the daily
practice of individualized medicine.[121]
Currently, the evolution of FISH-based technologies is moving towards a cell-
specifc measurement of the genome, transcriptome, and proteome. In this way,
a range of possibilities is opened for such techniques to be applied in genetic
diagnosis, which can bring important advances for leukemia diagnosis, rang-
ing from the resolution of cytogenetic and genomic abnormalities to epigenetic
alterations.

REFERENCES
1. Greaves, M.F. Biological models for leukaemia and lymphoma. IARC Sci. Publ. 2004,
157, 351–372.
2. Brown, C.M.S.; Larsen, S.R.; Iland, H.J.; Joshua, D.E.; Gibson, J. Leukaemias into the
21st century: Part 1: The acute leukaemias. Intern. Med. J. 2012, 42(11), 1179–1186.
3. Bloomfeld, C.D., Foon, K.A., Levine, E.G. Leukemias. In: Medical Oncology; Calabresi,
P., Schein, P.S., Eds. McGraw Hill, Inc, New York, NY, 1993, pp. 459–501.
4. Juliusson, G.; Hough, R. Leukemia. In: Tumors in Adolescents and Young Adults; Stark,
D.P., Vassal, G., Eds. Prog. Tumor. Res., Karger, Basel, 2016, pp. 43, 87–100.
5. Arber, D.A.; Orazi, A.; Hasserjian, R.; Thiele, J.; Borowitz, M.J.; Le Beau, M.M.;
Bloomfeld, C.D.; Cazzola, M.; Vardiman, J.W. The 2016 revision to the World Health
Organization classifcation of myeloid neoplasms and acute leukemia. Blood. 2016, 127,
2391–2405.
6. Silva, M.L.M.; Land, M. Anomalias Citogenéticas e Moleculares nas Leucemias da
Infância. In: Doenças genéticas em pediatria; Carakushansky, G., Ed. Guanabara
Koogan S.A., Rio de Janeiro, RJ, 2001, pp. 36, 350–362.
7. Wan, T.S.K. Cancer cytogenetics: Methodology revisited. Ann. Lab. Med. 2014, 34(6),
413–425.
8. Cortes, J.; Pavlovsky, C.; Saußele, S. Chronic myeloid leukaemia. Lancet Sem. 2021,
398, 1914–1926.
9. Udayakumar, A.M.; Alkindi, S.; Pathare, A.V.; Raeburn, J.A. Complex t(8;13;21)
(q22;q14;q22)-A novel variant of t(8;21) in a patient with acute myeloid leukemia (AML-
M2). Arch. Med. Res. 2008, 39, 252–256.
10. Mangan, J.K.; Speck, N.A. RUNX1 mutations in clonal myeloid disorders: From conven-
tional cytogenetics to next generation sequencing, a story 40 years in the making. Crit.
Rev. Oncog. 2011, 16, 77–91.
11. Braoudaki, M.; Tzortzatou-Stathopoulou, F. Clinical cytogenetics in pediatric acute leu-
kemia: An update. Clin. Lymph., Myel. Leuk. 2012, 12, 230–237.
12. Bint, S.; Davies, A.; Ogilvie, C. Multicolor banding remains an important adjunct to
array CGH and conventional karyotyping. Mol. Cytogenet. 2013, 6, 55.
FISH—in Leukemia Diagnostics 97

13. Bochtler, T.; Stölzel, F.; Heilig, C.E.; Kunz, C.; Mohr, B.; Jauch, A.; Janssen, J.W.;
Kramer, M.; Benner, A.; Bornhäuser, M.; Ho, A.D.; Ehninger, G.; Schaich, M.; Krämer,
A. Clonal heterogeneity as detected by metaphase karyotyping is an indicator of poor
prognosis in acute myeloid leukemia. J. Clin. Oncol. 2013, 31, 3898–3905.
14. Das, K.; Tan, P. Molecular cytogenetics: Recent developments and applications in can-
cer. Clin. Genet. 2013, 84, 315–325.
15. Gozzetti, A.; Le Beau, M.M. Fluorescence in situ hybridization: Uses and limitations.
Semin. Hematol. 2000, 37, 320–333.
16. Wan, T.S.K. Cancer cytogenetics: An introduction. In: Cancer Cytogenetics Methods
and Protocols; Wan, T.S.K. Ed. Humana Press, New York, NY, 2017, 5–7.
17. Gall, G.; Pardue, M.L. Formation and detection of RNA-DNA hybrid molecules in cyto-
genetical preparations. Proc. Natl. Acad. Sci. USA. 1969, 63, 378–381.
18. John, H.A.; Birnstiel, M.L.; Jones, K.W. RNA-DNA hybrids at the cytological level.
Nature. 1969, 223, 582–587.
19. Buongiorno-Nardelli, M.; Amaldi, F. Autoradiographic detection of molecular hybrids
between rRNA and DNA in tissue sections. Nature. 1970, 225, 946–948.
20. Coons, A.H.; Creech, H.J.; Jones, R.N. Immunological properties of an antibody con-
taining a fuorescent group. Proc. Soc. Exp. Biol. Med. 1941, 47, 200–202.
21. Rudkin, G.T.; Stollar, B.D. High resolution detection of DNA: RNA hybrids in situ by
indirect immunofuorescence. Nature. 1977, 265, 472–473.
22. Bauman, J.G.J.; Wiegant, J.; Borst, P.; Van Duijn, P. A new method for fuorescence
microscopical localization of specifc DNA sequences by in situ hybridization of fuoro-
chrome-labelled RNA. Experimental Cell Research. 1980, 128, 485–490.
23. Langer, P.R.; Waldrop, A.A.; Ward, D.C. Enzymatic synthesis of biotin-labeled poly-
nucleotides: Novel nucleic acid affnity probes. Proc. Natl. Acad. Sci USA. 1981, 78,
6633–6637.
24. Wolff, D.J.; Bagg, A.; Cooley, L.D.; Dewald, G.W.; Hirsch, B.A.; Jacky, P.B.; Rao, K.W.;
Rao, P.N.; Association for Molecular Pathology Clinical Practice Committee; American
College of Medical Genetics Laboratory Quality Assurance Committee. Guidance for
fuorescence in situ hybridization testing in hematologic disorders. J. Mol. Diagn. 2007,
9, 134–143.
25. Flis, S.; Chojnacki, T. Chronic myelogenous leukemia, a still unsolved problem: Pitfalls
and new therapeutic possibilities. Drug Des. Devel. Ther. 2019, 8, 825–843.
26. Andolina, J.R.; Neudorf, S.M.; Corey, S.J. How I treat childhood CML. Blood. 2012,
119, 1821–1830.
27. Khemka, R.; Gupta, M.; Jena, N.K. CML with megakaryocytic blast crisis: Report of 3
cases. Pathol. Oncol. Res. 2018, 25, 1253–1258.
28. Rozman, C.; Montserrat, E. Chronic lymphocytic leukemia. N. Engl. J. Med. 1995, 333,
1052–1057.
29. Kikushige, Y.; Ishikawa, F.; Miyamoto, T.; Shima, T.; Urata, S.; Yoshimoto, G.; Mori,
Y.; Iino, T.; Yamauchi, T.; Eto, T.; Niiro, H.; Iwasaki, H.; Takenaka, K.; Akashi, K. Self-
renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic
lymphocytic leukemia. Cancer Cell. 2011, 20, 246–259.
30. Calin, G.A.; Dumitru, C.D.; Shimizu, M.; Bichi, R.; Zupo, S.; Noch, E.; Aldler, H.;
Rattan, S.; Keating, M.; Rai, K.; Rassenti, L.; Kipps, T.; Negrini, M.; Bullrich, F.;
Croce, C.M. Frequent deletions and down-regulation of micro- RNA genes miR15 and
miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA. 2002, 99,
15524–15529.
31. Klein, U.; Lia, M.; Crespo, M.; Siegel, R.; Shen, Q.; Mo, T.; Ambesi-Impiombato, A.;
Califano, A.; Migliazza, A.; Bhagat, G.; Dalla-Favera, R. The DLEU2/miR-15a/16-1
98 Cytogenetics and Molecular Cytogenetics

cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leuke-
mia. Cancer Cell. 2010, 17, 28–40.
32. Onida, F.; Kantarjian, H.M.; Smith, T.L.; Ball, G.; Keating, M.J.; Estey, E.H.; Glassman,
A.B.; Albitar, M.; Kwari, M.I.; Beran, M. Prognostic factors and scoring systems in
chronic myelomonocytic leukemia: A retrospective analysis of 213 patients. Blood.
2002, 99, 840–849.
33. Such, E.; Cervera, J.; Costa, D.; Sole, F.; Vallespí, T.; Luño, E.; Collado, R.; Calasanz,
M.J.; Hernández-Rivas, J.M.; Cigudosa, J.C.; Nomdedeu, B.; Mallo, M.; Carbonell,
F.; Bueno, J.; Ardanaz, M.T.; Ramos, F.; Tormo, M.; Sancho-Tello, R.; del Cañizo,
C.; Gómez, V.; Marco, V.; Xicoy, B.; Bonanad, S.; Pedro, C.; Bernal, T.; Sanz, G.F.
Cytogenetic risk stratifcation in chronic myelomonocytic leukemia. Haematologica.
2011, 96, 375–383.
34. Ross, J.A.; Johnson, K.J.; Spector, G.L.; Kersey, J.H. Epidemiology of acute childhood
leukemia. In: Childhood Leukemia: A Practical Handbook; Reaman, G.H.; Smith, F.O.,
Eds. Springer, Berlin, Heidelberg, 2011, pp. 3–21.
35. Gilliland, D.; Jordan, C.; Felix, C. The molecular basis of leukemia. Am. Soc. Hematol.
2004, 80–97.
36. Lo-Coco, F.; Ammatuna, E. The biology of acute promyelocytic leukemia and its impact
on diagnosis and treatment. Am. Soc. Hematol. 2006, 514, 156–161.
37. Arceci, R., Aplenc, R. Acute myelogenous leukemia in children. In: Wintrobe’s Clinical
Hematology; Greer, J.P. Ed. Lippincott Williams & Wilkins, Philadelphia, PA, 2009,
pp. 1919–1932.
38. Bolouri, H.; Farrar, J.E.; Triche, T.Jr.; Ries, R.E.; Lim, E.L.; Alonzo, T.A.; Ma, Y.;
Moore, R.; Mungall, A.J.; Marra, M.A.; Zhang, J.; Ma, X.; Liu, Y.; Liu, Y.; Auvil, J.M.G.;
Davidsen, T.M.; Gesuwan, P.; Hermida, L.C.; Salhia, B.; Capone, S.; Ramsingh, G.;
Zwaan, C.M.; Noort, S.; Piccolo, S.R.; Kolb, E.A.; Gamis, A.S.; Smith, M.A.; Gerhard,
D.S.; Meshinchi, S. The molecular landscape of pediatric acute myeloid leukemia reveals
recurrent structural alterations and age-specifc mutational interactions. Nat. Med. 2018,
24, 103–112.
39. Raimondi, S.; Chang, M.N.; Ravindranath, Y.; Behm, F.G.; Gresik, M.V.; Steuber, C.P.;
Weinstein, H.J.; Carroll, A.J. Chromosomal abnormalities in 478 children with acute
myeloid leukemia: Clinical characteristics and treatment outcome in a cooperative
Pediatric Oncology Group Study: POG 8821. Blood. 1999, 94, 3707–3716.
40. Manola, K. Cytogenetics of pediatric acute myeloid leukemia. Europ J Haematol. 2009,
83, 391–405.
41. Mrozek, K.; Heerema, N.; Bloomfeld, C. Cytogenetics in acute leukemia. Blood
Reviews. 2004, 18, 115–134.
42. Pui, C.H.; Carroll, W.L.; Meshinchi, S.; Arceci, R.J. Biology, risk stratifcation, and
therapy of pediatric acute leukemias: An update. J Clin Oncol. 2011, 29, 551–565.
43. Arceci, R., Meshinchi, S. Biology of acute myeloid Leukemia. In: Childhood Leukemia:
A Practical Handbook; Reaman, G.H.; Smith, F.O., Eds. Springer, Berlin, Heidelberg,
2011, pp. 63–74.
44. Creutzig, U.; Zimmermann, M.; Ritter, J.; Reinhardt, D.; Hermann, J.; Henze, G.;
Jürgens, H.; Kabisch, H.; Reiter, A.; Riehm, H.; Gadner, H.; Schellong, G. Treatment
strategy and long term results in pediatric patients treated in four consecutive AML-
BFM trials. Leukemia. 2005, 19, 2030–2042.
45. Gamerdinger, U.; Teigler-Schlegel, A.; Pils, S.; Bruch, J.; Viehmann, S.; Keller, M.;
Jauch, A.; Harbott, J. Cryptic chromosomal aberrations leading to an AML1/ETO rear-
rangement are frequently caused by small insertions. Genes Chromosomes Cancer.
2003, 36, 261–272.
FISH—in Leukemia Diagnostics 99

46. Creutzig, U.; Kutny, M.A.; Barr, R.; Schlenk, R.F.; Ribeiro, R.C. Acute myelogenous
leukemia in adolescents and young adults. Pediatr. Blood Cancer. 2018, 65, e27089.
47. De Figueiredo, A.F.; Liehr, T.; Bath, S.; Binato, R.; De Souza, M.T.; De Matos, R.R.;
Salles, T.de, J.; Jordy, F.C.; Ribeiro, R.C.; Abdelhay, E.; Silva, M.L. A complex karyo-
type masked a cryptic variant t(8;21)(q22;q22) in a child with acute myeloid leukemia.
Leuk. Lymphoma. 2011, 52, 1593–1596.
48. Silva, M.L.M.; Raimondi, S.C.; Abdelhay, E.; Gross, M.; Mkrtchyan, H.; de Figueiredo,
A.F.; Ribeiro, R.C.; de Jesus Marques-Salles, T.; Sobral, E.S.; Gerardin Land, M.P.;
Liehr, T. Banding and molecular cytogenetic studies detected a CBFB-MYH11 fusion
gene that appeared as abnormal chromosomes 1 and 16 in a baby with acute myeloid
leukemia FAB M4-Eo. Cancer Genet. Cytogenet. 2008, 182, 56–60.
49. Nakase, K.; Wakita, Y.; Minamikawa, K.; Yamaguchi, T.; Shiku, H. Acute promyelo-
cytic leukemia with del(6)(p23). Leuk. Res. 2000, 24, 79–81.
50. Matos, R.R.C.; Mkrtchyan, H.; Amaral, B.A.S.; Liehr, T.; de Souza, M.T.; Ney-Garcia,
D.R.; Santos, N.; Marques-Salles, T.J.; Ribeiro, R.C.; Figueiredo, A.F.; Silva, M.L. An
unusual cytogenetic rearrangement originating from two different abnormalities in
chromosome 6 in a child with acute promyelocytic leukemia. Acta Haematol. 2013,
130, 23–26.
51. Meyer, C.; Burmeister, T.; Gröger, D.; Tsaur, G.; Fechina, L.; Renneville, A.; Sutton, R.;
Venn, N.C.; Emerenciano, M.; Pombo-de-Oliveira, M.S.; Barbieri Blunck, C.; Almeida
Lopes, B.; Zuna, J.; Trka, J.; Ballerini, P.; Lapillonne, H.; De Braekeleer, M.; Cazzaniga,
G.; Corral Abascal, L.; van der Velden, V.H.J.; Delabesse, E.; Park, T.S.; Oh, S.H.;
Silva, M.L.M.; Lund-Aho, T.; Juvonen, V.; Moore, A.S.; Heidenreich, O.; Vormoor,
J.; Zerkalenkova, E.; Olshanskaya, Y.; Bueno, C.; Menendez, P.; Teigler-Schlegel,
A.; Zur Stadt, U.; Lentes, J.; Göhring, G.; Kustanovich, A.; Aleinikova, O.; Schäfer,
B.W.; Kubetzko, S.; Madsen, H.O.; Gruhn, B.; Duarte, X.; Gameiro, P.; Lippert, E.;
Bidet, A.; Cayuela, J.M.; Clappier, E.; Alonso, C.N.; Zwaan, C.M.; van den Heuvel-
Eibrink, M.M.; Izraeli, S.; Trakhtenbrot, L.; Archer, P.; Hancock, J.; Möricke, A.;
Alten, J.; Schrappe, M.; Stanulla, M.; Strehl, S.; Attarbaschi, A.; Dworzak, M.; Haas,
O.A.; Panzer-Grümayer, R.; Sedék, L.; Szczepański, T.; Caye, A.; Suarez, L.; Cavé, H.;
Marschalek, R. The MLL recombinome of acute leukemias in 2017. Leukemia. 2018,
32, 273–284.
52. De Figueiredo, A.F.; Liehr, T.; Bath, S.; Binato, R.; Ventura, E.M.; de Souza, M.T.;
de Matos, R.R.; Ribeiro, R.C.; Abdelhay, E.; Silva, M.L. A new cryptic ins(11;1)
(q23;q21q31) detected in a t(1;8;11)(q21;p21;q23) in a baby with acute myeloid leukemia
FAB AML-M5. Blood Cells Mol. Dis. 2010, 45, 197–198.
53. Soupir, C.P.; Vergilio, J.A.; Dal Cin, P.; Muzikansky, A.; Kantarjian, H.; Jones, D.;
Hasserjian, R.P. Philadelphia chromosome-positive acute myeloid leukemia: A rare
aggressive leukemia with clinicopathologic features distinct from chronic myeloid leu-
kemia in myeloid blast crisis. Am. J. Clin. Pathol. 2007, 127, 642–650.
54. Cuneo, A.; Ferrant, A.; Michaux, J.L.; Demuynck, H.; Boogaerts, M.; Louwagie,
A.; Doyen, C.; Stul, M.; Cassiman, J.J.; Dal Cin, P.; Castoldi, G.; Van den Berghe,
H. Philadelphia chromosome-positive acute myeloid leukemia: Cytoimmunologic and
cytogenetic features. Haematologica. 1996, 81, 423–427.
55. Meshinchi, S.; Arceci, R.J. Prognostic factors and risk-based therapy in pediatric acute
myeloid leukemia. The Oncologist. 2007, 12, 341–355.
56. Von Lindern, M.; Fornerod, M.; van Baal, S.; Jaegle, M.; de Wit, T.; Buijs, A.; Grosveld,
G. The translocation t(6;9), associated with a specifc subtype of acute myeloid leuke-
mia, results in the fusion of two genes, DEK and CAN, and the expression of a chime-
ric, leukemiaspecifc DEK-CAN mRNA. Mol. Cell. Biol. 1992, 12, 1687–1697.
100 Cytogenetics and Molecular Cytogenetics

57. Martineau, M.; Berger, R.; Lillington, D.M.; Moorman, A.V.; Secker-Walker, L.M.
The t(6;11)(q27;q23) translocation in acute leukemia: A laboratory and clinical study
of 30 cases: EU concerted action 11q23 Workshop participants. Leukemia. 1998, 12,
788–791.
58. Balgobind, B.V.; Raimondi, S.C.; Harbott, J.; Zimmermann, M.; Alonzo, T.A.;
Auvrignon, A.; Beverloo, H.B.; Chang, M.; Creutzig, U.; Dworzak, M.N.; Forestier,
E.; Gibson, B.; Hasle, H.; Harrison, C.J.; Heerema, N.A.; Kaspers, G.J.; Leszl, A.;
Litvinko, N.; Nigro, L.L.; Morimoto, A.; Perot, C.; Pieters, R.; Reinhardt, D.; Rubnitz,
J.E.; Smith, F.O.; Stary, J.; Stasevich, I.; Strehl, S.; Taga, T.; Tomizawa, D.; Webb, D.;
Zemanova, Z.; Zwaan, C.M.; van den Heuvel-Eibrink, M.M. Novel prognostic sub-
groups in childhood 11q23/MLL-rearranged acute myeloid leukemia: Results of an
international retrospective study. Blood. 2009, 114, 2489–2496.
59. de Matos, R.R.C.; Ferreira, G.M.; Meyer, C.; Marschalek, R.; Larghero, P.; Ribeiro,
R.C.; Liehr, T.; Othman, M.; Bizarro, M.T.S.M.; Sobral da Costa, E.; Land, M.G.P.;
Abdelhay, E.; Binato, R.; Silva, M.L.M. KMT2A-MLLT1 and the novel SEC16A-
KMT2A in a cryptic 3-way translocation t(9;11;19) present in an infant with acute lym-
phoblastic leukemia. J. Pediatr. Hematol. Oncol. 2022, 44, e719–e722.
60. De Braekeleer, E.; Meyer, C.; Douet-Guilbert, N.; Morel, F.; Le Bris, M.J.; Berthou,
C.; Arnaud, B.; Marschalek, R.; Férec, C.; De Braekeleer, M. Complex and cryptic
chromosomal rearrangements involving the MLL gene in acute leukemia: A study of 7
patients and review of the literature. Blood Cells Mol. Dis. 2010, 44, 268–274.
61. De Figueiredo, A.F.; Vieira, T.P.; Liehr, T.; Bhatt, S.; de Souza, M.T.; Binato, R.;
Marques-Salles, T.deJ.; Carboni, E.; Ribeiro, R.C.; Silva, M.L.; Abdelhay, E. A rare
cryptic and complex rearrangement leading to MLL-MLLT10 gene fusion masked by
del(10)(p12) in a child with acute monoblastic leukemia (AML-M5). Leuk. Res. 2012,
36, e74–e77.
62. Ma, Z.; Morris, S.W.; Valentine, V.; Li, M.; Herbrick, J.A.; Cui, X.; Bouman, D.; Li, Y.;
Mehta, P.K.; Nizetic, D.; Kaneko, Y.; Chan, G.C.; Chan, L.C.; Squire, J.; Scherer, S.W.;
Hitzler, J.K. Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13q13) of
acute megakaryoblastic leukemia. Nat. Genet. 2001, 28, 220–221.
63. Carroll, A.; Civin, C.; Schneider, N.; Dahl, G.; Pappo, A.; Bowman, P.; Emami, A.;
Gross, S.; Alvarado, C.; Phillips, C.; Krischer, J.; Crist, W.; Head, D.; Gresik, M.;
Ravindranath, Y.; Weinstein, H. The t(1;22)(p13;q13) is nonrandom and restricted to
infants with acute megakaryoblastic leukemia: A Pediatric Oncology Group study.
Blood. 1991, 78, 748–752.
64. Dastugue, N.; Lafage-Pochitaloff, M.; Pagès, M.P.; Radford, I.; Bastard, C.; Talmant,
P.; Mozziconacci, M.J.; Léonard, C.; Bilhou-Nabéra, C.; Cabrol, C.; Capodano,
A.M.; Cornillet-Lefebvre, P.; Lessard, M.; Mugneret, F.; Pérot, C.; Taviaux, S.;
Fenneteaux, O.; Duchayne, E.; Berger, R.; Groupe Français d’Hematologie Cellulaire.
Cytogenetic profle of childhood and adult megakaryoblastic leukemia (M7): A study
of the Group Francais de Cytogenetique Hematologique (GFCH). Blood. 2002, 100,
618–626.
65. Capela de Matos, R.R.; Othman, M.A.K.; Ferreira, G.M.; Costa, E.S.; Melo, J.B.;
Carreira, I.M.; de Souza, M.T.; Lopes, B.A.; Emerenciano, M.; Land, M.G.P.; Liehr, T.;
Ribeiro, R.C.; Silva, M.L.M. Molecular approaches identify a cryptic MECOM rear-
rangement in a child with a rapidly progressive myeloid neoplasm. Cancer Genet. 2018,
221, 25–30.
66. Onciu, M. Acute lymphoblastic leukemia. Hematol. Oncol. Clin. North Am. 2009, 23,
655–674.
FISH—in Leukemia Diagnostics 101

67. Bhojwani, D.; Yang, J.J.; Pui, C.H. Biology of childhood acute lymphoblastic leukemia.
Pediatr. Clin. North. Am. 2015, 62, 47–60.
68. Pui, C.H. Recent research advances in childhood acute lymphoblastic leukemia. J.
Formos. Med. Assoc. 2010, 109, 777–787.
69. Vrooman, L.M.; Silverman, L.B. Treatment of childhood acute lymphoblastic leuke-
mia: Prognostic factors and clinical advances. Curr. Hematol. Malig. Rep. 2016, 11,
385–394.
70. Shago, M. Chromosome preparation for acute lymphoblastic leukemia. In: Cancer
Cytogenetics Methods and Protocols; Wan, T.S.K., Ed. Humana Press, New York, NY,
2017, pp. 19–31.
71. Greenought, A.; Dave, S.S. New clues to the molecular pathogenesis of Burkitt lym-
phoma revealed through next-generation sequencing. Curr. Opin. Hematol. 2014, 21,
326–332.
72. Swerdlow, S.H., Campo, E., Harris, N.L., Jaffe, E.S., Pileri, S.A.; Stein, H.; Thiele,
J.; Vardiman, J.W. (eds.). WHO Classifcation of Tumours of Haematopoietic and
Lymphoid Tissues. 4th Edition. IARC: Lyon; 2008.
73. Petit, B.; Mele, L.; Rack, K.; Camera, A.; Vekemans, M.C.; Bassan, R.; Pulsoni, A.;
Delannoy, A.; Pagano, L. Characteristics of secondary acute lymphoblastic leukemia
with L3 morphology in adult patients. Leuk Lymphoma. 2002, 43, 1599–1604.
74. De Souza, M.T.; Hassan, R.; Liehr, T.; Marques-Salles, T.J.; Boulhosa, A.M.; Abdelhay,
E.; Ribeiro, R.C.; Silva, M.L. Conventional and molecular cytogenetic characterization
of Burkitt lymphoma with bone marrow involvement in Brazilian children and adoles-
cents. Pediatr. Blood Cancer. 2014, 61, 1422–1426.
75. Chan, K.W. Acute lymphoblastic leukemia. Curr. Probl. Pediatr. Adolesc. Health
Care. 2002, 32, 40–49.
76. Kimura, S.; Mullighan, C.H. Molecular markers in ALL: Clinical implications. Best.
Pract. Res. Clin. Haematol. 2020, 33, 101193.
77. Ney-Garcia, D.R.; Liehr, T.; Emerenciano, M.; Meyer, C.; Marschalek, R.; Pombo-de-
Oliveira, M.do, S.; Ribeiro, R.C.; Poirot Land, M.G.; Macedo Silva, M.L. Molecular
studies reveal a MLL-MLLT3 gene fusion displaced in a case of childhood acute lym-
phoblastic leukemia with complex karyotype. Cancer Genet. 2015, 15, 1–4.
78. Garcia, D.R.; Arancibia, A.M.; Ribeiro, R.C.; Land, M.G.; Silva, M.L. Intrachromosomal
amplifcation of chromosome 21 (iAMP21) detected by ETV6/RUNX1 FISH screen-
ing in childhood acute lymphoblastic leukemia: A case report. Rev. Bras. Hematol.
Hemoter. 2013, 35, 2–4.
79. Ney-Garcia, D.R.; Liehr, T.; Bhatt, S.; de Souza, M.T.; de Matos, R.R.; Binato, R.;
Jordy, F.C.; Abdelhay, E.; Ribeiro, R.C.; Silva, M.L. Molecular cytogenetics stud-
ies reveal unexpected chromosomal inversion as variant of t(12;21)(p13;q22) in child
with B-cell precursor acute lymphoblastic leukemia. Leuk Lymphoma. 2012, 53,
342–344.
80. Mrozek, K.; Harper, D.P.; Aplan, P.D. Cytogenetics and molecular genetics of acute
lymphoblastic leukemia. Hematol. Oncol. Clin. N. Am. 2009, 23, 991–1010.
81. Sundaresh, A.; Williams, O. Mechanism of ETV6-RUNX1 leukemia. Adv. Exp. Med.
Biol. 2017, 962, 201–216.
82. Lange, B.J.; Smith, F.O.; Feusner, J.; Barnard, D.R.; Dinndorf, P.; Feig, S.; Heerema,
N.A.; Arndt, C.; Arceci, R.J.; Seibel, N.; Weiman, M.; Dusenbery, K.; Shannon, K.;
Luna-Fineman, S.; Gerbing, R.B.; Alonzo, T.A. Outcomes in CCG-2961, a Children’s
Oncology Group Phase 3 trial for untreated pediatric acute myeloid leukemia: A report
from the Children’s Oncology Group. Blood. 2008, 111, 1044–1053.
102 Cytogenetics and Molecular Cytogenetics

83. Martinez-Climent, J.A. Molecular cytogenetics of childhood hematological malignan-

cies. Leukemia. 1997, 11, 1999–2021.
84. Takeuchi, S.; Koike, M.; Seriu, T.; Bartram.; C.R.; Schrappe, M.; Reiter, A.; Park, S.;
Taub, H.E.; Kubonishi, I.; Miyoshi, I.; Koeffer, H.P. Frequent loss of heterozygosity
on the long arm of chromosome 6: Identifcation of two distinct regions of deletion in
childhood acute lymphoblastic leukemia. Cancer. Res. 1998, 58, 2618–2623.
85. Fischer, K.; Fröhling, S.; Scherer, S.W.; McAllister Brown, J.; Scholl, C.; Stilgenbauer,
S.; Tsui, L.C.; Lichter. P.; Döhner, H. Molecular cytogenetic delineation of deletions and
translocations involving chromosome band 7q22 in myeloid leukemias. Blood. 1997, 89,
2036–2041.
86. Koike, M.; Tasaka, T.; Spira, S.; Tsuruoka, N.; Koeffer, H.P. Allelotyping of acute
myelogenous leukemia: Loss of heterozygosity at 7q31.1 (D7S486) and q33–34
(D7S498, D7S505). Leuk. Res. 1999, 23, 307–310.
87. Betts, D.R.; Ammann, R.A.; Hirt, A.; Hengartner, H.; Beck-Popovic, M.; Kuhne, T.;
Nobile, L.; Cafisch, U.; Wacker, P.; Niggli, F.K. The prognostic signifcance of cytoge-
netic aberrations in childhood acute myeloid leukaemia: A study of the Swiss Paediatric
Oncology Group (SPOG). European J. Haematology. 2007, 78, 468–476.
88. Forestier, E.; Heim, S.; Blennow, E.; Borgström, G.; Holmgren, G.; Heinonen, K.;
Johannsson, J.; Kerndrup, G.; Andersen, M.K.; Lundin, C.; Nordgren, A.; Rosenquist,
R.; Swolin, B.; Johansson, B.; Nordic Society of Paediatric Haematology and Oncology
(NOPHO); Swedish Cytogenetic Leukaemia Study Group (SCLSG); NOPHO
Leukaemia Cytogenetic Study Group (NLCSG). Cytogenetic abnormalities in child-
hood acute myeloid leukaemia: A Nordic series comprising all children enrolled in the
NOPHO-93-AML trial between 1993 and 2001. Br. J. Haematol. 2003, 121, 566–577.
89. Hall, G.W. Childhood myeloid leukemias. Best. Pract. Res. Clin. Haematol. 2001, 14,
573–591.
90. Raimondi, S.; Chang, M. N.; Ravindranath, Y.; Behm, F.G.; Gresik, M.V.; Steuber, C.P.;
Weinstein, H.J.; Carroll, A.J. Chromosomal abnormalities in 478 children with acute
myeloid leukemia: Clinical characteristics and treatment outcome in a cooperative
Pediatric Oncology Group Study-POG 8821. Blood. 1999, 94, 3707–3716.
91. Heerema, N.A. 9p rearrangements in ALL. Atlas Genet. Cytogenet. Oncol. Haematol.
1999. http://AtlasGeneticsOncology.org/Anomalies/9prearrALLID1156.html [accessed
on 03/12/2022].
92. Wells, R.J.; Arthur, D.C.; Srivastava, A.; Heerema, N.A.; Le Beau, M.; Alonzo, T.A.;
Buxton, A.B.; Woods, W.G.; Howells, W.B.; Benjamin, D.R.; Betcher, D.L.; Buckley,
J.D.; Feig, S.A.; Kim, T.; Odom, L.F.; Ruymann, F.B.; Smithson, W.A.; Tannous, R.;
Whitt, J.K.; Wolff, L.; Tjoa, T.; Lampkin, B.C. Prognostic variables in newly diagnosed
children and adolescents with acute myeloid leukemia: Children’s Cancer Group Study
213. Leukemia. 2002, 16, 601–607.
93. Grimwade, D.; Walker, H.; Oliver, F.; Wheatley, K.; Harrison, C.; Harrison, G.; Rees,
J.; Hann, I.; Stevens, R.; Burnett, A.; Goldstone, A. The importance of diagnostic cyto-
genetics on outcome in AML: Analysis of 1,612 patients entered into the MRC AML 10
trial: The medical Research Council Adult and Children’s Leukaemia Working Parties.
Blood. 1998, 92, 2322–2333.
94. Mrozek, K. Acute myeloid leukemia with a complex karyotype. Semin. Oncol. 2008,
35, 365–377.
95. Orozco, F.; Appelbaum, J. Unfavorable, complex, and monosomal karyotypes: The
most challenging forms of acute myeloid leukemia. Oncology. 2012, 26, 1–10.
96. Al-Achkar, W.; Aljapawe, A.; Othman, M.A.K.; Wafa, A. A de novo acute myeloid
leukemia (AML-M4) case with a complex karyotype and yet unreported breakpoints.
Mol. Cytogenet. 2013, 6, 18.
FISH—in Leukemia Diagnostics 103

97. Ney Garcia, D.R.; de Souza, M.T.; de Figueiredo, A.F.; Othman, M.A.K.; Rittscher, K.;
Abdelhay, E.; Capela de Matos, R.R.; Meyer, C.; Marschalek, R.; Land, M.G.P.; Liehr,
T.; Ribeiro, R.C.; Silva, M.L.M. Molecular characterization of KMT2A fusion partner
genes in 13 cases of pediatric leukemia with complex or cryptic karyotypes. Hematol.
Oncol. 2017, 35, 760–768.
98. Betts, D.; Ammann, R.A.; Hirt, A.; Hengartner, H.; Beck-Popovic, M.; Kuhne, T.;
Nobile, L.; Cafisch, U.; Wacker, P.; Niggli, F.K. The prognostic signifcance of cytoge-
netic aberrations in childhood acute myeloid leukaemia: A study of the Swiss Paediatric
Oncology Group (SPOG). European Journal of Haematology. 2007, 78, 468–476.
99. Marchesi, F.; Annibali, O.; Cerchiara, E.; Tirindelli, M.C.; Avvisati, G. Cytogenetic
abnormalities in adult non-promyelocytic acute myeloid leukemia: A concise review.
Crit. Rev. Oncol. Hemat. 2011, 80, 331–346.
100. Ney-Garcia, D.; Vieira, T.P.; Liehr, T.; Bhatt, S.; de Souza, M.T.; de Figueiredo, A.F.;
Ribeiro, R.C.; Silva, M.L. A case of childhood T cell acute lymphoblastic leukemia
with a complex t(9;9) and homozygous deletion of CDKN2A gene associated with a
Philadelphia-positive minor subclone. Blood Cells, Mol. Dis. 2012, 50, 131–133.
101. Cui, C.; Shu, W.; Li, P. Fluorescence in situ hybridization: Cell-based genetic diagnos-
tic and research applications. Front. Cell. Dev. Biol. 2016, 4, 89.
102. Bishop, R. Applications of fuorescence in situ hybridization (FISH) in detecting
genetic aberrations of medical signifcance. Bioscience Horizons: The International
Journal of Student Research. 2010, 3, 85–95.
103. Nordgren, A. Hidden aberrations diagnosed by interphase fuorescence in situ hybrid-
ization and spectral karyotyping in childhood acute lymphoblastic leukaemia. Leuk.
Lymphoma. 2003, 44, 2039–2053.
104. Capela de Matos, R.R.; Othman, M.A.K.; Ferreira, G.M.; Monteso, K.; de Souza, M.T.;
Rouxinol, M.; Melo, J.B.; Carreira, I.M.; Abdelhay, E.; Liehr, T.; Ribeiro, R.C.; Silva,
M. Somatic homozygous loss of SH2B3, and a non-Robertsonian translocation t(15;21)
(q25.3;q22.1) with NTRK3 rearrangement, in an adolescent with progenitor B-cell
acute lymphoblastic leukemia with the iAMP21. Cancer Genet. 2021, 262–263, 16–22.
105. Testa, J.R.; Misawa, S.; Ogrema, N.; van Slaten, K.; Wiernik, P.H. Chromosomal altera-
tions in acute leukemia patients: Studies with improved culture methods. Cancer Res.
1985, 45, 430–434.
106. Hungerford, D.A. Leukocytes cultures from small in occula of whole blood and the
preparation of metaphase chromosomes by treatament with hypotonic (KCl). Stain
Technol., 1965, 40, 333–338.
107. Gisselsson, D. Cytogenetic methods. In: Cancer Genetics; Heim, S.; Mitelman, F., Eds.
John Wiley & Sons, Inc, New Jersey, NY, 2009, p. 18.
108. Seabright, M. A rapid banding technique for human chromosomes. Lancet. 1971, 2,
971–972.
109. Guerra, M., Ed. FISH Conceito e aplicações na citogenética. Sociedade Brasileira de
Genética, Ribeirão Preto, SP; 2004.
110. Tallman, M.S.; Altman, J.K. How I treat acute promyelocytic leukemia. Blood. 2009,
114, 5126–5135.
111. Capela de Matos, R.R.; de Figueiredo, A.F.; Liehr, T.; Alhourani, E.; De Souza, M.T.;
Binato, R.; Ribeiro, R.C.; Silva, M.L. A novel three-way variant t(8;13;21)(q22;q33;q22)
in a child with acute myeloid leukemia with RUNX1/RUNX1T1: The contribution
of molecular approaches for revealing t(8;21) variants. Acta Haematol. 2015, 134,
243–245.
112. Capela de Matos, R.R.; Ney-Garcia, D.R.; Cifoni, E.; Othman, M.A.K.; Tavares de
Souza, M.; Carboni, E.K.; Ferreira, G.M.; Liehr, T.; Ribeiro, R.C.; Macedo Silva, M.L.
GAS6 oncogene and reverse MLLT3-KMT2A duplications in an infant with acute
104 Cytogenetics and Molecular Cytogenetics

myeloid leukemia and a novel complex hyperdiploid karyotype: Detailed high-resolu-

tion molecular cytogenetic studies. Cytogenet. Genome Res. 2017, 152, 33–37.
113. Bilhou-Nabera, C. t(11;14)(p13;q11) TRD/LMO2t(7;11)(q35;p13) TRB/LMO2.
Atlas Genet. Cytogenet. Oncol. Haematol. 1998. http://atlasgeneticsoncology.org/
haematological/1070/t(11;14)(p13;q11)-trd-lmo2t(7;11)(q35;p13)-trb-lmo2 [accessed on
03/15/2022].
114. Bacher, U.; Schnittger, S.; Haferlach, C.; Haferlach, T. Molecular diagnostics in acute
leukemias. Clin. Chem. Lab. Med. 2009, 47, 1333–1341.
115. Jerez, A.; Sugimoto, Y.; Makishima, H.; Verma, A.; Jankowska, A.M.; Przychodzen,
B.; Visconte, V.; Tiu, R.V.; O’Keefe, C.L.; Mohamedali, A.M.; Kulasekararaj, A.G.;
Pellagatti, A.; McGraw, K.; Muramatsu, H.; Moliterno, A.R.; Sekeres, M.A.; McDevitt,
M.A.; Kojima, S.; List, A.; Boultwood, J.; Mufti, G.J.; Maciejewski, J.P. Loss of het-
erozygosity in 7q myeloid disorders: Clinical associations and genomic pathogenesis.
Blood. 2012, 119, 6109–6118.
116. Joslin, J.M.; Fernald, A.A.; Tennant, T.R.; Davis, E.M.; Kogan, S.C.; Anastasi, J.;
Crispino, J.D.; Le Beau, M.M. Haploinsuffciency of EGR1, a candidate gene in the
del(5q), leads to the development of myeloid disorders. Blood. 2007, 110, 719–726.
117. De Figueiredo, A.F.; Capela de Matos, R.R.; Moneeb, M.A.K.; Liehr, T.; da Costa, E.S.;
Land, M.G.; Ribeiro, R.C.; Abdelhay, E.; Silva, M.L. Molecular cytogenetic studies
characterizing a novel complex karyotype with an uncommon 5q22 deletion in child-
hood acute myeloid leukemia. Mol. Cytogenet. 2015, 8, 62.
118. Baliakas, P.; Hadzidimitriou, A.; Sutton, L.A.; Rossi, D.; Minga, E.; Villamor, N.;
Larrayoz, M.; Kminkova, J.; Agathangelidis, A.; Davis, Z.; Tausch, E.; Stalika, E.;
Kantorova, B.; Mansouri, L.; Scarfò, L.; Cortese, D.; Navrkalova, V.; Rose-Zerilli,
M.J.; Smedby, K.E.; Juliusson, G.; Anagnostopoulos, A.; Makris, A.M.; Navarro, A.;
Delgado, J.; Oscier, D.; Belessi, C.; Stilgenbauer, S.; Ghia, P.; Pospisilova, S.; Gaidano,
G.; Campo, E.; Strefford, J.C.; Stamatopoulos, K.; Rosenquist, R.; European Research
Initiative on CLL (ERIC). Recurrent mutations refne prognosis in chronic lymphocytic
leukemia. Leukemia. 2015, 29, 329–336.
119. Seifert, H.; Mohr, B.; Thiede, C.; Oelschlägel, U.; Schäkel, U.; Illmer, T.; Soucek, S.;
Ehninger, G.; Schaich, M.; Study Alliance Leukemia (SAL). The prognostic impact of
17p (p53) deletion in 2272 adults with acute myeloid leukemia. Leukemia. 2009, 23,
656–663.
120. Stengel, A.; Schnittger, S.; Weissmann, S.; Kuznia, S.; Kern, W.; Kohlmann, A.;
Haferlach, T.; Haferlach, C. TP53 mutations occur in 15.7% of ALL and are associated
with MYC-rearrangement, low hypodiploidy, and a poor prognosis. Blood. 2014, 124,
251–258.
121. Wan, T.S.K.; Ma, E.S.K. Molecular cytogenetics: An indispensable tool for cancer
diagnosis. Chang Gung Med. J. 2012, 35, 96–110.
122. Xu, F., Li, P. Cytogenomic abnormalities and dosage-sensitive mechanisms for intel-
lectual and developmental disabilities. In: Developmental Disabilities: Molecules
Involved, Diagnosis and Clinical Care; Salehi, A., Ed. InTech, Rijeka, 2013, pp. 1–30.
123. Martin, C.L.; Warburton, D. Detection of chromosomal aberrations in clinical practice:
From karyotype to genome sequence. Annu. Rev. Genomics Hum. Genet. 2015, 16,
309–326.
 8 FISH—in Tissues
Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 105
Samples/Tissues ..................................................................................................... 106
Description of Methods.......................................................................................... 106
FISH on FFPE-Tissue Sections (No Nuclear Extraction) ............................ 106
How to Extract Nuclei from FFPE-Tissue Sections ..................................... 107
Yields ..................................................................................................................... 108
Conclusions ............................................................................................................ 108
Acknowledgments.................................................................................................. 108
References .............................................................................................................. 108

INTRODUCTION
Application of fuorescence in situ hybridization (FISH) in tissues refers here to stud-
ies done in interphase cells derived from various kinds of solid tissues from the
human body. These tissues are normally fxed in one of two ways: (i) formalin-fxed/
paraffn-embedded (FFPE) tissue,[1] or (ii) cryofxed ones.[2] Especially the pathology
of solid tumors,[3–5] and more exceptionally postmortem analysis of aborted fetus-
esare done by FISH.[6] As in FFPE sections embedded after long fxations times in
unbuffered formalin may contain highly degraded DNA not suited for FISH in most
clinics now FFPE and cryofxation are done in parallel.[7]
Of the four types of FISH-probes, whole chromosome paints, partial chromo-
some paints, centromeric probes (CPs) and locus-specifc probes (LSPs) only the
latter two are suited for routine interphase-FISH studies.[8] This is as only CPs and
LSPs lead to well-defned and relatively small signals in the interphase nucleus.
Thus, such signals can be counted to determine copy numbers of chromosomes or
chromosomal regions. Also a smart use of several LSPs or LSPs with CPs labeled
by different fuorochromes enables not only proof of deletions, duplications or even
amplifcations, and translocations or inversions may be picked up on the single-cell
level.[9]
Most FISH-studies done on tissues in pathology stick to sectioned and mounted
FFPE sections[1–5]; this has the disadvantage of partially overlapping, as well as
incomplete, cut nuclei, which cannot be evaluated properly and lead to artifcial loss
of signals.[10–11] However, it is argued that invasive and small tumor pieces could
be identifed better on sections.[12] Thus, the alternative approach where a nuclear

DOI: 10.1201/9781003223658-8 105

106 Cytogenetics and Molecular Cytogenetics

extraction technique[1, 2, 13] is applied is not used much in routine pathology, but has
been shown to lead to same results with lower cut-off rates; after nuclear extraction,
these are non-overlapping, and all incomplete nuclei have been discarded during the
extraction procedure.[1–2, 6, 14–16]
Here a commercial standard protocol for handling of FFPE sections is described;
also, a procedure how to extract nuclei from a cryosection, which can be used in
FISH later. Protocols for nuclear extraction from FFPE and a block of cyrofxed
material were previously described.[16]

SAMPLES/TISSUES
Each kind of solid human tissue, which can be either cryo- or FFPE-fxed, is suited
for this kind of FISH-study. It is not part of this protocol to describe the production
of a cryo- or FFPE-block or how to cut the slices and mount on glass-slides. Slices
may be differently thick—normally between from 3 to 8 µm. Also, after cutting
and thawing of cryo-sections in most cases a fxation with 3% paraformaldehyde is
necessary.[16]

DESCRIPTION OF METHODS
FISH ON FFPE-TISSUE SECTIONS (NO NUCLEAR EXTRACTION)
(Here a modifcation of the ZytoVision protocol is described—www.zytovision.
com/)

1. Incubate slides with FFPE-sections on slides on a heating plate at 70.5°C

for 10 min.
2. Immerse them in NeoClear (Merck, Darmstadt, Germany) two times for
10 min to deparaffnize the sections (room temperature = RT).
3. Rehydrate the sections in an ethanol series 100%, 95%, 70% for 2 min,
each; fnally put them for 2 min in distilled water (all at RT).
4. Apply “Heat Pretreatment Solution Citric” (ZytoVision, Bremerhaven,
Germany) preheated for ~1 h at 99°C. Put max 2–3 slides in a 100 ml
cuvette and incubate in a water bath at 99°C for 17 min.
5. Then transfer slides in distilled water (RT) and leave for 2 min.
6. Repeat step 5, remove slides from water, and dry carefully the surface and
the underneath of the slide with an absorbent paper, with exception for the
sections themselves.
7. Transfer slides in a humid chamber at 37°C and cover sections with a
suffcient amount of “Pepsin Solution” (ZytoVision, Bremerhaven,
Germany)—incubate for 5 min.
8. Take slides and let solution fow off on an absorbent paper and immediately
transfer to “Wash Buffer SSC” (ZytoVision, Bremerhaven, Germany), RT,
for 5 min.
9. Transfer to distilled water (at RT) for 1 min and then dehydrate section
in an ethanol series 70%, 95%, 100%, 2 min each. Then let slides air dry
putting them perpendicular.
FISH—in Tissues 107

10. Adjust FISH probe mix (ZytoVision, Bremerhaven, Germany) according

to size of section and size of coverslip. Seal coverslips by rubber cement
and let rubber cement dry for ~20 min (best in dark conditions).
11. Co-denature slides and FISH-probe at 74.5°C for 10 min and transfer then
to humid chamber at 37°C. Incubate overnight.
12. Mix 10 ml of “Wash Buffer A” (ZytoVision, Bremerhaven, Germany) with
250 ml of distilled water, distribute on three 100 ml cuvettes and heat to
38°C in a water bath.
13. Remove rubber cement from slide by forceps and put in frst of three pre-
pared cuvettes; there remove carefully the coverslip.
14. Transfer to second cuvette (38°C) and leave for 5 min.
15. Transfer to third cuvette (38°C) and leave for 5 min.
16. Repeat step 9.
17. Put 1 drop of “DAPI/DuraTect-Solution (ultra)” (ZytoVision, Bremerhaven,
Germany) on a 22×22 coverslip wish is laying on the lab table. Approach
the coverslip and drop with the slide, which is upside down and let fuid
spread between slide and coverslip.
18. Start evaluation about 15 min after coverslip was added.

HOW TO EXTRACT NUCLEI FROM FFPE-TISSUE SECTIONS

(a) Perform steps 1 to 6 as described previously.
(b) Latest at this step, but maybe better before starting with step 1 tissue parts
not to be analyzed (e.g. normal tissue) is to be removed.
(c) Cover tissue with proteinase K solution (5 mg proteinase K (Roche, Basel,
Switzerland, #03115887001), 50 µl 1 M Tris-HCl (pH 7.5), 20 µl 0.5 M
EDTA (pH 7.0), 2 µl 5 M NaCl, make up to 1 ml with fltered double-
distilled water; make fresh as required).
(d) Incubate for 1 h in a humid chamber (37°C). Do not use a coverslip.
(e) Fluid with disaggregated tissue is collected with a micro-pipette and fl-
tered via a 55 µm nylon mesh (Nytal 55, SEFAR-AG, Heiden, Switzerland
#3A07-0049-102-00). Fluid and nuclei pass through the mesh by gravity;
wash out the mesh 4 ml 1×PBS; collect all with a 15 ml plastic tube.
(f) Centrifuge the nuclei down at 850×g for 8 min and discard supernatant
apart from ~300µl.
(g) Add 4 ml 1×PBS and repeat step f.
(h) Dilute nuclei in remainder 300 µl of 1×PBS.
(i) Put a suspension-drop of on a dry and clean slide; evaporate the fuid from
the slide on a 40°C heating plate (~5–10 min), and store suspension at 4°C.
(j) After overnight drying of the slide, fx in 0.1% formalin buffer for 10 min (RT).
(k) Finalize by washing the slides in 1×PBS, distilled water and an ethanol
series (70%, 90%, 100%) 1 min each and air dry.
(l) After checking density of nuclei under a phase contrast light microscope,
the region with best nuclei density can be marked with a diamond pencil
and the slide can be used for FISH. Here a standard FISH protocol can be
used (excluding pepsin pretreatment) like e.g. for peripheral blood lym-
phocyte suspension.
108 Cytogenetics and Molecular Cytogenetics

FIGURE  8.1 Typical results as obtained after FISH on FFPE-tissue sections (without
nuclear extraction) are shown.
A) A sarcoma cell with MDM2 amplifcation and a normal cell from same section are shown
after application of ZytoLight ® SPEC MDM2/CEN 12 Dual Color Probe (ZytoVision,
Bremerhaven, Germany, # Z-2013–50).
B) A Dermatofbrosarcoma protuberans (DFPS) cell with t(17;22)(q21.33;q13.1) (one red, one
green and two fusion signals) and a normal cell are visible—probe applied was ZytoLight
® SPEC COL1A1/PDGFB Dual Color Dual Fusion Probe (ZytoVision, Bremerhaven,
Germany, # Z-2116–50).

YIELDS
Results of FISH on FFPE sections fxed on slides are shown in Figure 8.1. As stan-
dard in interphase FISH analyses, it is not suffcient to evaluate 1 to 5 nuclei only. In
pathology, 50 nuclei of tumor tissue is taken into account.[17–18]

CONCLUSIONS
Even though it has been proven that FISH results from archived, cryo- or FFPE-
fxed tissues are at least as reliable, if not even better, when instead of tissue-sections
including cut and overlaying nuclei, extracted nuclei are studied, most labs stick to
sections. This is most likely due to habit and practice but also the argument that after
nuclear extraction it is no longer reproducible where exactly the evaluated nuclei are
derived from in the original section.

ACKNOWLEDGMENTS
Figure  8.1 was provided by Stefanie Kankel, Institute of Human Genetics, Jena
University Hospital, Jena, Germany; sections were provided by the Institute for
Forensic Medicine FSU, Pathology Section, Jena University Hospital, Jena, Germany.

REFERENCES
1. Liehr, T.; Grehl, H.; Rautenstrauss, B. FISH analysis of interphase nuclei extracted from
paraffn-embedded tissue. Trends Genet. 1995, 11, 377–378.
FISH—in Tissues 109

2. Liehr, T.; Grehl, H.; Rautenstrauss, B. A rapid method for FISH analysis on interphase
nuclei extracted from cryofxed tissue. Trends Genet. 1996, 12, 505–506.
3. Wolfe, K.Q.; Herrington, C.S. Interphase cytogenetics and pathology: A tool for diagno-
sis and research. J. Pathol. 1997, 181, 359–361.
4. Fuller, C.E.; Perry, A. Fluorescence in situ hybridization (FISH) in diagnostic and inves-
tigative neuropathology. Brain Pathol. 2002, 12, 67–86.
5. Lim, A.S.; Lim, T.H. Fluorescence in situ hybridization on tissue sections. Methods Mol.
Biol. 2017, 1541, 119–125.
6. Fickelscher, I.; Starke, H.; Schulze, E.; Ernst, G.; Kosyakova, N.; Mkrtchyan, H.;
MacDermont, K.; Sebire, N.; Liehr, T. A further case with a small supernumerary
marker chromosome (sSMC) derived from chromosome 1: Evidence for high variability
in mosaicism in different tissues of sSMC carriers. Prenat. Diagn. 2007, 27, 783–785.
7. Long, A.A.; Komminoth, P.; Lee, E.; Wolfe, H.J. Comparison of indirect and direct in-
situ polymerase chain reaction in cell preparations and tissue sections: Detection of viral
DNA, gene rearrangements and chromosomal translocations. Histochemistry. 1993, 99,
151–162.
8. Liehr, T. Molecular cytogenetics in the era of chromosomics and cytogenomic
approaches. Front Genet. 2021, 12, 720507.
9. Iourov, I.; Vorsanova, S.; Yurov, Y. (Eds.). Human Interphase Chromosomes: Biomedical
Aspects. Springer, Berlin; 2020.
10. Dhingra, K.; Sahin, A.; Supak, J.; Kim, S.Y.; Hortobagyi, G.; Hittelman, W.N.
Chromosome in situ hybridization on formalin-fxed mammary tissue using non-
isotopic: Non-fuorescent probes: Technical considerations and biological implications.
Breast Cancer Res. Treat. 1992, 23, 201–210.
11. Liehr, T.; Stübinger, A.; Thoma, K.; Tulusan, H.A.; Gebhart, E. Comparative interphase
cytogenetics using FISH on human ovarian carcinomas. Anticancer Res. 1994, 14,
183–188.
12. Köpf, I.; Hanson, C.; Delle, U.; Verbiené, I.; Weimarck, A. A rapid and simplifed tech-
nique for analysis of archival formalin-fxed, paraffn-embedded tissue by fuorescence
in situ hybridization (FISH). Anticancer Res. 1996, 16, 2533–2536.
13. Hedley, D.W.; Friedlander, M.L.; Taylor, I.W.; Rugg, C.A.; Musgrove, E.A. Method for
analysis of cellular DNA content of paraffn-embedded pathological material using fow
cytometry. J. Histochem. Cytochem. 1983, 31, 1333–1335.
14. Qian, J.; Bostwick, D.G.; Takahashi, S.; Borell, T.J.; Brown, J.A.; Lieber, M.M.; Jenkins,
R.B. Comparison of fuorescence in situ hybridization analysis of isolated nuclei and rou-
tine histological sections from paraffn-embedded prostatic adenocarcinoma specimens.
Am. J. Pathol. 1996, 149, 1193–1199.
15. Köpf, I.; Hanson, C.; Delle, U.; Verbiené, I.; Weimarck, A. A rapid and simplifed tech-
nique for analysis of archival formalin-fxed, paraffn-embedded tissue by fuorescence
in situ hybridization (FISH). Anticancer Res. 1996, 16, 2533–2536.
16. Liehr, T. Characterization of archived formalin-fxed/paraffn-embedded or cryofxed
tissue, including nucleus extraction. In: Fluorescence In Situ Hybridization (FISH):
Application Guide. 2nd Edition. Springer, Berlin, 2017, pp. 201–208.
17. Wayne, P.A. Fluorescence In Situ Hybridization Methods for Clinical Laboratories‑
Aproved Guidelines. 2nd Edition. Clinical Laboratory Standards Institute MM07-A2;
2013.
18. Wiktor, A.E.; Van Dyke, D.L.; Stupca, P.J.; Ketterling, R.P.; Thorland, E.C.; Shearer,
B.M.; Fink, S.R.; Stockero, K.J.; Majorowicz, J.R.; Dewald, G.W. Preclinical validation
of fuorescence in situ hybridization assays for clinical practice. Genet. Med. 2006, 8,
16–23.
 9 FISH—in Human
Sperm and Infertility
Martina Rincic and Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 111
Indications .............................................................................................................. 112
Description of Methods.......................................................................................... 112
Sample Preparation ....................................................................................... 112
Results and Interpretation ...................................................................................... 113
Limitations and Pitfalls .......................................................................................... 114
Troubleshooting ..................................................................................................... 114
Conclusions ............................................................................................................ 115
References .............................................................................................................. 115

INTRODUCTION
According to a generally agreed defnition, infertility is the inability to have off-
spring despite active sexual intercourse of a couple for at least one year. This
problem affects ~12% of the population in reproductive age; one in six couples
needs the help of assisted reproduction techniques (ARTs) to conceive a child.[1]
Pre-implantation genetic testing (PGT) as an alternative to prenatal diagnosis was
introduced over 30 years ago. PGT performed together with ARTs aims to reduce
the transmission of genetic or chromosomal abnormality. Recently, the European
Society of Human Reproduction and Embryology (ESHRE) has subdivided PGT
into (i) PGT-M (for the monogenic disorder), (ii) PGT-SR (for structural rearrange-
ments), which causes miscarriage, and (iii) PGT-A (for aneuploidy screening).[2]
Despite all technical advancements in the feld of molecular genetics, according to
the latest ESHRE recommendations fuorescence in situ hybridization (FISH) and
single nucleotide polymorphism (SNP) array are recommended as techniques of
choice for PGT-SR.[3]
Infertility is a complex issue where male infertility factors are relatively com-
mon conditions, affecting at least 6% of men of reproductive age. There are many
causes known from changes in gross semen parameters (for instance, low sperm
counts or compromised motility) to more complex and severe factors. In general,
gametes of infertile individuals tend to display higher rates of chromosomal abnor-
malities than those seen in fertile persons, resulting fnally in aneuploidy embryos.
Ideally, clinical diagnosis of gamete aneuploidy should include analysis of all

DOI: 10.1201/9781003223658-9 111

112 Cytogenetics and Molecular Cytogenetics

human autosomes and sex chromosomes. On the other hand, that type of analysis
is time-consuming and with a high cost, and is often unfeasible. Since most aneu-
ploid embryos do not survive the early stages of development, the aneuploidies that
are of major clinical importance are those that can be non-lethal, and thus compat-
ible with survival. They include autosomal trisomies, such as Trisomy 13 (Pätau
syndrome), Trisomy 18 (Edwards syndrome), and Trisomy 21 (Down syndrome),
and aneuploidies of the sex chromosomes, such as e.g. Turner- and Klinefelter-
syndrome (45,X and 47,XXY).
In 1978, direct cytogenetic analysis of the human sperm cells was introduced.[4]
Given, that this original technique was diffcult to perform, time-consuming, expen-
sive, and above all yields only a small number of karyotypes, interphase FISH was
an elegant solution to replace it. Nowadays FISH on human sperm is most commonly
used to determine the proportion of aneuploidy present in autosomes and sex chro-
mosomes of infertile men.[1]

INDICATIONS
Generally, FISH analysis of human sperm is used to perform a basic study of
aneuploidy. According to literature, a FISH sperm analysis is indicated in cases of
(i)  recurrent pregnancy loss, (ii) repeated in vitro fertilization failure, (iii) abnor-
mal seminal parameters including count, motility, and morphology, and (iv) genetic
aberrations (such as but not limited to, Robertsonian and reciprocal translocations,
chromosome inversions, ring chromosomes, and numerical sex chromosome aberra-
tions).[5, 6] Additionally, FISH analysis of human sperm is indicated in men exposed
to potential mutagens, and to evaluate the impact of lifestyle factors on sperm aneu-
ploidy rate.[7–9]

DESCRIPTION OF METHODS
Considering the diverse causes of man infertility and referral reasons starting sam-
ple can be ejaculated, epididymal, or testicular-derived sperm. As already men-
tioned, the chromosomes that are generally considered for analyses are 13, 18, 21,
X, and Y, given that those aneuploidies are compatible with life. Nevertheless, all
chromosomes can be studied by us of probes specifc for each individual chromo-
some. The technical procedure of interphase FISH on human sperm nuclei has
several phases: A) sample preparation, B) hybridization, C) post-hybridization
washes, and D) visualization.[10, 11] As steps B–D are following standard conditions
(as e.g. described by the provider of corresponding FISH probes), they are not
further detailed here.

SAMPLE PREPARATION
As sperms (including their DNA) are extremely condensed, being caused by exten-
sive intermolecular disulfde cross-links, they must be decondensed prior to the
FISH procedure. To allow DNA probes access to the chromatin Otto’s solution is
FISH—in Human Sperm and Infertility 113

applied {100 ml Tris-HCl buffer 10 mmol/1, pH 7.4, 220 mg KCl (30 mmol/1), 100
mg MgCl2×6H2O (5 mmol/l), and 46 mg dithioerythritol (3 mmol/1)}.

1. Suspend ejaculate/sperm sample in 8 ml PBS; use appropriate centrifuge

tubes. Centrifuge at 1,250 rpm for 10 min. Remove supernatant carefully
and repeat washing two times.
2. After resuspending the pellet in ~1–2 ml of PBS vortex, add 20 drops of
Otto’s solution (drop by drop); then suspend in 8 ml Otto’s solution and
incubate at 37°C for 30 min.
3. Centrifuge at 1,250 rpm for 10 min and remove supernatant carefully.
4. After resuspending the pellet in ~1–2 ml of Otto’s solution, add 20 drops
of ice-cold, freshly prepared Carnoy’s fxative (3:1 methanol: acetic acid—
drop by drop) while mixing on a vortexer, then add fxative to obtain a fnal
volume of 8 ml.
5. Repeat step 3*; add 4ml fxative.
6. Repeat step 5 two times.
7. Drop suspension on the slide and let air dry.
* If only a small pellet is visible, drop the sample onto a slide immediately.

In order to achieve good FISH-results, pretreatment of now decondensed cells with

pepsin is recommended:

1. Add 50 µl pepsin solution (10%) to a Coplin jar with 100 ml of distilled

H2O pre-warmed at 37°C in a water bath; mix thoroughly. Place slides and
incubate for 8 min in a water bath at 37°C.
2. Incubate 1×2 min in 1×PBS in a Coplin jar.
3. Incubate 10 min in 1% formaldehyde in PBS-MgCl2.
4. Repeat step 2.
5. Dehydrate the slides in ethanol (70%, 90%, 100%) for 2 min each and let
slides air-dry.

RESULTS AND INTERPRETATION

As reported in the literature, apparently healthy men exhibit intra-individual and
inter-individual disomy and diploidy rates in sperm.[12, 13] Besides needing to deter-
mine cut-off rates in sperm derived from fertile males, in general, interphase FISH
can also lead to artifcially induced signals (see section later). Thus, to obtain reliable
results, usually about 1,000 to 10,000 sperm nuclei per patient have to be analyzed
and aligned with cut-off rates. Additionally, signal scoring criteria can aid objective
analysis. These criteria include: (i) overlapped spermatozoa or sperm heads without a
well-defned boundary are not counted, (ii) in cases of disomy or diploidy all signals
should have the same intensity and be separated from each other by a distance longer
than the diameter of each signal, and (iii) nullisomies are not directly scored.[14]
Interpretation of abnormal fndings from FISH sperm analysis can be evalu-
ated using the quantitative or the qualitative approach. The quantitative approach
114 Cytogenetics and Molecular Cytogenetics

interprets FISH results as a numerical value (a score, essentially) that would indicate
the patient’s “degree of risk”. This approach has some shortcomings that are the fea-
tures intrinsic to this technique. It is very diffcult (if not impossible) to study simul-
taneously all the chromosomes of the karyotype (normally only X, Y, 13, 18, and
21 are analyzed), and also not all possible chromosomal abnormalities can be regis-
tered. Accordingly, this method tends to underestimate the extent of anomaly rates.
In the qualitative approach, signifcant increases in aneuploidy count would have
to be interpreted as evidence that there are meiotic defects, thus indicating that the
quality of the spermatogenesis is not at optimum. Such an approach has the potential
to identify most “at-risk” patients and should be favored over the quantitative one.[15]

LIMITATIONS AND PITFALLS

Shortcomings of FISH sperm analysis are (i) the inability to access structural chro-
mosomal aberrations and (ii) the fact that sub-regions of chromosomes are studied,
as locus-specifc probes are normally used. Besides, interphase FISH analysis can
lead to false positive results as decreased or increased numbers of signals. The sig-
nal number may be decreased due to (a) insuffcient tissue penetrability resulting in
reduced hybridization effciency, (b) a certain number of signals that are physically
so close together that they appear as one, and (c) analysis of damaged or overlapping
nuclei. On the other side, signal number may be increased by (I) signal splitting due
to dispersed or damaged DNA, and (II) signals caused by artifacts such as bacteria
or background fuorescence.

TROUBLESHOOTING
The most common technical points that can interfere with the quality of FISH in
human sperm are the following: no or weak signals, too much background, and
diffuse signals and cells. Reasons for signal loss may be too short denaturation or
hybridization time. Another possibility could be too stringent washing conditions
after hybridization. An increase in salt concentration or decrease of temperature
could resolve this, while higher dextran sulfate concentration and increase in probe
concentration could lead to better signals.
Usually, too much background is the result of some kind of “dirtiness” on slide
surface and can be reduced during hybridization or post-hybridization washing by
increasing the temperature or decreasing salt concentration. Additionally, the appli-
cation of directly labeled probes reduces background fuorescence. During the wash-
ing steps, it is important to prevent the slide surfaces from drying out; otherwise
background problems may arise. In addition, slides can be dirty at delivery and may
need some cleaning before use in FISH-procedure/before dropping cell suspension
on them.
To resolve diffuse signals and cells usually decreasing denaturation time and
temperature by 2°C, and/or shortening decondensation time will help to settle this
issue. Pepsin pretreatment conditions, as well as the denaturation time of the target
DNA, should be tested in each laboratory at the introduction of this kind of test.
FISH—in Human Sperm and Infertility 115

If pepsin concentration is too stringent, it can result in clean slides without any
remaining nuclei.

CONCLUSIONS
FISH is the fastest and most accurate test to assess the aneuploidy of chromosomes in
sperm nuclei on a single-cell level. Clinically, the result of FISH sperm analysis can
be used in counseling couples affected by male infertility, to come to an informed
and consented choice regarding their further reproductive plans.

REFERENCES
1. Chandra, A.; Martinez, G.M.; Mosher, W.D.; Abma, J.C.; Jones, J. Fertility, family plan-
ning, and reproductive health of U.S. women: Data from the 2002 National Survey of
Family Growth. Vital Health Stat. 2005, 23, 1–160.
2. Zegers-Hochschild, F.; Adamson, G.D.; Dyer, S.; Racowsky, C.; de Mouzon, J.; Sokol,
R.; Rienzi, L.; Sunde, A.; Schmidt, L.; Cooke, I.D.; Simpson, J.L.; van der Poel, S. The
international glossary on infertility and fertility care, 2017. Hum. Reprod. 2017, 32,
1786–1801.
3. ESHRE PGT-SR/PGT-A Working Group; Coonen, E.; Rubio, C.; Christopikou, D.;
Dimitriadou, E.; Gontar, J.; Goossens, V.; Maurer, M.; Spinella, F.; Vermeulen, N.; De
Rycke, M. ESHRE PGT Consortium good practice recommendations for the detection
of structural and numerical chromosomal aberrations. Hum. Reprod. Open. 2020, 2020,
hoaa017.
4. Rudak, E.; Jacobs, P.A.; Yanagimachi, R. Direct analysis of the chromosome constitu-
tion of human spermatozoa. Nature. 1978, 274, 911–913.
5. Ramasamy, R.; Besada, S.; Lamb, D.J. Fluorescent in situ hybridization of human
sperm-diagnostics.; indications.; and therapeutic implications. Fertil. Steril. 2014, 102,
1534–1539.
6. Fakhrabadi, M.P.; Kalantar, S.M.; Montazeri, F.; Ashkezari, M.D.; Fakhrabadi, M.P.;
Yazd, S.S.N. FISH-based sperm aneuploidy screening in male partner of women with a
history of recurrent pregnancy loss. Middle East Fert. Soc. J. 2020, 25, 23.
7. Saad, A.A.; Hussein, T.; El-Sikaily, A.; Abdel-Mohsen, M.A.; Mokhamer, E.H.; Youssef,
A.I.; Mohammed, J. Effect of polycyclic aromatic hydrocarbons exposure on sperm
DNA in idiopathic male infertility. J. Health Pollut. 2019, 9, 190309.
8. Srám RJ.; Binková, B.; Rössner, P.; Rubes, J.; Topinka, J.; Dejmek, J. Adverse repro-
ductive outcomes from exposure to environmental mutagens. Mutat. Res. 1999, 428,
203–215.
9. Robbins, W.A.; Vine, M.F.; Truong, K.Y.; Everson, R.B. Use of fuorescence in situ
hybridization (FISH) to assess effects of smoking, caffeine, and alcohol on aneuploidy
load in sperm of healthy men. Environ. Mol. Mutagen. 1997, 30, 175–183.
10. Sarrate, Z.; Anton, E. Fluorescence in situ hybridization (FISH) protocol in human
sperm. J. Vis. Exp. 2009, (31), 1405.
11. Rauch, A. Human sperm cells. In: FISH Technology; Rautenstrauss, B.W.; Liehr, T. Eds.
Springer, Berlin, Heidelberg, 2002, pp. 127–137.
12. García-Mengual, E.; Triviño, J.C.; Sáez-Cuevas, A.; Bataller, J.; Ruíz-Jorro, M.; Vendrell,
X. Male infertility: Establishing sperm aneuploidy thresholds in the laboratory. J. Assist.
Reprod. Genet. 2019, 36, 371–381.
116 Cytogenetics and Molecular Cytogenetics

13. Schultz, H.; Mennicke, K.; Schlieker, H.; Al-Hasani, S.; Bals-Pratsch, M.; Diedrich, K.;
Schwinger, E. Comparative study of disomy and diploidy rates in spermatozoa of fertile
and infertile men: A donor-adapted protocol for multi-colour fuorescence in situ hybrid-
ization (FISH). Int. J. Androl. 2000, 23, 300–308.
14. Blanco, J.; Egozcue, J.; Vidal, F. Incidence of chromosome 21 disomy in human sper-
matozoa as determined by fuorescent in-situ hybridization. Hum. Reprod. 1996, 11,
722–726.
15. Sarrate, Z.; Vidal, F.; Blanco, J. Role of sperm fuorescent in situ hybridization studies
in infertile patients: Indications.; study approach.; and clinical relevance. Fertil. Steril.
2010, 93, 1892–1902.
 10 FISH—in Spontaneously
Aborted Products
of Conception
Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 117
Samples/Tissues ..................................................................................................... 118
Description of Methods.......................................................................................... 118
Yields ..................................................................................................................... 119
Conclusions ............................................................................................................ 119
References .............................................................................................................. 119

INTRODUCTION
Infertility is a major problem in Western countries, but also—either less expressed
or less registered—in all human societies around the world. Besides the inability to
conceive, some couples experience (repeated) abortions at early, middle or late preg-
nancy stages.[1] Such couples should attract gynecologist’s attention during routine
patient admission, and these couples should be offered (cyto)genetic studies for both
partners.[2] In a subset of these cases, a gonosomal mosaic, a small supernumerary
marker chromosome or a balanced rearrangement can be traced, as being causative
for the problem.[3, 4] Besides, and especially in cases where no such conclusive results
can be found, the spontaneously aborted products of conception (PoCs) themselves
should be studied genetically.[5] Chromosomal aberrations identifed in these PoCs
can be important (i) to make such couples better understand why an abortion has
happened,[6] and (ii) also as they can be a hint on a cryptic gonadal mosaicism being
present in male or female partner.[7]
In some settings, DNA extraction from PoCs and subsequent chromosome-
microarray,[8] multiplex ligation-dependent probe amplifcation (MLPA),[9] or simple
STR (= short tandem repeat DNA) -analyses (own unpublished data) may be used
to fnd genomic imbalances, that have being causative for the abortion. Another
straightforward method is tissue cultivation from PoCs and subsequent chromosome
analyses by GTG-banding[5, 10, 11]; alternatively and if available, also formalin-fxed
formalin embedded material of PoCs may be analyzed by molecular cytogenetics.[12]
However, the latter is missing the opportunity to possibly get an informative banding
cytogenetic result. Furthermore, as PoC-cell-cultivation is prone to culture failure,

DOI: 10.1201/9781003223658-10 117

118 Cytogenetics and Molecular Cytogenetics

it has been shown that interphase molecular cytogenetics may be a way out here to
obtain nonetheless informative results.[5]

SAMPLES/TISSUES
Cytogenetic preparations with and without metaphase spreads, but at least contain-
ing suffcient interphase nuclei from PoCs are used for the here described analyses.
Preparation is done using standard techniques.[13]

DESCRIPTION OF METHODS
The method applied here is a standard fuorescence in situ hybridization (FISH)
approach—see e.g.[14] Commercially available probes, either centromere- or locus-
specifc ones, are applied. Preferentially used are centromeric probes, still, for
chromosomes 13 and 21, and 14 and 22 only the probes D13/21Z1 and D14/22Z1
staining both corresponding chromosome pairs are available. In addition, problems
may arise with centromeric probe D1Z7 in 1p11.1–1q11 being identical to D5Z2 in
5p11-q11.1 and D19Z3 in 19q11; chromosome 1 may be identifed by D1Z5 in 1q11-
q12, but additional (derivatives of) chromosomes 5 and 19 cannot be indubitably
distinguished in metaphase and not at all in interphase.[15, 16] Accordingly, com-
mercially available locus-specifc probes can be selected if these chromosomes
shall be studied.
As the available amount of PoC-material normally is restricted, at least two- to
multicolor-FISH settings are recommended. According to available commercially
available probes applied multicolor-FISH probe sets may be put together (e.g.
Table 10.1). In a study from 2017 we had an ~37% detection rate of abortion causing
chromosomal aneuploidies when concentrating on chromosomes 13/21, 14/22, 15,
16, 17, 18, X and Y. Suited probe sets which are composed of commercially avail-
able probes from Abbott/Vysis and Cytocell are listed in Table 10.1. Probe sets 1
to 3 (Table 10.1) should be applied subsequently to samples to be tested. In case of
fve signals for D13/21Z1 or D14/22Z1 probes, the result needs to be checked by
locus-specifc (LSI) probes, as listed in Table 10.1. The latter is necessary, as it can
easily be that instead of a trisomy 13, 14, 21 or 22 suited chromosome-specifc LSI
probes do not reveal an aberrant signal pattern. This may be due to chromosomal
heteromorphisms, which can lead to additional signals in the centromeric regions
of another chromosome.[17] Additionally, such a result can be due to a partial tri-
somy of one of the four chromosomes in the sense of a small supernumerary marker
chromosome.[15]
For the here-suggested set-up and composition of probe sets (Table 10.1), com-
mercially available probes are suggested. Thus, the established lab internal standard
FISH-procedure may be used, or the protocol as suggested by the provider of the
commercial probes can be applied. It has to be tested under each individual lab-con-
dition if a pretreatment with RNAse and/or pepsin is necessary or can be skipped;
this is dependent on the plasma load of interphase nuclei after cytogenetic work-up
of PoCs.
FISH—in Spontaneously Aborted Products of Conception 119

TABLE 10.1
Probe Sets Adapted from[5] Consisting of Commercially Available Probes Are
Listed; With This Setting Polyploidies, Gonsomal Aberrations and Numerical
Aberrations of Chromosomes 13, 14, 15, 16, 17, 21 and 22 Can Be Accessed.
Probe Set Probes Location Fluorochrome
1 D13/21Z1 13p11.1-q11 and SpectrumGreen (SG)
D15Z3 21p11.1-q11.1 TexasRed (TR)
D18Z1 15q11 Diethylaminocoumarin
18p11.1-q11.1 (DEAC)
2 D14/22Z1 14p11.1-q11.1 and SG
D16Z2 22p11.1-q11.1 TR
16p11.1-q11.1
3 D17Z1 17p11.1-q11.1 SpectrumOrange (SO)
DXZ1 Xp11.1-q11.1 DEAC
Yq12 Yq12 SG
if D13/21Z1 gives 5 signals LSI 13/21 13q14 and 21q22.13-q22.2 SG and SO
if D14/22Z1 gives 5 signals LSI D22S75/ 22q11.2 and 22q13.3 SO and SG
ARSA

YIELDS
Studying PoCs with the suggested probe sets (Table 10.1) identifed in a pilot study
of 286 PoCs chromosomal abnormalities in 106 cases. Polyploidy was present in
23/106 cases, sex-chromosome abnormalities in 37/106 cases and trisomies in the
remainder 46 cases. Twenty-two of the latter 46 cases were trisomy 16.[5]

CONCLUSIONS
Taking advantage of interphase FISH to unravel reasons of (repeated) abortions is
still only scarcely used in human genetic labs. The possibilities to obtain a diagnosis
on the reasons for an abortion are still by far not exhausted in routine settings; not
only the study referred to here,[5] but also other authors highlighted this option,[5, 8–12]
which among others provides also the advantage of a single cell specifc evaluation,
and thus to detect mosaic conditions, too.

REFERENCES
1. Flyckt, R.; Falcone, T. Infertility: A practical framework. Cleve Clin. J. Med. 2019, 86,
473–482.
2. Larsen, E.C.; Christiansen, O.B.; Kolte, A.M.; Macklon, N. New insights into mecha-
nisms behind miscarriage. BMC Med. 2013, 11, 154.
3. Liehr, T. Small supernumerary marker chromosomes detected in connection with infer-
tility. Zhonghua Nan. Ke. Xue. 2014, 20, 771–780.
120 Cytogenetics and Molecular Cytogenetics

4. Manvelyan, M.; Schreyer, I.; Höls-Herpertz, I.; Köhler, S.; Niemann, R.; Hehr, U.; Belitz,
B.; Bartels, I.; Götz, J.; Huhle, D.; Kossakiewicz, M.; Tittelbach, H.; Neubauer, S.;
Polityko, A.; Mazauric, M.L.; Wegner, R.; Stumm, M.; Küpferling, P.; Süss, F.; Kunze,
H.; Weise, A.; Liehr, T.; Mrasek, K. Forty-eight new cases with infertility due to bal-
anced chromosomal rearrangements: Detailed molecular cytogenetic analysis of the 90
involved breakpoints. Int. J. Mol. Med. 2007, 19, 855–864.
5. Tkach, I.R.; Huleyuk, N.L.; Zastavna, D.V.; Weise, A.; Liehr, T.; Ciszkowicz, E.; Tyrka,
M. Chromosomal aberrations in spontaneously aborted products of conception from
Ukraine. Biopolymers Cell. 2017, 33, 424–433.
6. Griebel, C.P.; Halvorsen, J.; Golemon, T.B.; Day, A.A. Management of spontaneous
abortion. Am. Fam. Physician. 2005, 72, 1243–1250.
7. Kamel, A.K.; Abd El-Ghany, H.M.; Mekkawy, M.K.; Makhlouf, M.M.; Mazen, I.M.; El
Dessouky, N.; Mahmoud, W.; Abd El Kader, S.A. Sex chromosome mosaicism in the
gonads of DSD patients: A karyotype/phenotype correlation. Sex. Dev. 2015, 9, 279–288.
8. Gao, J.; Liu, C.; Yao, F.; Hao, N.; Zhou, J.; Zhou, Q.; Zhang, L.; Liu, X.; Bian, X.; Liu, J.
Array-based comparative genomic hybridization is more informative than conventional
karyotyping and fuorescence in situ hybridization in the analysis of frst-trimester spon-
taneous abortion. Mol. Cytogenet. 2012, 5, 33.
9. Kim, J.W.; Lyu, S.W.; Sung, S.R.; Park, J.E.; Cha, D.H.; Yoon, T.K.; Ko, J.J.; Shim, S.H.
Molecular analysis of miscarriage products using multiplex ligation-dependent probe
amplifcation (MLPA): Alternative to conventional karyotype analysis. Arch. Gynecol.
Obstet. 2015, 291, 347–354.
10. Vorsanova, S.G.; Kolotii, A.D.; Iourov, I.Y.; Monakhov, V.V.; Kirillova, E.A.; Soloviev,
I.V.; Yurov, Y.B. Evidence for high frequency of chromosomal mosaicism in spontane-
ous abortions revealed by interphase FISH analysis. J. Histochem. Cytochem. 2005, 53,
375–380.
11. Jobanputra, V.; Sobrino, A.; Kinney, A.; Kline, J.; Warburton, D. Multiplex interphase
FISH as a screen for common aneuploidies in spontaneous abortions. Hum. Reprod.
2002, 17, 1166–1170.
12. Russo, R.; Sessa, A.M.; Fumo, R.; Gaeta, S. Chromosomal anomalies in early spontane-
ous abortions: Interphase FISH analysis on 855 FFPE frst trimester abortions. Prenat.
Diagn. 2016, 36, 186–191.
13. Weise, A.; Liehr, T. Pre- and postnatal diagnostics and research on peripheral
blood, bone marrow chorion, amniocytes, and fbroblasts. In: Fluorescence In Situ
Hybridization (FISH): Application Guide; Liehr, T., Ed. 2nd Edition. Springer, Berlin,
2017, pp. 171–180.
14. Weise, A.; Liehr, T. Background. In: Fluorescence In Situ Hybridization (FISH):
Application Guide; Liehr, T., Ed. 2nd Edition. Springer, Berlin, 2017, pp. 1–14.
15. Nietzel, A.; Rocchi, M.; Starke, H.; Heller, A.; Fiedler, W.; Wlodarska, I.; Loncarevic,
I.F.; Beensen, V.; Claussen, U.; Liehr, T. A new multicolor-FISH approach for the charac-
terization of marker chromosomes: Centromere-specifc multicolor-FISH (cenM-FISH).
Hum. Genet. 2001, 108, 199–204.
16. Liehr, T. Benign & Pathological Chromosomal Imbalances Microscopic and
Submicroscopic Copy Number Variations (CNVs) in Genetics and Counseling. Academic
Press, New York, 2014.
17. Liehr, T.; Ziegler, M. Rapid prenatal diagnostics in the interphase nucleus: Procedure
and cut-off rates. J. Histochem. Cytochem. 2005, 53, 289–291.
 11 FISH—Characterization
of Chromosomal
Alterations,
Recombination, and
Outcomes after
Segregation
Ilda Patrícia Ribeiro, Eunice Matoso,
Joana Barbosa Melo, Ana Jardim,
Thomas Liehr and Isabel Marques Carreira

CONTENTS
Introduction............................................................................................................ 122
Chromosomal Alterations ...................................................................................... 124
Numerical Alterations................................................................................... 125
Unbalanced Structural Rearrangements ....................................................... 129
Balanced Structural Rearrangements............................................................ 135
Case Problems........................................................................................................ 138
Clinical Case 1 ....................................................................................................... 138
Clinical Information...................................................................................... 138
Techniques Performed .................................................................................. 138
Results........................................................................................................... 138
Questions ...................................................................................................... 138
Curiosity ....................................................................................................... 139
Conclusion .................................................................................................... 140
Clinical Case 2 ....................................................................................................... 140
Clinical Information...................................................................................... 140
Techniques Performed .................................................................................. 140
Results Twin 1............................................................................................... 140
Results Twin 2............................................................................................... 140
Questions ...................................................................................................... 140
Conclusion .................................................................................................... 142

DOI: 10.1201/9781003223658-11 121

122 Cytogenetics and Molecular Cytogenetics

Clinical Case 3 ....................................................................................................... 143

Clinical Information...................................................................................... 143
Techniques Performed .................................................................................. 143
Results........................................................................................................... 143
Questions ...................................................................................................... 143
Curiosity ....................................................................................................... 145
Conclusion .................................................................................................... 145
Clinical Case 4 ....................................................................................................... 145
Clinical Information...................................................................................... 145
Techniques Performed .................................................................................. 145
Results........................................................................................................... 146
Questions ...................................................................................................... 146
Conclusion .................................................................................................... 147
Conclusions............................................................................................................ 147
Dedication .............................................................................................................. 148
References.............................................................................................................. 148

INTRODUCTION
The process leading to the formation of haploid gametes from diploid cells through
one round of DNA replication and two rounds of chromosome segregation is called
meiosis, or more precisely Meiosis I (MI) and Meiosis II (MII).[1] Both meiosis and
mitosis begin with replication of DNA in order to originate a cell with four chroma-
tids of each type of chromosome, two from the mother and two from the father.[2]
During the frst meiotic division (MI), the separation of maternal and paternal copies
of each chromosome occurs: this is the so-called reductional division. It is in the sec-
ond meiotic division (MII) that the separation of sister chromatids occurs: this is the
equational division (very similar to mitosis). The resulting four chromatids are dis-
tributed to four different nuclei, which occurs simultaneously for all chromosomes
in the two rounds of chromosome segregation.[2] During the meiosis process, there
is not only a reduction of chromosome number to a haploid set but also the aleatoric
segregation of maternal and paternal chromosomes, and genetic combinations in the
offspring due to exchange of genetic material in maternal and paternal homologues
through recombination.[3] The reduction in chromosome number is pivotal to restore
diploid chromosome complement in the subsequent generation through the union of
two gametes during sexual reproduction, ensuring continuity of the species.[4]
There are three signifcant deviations in MI in comparison to mitosis:

(i) homologous chromosomes are physically linked together through chi-

asmata, the products of homologous recombination that occurred at
pachytene;
(ii) sister kinetochores attach to microtubules from opposite spindle pole
(mono-oriented); and
(iii) cohesion is lost in a step-wise manner on chromosome arms during the
frst division but preserved at the pericentromere.[5]
FISH—Characterization of Chromosomal Alterations 123

Malfunction in these three building blocks of chromosome segregation presents as

consequence in an inequitable distribution of sister chromatids, and thus in numeri-
cal alterations.[6] Therefore, the complex behavior of chromosomes during the two
meiotic divisions pave the way for errors to occur. Errors in homologous chromosome
segregation during either meiosis or mitosis can lead to an aberrant chromosome
number, which is termed aneuploidy (occurrence of one or more extra or missing
chromosomes leading to an unbalanced chromosome complement); this could occur
in the postnatal, prenatal, or preimplantation stage, having signifcant clinical con-
sequences. The identifcation of these chromosomal errors and the understanding of
the molecular and cellular mechanisms involved in this process is pivotal for preven-
tion strategies, genetic counseling, and personalized clinical treatment.[7] Frequently
aneuploidy involves the whole chromosome, but partial aneuploidy is also com-
mon due to chromosome breakage and rearrangement.[8] Chromosomal alterations
distributed throughout the whole body (constitutional) could occur during gamete
formation, at fertilization, or in the embryo before implantation.[8] Usually, aneu-
ploidy is the result of a failure to segregate chromosomes in cell division, MI or
MII that includes non-disjunction, premature disjunction, or anaphase lag.[9] Most
chromosome-missegregation events in human meiosis occur because of incorrect
segregation of homologues during MI (non-disjunction).[10] A non-disjunction during
MI of oocyte formation is more frequent than in sperm; however, in the majority of
possible sex chromosome aneuploidies, a paternal meiotic non-disjunction accounts
for 50% of cases.[11] Human female meiosis is error prone, presenting an aneuploidy
rate of about 15% to 30–70%, depending on maternal age, compared with 1–4%
in sperm.[12, 13] So, human aneuploidy has often maternal origin, with exception for
47,XYY (presence of an extra Y-chromosome) and 45,X (a missing sex chromo-
some), which are often due to paternal or postfertilisation errors.[6] These discrepan-
cies could be explained by the timing differences in female and male meiosis. Live
cell imaging screening of mammalian oocytes showed that chromosome segregation
defects are mostly due to an error-prone chromosome-mediated spindle assembly
and kinetochore splitting.[14] It is also important to stress that the consequences are
different depending on which meiotic cycle is involved in non-disjunction, leading
for example to a disomic gamete. If non-disjunction occurs in MI, the gamete would
have 24 chromosomes from both grandparents (paternal and maternal) homologs,
but if occurs in MII, the gamete would have 24 chromosomes with both copies exclu-
sively from the paternal or maternal homolog.[9] Accordingly, segregation of chromo-
somes in human meiosis is often error-prone, having as a consequence a numerically
unbalanced chromosome complement. Additionally, chromosome segregation errors
could also emerge during mitotic cell divisions after fertilization, having as conse-
quence mosaicism—the presence of both aneuploid and normal diploid cells within
one individual.[6] Mosaicism is observed in 1–2% of pregnancies undergoing chori-
onic villus sampling and in 0.1% of amniocenteses, and it can result in a less severe
phenotype.[6] However, this phenotype/genotype interpretation is especially diffcult
in prenatal diagnosis, since we cannot deduce at which stage of the development the
error occurred. Although mosaicism can encompass either autosomes or gonosomes
it is more common in sex chromosomes, being observed in about 0.2% of fetuses
prenatally.[15] The clinical phenotype of mosaicism varies with the percentage and
124 Cytogenetics and Molecular Cytogenetics

tissue distribution of the aneuploid cells. A distinct event is the chimerism, where
different cell lines are originated from more than one zygote.

CHROMOSOMAL ALTERATIONS
In 1956, a new era of clinical cytogenetics started with the identifcation of the exact
chromosome number in humans, and therefore the description of several chromo-
somal syndromes with altered chromosome numbers.[15] Chromosomal alterations
could be categorized as numerical or structural and could involve more than one
chromosome. Numerical chromosomal alterations are more common than struc-
tural ones and represent a deviation from the normal diploid number for a given
species. This category can be subdivided in two groups: (i) aneuploidy (a group in
which individual chromosomes may be either missing or present in one or more
additional copies) and (ii) polyploidy (here whole haploid sets of chromosomes may
be added or lost, as triploidy (3n) in human with 69 chromosomes, or tetraploidy
(4n) in human 92 chromosomes).[16] Triploidy or tetraploidy are lethal disorders often
observed in spontaneous abortions and rarely in newborns with a reduced survival
time. Triploidy is one of the most common chromosome abnormalities occurring in
~1% of recognized conceptions, and 10% of recognized miscarriages.[17] Triploidy
can refect the double contribution coming from the father (di-andry) or the mother
(di-gyny). Diandry is usually the consequence of two sperm simultaneously fertil-
izing the ovum.[18–20] Digyny could be the result of nondisjunction of the entire set at
either the MI or MII in oogenesis, or a polar body may fail to be extruded.[21]
Structural rearrangements result from chromosome breakage and reunion within a
single chromosome or between two or more non-homologous chromosomes originat-
ing either balanced or unbalanced complements.[15] Balanced rearrangement means
that there is usually no alteration in chromosome content, while unbalanced ones lead
to gains or losses of chromosomal material. At birth, structural rearrangements (bal-
anced and unbalanced) were reported in about 1/400 infants.[22] Signifcant clinical
phenotypes were observed in carriers of unbalanced rearrangements, as a consequence
of loss and/or duplication of genetic material. Some examples of unbalanced rearrange-
ments are deletions, duplications, rings and isochromosomes, and of balanced rear-
rangements are inversions (paracentric and pericentric), insertions (direct or inverted),
and translocations (reciprocal and Robertsonian).[15] Additionally, we also have small
marker chromosomes (SMCs), which are usually supernumerary (sSMCs).
Balanced as well as unbalanced structural abnormalities can be inherited from
a carrier parent or could arise as de novo. For inherited balanced structural rear-
rangements, the risk for a clinical phenotype is low; however, for de novo rearrange-
ments, this risk is higher, even if the rearrangement looks balanced: submicroscopic
deletions/duplications at the breakpoints and alterations in the genes or regulatory
elements near to the breakpoints cannot be excluded.[22] So, the management of bal-
anced rearrangements identifed in prenatal cases is diffcult, especially with a de
novo origin. It is important to highlight that a balanced carrier (heterozygote) could
present with diffculties in reproduction, namely infertility, spontaneous abortions,
or abnormal offspring, which is a result of anomalous pairing and segregation at
prophase I.[22]
FISH—Characterization of Chromosomal Alterations 125

Moreover, chromosomal alterations can also be categorized as constitutional or

acquired. Constitutional chromosomal alterations occur during gametogenesis or
early embryogenesis, affecting all, or great fraction of an organism’s cells,[11] with an
estimated incidence of 20–50% of all human conceptions.[7] Acquired chromosomal
abnormalities occur postnatally during adulthood, affecting a single clone of cells
with a specifc distribution in the body, and are usually associated with cancer.
Human cytogenetic nomenclature describing each type of identifed chromo-
somal alterations was developed by an expert committee and is published in col-
laboration with Cytogenetic and Genome Research (formerly: Cytogenetics and
Cell Genetics) since 1963. The current version was prepared and updated by the
International Standing Committee on Human Cytogenomic Nomenclature (ISCN)
and adopted in 2020.[23]
Limited due to chromosome resolution at the level of chromosome banding
(3–5 Mb), on the basis of recognition and interpretation, masked or cryptic altera-
tions may be diffcult if not impossible to ascertain. The advent of molecular cyto-
genetics, particularly the fuorescence in situ hybridization (FISH) technique, have
overcome, in the past 3 decades, the gap between conventional banding cytogenetics
and molecular biology. Specifc nucleic acid sequences could be fuorescent-labeled
and located in interphase cells or metaphase chromosomes, being an important tool
in clinical genetic laboratories. The orientation must be given by the clinic or by a
previous cytogenetic analysis, which will indicate what probe(s)/sequence(s) to use
and screen. Meanwhile also chromosome microarray approaches are an indication
for further FISH-tests.
Numerous and diverse DNA sequences have been developed and used as probes:
(i) unique sequence; (ii) repetitive sequence (such as α-satellite and telomeric DNA);
(iii) locus-specifc DNA obtained by PCR amplifcation, large genomic DNA
sequences cloned into cosmids, bacterial artifcial chromosomes (BACs), P1-derived
artifcial chromosomes (PACs), yeast artifcial chromosomes (YACs); and (iv) chro-
mosome band or arm specifc sequences generated by microdissection or as DNA
libraries established by chromosome fow sorting.[24]
The application of diverse strategies, with combination of specifc probe sets in
multicolor combinations, has increased the characterization of simple to complex chro-
mosomal aberrations. With FISH, genetic alterations can be analyzed at the single-cell
level, screening simultaneously different chromosomes and loci, and determine mosa-
icism or clonal variability. Resolution of FISH largely depends of the target material: at
metaphase level varies between 2–5 Mb, but in interphase nuclei varies between 2 Mb
and 50 kb. In fber-FISH, the resolution can achieve 5–500 kb.[24]

NUMERICAL ALTERATIONS
Aneuploidy can affect any one of the 23 pairs of chromosomes. In humans, aneu-
ploidy leads to about 35% of miscarriages and 4% of stillbirths.[10] Aneuploidy is
also present in around 30–60% of embryos, in 30–70% of oocytes and in 0.3% of
newborns (with the most common abnormalities being trisomy 21 and sex chro-
mosome trisomies), facing developmental disabilities and intellectual disability.[10]
Regarding human aneuploidy, examples of trisomic conditions compatible with
126 Cytogenetics and Molecular Cytogenetics

FIGURE 11.1 Representative results of GTG-banding—karyograms of

A) Down syndrome: 47,XX,+21.
B) Down syndrome: 46,XY,der(14;21)(q10;q10),+21.
C) Edwards syndrome: 47,XY,+18.
D) Pätau syndrome: 47,XY,+13.

full-term pregnancies are the Down (trisomy 21), Edwards (trisomy 18), and Pätau
(trisomy 13) syndromes (Figure 11.1).
Down syndrome is one of the best-described and most frequent chromosome
disorders that causes intellectual disability. The incidence of Down syndrome
is approximately 1/600 newborns, being originated in almost 94% of cases from
meiotic nondisjunction.[15, 20] In approximately 95% of cases, the extra chromo-
some 21 is of maternal origin, and around 80% of those are due to an error during
MI.[15] Nevertheless, almost 4% of Down syndrome patients present an unbalanced
Robertsonian translocation involving chromosome 21 and the long arm of chromo-
somes 13, 14, 15, 21, or 22.[15] It is important to highlight that if a parent is a balanced
21/21 isochromosome carrier, this represents a 100% risk of having offspring with
Down syndrome. Female carriers of balanced 14/21 or 21/22 Robertsonian translo-
cations have a 10–15% risk for unbalanced Down syndrome offspring in comparison
to 5% for male carriers.[15] These translocations could be de novo or inherited from a
balanced carrier parent (usually the mother). Additionally, mosaicism for trisomy 21
is also the cause of Down syndrome in almost 2% of the cases.[15]
FISH—Characterization of Chromosomal Alterations 127

Edwards syndrome has a frequency of about 1/6000 live births and a poor post-
natal survival estimate (~90% die in the frst 6 months).[20] Few cases of trisomy 18
mosaicism were reported, but some patients with unbalanced translocations involv-
ing all or most of chromosome 18 long arm were also described presenting with
trisomy 18 features.[15]
Pätau syndrome has an estimated incidence of about 1/12,000 live births, and
90% of these patients rarely survive the newborn period.[20] The origin of the extra
chromosome 13 is frequently from a maternal meiotic non-disjunction error, but may
also arise from an unbalanced Robertsonian translocation involving chromosome
13 (in ~20% of the cases) or yet from trisomy 13 mosaicism, which is a rare event
and presents a less severe clinical phenotype.[15]
Likewise, aneuploidies involving sex chromosomes are also a possible consequence
of errors in chromosome segregation but present less severe phenotypes in comparison
with somatic trisomies. The gene dosage imbalances for genes encoded in sex chro-
mosomes are lower than for the genes encoded on somatic chromosomes, and besides
that there is the inactivation process of the extra X chromosome(s), if there is more than
one present per cell.[25] Examples of sex chromosome abnormalities are Turner (45,X),
triple X (XXX), Klinefelter (47,XXY) and 47,XYY syndromes (Figure 11.2).[25]

FIGURE 11.2 Representative results of GTG-banding—karyograms of

A) Turner syndrome: 45,X.
B) Triple X syndrome: 47,XXX.
C) Klinefelter syndrome: 47,XXY.
D) ‘XYY’ syndrome: 47,XYY.
128 Cytogenetics and Molecular Cytogenetics

Turner syndrome frequency is around 1/1800 newborn females, with approxi-

mately 50% of patients a having 45,X karyotype.[15, 20] Other carriers of this syndrome
can have isochromosomes, short-arm deletions or rings involving X-chromosome.[6]
Mosaicism is also a possible cause for this syndrome in 30% of patients, which pres-
ent both a 45,X cell lineage and a 46,XX cell lineage and/or one containing a rear-
ranged X-chromosome. [26] The origin of monosomy X seems to be primarily an
error in male meiosis, as the single X-chromosome derives from the mother in 80%
of 45,X cases.[6] Although monosomy X is associated with infertility some carriers
have healthy babies, supporting the theory of mosaicism and the presence of a nor-
mal cell line in the gonads.
Klinefelter syndrome has a frequency of almost 1/500 newborn males and the
most common karyotype is 47,XXY.[15, 20] Mosaicism is also detected in few patients,
which encompasses normal 46,XY cells combined with cells with two or more
X chromosomes. Variants of Klinefelter syndrome are also described, involving
more than two X-chromosomes and multiple Y-chromosomes, such as 48,XXYY,
48,XXXY, and 49,XXXXY, that are associated to an increased severity of pheno-
type.[15] This syndrome can be of maternal or paternal origin resulting from a meiotic
non-disjunction error.[6]
Trisomy X syndrome frequency is around 1/900 in newborn females, originating
most often from a nondisjunction error in maternal MI.[6, 20] Carriers of this chromo-
somal alterations are often fertile and with no obvious phenotypic alterations.
All the previously discussed numerical chromosomal alterations are easily diag-
nosed following conventional banding cytogenetics that can take 3 to 12 days for a
fnal report, depending whether peripheral blood, amniotic fuid or fbroblasts are
used as biological material. Every time that a rapid diagnosis of an aneuploidy is
needed, a specifc FISH analysis can be done in a native, not in vitro cultivated bio-
logical material, such as native amniocytes, and a result can be obtained in 24 hours.
However, although we can have this rapid diagnosis, for example, for a trisomy 21
(Figure 11.3), we cannot ascertain through this rapid test whether the trisomy 21 is

FIGURE 11.3 FISH in native amniocyte-nuclei showing hint of a trisomy 21: 3 SpectrumOrange

signals of locus-specifc probes for the long arm of chromosome 21 and 2 SpectrumGreen sig-
nals (= normal) for locus-specifc probes for the long arm of chromosome 13.
FISH—Characterization of Chromosomal Alterations 129

associated with a Robertsonian translocation or any other structural rearrangement.

In order to have that information, a karyotype has to be done.

UNBALANCED STRUCTURAL REARRANGEMENTS

In cases with unbalanced structural chromosomal rearrangements, there is a disequi-
librium compared to normal gene dosage of a particular genomic region that affects
the chromosomal structure. Commonly, they have an impact on the phenotype of the
carrier and can be detected either by banding cytogenetics (if they are larger than
3–5 Mb) or by FISH analysis (especially, when there is a clinical diagnosis that needs
to be confrmed). Unbalanced structural rearrangements include (a) deletions (del),
(b) duplications (dup), (c) ring chromosomes (r), (d) isochromosomes (i), or (e) small
supernumerary marker chromosomes (sSMCs) (Figure 11.4).
Deletion results from loss of chromosomal material of a specifc chromosome.
The clinical phenotype is dependent on size, gene content, and location of deleted
sequences within the genome. There are two different types of deletions character-
ized by the number of chromosome breaks involved. (i) A terminal deletion results
from a single break in a distal part of one chromosome, and the loss of the distal
acentric segment. Cri-du-chat syndrome is an example that is caused by a terminal
deletion in the short arm of chromosome 5 (Figure  11.5). (ii) An interstitial dele-
tion results from two breaks in a single chromosome and loss of the acentric frag-
ment between the two breaks.[22] Moreover, a deletion may be due to segregation and
recombination of a balanced rearrangement, i.e. translocation, inversion and inser-
tion (see later). If deletions involve segments smaller than 3–5 Mb, they might not be
detected by banding cytogenetics. In these cases, clinical information can give a clue
towards which FISH probe to use (locus specifc), to make, confrm, or dismiss the
clinical suspicion of a corresponding diagnosis (Figure 11.6).

FIGURE  11.4 Schematic representation on formation of duplication, deletion, of isochro-

mosomes of long and short chromosome arms, ring chromosomes, and small supernumerary
marker chromosomes (sSMCs).
130 Cytogenetics and Molecular Cytogenetics

FIGURE  11.5 Cri-du-chat syndrome is characterized by a deletion in the short arm of a

chromosome 5.
A) GTG-banding karyogram 46,XX,del(5)(p14.1) of a corresponding patient.
B) GTG-banded pair of chromosomes 5, showing del(5)(p14.1) in higher magnifcation.
C) After FISH, using a locus-specifc probe for 5p15.2 (loci D5S817/D5S2875, Q-Biogene,
SpectrumRed) and control probe for 5q31 region (locus D5S89 SpectrumGreen) reveals a
heterozygote deletion on one chromosome 5 (arrow).

Duplication results in extra copies of a chromosomal region and consequently

in higher copy numbers of corresponding genes, which may affect phenotype by
modifying gene dosage. A tandem and an inverted duplication results from dupli-
cated sections adjacent to the original ones. Furthermore, duplications may result
from insertion or addition of the duplicated region in a completely different genomic
environment.[27] Duplications often originate from unequal crossing over, especially
in genomic regions with repetitive DNA sequences. Additionally, duplications arise
after recombination and/or segregation of structural balanced rearrangements,
namely translocations, inversions, and insertions.[22] Interestingly, almost 5% of
the human genome includes duplicated sequences also known as low-copy repeats,
having a link with genomic disorders since highly homologous fanking repeats
predisposes these regions to recurrent rearrangement by nonallelic homologous
recombination, which could lead to deletion, duplication, or inversion.[28] Cat eye
syndrome, a common example of tetrasomy of the proximal long arm of chromo-
some 22 due to an extra dicentric chromosome, is associated with coloboma of the
eye, intellectual impairment, and anal atresia (Figure 11.7).[22]
Ring chromosomes usually form by two terminal breaks in both arms of the same
chromosome, followed by fusion of the broken ends, with loss of the distal acen-
tric genetic material.[29] Alternative mechanisms were also described, namely the
fusion of subtelomeric sequences or telomere–telomere fusion with no deletion, the
terminal deletion and a contiguous inverted duplication due to an inv-dup-del rear-
rangement.[30] There are three most common types of ring chromosomes: (i) large
rings with minimal loss from the terminal segments of the short and long arms,
(ii) very small rings as extra chromosomes in the karyotype, and (iii) rings of the
X-chromosome that are detected in females with Turner syndrome characteristics.[22]
FISH—Characterization of Chromosomal Alterations 131

FIGURE 11.6 FISH-detection of microdeletion syndromes with corresponding suited com-

mercially available probes is shown; derivative chromosomes are highlighted by arrowheads.
A) DiGeorge syndrome: specifc probe D22S75 (22q11.2 SpectrumRed) and control ARSA
(22q13.3 SpectrumGreen).
B) Williams-Beuren syndrome: specifc probe ELN (7q11.23 SpectrumGreen) and control
TWIST1 (7p21.1 SpectrumRed).
C) Wolf-Hirschhorn syndrome: specifc probe (SpectrumRed) and control (SpectrumGreen).
D) Prader-Willi syndrome: specifc probe SNRPN (15q11.2 SpectrumRed) and control PML
(15q24.1 SpectrumGreen). This probe can also be used for Angelman syndrome patients.
E) Miller-Diecker syndrome: specifc probe LIS1 (17p13.3 SpectrumRed) and control RAI1
(17p11.2 SpectrumGreen).
F) Smith-Magenis syndrome: specifc probe RAI1 (17p11.2 SpectrumGreen) and control
LIS1 (17p13.3 SpectrumRed).
132 Cytogenetics and Molecular Cytogenetics

FIGURE  11.7 Characterization of a small supernumerary marker chromosome (sSMC) is

depicted:
A) Karyogram of 6 year old boy with cat eye syndrome: GTG-banding revealed a karyotype
47,XY,+mar. The marker has two AgNOR positive and two C-band positive regions.
B) Partial metaphase after FISH highlighting the two normal 22 chromosomes and the sSMC
(white arrow). The SMC does not present signals for D22S75 (red), a probe located in the
DiGeorge syndrome region.
C) Partial metaphase showing one normal 22 chromosome and the sSMC (green circle) with
two signals for the centromeric probe specifc for chromosomes 14/22.
D) Normal 22 chromosomes and the sSMC(22) showing the presence of 2 centromeres
(cep14/22 in green), signals for BAC RP11–172D7 in 22q11.21 (cat eye syndrome critical
region), and two signals corresponding to acrocentric arms (midi54).
E) One signal in the marker for BAC RP11–81B3 in 22q11.21 and absence of signal for BAC
RP11–1058B20 in 22q11.22 further confrmed that the sSMC was a typical cat eye syn-
drome marker.

It may be possible to characterize them by FISH using subtelomeric probes

(Figure 11.8). Ring chromosomes have been described for all human chromosomes,
although 50% of them arise from acrocentric chromosomes.[31] Ring chromosomes
present variable sizes according to the fraction of material lost and are frequently
unstable during cell division, being rarely transmitted to offspring.[15] A great major-
ity of these chromosomes arise de novo (99%), although a few cases of inherited ring
chromosomes have been reported,[32, 33] which are mostly of maternal origin (90%)
because ring chromosomes induce (male) infertility.[31] The phenotype of ring chro-
mosome carriers is very variable depending on the chromosome involved, the size
FISH—Characterization of Chromosomal Alterations 133

FIGURE  11.8 Characterization of a ring chromosome derived from chromosome 21 is

shown:
A) Karyogram of a case with 46,XX,r(21)(p11.2q22.3).
B) Metaphase FISH using a centromeric probe for chromosomes 13/21 (D13Z1/D21Z1) in red
confrmed chromosome 13 or 21 origin of the ring.
C) Dual color FISH using probes for RB1 (13q14.2 green) and DSCR1 (21q22 red) suggests a
chromosome 21 ring origin.
D) Dual color FISH using probes for GS10K2-T7 (4p16.3 green) and A224XH1 (4q35.2 red)
and a probe for 21q22 (blue) confrmed chromosome 21 ring origin.

of the deleted acentric fragment and consequently the gene content and also due to
ring chromosome instability, but usually overlaps that of the syndromes associated
with deletion of both ends of the corresponding chromosome.[34] So, besides dele-
tions that occur by ring formation, ring instability can also lead to other genomic
imbalances with gain or loss of genetic material and consequently to distinct phe-
notypes. Sister chromatid exchanges happening during mitosis in patients with ring
chromosomes result in secondary chromosomal abnormalities like dicentric rings,
interlocked rings, and other structural alterations or even ring chromosome loss lead-
ing to monosomic cells.[30]
Isochromosomes are composed of two copies of the same arm of one chromo-
some (mirror image of one of the arms of the chromosome). Some mechanisms
have been suggested for their formation, like transversal mis-division of the
134 Cytogenetics and Molecular Cytogenetics

FIGURE 11.9 A case with mosaicism involving a cell line 45,X and fve cell lines present-
ing different dicentric Y chromosomes with the breakpoints in the terminal segment of the
short arm, and another cell line with two derivative Y chromosomes is characterized here.
A) Karyogram with a large dicentric Y chromosome (circle).
B) Enlarged sex-chromosomes of the case (GTG-banded).
C) CBG banding staining Yq12 two times on the der(Y)—arrow.
D–I) FISH analysis using different probes as shown in the pictures.

centromere during MII, sister chromatid breakage and reunion near the centro-
mere, or exchange between homologues during meiosis.[15] Isochromosomes X are
often observed in Turner syndrome individuals, and represent the most frequent
isochromosomes observed, that are, in fact, in the majority of cases dicentric.[15]
Isochromosome Yp, that results in duplication of the short arm and loss of the long
arm, has been reported to be linked to azoospermia (Figure 11.9).[35] Autosomal
isochromosomes occur more often in acrocentric chromosomes with loss of the
short arms.
Small supernumerary marker chromosomes (sSMCs) are structurally abnor-
mal chromosomes that cannot be unambiguously identifed or characterized by
conventional banding cytogenetics alone.[23] These sSMCs are equal to or smaller
in size than a chromosome 20 of the same metaphase spread.[36] The origin and
composition of an sSMC is recognizable by molecular cytogenetics analysis. They
are seen in different shapes like ring, centric minute, and inverted duplication,
and could be centric, or acentric, complex (= deriving from more than one chro-
mosome), being discontinuous due to formation via chromothripsis and being
present in mosaic.
FISH—Characterization of Chromosomal Alterations 135

BALANCED STRUCTURAL REARRANGEMENTS

Up to now, we described chromosomal alterations (numerical and unbalanced) that
normally have an impact on the phenotype. Now we will focus on balanced rear-
rangements, which are often diagnosed after birth of a dysmorphic child, after a pre-
natal ultrasound of a fetus with morphological alterations, or after studying couples
with infertility. The balanced chromosomal alterations comprise three types: inver-
sions (inv), translocations (t), and insertions (ins) (Figure 11.10).
Inversions can be pericentric, when a single break occurs in each arm of the same
chromosome, or paracentric, with two breaks in the same arm of a chromosome
(Figure 11.11 A). Often, inversion carriers are clinically not affected; however, the off-
spring has an increased risk of partial duplications and deletions due to the formation
of an inversion loop during chromosome pairing in prophase I of meiosis.[15] So, the
inversion loop is composed of four chromatids, two normal and two inverted strands,
leading to abnormal gametes when crossing-over events happen within this loop.[22]
The chromatids resulting from recombination in a paracentric inversion are one nor-
mal, one with the same inversion as the progenitor, a dicentric, and an acentric one.
For pericentric inversions, recombination also leads to four types of chromatids: one
normal, one with the same inversion as the progenitor, and two with unbalances (distal
deletion and duplication in a reverse manner in relation to each other).
Translocations are common (1:500) and arise from the exchange of chromosomal
segments, usually between two non-homologous chromosomes. Translocations can
be grouped into reciprocal and Robertsonian ones (Figure  11.11 B), and if more
than two chromosomes are involved we have a complex rearrangement. Reciprocal
translocations originate in the exchange of broken-off segments between two dif-
ferent chromosomes; frequently carriers are normal but have an enhanced risk of
unbalanced offspring, which depends on the translocation components’ segregation,
position of breakpoints, and centromere location.[15] Homologues pairing at meiosis
is different in translocation carriers, since the two derivative chromosomes and their
two normal homologues pair to form a cross-shaped quadrivalent (four chromatids)
at pachytene with each homologous segment pairing with its counterpart.[22]
There are fve basic segregation patterns from a reciprocal translocation quadri-
valent[20, 22]:

• alternate segregation: normal and translocation chromosomes move to

opposite poles, originating in balanced gametes.
• adjacent I segregation: adjacent nonhomologous centromeres move to the
same pole, leading to a zygote with partial trisomy of one chromosome and
partial monosomy of the other, when fertilized by a normal haploid gamete.
• adjacent II segregation: adjacent homologous centromeres move to the same
pole, leading to large amounts of unbalanced chromatin, often incompatible
with embryonic survival.
• 3: 1 segregation: three of the four chromosomes move to one pole and only
one moves to the opposite pole.
• 4: 0 segregation: four chromosomes move together to one pole; this is pos-
sible, but not compatible with live.
136 Cytogenetics and Molecular Cytogenetics

FIGURE  11.10 Karyograms of cases with structural chromosomal aberrations (in parts
with schematic depictions):
A) pericentric inversion: 46,XX,inv(1)(p22.1q44).
B) paracentric inversion: 46,XY,inv(1)(q23.1q32.1).
C) reciprocal translocation: 46,XY,t(7;12)(p13;q22).
D) Robertsonian translocation: 45,XX,der(13;14)(q10;q10).
E) Insertion: 46,XX,ins(12;18)(q24.31;q21.3q23).
FISH—Characterization of Chromosomal Alterations 137

FIGURE 11.11 Schematic representation of

A) paracentric and pericentric inversion.
B) reciprocal and Robertsonian translocation
C) inverted and direct insertions.

Robertsonian translocations affect two acrocentric chromosomes, losing both of

their short ‘p’ arms and joining near their centromeres to originate a single chro-
mosome (carriers with 45 chromosomes); the fused chromosome can also include
two centromeres (dicentric chromosome).[15] There seems to be no phenotype in
Robertsonian translocation carriers since the genetic material lost is present in each
of the short arms of acrocentric chromosomes, and a functional cell only needs six
to eight of these regions.
Since in Robertsonian translocations only ‘three’ chromosomes are implicated,
a trivalent is formed at pachytene, resulting in segregation of six types of gametes,
i.e. two normal and four with trisomies or monosomies when fertilized by a normal
gamete.[20, 22] The risk of unbalanced offspring is higher in female carriers since
males often are infertile. The most common Robertsonian translocation involves
chromosomes 13 and 14, followed by chromosomes 14 and 21.[26, 37]
Insertions detected by banding cytogenetics are quite rare. They result from three
breaks, being the segment from one chromosome inserted into another chromosome,
138 Cytogenetics and Molecular Cytogenetics

in a directed or inverted orientation (Figure 11.11 C). The incidence of microscopi-

cally visible insertions was estimated to be around 1 in 80,000 live births.[38] More
recently, using array-comparative genomic hybridization (aCGH) and FISH, the
incidence of insertion events was estimated to be about 1 in 500 or 1 in 563 individu-
als analyzed.[39, 40] In 2012, Nowakowska and colleagues[41] demonstrated that around
2.1% of apparently de novo, interstitial copy number variants (CNVs) were in fact
imbalances that resulted from parents with balanced insertions. Although insertion
carriers are clinically normal, their offspring has an enhanced risk of partial mono-
somy or partial trisomy of the inserted segment. These cases need to be confrmed
by FISH techniques, usually by whole chromosome painting (wcp) probes.

CASE PROBLEMS
Selected clinical cases will be presented as examples of karyotyping and FISH appli-
cations in postnatal and in prenatal diagnosis.
For all clinical post and prenatal cases, three questions will be answered:

i) What kind of chromosomal alteration was found? How to interpret the

results? What is the most probable mechanism of origin of the alteration?
ii) What is the origin of the chromosomal alteration?
iii) What is the future strategy for the family?

CLINICAL CASE 1
CLINICAL INFORMATION
A 6-year-old girl with microcephaly, moderate to severe psychomotor developmental
delay, and prenatal history of intrauterine growth restriction was studied.

TECHNIQUES PERFORMED
G banding cytogenetics;
FISH for DiGeorge critical region and for the subtelomeric regions.

RESULTS
The combined result for this case was 46,XX.ish del(22)(q13.33)(D22S1726-).

QUESTIONS
i) What kind of chromosomal alteration was found? How to interpret the
results? What is the most probable mechanism of origin of the alteration?

The high resolution karyotype was normal (Figure  11.12 A) as well as the FISH
results for the DiGeorge critical region in 22q11.2 (Figure 11.12 B).
FISH—Characterization of Chromosomal Alterations 139

FIGURE 11.12 Example of a cryptic terminal deletion:

A) Seemingly normal karyogram, 46,XX.
B) However, only after FISH, using the depicted probe sets, a subtelomeric deletion is obvi-
ous when applying a telomere-near probe for chromosome 22 (arrow).

When the subtelomeric FISH probes—ToTelVysion (Vysis ToTelVysion Multi-

Color FISH Probes)—were applied, a subtelomeric deletion on 22q13.33 was
observed (Figure 11.12 B).

CURIOSITY
Deletion 22q13 has been accidentally diagnosed by FISH in several individuals pri-
marily analyzed for 22q11.3 deletion syndrome. Numerous commercially available
FISH assays for 22q11.3 deletion syndrome use arylsulfatase A (ARSA) gene as the
control probe. ARSA maps to 22q13 and therefore detects deletions of this region.
The ARSA probe successfully detects the majority of 22q13.3 deletions; however,
microdeletions distal to ARSA may require FISH analysis using the 22q subtelomere
probes,[42] as happened in our case.

ii) What is the origin of the chromosomal alteration?

In order to establish the most probable mechanism of origin of this alteration, both
parents were also analyzed by karyotyping and FISH techniques. This did not reveal
any numerical or structural alteration (data not shown), and therefore this terminal
deletion was classifed as de novo.

iii) What is the future strategy for the family?

This couple was referred for genetic counselling. Monitoring and testing future
pregnancies was recommended.
140 Cytogenetics and Molecular Cytogenetics

CONCLUSION
This terminal deletion of the long arm of chromosome 22 (22q13.33-qter) seems to
explain the clinical phenotype of the girl. The deletion 22q13.3 syndrome or Phelan-
McDermid syndrome is characterized by neonatal hypotonia, global developmental
delay, normal to accelerated growth, absent to severely delayed speech, and minor
dysmorphic features. The loss of 22q13.3 can result from simple deletion, transloca-
tion, ring chromosome formation, and less commonly structural changes affecting
the long arm of chromosome 22, specifcally the region containing the SHANK3
gene.[43] Almost 80% of individuals with Phelan-McDermid syndrome have de novo,
simple deletions of 22q13,[44] as observed in our case.

CLINICAL CASE 2
CLINICAL INFORMATION
A woman in the 15th week of twin pregnancy did trophoblast biopsy as sonography
diagnosed hygroma in one of fetus and a borderline nuchal translucency in the other
fetus.

TECHNIQUES PERFORMED
QF-PCR for aneuploidy screening;
G banding cytogenetics;
FISH technique using subtelomeric probes for chromosomes 11 and 18.

RESULTS TWIN 1
QF-PCR for aneuploidy screening was performed with a normal result (data not
shown).
The combined result (Figure 11.13 A and B) for this case was:
46,X Y,der(18)t(11;18)(p15.3;p11.2).ish der(18)t(11;18)(p15.5;p11.32)
(D11S2071+,D18S552-)

RESULTS TWIN 2
QF-PCR for aneuploidy screening was performed with a normal result (data not
shown).
The combined result (Figure  11.13 C—FISH not shown) for this case was:
46,XX,t(11;18)(p15.5;p11.23).ish t(11;18)(p15.5-,p11.32+;p11.32-,p15.5+)(D11S2071-,
D18S552+; D18S552-, D11S2071+)

QUESTIONS
i) What kind of chromosomal alteration was found? How to interpret the
results? What is the most probable mechanism of origin of the alteration?
FISH—Characterization of Chromosomal Alterations 141

FIGURE 11.13 A part of a family with t(11;18) is shown.

A) karyogram of twin 1: 46,XY,der(18)t(11;18)(p15.3;p11.2).
B) FISH result of twin 1 using subtelomeric probes for chromosomes 11 and 18, confrming
presence of the der(18)t(11;18)(p15.3;p11.2).
C) In twin 2, 46,XY,t(11;18)(p15.3;p11.2)mat was found (FISH shown).
142 Cytogenetics and Molecular Cytogenetics

The G-banded metaphases of the trophoblast biopsy cells revealed, in twin 1, a male
fetus with a chromosome 18 derivative from a translocation involving chromosomes
11 and 18 (Figure 11.13 A) and, in twin 2, a female fetus with an apparently bal-
anced reciprocal translocation involving the same chromosomes (Figure 11.13 C).
The structural alterations identifed in both twins were confrmed by FISH technique
using subtelomeric probes, for 11p15.5 and 18p11.32 (twin 1—Figure 11.13 B; twin
2—not shown). It is important to stress that the result for twin 1 does not contradict
that obtained with the screening for aneuploidies by the QF-PCR technique, since
this technique is not suitable for the detection of partial aneuploidies in the tested
chromosomes, such as this one identifed in chromosome 18.
In order to establish the most probable mechanism of origin of the alteration, the
study of the parents was recommended.

ii) What is the origin of the chromosomal alteration?

The study (G-banding from peripheral blood) of both parents revealed a normal
karyotype for the mother, and the presence of a reciprocal translocation involving
chromosomes 11 and 18 in the father, 46,XY,t(11;18)(p15.3;p11.2) (data not shown).
Regarding the most probable biological mechanism, it can be concluded that the
male progenitor is a carrier of a structural chromosomic rearrangement, a reciprocal
translocation with breakpoints in 11p15.3 and 18p11.2, which has no implication in
the phenotype of the carrier. However, during spermatogenesis there is a risk for the
offspring to inherited imbalances as a result of anomalous segregations. In this case,
the identifed structural chromosomal rearrangement in fetus 1 is likely to be due
to an adjacent I segregation, having the fetus a derivative chromosome 18 inherited
from the paternal translocation. Fetus 2 is the result of an alternate segregation as it
carries the same translocation as the father.

iii) What is the future strategy for the family?

This couple was offered genetic counselling where it was well explained the conse-
quence of this reciprocal translocation in this phenotypically normal father for future
pregnancies, and the need of family studies (parents, brothers, sisters, and other fam-
ily members at risk). To all carriers, invasive prenatal diagnosis should be offered.
As chromosome 11 is involved in the rearrangement also the hint should be given,
that this imprinting associated chromosome is slightly more likely to be involved in
uniparental disomy (UPD) due to the t(11;18). Thus, prenatal UPD test may also be
considered in future pregnancies.

CONCLUSION
This clinical case shows the implications for the offspring of a balanced structural
rearrangement in one of the progenitors and the need to offer to this couple prena-
tal diagnosis in future gestations and genetic counselling to explain the implica-
tions of these genetic fndings, the risk of recurrence, and the need to offer family
FISH—Characterization of Chromosomal Alterations 143

studies to other relatives. In this clinical case, the male progenitor is a carrier of a
reciprocal translocation between chromosomes 11 and 18, and the unbalanced struc-
tural chromosomal rearrangement involving partial monosomy of the short arm of
chromosome 18 (p11.2-pter) and partial trisomy of the short arm of chromosome 11
(p15.3-pter) observed in twin 1 was originated by an adjacent 1 segregation during
spermatogenesis. Twin 2, on the other hand, was a balanced carrier like the male
progenitor, and resulted from an alternate type of segregation. The imbalances iden-
tifed in twin 1 are most probably the cause of the ultrasound alterations detected and
consequently are correlated to the clinical phenotype.

CLINICAL CASE 3
CLINICAL INFORMATION
A pregnant woman in 13th week of gestation had a sonographic diagnosis of holo-
prosencephaly in the fetus, and thus amniotic fuid was collected.

TECHNIQUES PERFORMED
FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes;
G banding cytogenetics;
FISH technique using whole chromosome painting probe for chromosome 21
(WCP21).

RESULTS
FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes
was performed with a normal result (Figure 11.14 A-3). The combined result of G
banding and molecular cytogenetics revealed two partial monosomies involving
the terminal region of chromosome 13q32-qter and the proximal region of chro-
mosome 21pter-q22.1. Karyotype: 45,XY,der(13)t(13;21)(q32;q22.1),−21.ish t(13;21)
(q32;q22.1)(wcp21+;wcp21+).

QUESTIONS
i) What kind of chromosomal alteration was found? How to interpret the
results? What is the most probable mechanism of origin of the alteration?

G-banding in amniotic fuid cells revealed a male fetus with 45 chromosomes due
to a monosomy of chromosome 21 and also a structurally altered chromosome 13
(Figure 11.14 A-1). By FISH technique using WCP21, it was possible to characterize
the derivative chromosome 13 as an unbalanced structural chromosomal rearrange-
ment involving chromosome 13 and 21 (Figure 11.14 A-1 and 11.14 A-2). This structural
rearrangement was also confrmed in fbroblast cells of the fetus (results not shown)
and seems to be the origin of the detected ultrasound anomaly, being consequently
144 Cytogenetics and Molecular Cytogenetics

FIGURE 11.14 Case of a partial monosomy 21:

A) 1) Fetal GTG-banding seems to show a monosomy 21 and a derivative chromosome 13
(derivative chromosome 13 appears on the left of chromosome 13 pair); 2) FISH using a
whole chromosome paint for chromosome 21 showing one chromosome 21 and also that
there is chromosome 21 material on another chromosome; 3) Previous FISH quick test in
native amniocyte nuclei gave a normal result for locus specifc probes in 21q22.13~22.2.
B) 1) Paternal GTG-banding revealed a karyotype of 46,XY,t(13;21)(q32;q22.1). 2) This
result was confrmed by FISH.
C) Schematic depiction of balanced structural rearrangement in the father and the unbal-
anced structural rearrangement in the fetus and the location of FISH probe used for the
rapid detection of aneuploidies.
FISH—Characterization of Chromosomal Alterations 145

correlated with the clinical phenotype. In order to ascertain the origin of this structural
alteration, both parents were also analyzed by conventional cytogenetics and FISH.

CURIOSITY
Monosomy 21 as seemingly present in GTG-banding is not compatible with life,
and such cases abort spontaneously in very early embryogenesis; thus, here a cryptic
rearrangement should be suspected by the clinical laboratory geneticists.

ii) What is the origin of the chromosomal alteration?

Both parents, phenotypically normal, were analyzed by G-banded metaphases

obtained from peripheral blood that revealed a normal karyotype for the mother
(46,XX), and the presence of a reciprocal translocation involving chromosomes 13
and 21 in the fathers’ karyotype, which was also confrmed by FISH: 46,XY,t(13;21)
(q32;q22.1).ish t(13;21)(wcp21+;wcp21+) (Figure 11.14 B-1 and 11.14 B-2).

iii) What is the future strategy for the family?

This couple accepted genetic counselling so that the consequences of this translo-
cation in this phenotypically normal father for future pregnancies and the need for
family studies (parents, brothers, sisters, and other family members at risk) were out-
lined. Also all carriers in the family should be offered invasive prenatal diagnosis,
when expecting a child.

CONCLUSION
It was possible to infer that the alteration of the fetus was inherited from the male
progenitor (Figure  11.14 C). The explanation for the normal result obtained using
FISH for the rapid detection of aneuploidies is because this fetus carries an unbal-
anced structural chromosomal rearrangement involving chromosomes 13 and 21 and
the FISH probe used for rapid detection of aneuploidies is located more distally at
21q22.13-q22.2; and since this region is present in the derivative chromosome 13, the
FISH study showed two signals for chromosome 21.

CLINICAL CASE 4
CLINICAL INFORMATION
In a 14-month-old girl, hypotonia due to damage to the 1st neuron, agenesis of the
corpus callosum, and macrocephaly were diagnosed. Peripheral blood was taken for
cytogenetic analyses.

TECHNIQUES PERFORMED
G banding cytogenetics;
146 Cytogenetics and Molecular Cytogenetics

FISH using subtelomeric probes, VIJyRM2000 and MS607 (Vysis-Totelvysion)

for 7q36.3 and 22q13.3, respectively.

RESULTS
Karyotyping showed a normal result, 46,XX (data not shown).
FISH using subtelomeric probes for 7q36.3 and 22q13.3 revealed a partial mono-
somy of the terminal region of the long arm of chromosome 22 and a partial trisomy
of the terminal region of the long arm of chromosome 7, 46,XX.ish der(22)t(7;22)
(q36.3;q13.3)(MS607-,VIJyRM2000+) (Figure 11.15).

QUESTIONS
i) What kind of chromosomal alteration was found? How to interpret the
results? What is the most probable mechanism of origin of the alteration?

The presence of a derivative chromosome 22 from a translocation involving chro-

mosomes 22 and 7 (Figure 11.15 A) seems to be the origin of the phenotype of this
girl. Since these chromosomal alterations are submicroscopic, it is not possible to
detect them by G banding and, therefore, in these cases, molecular cytogenetics
is essential for the diagnosis. This proband had an older brother with severe psy-
chomotor delay, short stature, microcephaly, choanal atresia, and hypogenitalism,

FIGURE  11.15 Family with t(7;22): Banding and molecular cytogenetics results obtained
are shown for
A) the affected daughter.
B) the affected brother.
C) the carrier mother.
FISH—Characterization of Chromosomal Alterations 147

which was also referred for G banding and molecular cytogenetic study. These
studies revealed a partial monosomy of the terminal region of chromosome 7q
and a partial trisomy of the terminal region of the chromosome 22q, 46,XY.ish
der(7)t(7;22)(q36.3;q13.3)(VIJyRM2000-,MS607+). The presence of this deriva-
tive 7 from a translocation involving chromosomes 7 and 22 seems to explain the
phenotype of this boy. These alterations are the exact opposite of those observed
in the sister (Figure 11.15 B).

ii) What is the origin of the chromosomal alteration?

Peripheral blood of both parents, phenotypically normal, was analyzed by G-banding

and FISH. These studies revealed a normal karyotype in the father (46,XY) and
the presence of a reciprocal translocation involving chromosomes 7 and 22 in the
mother, 46,XX.ish t(7;22)(q36;q13.3)(MS607+,VIJyRM2000+) without impact on
the phenotype. It was possible to infer that the chromosomal alterations in the girl
and her brother were inherited from the female progenitor (Figure 11.15 C) due to an
anomalous segregation (adjacent segregation) during meiosis.

iii) What is the future strategy for the family?

Once again, genetic counselling was offered and all implications for the specifc
situation explained further, including prenatal diagnosis for future pregnancies and
the need for additional studies offered to relatives of the carrier. As chromosome 7
is involved in the rearrangement, also the hint should be given that this imprinting
associated chromosome is slightly more likely to be involved in UPD due to the
t(7;22). Thus, prenatal UPD test may also be considered in future pregnancies.

CONCLUSION
These two different unbalanced chromosomal structural rearrangements in these
siblings involving the same two chromosomes (7 and 22) were inherited from the
female progenitor and seem to explain their respective phenotypes.

CONCLUSIONS
The complex behavior of chromosomes during the two meiotic divisions is error-
prone. Aneuploidy can affect any chromosome; however, only trisomies of the sex
chromosomes, of autosomes 13, 18, or 21, and monosomy of the X chromosome,
are compatible with survival up to the end of pregnancy, the remainder having been
eliminated by natural selection. Structural alterations originated from chromosomal
breakage and rejoining could affect the number, distribution, and expression of
genes with a signifcant impact in the offspring phenotype. Couples at particularly
increased risk of having offspring with a numerical or structural alteration are those:

• where one partner carries a chromosomal rearrangement such as a translo-

cation or an inversion, as described earlier.
148 Cytogenetics and Molecular Cytogenetics

• where one partner has gonadal or germinal mosaic for a trisomic cell line.
Couples with various conceptions involving the same trisomy give a clear
evidence of a gonadal mosaicism for a trisomic cell line affecting the pri-
mordial germ cells.

Such chromosomal alterations and their mechanisms of origin can be identifed

through banding cytogenetic studies, but in many situations need to be comple-
mented by FISH, depending on the clinical suspicion or on the previous study of
the karyotype or from both. It is important to stress that through the diagnosis of
both numerical and structural alterations (balanced and unbalanced) by karyotyp-
ing, the recurrence risk can be calculated for each specifc situation. Genetic coun-
selling must be offered to these couples and, if appropriate, prenatal diagnosis can
be recommended ensuring, that a future pregnancy is chromosomally balanced. For
couples that presented with repeated early miscarriages or primary infertility, a pre-
implantation diagnosis with selective transfer of embryos might be appropriate.

DEDICATION
To all our patients and their families, to everyone our laboratory (Cytogenetics and
Genomics Laboratory of the Faculty of Medicine of the University of Coimbra) and
clinical colleagues, to our students

REFERENCES
1. Pezza, R.J. Mechanisms of chromosome segregation in meiosis: New views on the old
problem of aneuploidy. FEBS J. 2015, 282, 2424–2425.
2. Petronczki, M.; Siomos, M.F.; Nasmyth, K. Un ménage à quatre: The molecular biology
of chromosome segregation in meiosis. Cell. 2003, 112, 423–440.
3. Greaney, J.; Wei, Z.; Homer, H. Regulation of chromosome segregation in oocytes and
the cellular basis for female meiotic errors. Hum. Reprod. Update. 2018, 24, 135–161.
4. Villeneuve, A.M.; Hillers, K.J. Whence meiosis? Cell. 2001, 106, 647–650.
5. Duro, E.; Marston, A.L. From equator to pole: Splitting chromosomes in mitosis and
meiosis. Genes Dev. 2015, 29, 109–122.
6. Storchova, Z. Chromosomal numerical aberrations. In: eLS. John Wiley & Sons, Ltd,
Chichester, 2016.
7. Queremel Milani, D.A.; Tadi, P. Genetics, Chromosome Abnormalities. Treasure Island,
FL, StatPearls Publishing; 2022.
8. Delhanty, J.D. Origins of human aneuploidy. In: eLS. John Wiley & Sons, Ltd, Chichester,
2018.
9. Mahdieh, N.; Rabbani, B. An overview of mutation detection methods in genetic disor-
ders. Iran J. Pediatr. 2013, 23, 375–388.
10. Hassold, T.; Hunt, P. To err (meiotically) is human: The genesis of human aneuploidy.
Nat. Rev. Genet. 2001, 2, 280–291.
11. McFadden, D.E.; Friedman, J.M. Chromosome abnormalities in human beings. Mutat.
Res. 1997, 396, 129–140.
12. Nagaoka, S.I.; Hassold, T.J.; Hunt, P.A. Human aneuploidy: Mechanisms and new
insights into an age-old problem. Nat. Rev. Genet. 2012, 13, 493–504.
13. Ottolini, C.S.; Newnham, L.; Capalbo, A.; Natesan, S.A.; Joshi, H.A.; Cimadomo, D.;
Griffn, D.K.; Sage, K.; Summers, M.C.; Thornhill, A.R.; Housworth, E.; Herbert, A.D.;
FISH—Characterization of Chromosomal Alterations 149

Rienzi, L.; Ubaldi, F.M.; Handyside, A.H.; Hoffmann, E.R. Genome-wide maps of
recombination and chromosome segregation in human oocytes and embryos show selec-
tion for maternal recombination rates. Nat. Genet. 2015, 47, 727–735.
14. Holubcová, Z.; Blayney, M.; Elder, K.; Schuh, M. Human oocytes: Error-prone chro-
mosome-mediated spindle assembly favors chromosome segregation defects in human
oocytes. Science. 2015, 348, 1143–1147.
15. Luthardt, F.W.; Keitges, E. Chromosomal syndromes and genetic disease. In: eLS. John
Wiley & Sons, Ltd, Chichester, 2001.
16. McFeely, R.A. Chromosome abnormalities. Vet. Clin. North Am. Food Anim. Pract.
1993, 9, 11–22.
17. McFadden, D.E.; Robinson, W.P. Phenotype of triploid embryos. J. Med. Genet. 2006,
43, 609–612.
18. Zaragoza, M.V.; Surti, U.; Redline, R.W.; Millie, E.; Chakravarti, A.; Hassold, T.J.
Parental origin and phenotype of triploidy in spontaneous abortions: Predominance of
diandry and association with the partial hydatidiform mole. Am. J. Hum. Genet. 2000,
66, 1807–1820.
19. McFadden, D.E.; Langlois, S. Parental and meiotic origin of triploidy in the embryonic
and fetal periods. Clin. Genet. 2000, 58, 192–200.
20. Gardner, R.J.M.; Amor, D.J. Gardner and Sutherland’s Chromosome Abnormalities and
Genetic Counseling. 5th Edition. Oxford University Press, Oxford; 2018.
21. Filges, I.; Manokhina, I.; Peñaherrera, M.S.; McFadden, D.E.; Louie, K.; Nosova, E.;
Friedman, J.M.; Robinson, W.P. Recurrent triploidy due to a failure to complete maternal
meiosis II: Whole-exome sequencing reveals candidate variants. Mol. Hum. Reprod.
2015, 21, 339–346.
22. Moore, C.M.; Best, R.G. Chromosomal genetic disease: Structural aberrations. In eLS.
John Wiley & Sons, Ltd, Chichester, 2001.
23. ISCN 2020. An International System for Human Cytogenomic Nomenclature (2020);
McGowan-Jordan J.; Hastings, R.J.; Moore, S., Eds. Karger, Basel; 2020.
24. Speicher, M.R.; Carter, N.P. The new cytogenetics: Blurring the boundaries with molec-
ular biology. Nat. Rev. Genet. 2005, 6, 782–792.
25. Potapova, T.; Gorbsky, G.J. The consequences of chromosome segregation errors in
mitosis and meiosis. Biology (Basel). 2017, 6, 12.
26. Hook, E.B.; Cross, P.K. Rates of mutant and inherited structural cytogenetic abnormali-
ties detected at amniocentesis: Results on about 63,000 fetuses. Ann. Hum. Genet. 1987,
51, 27–55.
27. Clancy, S.; Shaw, K. DNA deletion and duplication and the associated genetic disorders.
Nat. Educ 2008, 1, 23.
28. Sharp, A.J.; Locke, D.P.; McGrath, S.D.; Cheng, Z.; Bailey, J.A.; Vallente, R.U.; Pertz,
L.M.; Clark, R.A.; Schwartz, S.; Segraves, R.; Oseroff, V.V.; Albertson, D.G.; Pinkel, D.;
Eichler, E.E. Segmental duplications and copy-number variation in the human genome.
Am. J. Hum Genet. 2005, 77, 78–88.
29. Sigurdardottir, S.; Goodman, B.K.; Rutberg, J.; Thomas, G.H.; Jabs, E.W.; Geraghty,
M.T. Clinical, cytogenetic, and fuorescence in situ hybridization fndings in two cases of
“complete ring” syndrome. Am. J. Med. Genet. 1999, 87, 384–390.
30. Guilherme, R.S.; Meloni, V.F.; Kim, C.A.; Pellegrino, R.; Takeno, S.S.; Spinner, N.B.;
Conlin, L.K.; Christofolini, D.M.; Kulikowski, L.D.; Melaragno, M.I. Mechanisms of
ring chromosome formation, ring instability and clinical consequences. BMC Med.
Genet. 2011, 12, 171.
31. Caba, L.; Rusu, C.; Plăiaşu, 5th.; Gug, G.; Grămescu, M.; Bujoran, C.; Ochiană, D.;
Voloşciuc, M.; Popescu, R.; Braha, E.; Pânzaru, M.; Butnariu, L.; Sireteanu, A.; Covic,
150 Cytogenetics and Molecular Cytogenetics

M.; Gorduza, E. Ring autosomes: Some unexpected fndings. Balkan J. Med. Genet.
2012, 15, 35–46.
32. Knijnenburg, J.; van Haeringen, A.; Hansson, K.B.; Lankester, A.; Smit, M.J.; Belfroid,
R.D.; Bakker, E.; Rosenberg, C.; Tanke, H.J.; Szuhai, K. Ring chromosome formation as
a novel escape mechanism in patients with inverted duplication and terminal deletion.
Eur. J. Hum. Genet. 2007, 15, 548–545.
33. Yip, M.Y. Autosomal ring chromosomes in human genetic disorders. Transl Pediatr.
2015, 4, 164–174.
34. Schinzel, A. Catalogue of Unbalanced Chromosome Aberrations in Man Berlin. Walter
de Gruyter. Basel; 2001.
35. Hemmat, M.; Hemmat, O.; Boyar, F.Z. Isochromosome Yp and jumping translocation of
Yq resulting in fve cell lines in an infertile male: A case report and review of the litera-
ture. Mol. Cytogenet. 2013, 6, 36.
36. Liehr, T.; Claussen, U.; Starke, H. Small supernumerary marker chromosomes (sSMC)
in humans. Cytogenet Genome Res. 2004, 107, 55–67.
37. Therman, E.; Susman, B.; Denniston, C. The nonrandom participation of human acro-
centric chromosomes in Robertsonian translocations. Ann. Hum. Genet. 1989, 53,
49–65.
38. Van Hemel, J.O.; Eussen, H.J. Interchromosomal insertions: Identifcation of fve cases
and a review. Hum Genet. 2000, 107, 415–432.
39. Neill, N.J.; Ballif, B.C.; Lamb, A.N.; Parikh, S.; Ravnan, J.B.; Schultz, R.A.; Torchia,
B.S.; Rosenfeld, J.A.; Shaffer, L.G. Recurrence, submicroscopic complexity, and poten-
tial clinical relevance of copy gains detected by array CGH that are shown to be unbal-
anced insertions by FISH. Genome Res. 2011, 21, 535–544.
40. Kang, S.H.; Shaw, C.; Ou, Z.; Eng, P.A.; Cooper, M.L.; Pursley, A.N.; Sahoo, T.; Bacino,
C.A.; Chinault, A.C.; Stankiewicz, P.; Patel, A.; Lupski, J.R.; Cheung, S.W. Insertional
translocation detected using FISH confrmation of array-comparative genomic hybrid-
ization (aCGH) results. Am. J. Med. Genet. A. 2010, 152, 1111–1126.
41. Nowakowska, B.A.; de Leeuw, N.; Ruivenkamp, C.A.; Sikkema-Raddatz, B.; Crolla,
J.A.; Thoelen, R.; Koopmans, M.; den Hollander, N.; van Haeringen, A.; van der Kevie-
Kersemaekers, A.M.; Pfundt, R.; Mieloo, H.; van Essen, T.; de Vries, B.B.; Green, A.;
Reardon, W.; Fryns, J.P.; Vermeesch, J.R. Parental insertional balanced translocations
are an important cause of apparently de novo CNVs in patients with developmental
anomalies. Eur. J. Hum. Genet. 2012, 20, 166–170.
42. Durand, C.M.; Betancur, C.; Boeckers, T.M.; Bockmann, J.; Chaste, P.; Fauchereau, F.;
Nygren, G.; Rastam, M.; Gillberg, I.C.; Anckarsäter, H.; Sponheim, E.; Goubran-Botros,
H.; Delorme, R.; Chabane, N.; Mouren-Simeoni, M.C.; de Mas, P.; Bieth, E.; Rogé, B.;
Héron, D.; Burglen, L.; Gillberg, C.; Leboyer, M.; Bourgeron, T. Mutations in the gene
encoding the synaptic scaffolding protein SHANK3 are associated with autism spec-
trum disorders. Nat. Genet. 2007, 39, 25–27.
43. Phelan, M.C. Deletion 22q13.3 syndrome. Orphanet. J. Rare Dis. 2008, 3, 14.
44. Cammarata-Scalisi, F.; Callea, M.; Martinelli, D.; Willoughby, C.E.; Tadich, A.C.;
Araya Castillo, M.; Lacruz-Rengel, M.A.; Medina, M.; Grimaldi, P.; Bertini, E.; Nevado,
J. Clinical and genetic aspects of Phelan-McDermid syndrome: An interdisciplinary
approach to management. Genes (Basel). 2022, 13, 504.
 12 Multicolor-FISH—
Methods and
Applications
Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 151
Samples/Tissues ..................................................................................................... 152
Description of Methods.......................................................................................... 152
Yields ..................................................................................................................... 154
Conclusions ............................................................................................................ 154
References .............................................................................................................. 154

INTRODUCTION
Since their establishment in routine (cyto-)genetic diagnostics in the mid-1990s, mul-
ticolor fuorescence in situ hybridization (mFISH) approaches have become indis-
pensable for the identifcation and description of chromosomal rearrangements. The
subsequent exact characterization of chromosomal breakpoints is of superior clinical
impact and is the requisite condition for further molecular investigations aimed at
the identifcation of disease-related genes.[1, 2] Thus, various approaches for a dif-
ferentiation of chromosomal subbands based on mFISH assays were established.[3]
Here an overview is presented on available mFISH approaches, with special focus
on mFISH-banding methods including their advantages, limitations, and possible
applications.
Although the G-bands by trypsin using Giemsa (GTG) banding technique is still
the starting point of most (molecular) cytogenetic test requests, its technical restric-
tions are well known (e.g., only chromosome morphology combined with a black and
white banding pattern can be evaluated). Nonetheless, a quick and not costly over-
view on eventual gross changes within the human whole genome can be achieved by
that cytogenetic banding approach.[2]
mFISH methods using all 24 human whole chromosome painting probes simulta-
neously, such as multiplex-FISH (M-FISH), spectral karyotyping (SKY), combined
ratio labeling FISH (COBRA-FISH), and others,[3] reach their limits in the identi-
fcation of intrachromosomal rearrangements (such as small interstitial deletions,
duplications, and inversions without change of the centromeric index) and when
exact characterization of breakpoints is required. These limitations were overcome

DOI: 10.1201/9781003223658-12 151

152 Cytogenetics and Molecular Cytogenetics

by the development of new mFISH probe sets during the last decades. There were
many tumor-[3, 4] and clinical-cytogenetics[2, 3] -oriented probe sets established for the
human genome, all of them being suited for any kind of imaginative research appli-
cation,[2, 5] but also mFISH-probe sets for animal,[3] plant,[3] or even bacteria.[3, 5] Here
we concentrate on human mFISH probe sets suited for FISH-banding.[3, 6]
FISH banding methods are defned as “any kind of FISH technique, which pro-
vides the possibility to characterize simultaneously several chromosomal subregions
smaller than a chromosome arm (excluding the short arms of the acrocentric chro-
mosomes)”.[6] In contrast to the standard cytogenetic chromosome banding tech-
niques, giving a protein-related banding pattern,[2] the FISH banding techniques are
DNA-mediated.

SAMPLES/TISSUES
In routine settings, mFISH-banding is applicable in each cytogenetic preparation
derived from dividing cells on metaphase spreads. These are normally tumor cells,
cells derived from bone marrow, blood or fbroblasts (normally limited to fetal tis-
sues including chorion, amnion, and abortion material or skin fbroblasts in postna-
tal cases).[5] In research, also interphase cells from each tissue, including archival
formalin-fxed paraffn embedded or cryofxed tissues.[3, 5]

DESCRIPTION OF METHODS
Five different FISH banding methods are available at present, which differ in their
probe composition as well as in their banding resolution:

1. The cross-species color banding (Rx-FISH) or Harlequin FISH probe set

provides the lowest resolution of 80–90 bands per haploid human karyotype
and consists of fow-sorted gibbon chromosomes.[7] A set of 110 human–
hamster somatic cell hybrids (split into two pools and labeled with two fuo-
rochromes), when hybridized to human chromosomes, leads to about 100
“bars” along the genome. This pattern has been called “somatic cell hybrid-
based chromosome bar code”.[8] A combination of the latter and the afore-
mentioned Rx-FISH probe set results in 160-chromosome region-specifc
DNA-mediated bands in human karyotypes.[8]
2. Spectral color banding (SCAN) was described exemplarily for the two
chromosomes 3 and 10. E.g. eight microdissection libraries were created
along chromosome 10 with the goal to obtain a banding pattern similar to
GTG banding at the 300-band level.[9]
3. A chromosome can be characterized as well by a specifc signal pattern
produced by locus-specifc probes. The frst attempts to label each chromo-
some by subregional DNA probes in different colors were performed by
Lichter et  al.[10] and Lengauer et al.[11] Such probe sets are called chromo-
some bar codes (CBCs) and reached yet a resolution of up to 400 bands per
haploid karyotype, depending on the number of the applied probes.[12]
4. The Interspersed PCR multiplex FISH (IPM-FISH) approach[13] has
an approximate resolution of 400 bands per haploid karyotype, mainly
Multicolor-FISH—Methods and Applications 153

dependent on chromosome quality. In IPM-FISH, whole chromosome

painting probes are used, which are modifed by interspersed PCR, leading
to a 24-color FISH painting plus an R-band-like pattern.
5. The high-resolution multicolor banding (MCB) technique is based on over-
lapping microdissection libraries producing fuorescence profles along the
human chromosomes, and was initially described exemplarily for chromo-
some 5 in 1999 (Figure 12.1).[14] MCB allows the differentiation of chromo-
some region-specifc areas at the band and subband levels at a resolution of
550 bands per haploid karyotype. As the number of pseudocolored bands per
chromosome can freely be assigned using the isis software (MetaSystems,
Altlussheim, Germany), a resolution higher than that of GTG banding of the
corresponding chromosome can be achieved (e.g., up to 10 MCB bands for
chromosome 22 equal 800 bands per total haploid karyotype). An MCB set
of approximately 140 microdissection libraries covering the entire human
genome was described in 2002.[15] Also, the simultaneous use of all human
MCB libraries in one hybridization step for the characterization of complex
karyotypes is possible by mMCB.[16] MCB has been applied in hundreds of
studies yet and is also available a murine variant.[17, 18]

FIGURE  12.1 MCB pseudo-color pattern for all 24 human chromosomes in a ~380-band
level. Two homologue autosomes and one gonosome each are presented. The chromosomes
depicted here have been put together from 24 different MCB experiments.
154 Cytogenetics and Molecular Cytogenetics

FISH banding approaches such as the CBC-technique using locus specifc probes,
region-specifc human–hamster somatic cell hybrids, or nonoverlapping microdis-
section libraries (SCAN) have, per defnition, the disadvantage that unstained and
non-informative gaps are left along the chromosome. Such gaps can cause problems,
as breakpoints within the unstained gaps cannot exactly be described. Conversely,
techniques based on locus-specifc probes would theoretically provide the advan-
tage that chromosomal breakpoints could be defned very exactly by the correspond-
ing breakpoint-spanning or fanking clones. The IPM-FISH approach, the Rx-FISH
technique, the Rx-FISH combined with somatic cell hybrids, and the MCB methods
provide the advantage of leaving no non-informative gaps.
The MCB probe sets are commercially available as m-banding (MetaSystems,
Altlussheim, Germany) or are available on request at [email protected].
de. Besides, probes can be prepared as previously described using chromosome
microdissection.[15]

YIELDS
Only above mentioned FISH banding probe sets (1), (4), and (5) are fnished for
the whole human genome and can be applied to achieve comprehensive informa-
tion in one single hybridization step. Concerning banding resolution, MCB has
the highest and most fexible one. Additionally, according to the question to be
studied, it can be chosen if only selected chromosomes or the whole genome shall
be hybridized.

CONCLUSIONS
The introduction of FISH banding methods was a great step forward for molec-
ular cytogenetic diagnostics. However, none of the mentioned new methods can,
for technical reasons, ever be fully informative for itself. The cytogeneticist always
has to double check the results obtained in FISH banding with those achieved by
other approaches, such as GTG banding, M-FISH or SKY, chromosomal microarray
analyses (CMA), sequencing or others. Examples for that modus operandi can be
found in the literature.[4] Nevertheless, the goal must be to achieve fully informative
cytogenetic results in a minimum of time and with a minimum of FISH experiments.
Thus, further developments with respect to probe combinations (such as M-FISH/
SKY with FISH banding methods) will be necessary and are on the way.[19] To per-
form and evaluate such complex experiments, which then will have to be based on up
to nine fuorochromes, further technical developments in microscopy and computer
software would be needed.

REFERENCES
1. Liehr, T. Molecular cytogenetics in the era of chromosomics and cytogenomic
approaches. Front. Genet. 2021, 12, 720507.
2. Liehr, T. Cytogenomics. Academic Press, New York; 2021.
Multicolor-FISH—Methods and Applications 155

3. Liehr, T. Basics and literature on multicolor fuorescence in situ hybridization applica-

tion. http://cs-tl.de/DB/TC/mFISH/5-wcp-oth.html [accessed on 01/12/2022].
4. Liehr, T.; Othman, M.A.; Rittscher, K.; Alhourani, E. The current state of molecular
cytogenetics in cancer diagnosis. Expert Rev. Mol. Diagn. 2015, 15, 517–526.
5. Liehr, T. Fluorescence In Situ Hybridization (FISH): Application Guide. 2nd Edition.
Springer, Berlin; 2017.
6. Liehr, T.; Starke, H.; Heller, A.; Kosyakova, N.; Mrasek, K.; Gross, M.; Karst, C.;
Steinhaeuser, U.; Hunstig, F.; Fickelscher, I.; Kuechler, A.; Trifonov, V.; Romanenko,
S.A.; Weise, A. Multicolor fuorescence in situ hybridization (FISH) applied to FISH-
banding. Cytogenet. Genome. Res. 2006, 114, 240–244.
7. Müller, S.; Neusser, M.; Wienberg, J. Towards unlimited colors for fuorescence in-situ
hybridization (FISH). Chromosome Res. 2002, 10, 223–232.
8. Müller, S.; Rocchi, M.; Ferguson-Smith, M.A.; Wienberg, J. Toward a multicolor chro-
mosome bar code for the entire human karyotype by fuorescence in situ hybridization.
Hum. Genet. 1997, 100, 271–278.
9. Liehr, T. About SCAN. http://cs-tl.de/DB/TC/mFISH/6-banding.html#5 [accessed on
01/12/2022].
10. Lichter, P.; Tang, C.J.; Call, K.; Hermanson, G.; Evans, G.A.; Housman, D.; Ward, D.C.
High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid
clones. Science 1990, 247, 64–69.
11. Lengauer, C.; Speicher, M.R.; Popp, S.; Jauch, A.; Taniwaki, M.; Nagaraja, R.; Riethman,
H.C.; Donis-Keller, H.; D’Urso, M.; Schlessinger, D.; Cremer, T. Chromosomal bar codes
produced by multicolor fuorescence in situ hybridization with multiple YAC clones and
whole chromosome painting probes. Hum. Mol. Genet. 1993, 2, 505–512.
12. Liehr, T. About CBC. http://cs-tl.de/DB/TC/mFISH/6-banding.html#3 [accessed on
01/12/2022].
13. Aurich-Costa, J.; Vannier, A.; Gregoire, E.; Nowak, F.; Cherif, D. IPM-FISH, a new
M-FISH approach using IRS-PCR painting probes: Application to the analysis of seven
human prostate cell lines. Genes Chromosomes Cancer. 2001, 30, 143–160.
14. Chudoba, I.; Plesch, A.; Lörch, T.; Lemke, J.; Claussen, U.; Senger, G. High resolution
multicolor-banding: A new technique for refned FISH analysis of human chromosomes.
Cytogenet. Cell Genet. 1999, 84, 156–160.
15. Liehr, T.; Heller, A.; Starke, H.; Rubtsov, N.; Trifonov, V.; Mrasek, K.; Weise, A.;
Kuechler, A.; Claussen, U. Microdissection based high resolution multicolor banding for
all 24 human chromosomes. Int. J. Mol. Med. 2002, 9, 335–339.
16. Weise, A.; Heller, A.; Starke, H.; Mrasek, K.; Kuechler, A.; Pool-Zobel, B.-L.; Claussen,
U.; Liehr, T. Multitude multicolor chromosome banding (mMCB): A comprehensive one-
step multicolor FISH banding method. Cytogenet. Genome Res. 2003, 103, 34–39.
17. Leibiger, C.; Kosyakova, N.; Mkrtchyan, H.; Glei, M.; Trifonov, V.; Liehr, T. First molec-
ular cytogenetic high resolution characterization of the NIH 3T3 cell line by murine
multicolor banding. J. Histochem. Cytochem. 2013, 61, 306–312.
18. Liehr, T. About MCB. http://cs-tl.de/DB/TC/mFISH/6-banding.html#6 [accessed on
01/12/2022].
19. Zhang, C.; Cerveira, E.; Rens, W.; Yang, F.; Lee, C. Multicolor fuorescence in situ
hybridization (FISH) approaches for simultaneous analysis of the entire human genome.
Curr. Protoc. Hum. Genet. 2018, 99, e70.
 13 FISH—Centromere-
and Heterochromatin-
Specifc Multicolor
Probe Sets
Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 157
Samples to Be Characterized ................................................................................. 158
Centromere- and Heterochromatin-Specifc FISH Approaches ............................. 158
Yields ..................................................................................................................... 160
Conclusions ............................................................................................................ 160
Acknowledgments.................................................................................................. 160
References .............................................................................................................. 160

INTRODUCTION
The human genome project has been declared to be fnished by 2003.[1] However, in
the almost two decades since then, it has been a well-known truism that there was
almost 10% of the DNA-sequence still not accessed, yet.[2] This affects especially
the highly repetitive DNA-stretches, mainly being concentrated in the highly variant
heterochromatic parts of the human genome.[3] This gap was just recently closed by
Evan Eichler and colleagues for a single genome.[2] Nonetheless, heterochromatic
regions of the human genome are understudied and cannot be accessed by standard
molecular genetic approaches, including sequencing, as of now.[4]
Thus, it is also not surprising that since 1996 different fuorescence in situ hybrid-
ization (FISH) probe sets were available to characterize euchromatic material along
marker or derivative chromosomes.[5] However until 2001, whole genome–oriented
heterochromatic FISH-probe sets were not established; the frst then available one
was specifc for all human centromeres.[6] It even lasted until 2012 until a probe set
for all large heterochromatic regions in the human genome was developed.[7] The
two mentioned probe sets are suited for the one-step-characterization of the chro-
mosomal origin in small supernumerary marker chromosomes (sSMCs),[8, 9] or larger
derivative chromosomes with rearrangements including human hererochromatin.[10]

DOI: 10.1201/9781003223658-13 157

158 Cytogenetics and Molecular Cytogenetics

Here, the corresponding centromere-specifc multicolor FISH (cenM-FISH),[6] and

the heterochromatin-M-FISH (HCM-FISH) probe sets are introduced.[7]

SAMPLES TO BE CHARACTERIZED
In about 50–70% of cases with sSMCs, the derivatives are known to originate from
chromosome 15.[8, 9] Among the remaining cases, there is: 1) a great variation in chro-
mosomal and parental origin, 2) possibly a mosaicism, 3) the possibility of genomic
imprinting effects, and 4) of homozygosity of autosomal recessively inherited muta-
tions in uniparental isodisomy (UPD). As great dissimilarities in their clinical out-
comes are reported, too, the characterization of prenatally detected, particularly de
novo sSMCs, is of special interest for genetic counseling and appropriate medical
care. Characterization of one or more sSMCs in a patient should be followed by test-
ing for UPD, as the latter can cause clinical signs and symptoms even in heterochro-
matic sSMC cases, as well.[8, 9, 11, 12]
Many sSMCs consist only of heterochromatic material, and here it is important to
distinguish those from such cases with potentially deleterious euchromatic material
on them.[8, 9] Besides, there are other incidences where larger derivatives may either
include enlarged heterochromatin (chromosomal heteromorphisms), which may be
cytogenetically similar to deleterious unbalanced rearrangements.[3, 10]

CENTROMERE- AND HETEROCHROMATIN-SPECIFIC

FISH APPROACHES
To characterize an sSMC, whole chromosome painting probe–based FISH
approaches, such as multiplex FISH (M-FISH) or spectral karyotyping (SKY),[5]
are well suited as long as the marker is larger than the short arm of chromosome
16. If the sSMC is smaller, and mainly heterochromatic, then in that way often no
result may be obtained.[8, 9] Thus, for rapid characterization of such sSMCs, a mul-
ticolor FISH technique was established, allowing the unambiguous one-step identi-
fcation of all human centromeric regions, excluding chromosomes 13 and 21 (due
to same alphid DNA-sequences on these chromosomes). The so-called cenM-FISH
(Figure 13.1) is based on all available centromere-specifc DNA probes, labeled in
fve different fuorochromes.[6]
sSMCs are initially detected by GTG banding analysis and can be characterized
for the presence of an acrocentric chromosome-derived short arm by nucleolus
organizing region (NOR) staining. According to the NOR staining result (NOR-
negative or NOR-positive), the origin of SMCs can be determined by application
of all human centromeres in one experiment (i.e., cenM-FISH[6]) or by the acrocen-
tric centromere-specifc multicolor FISH (acro-cenM-FISH) probe set,[13] respec-
tively. The latter consists of a probe specifc for the acrocentric human p-arms, a
NOR-specifc probe, a probe specifc for Yq12, and all centromere-specifc probes
for 13/21, 14/22, 15, and 22. These two approaches were successfully applied in
about 1,500 cases with SMCs up to present.[8] In general, neocentromeric sSMCs
are not stained by any of the two aforementioned approaches; in this case, other
Multicolor-FISH—Centromere- and Heterochromatin-Specifc 159

FIGURE 13.1 An sSMC was found in a clinically normal female.

A) CenM-FISH characterized the sSMC (arrow) as chromosome 2 derived.
B) SubcenM-FISH revealed that, irrespective of its small size, the sSMC lead to a partial
trisomy 2p11.2 to 2q11.2. According to,[8] there are overall eleven cytogenetically compa-
rable cases without any clinical signs. This case is # 02-O-p11.2/3–2.[8]

techniques for their characterization, such as M-FISH, microdissection of sSMCs,

or chromosome microarray (CMA) have to be performed. About 130 cases (~1.8%
of all reported sSMCs) with neocentromeres are reported in the literature.[8, 9] After
determination of the origin of an sSMC with centromeric DNA, the most important
question to address is if there is euchromatic material of the corresponding chro-
mosome on it, or not. This question for partial trisomy can be studied by hybrid-
izing whole chromosome painting or chromosome arm-specifc probes. Another
possibility is the use of a probe set specifc for the pericentric region of all human
chromosomes, the centromere-near multicolor FISH (subcenM-FISH) probe set. A
chromosome-specifc subcenM-FISH probe set consists of a centromere-specifc
satellite probe, one centromere-near locus-specifc probe in the long arm and the
short arm (excluding the acrocentric chromosomes), and chromosome arm-specifc
probes.[14] On the other hand, CMA can also be the initial hint that a patient is not
carrier of a simple intra-chromosomal duplication or unbalanced translocation but
of an sSMC.
In some cases, the centromeric region of a heterochromatic sSMC can be so small
that it hardly stains by a probe contained in the cenM-FISH-probe set. Then the
HCM-FISH probe set may be helpful, as it contains probes specifc for 1q12, 9p12/
q13, 9q12, NOR, 15p11.2, 16q11.2, Yq12, and all acrocentric p-arms.[7]
All mentioned probe sets are available on request at [email protected]
jena.de. Besides, probes can be prepared as previously described,[6, 7, 13, 14] using com-
mercially available locus specifc probes and/or chromosome microdissection.[5]
160 Cytogenetics and Molecular Cytogenetics

YIELDS
In Figure  13.1, the possible yield of application of (sub-)cenM-FISH probe sets is
shown—i.e. the characterization of origin and content of an sSMC. As summarized
elsewhere, only molecular cytogenetics can reliably characterized sSMCs, especially
in infertile patients.[15]

CONCLUSIONS
In summary, nowadays the detection of an sSMC does not stop after GTG banding,
NOR staining, and exclusion of a chromosome 15 origin. It can be exactly described
after (acro)cenM-FISH and/or HCM-FISH, subcenM-FISH provides information on
additional euchromatin, and molecular genetics uncovers eventually present UPD.
CMA can only be applied in euchromatic sSMCs without mosaicism.[15] Meanwhile,
a relatively good correlation of sSMC content and clinics is available,[8] being
extremely helpful in genetic counseling.

ACKNOWLEDGMENTS
The case shown in Figure 13.1 was provided by Drs. Cramer and Hickmann, Essen,
Germany.

REFERENCES
1. https://en.wikipedia.org/wiki/Human_Genome_Project [accessed on 01/12/2022].
2. Nurk, S.; Koren, S.; Rhie, A.; Rautiainen, M.; Bzikadze, A.V.; Mikheenko, A.; Vollger,
M.R.; Altemose, N.; Uralsky, L.; Gershman, A.; Aganezov, S.; Hoyt, S.J.; Diekhans, M.;
Logsdon, G.A.; Alonge, M.; Antonarakis, S.E.; Borchers, M.; Bouffard, G.G.; Brooks,
S.Y.; Caldas, G.V.; Cheng, H.; Chin, C.-S.; Chow, W.; de Lima, L.G.; Dishuck, P.C.;
Durbin, R.; Dvorkina, T.; Fiddes, I.T.; Formenti, G.; Fulton, R.S.; Fungtammasan, A.;
Garrison, E.; Grady, P.G.S.; Graves-Lindsay, T.-A.; Hall, I.M.; Hansen, N.F.; Hartley,
G.A.; Haukness, M.; Howe, K.; Hunkapiller, M.W.; Jain, C.; Jain, M.; Jarvis, E.D.;
Kerpedjiev, P.; Kirsche, M.; Kolmogorov, M.; Korlach, J.; Kremitzki, M.; Li, H.; Maduro,
V.V.; Marschall, T.; McCartney, A.M.; McDaniel, J.; Miller, D.E.; Mullikin, J.C.; Myers,
E.W.; Olson, N.D.; Paten, B.; Peluso, P.; Pevzner, P.A.; Porubsky, D.; Potapova, T.; Rogaev,
E.I.; Rosenfeld, J.A.; Salzberg, S.L.; Schneider, V.A.; Sedlazeck, F.J.; Shafn, K.; Shew,
C.J.; Shumate, A.; Sims, Y.; Smit, A.F.A.; Soto, D.C.; Sović, I.; Storer, J.M.; Streets, A.;
Sullivan, B.A.; Thibaud-Nissen, F.; Torrance, J.; Wagner, J.; Walenz, P.P.; Wenger, A.;
Wood, J.M.D.; Xiao, C.; Yan, S.M.; Young, A.C.; Zarate, S.; Surti, U.; McCoy, R.C.;
Dennis, M.Y.; Alexandrov, I.A.; Gerton, J.L.; O’Neill, R.J.; Timp, W.; Zook, J.M.;
Schatz, M.C.; Eichler, E.E.; Miga, K.H.; Phillippy, A.M. The complete sequence of a
human genome. bioRxiv. 2021. preprint doi: https://doi.org/10.1101/2021.05.26.445798.
3. Liehr, T. Benign & Pathological Chromosomal Imbalances, 1st Edition Microscopic
and Submicroscopic Copy Number Variations (CNVs) in Genetics and Counseling.
Academic Press, New York; 2014.
4. Liehr, T. Repetitive elements in humans. Int. J. Mol. Sci. 2021, 22, 2072.
5. Liehr, T. Molecular cytogenetics in the era of chromosomics and cytogenomic
approaches. Front. Genet. 2021, 12, 720507.
Multicolor-FISH—Centromere- and Heterochromatin-Specifc 161

6. Nietzel, A.; Rocchi, M.; Starke, H.; Heller, A.; Fiedler, W.; Wlodarska, I.; Loncarevic,
I.F.; Beensen, V.; Claussen, U.; Liehr, T. A new multicolor-FISH approach for the charac-
terization of marker chromosomes: Centromere-specifc multicolor-FISH (cenM-FISH).
Hum. Genet. 2001, 108, 199–204.
7. Bucksch, M.; Ziegler, M.; Kosayakova, N.; Mulatinho, M.V.; Llerena, J.C. Jr.; Morlot,
S.; Fischer, W.; Polityko, A.D.; Kulpanovich, A.I.; Petersen, M.B.; Belitz, B.; Trifonov,
V.; Weise, A.; Liehr, T.; Hamid, A.B. A new multicolor fuorescence in situ hybridiza-
tion probe set directed against human heterochromatin: HCM-FISH. J. Histochem.
Cytochem. 2012, 60, 530–536.
8. Liehr, T. Small supernumerary marker chromosomes. http://cs-tl.de/DB/CA/sSMC/0-
Start.html [accessed on 01/12/2022].
9. Liehr, T. Small Supernumerary Marker Chromosomes (sSMC): A Guide for Human
Geneticists and Clinicians: With Contributions by UNIQUE (The Rare Chromosome
Disorder Support Group). Springer, Berlin; 2012.
10. Liehr, T. Cases with heteromorphisms. http://cs-tl.de/DB/CA/HCM/0-Start.html
[accessed on 01/12/2022].
11. Liehr, T. Uniparental Disomy (UPD) in Clinical Genetics: A Guide for Clinicians and
Patients. Springer, Berlin; 2014.
12. Liehr, T. Cases with uniparental disomy. http://cs-tl.de/DB/CA/UPD/0-Start.html
[accessed on 01/12/2022].
13. Trifonov, V.; Seidel, J.; Starke, H.; Prechtel, M.; Beensen, V.; Ziegler, M.; Hartmann, I.;
Heller, A.; Nietzel, A.; Claussen, U.; Liehr, T. Enlarged chromosome 13 p-arm hiding a
cryptic partial trisomy 6p22.2-pter. Prenat. Diagn. 2003, 23, 427–430.
14. Weise, A.; Liehr, T. Subtelomeric and/or subcentromeric probe sets. In: Fluorescence
In Situ Hybridization (FISH): Application Guide; Liehr, T., Ed. 2nd Edition. Springer,
Berlin, 2017, pp. 261–269.
15. Liehr, T.; Hamid Al-Rikabi, A.B. Impaired spermatogenesis due to small supernumerary
marker chromosomes: The reason for infertility is only reliably ascertainable by cytoge-
netics. Sex. Dev. 2018, 12, 281–287.
 14 FISH—Detection
of Individual Radio
Sensitivity
Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 163
Samples/Tissues ..................................................................................................... 164
Description of Methods.......................................................................................... 165
One- to Three-Color FISH ............................................................................ 165
24-Color FISH .............................................................................................. 166
Multicolor Banding ....................................................................................... 166
Specifc Methodological Details for M-FISH Evaluation...................................... 166
Yields ..................................................................................................................... 167
Conclusions ............................................................................................................ 167
References .............................................................................................................. 167

INTRODUCTION
In radiotherapy, alongside tumor control, the prevention of severe treatment-related
side effects is a major concern. Although the majority of radio-oncological patients
tolerate a standard treatment protocol, toxic side effects can be detected in 0.2–10%
of the cases.[1, 2] The latter can be a result of increased individual radio sensitiv-
ity, caused by a combination of exogenous and endogenous factors including genetic
reasons. However, most of the exact underlying still mechanisms remain unknown.
Only in few cases is increased radio sensitivity a result of an identifed single gene
mutation, e.g., in ataxia telangiectasia (AT) or Nijmegen breakage syndrome (NBS)
patients, OMIM #208900 and #251260,[3] but in the majority, it is supposed to be
modulated by a mixture of different genes.[4]
To have a chance to detect a possible overreaction prior to therapy, the availabil-
ity of a predictive test system has been the aim for some decades. Although different
approaches were tested (e.g., clonogenic cell survival,[5] G2-assay,[6] comet assay[7]),
there is still no reliable routine assay to identify hypersensitive or less sensitive patients
to adjust their therapeutic dose. It is known that chromosomal aberrations 1) are indica-
tors of a previous exposure to irradiation and 2) can be used to estimate radio sensitiv-
ity. Previous studies demonstrated that molecular cytogenetic methods are superior to
conventional cytogenetic analysis in detection and characterization of aberrations. At

DOI: 10.1201/9781003223658-14 163

164 Cytogenetics and Molecular Cytogenetics

this, the frequency of breaks and the occurrence of specifc aberration types refect
individual sensitivity to radiation. Especially complex chromosomal aberrations (CCR)
were identifed as indicators for increased individual radio sensitivity.
To detect individual radio sensitivity by fuorescence in situ hybridization (FISH),
three approaches are presently available: i) painting of one up to three different chro-
mosomes in one or different colors, ii) painting of all 24 human chromosomes in
different colors (24-color FISH), and iii) FISH-based chromosome banding for the
detection of intra-chromosomal rearrangements.

SAMPLES/TISSUES
Our own studies have focused on the analysis of in vitro-induced chromosomal
aberrations in NBS and AT homozygote and heterozygote individuals compared
with normal reacting controls (Figures 14.1 and 14.2).[8, unpublished data] The aim was to

FIGURE  14.1 Example of a 24-color FISH result: karyogram of an NBS-heterozygote

person (pseudocolor representation) after in vitro irradiation with 2.0 Gy and scheme of
pseudocolors for each individual chromosome. The aberration can be described (nomencla-
ture modifed according to the ISCN 2020[29]) as follows: t(1;2),dic(1;14),ace(1),dic(3;9), ace
t(3;7),del(7). This result can be summarized as seven breaks per mitosis. The translocation
between chromosomes 3, 7, and 9 forms a complex chromosomal rearrangement (CCR).
Abbreviations: p=short arms of the acrocentric chromosomes #13, #14, #15, #21, and #22
consisting of repetitive DNA, pseudocolored in a different paint; h = heterochromatic DNA,
which is polymorphic and can be present at #1, #9, and #16. (Stars indicate centromere local-
ization.) Images were captured with the ISIS3 digital FISH imaging system (MetaSystems,
Altlussheim, Germany) using a PCO VC45 CCD camera (PCO, Kehl, Germany) on an
Axioplan 2 microscope (Zeiss, Jena, Germany).
FISH—Detection of Individual Radio Sensitivity 165

FIGURE 14.2 For the graphic depicted here, four normal controls, two Nijmegen breakage
syndrome (NBS), and two ataxia telangiectasia (AT) heterozygotes (NBS-het, AT-het) plus
one NBS and one AT homozygote (NBS-hm, AT-hm) were included. (A) shows the average
number of breaks per mitosis, while (B) presents the average number of CCR occurring in
each mitosis for each of the fve groups. Three different in vitro irradiation doses were ana-
lyzed per patient. The applied doses were 0.0, 0.7, and 2.0 Gy; in the AT-hm patient, instead
of 2.0 Gy, 1.4 Gy was used. A clear differentiation was possible in both diagrams between
normal controls and the homozygote patients, as well as between controls and AT-het.

demonstrate that a difference in radio sensitivity which was already known between
these individuals is also detectable using the 24-color FISH technique. Thus, the
increased radio sensitivity of NBS and AT patients can serve as a positive control in
a predictive assay.

DESCRIPTION OF METHODS
ONE- TO THREE-COLOR FISH
FISH applying one up to three whole-chromosome painting (wcp) probes simulta-
neously was used to detect radiation-induced chromosome instability in peripheral
166 Cytogenetics and Molecular Cytogenetics

blood lymphocytes and fbroblasts.[9–16] However, in the analyses, the used chromo-
somes are selected by chance, although a random distribution of chromosomal rear-
rangements along the chromosomes is up to now still discussed controversially.[17–18]
To avoid this problem and to obtain an overview of the whole karyotype, 24-color
FISH can be applied using a probe mix of the 24 different human wcp probes.[8, 9, 19, 20]

24-COLOR FISH
This method, frst described in 1996,[21, 22] allows the simultaneous visualization of
all chromosomes within a metaphase in different specifc colors. Nearly all inter-
chromosomal aberrations (as reciprocal and nonreciprocal translocations, complex
rearrangements, ring chromosomes, acentric fragments, dicentric fragments, or
insertions) can be detected and defned in more detail.
24-color FISH has been used, for example, for studies on normal peripheral blood
lymphocytes irradiated in vitro,[8, 19] on radiotherapy-induced residual chromosomal
damage in peripheral lymphocytes,[9] on bone marrow cells of Chernobyl victims,[19]
and for analysis of chromosomal aberrations after irradiation with ionizing alpha
particles. The latter was also used for investigations on the formation of CCR.[23]

MULTICOLOR BANDING
FISH methods using all 24 human whole-chromosome painting probes simultane-
ously reach their limits in identifcation of intra-chromosomal rearrangements (such
as small interstitial deletions, duplications, and inversions without change of the cen-
tromeric index) and when exact characterization of breakpoints is required. These
limitations have been overcome by the development of FISH-banding methods dur-
ing the last decades.[25] In the meantime, one of these approaches, the multicolor
banding (MCB) technique (or mBAND),[26] was also used for the analysis of X-ray-
induced aberrations.[27, 28] However, only a probe set for chromosome 5 was applied,
demonstrating that intra chromosomal aberrations are present in a considerable por-
tion in radiation-induced changes.

SPECIFIC METHODOLOGICAL DETAILS FOR M-FISH EVALUATION

In the following, the criteria used in our own studies are specifed; overall, these
require an experienced cytogeneticist. After the 24-color FISH procedure including
hybridization, posthybridization washes, and detection, at least 100 metaphases per
irradiation dose and patient/proband have to be acquired. This can be performed
flter-based (m-FISH),[21] or spectracube-based (SKY).[26] All captured metaphases
have to be karyotyped. For detailed evaluation of each metaphase, it is not enough
to rely only on the pseudocolor functions of the used software, but for unambigu-
ous results, one has to check the different fuorochrome channels or hybridization
profles. Then, aberration types and involved chromosomes have to be registered
in detail. All occurring aberrations have to be classifed into reciprocal and nonre-
ciprocal translocations, ring chromosomes, acentric fragments, dicentric fragments,
FISH—Detection of Individual Radio Sensitivity 167

inversions, insertions, and complex rearrangements. Translocations, insertions, and

complex rearrangements are visible because of a color change along a rearranged
chromosome. Dicentrics and ring chromosomes can easily be identifed using the
inverted DAPI (4′,6-diamidino-2-phenylindole) picture. Complex chromosomal rear-
rangements (CCR) consist, per defnition, of at least two chromosomes with three or
more breaks.[24]
For further evaluation, the frequency of break events constituting the observed
aberrations was estimated as the minimal number of breaks considered to be nec-
essary for producing the aberrations in each metaphase. The total number of break
events in each patient/control and irradiation dose was summed up and divided by
the number of metaphases analyzed to obtain the average rate of breaks per mitosis
(B/M). The radiosensitivity of lymphocytes is expressed as number of radiation-
induced B/M and CCR/M after each irradiation dose, subtracted by the 0.0-Gy
control value to correct the infuence of spontaneous basic aberration frequencies.[8]

YIELDS
The 24-color FISH is a highly informative approach concerning the characterization
of radiation-induced chromosomal rearrangements, but is based on a very sophisti-
cated and time-consuming evaluation procedure. A typical result for one metaphase
and a summary of obtained data are shown in Figures 14.1 and 14.2.

CONCLUSIONS
In summary, genetically determined intrinsic radiosensitivity can be detected and
quantifed by FISH approaches. Twenty-four-color FISH can be used as well as the
three-color FISH. FISH is as reliable but more informative than conventional cytoge-
netic data.[10, 11] As stated before, the 24-color FISH approach requires a very sophis-
ticated evaluation. However, data obtainable by this technique are indispensable to
decide whether there exist any hot spots for radiation-induced breakpoints. If so,
such hot spots could be characterized in detail by FISH-banding methods,[25] and
in the following, locus-specifc breakpoint-spanning probes could be applied in the
future for quick detection of individual radio sensitivity, possibly even in interphase
cytogenetics.

REFERENCES
1. Roberts, S.A.; Spreadborough, A.R.; Bulman, B.; Barber, J.B.; Evans, D.G.; Scott, D.
Heritability of cellular radiosensitivity: A marker of low-penetrance predisposition
genes in breast cancer? Am. J. Hum. Genet. 1999, 65, 784–794.
2. Rogers, P.B.; Plowman, P.N.; Harris, S.J.; Arlett, C.F. Four radiation hypersensitiv-
ity cases and their implications for clinical radiotherapy. Radiother. Oncol. 2000, 57,
143–154.
3. OMIM. www.ncbi.nlm.nih.gov/omim/ [accessed on 01/12/2022].
4. Turesson, I.; Nyman, J.; Holmberg, E.; Oden, A. Prognostic factors for acute and late skin
reactions in radiotherapy patients. Int. J. Radiat. Oncol. Biol. Phys. 1996, 36, 1065–1075.
168 Cytogenetics and Molecular Cytogenetics

5. Cole, J.; Arlett, C.F.; Green, M.H.; Harcourt, S.A.; Priestley, A.; Henderson, L.; Cole, H.;
James, S.E.; Richmond, F. Comparative human cellular radiosensitivity: II. The survival
following gamma-irradiation of unstimulated (G0) T-lymphocytes, T-lymphocyte lines,
lymphoblastoid cell lines and fbroblasts from normal donors, from ataxia-telangiectasia
patients and from ataxia-telangiectasia heterozygotes. Int. J. Radiat. Biol. 1988, 54,
929–943.
6. Sanford, K.K.; Parshad, R.; Price, F.M.; Jones, G.M.; Tarone, R.E.; Eierman, L.; Hale,
P.; Waldmann, T.A. Enhanced chromatid damage in blood lymphocytes after G2 phase
x irradiation, a marker of the ataxia-telangiectasia gene. J. Natl. Cancer Inst. 1990, 8,
1050–1054.
7. Hovhannisyan, G.G. Fluorescence in situ hybridization in combination with the comet
assay and micronucleus test in genetic toxicology. Mol. Cytogenet. 2010, 3, 17.
8. Kuechler, A.; Neubauer, S.; Grabenbauer, G.G.; Claussen, U.; Liehr, T.; Sauer, R.; Wendt,
T.G. Is 24-color FISH detection of in-vitro radiation-induced chromosomal aberrations
suited to determine individual intrinsic radiosensitivity? Strahlenther. Onkol. 2002, 178,
209–215.
9. Kuechler, A.; Dreidax, M.; Pigorsch, S.U.; Liehr, T.; Claussen, U.; Wendt, T.G.; Dunst,
J. Residual chromosomal damage after radiochemotherapy with and without amifostine
detected by 24-color FISH. Strahlenther. Onkol. 2003, 179, 493–498.
10. Neubauer, S.; Arutyunyan, R.; Stumm, M.; Dörk, T.; Bendix, R.; Bremer, M.; Varon, R.;
Sauer, R.; Gebhart, E. Radiosensitivity of ataxia telangiectasia and Nijmegen breakage
syndrome homozygotes and heterozygotes as determined by three-color FISH chromo-
some painting. Radiat. Res. 2002, 157, 312–321.
11. Gebhart, E.; Neubauer, S.; Schmitt, G.; Birkenhake, S.; Dunst, J. Use of three-color
chromosome in situ suppression technique for the detection of past radiation exposure.
Radiat. Res. 1996, 145, 47–52.
12. Neubauer, S.; Dunst, J.; Gebhart, E. The impact of complex chromosomal rearrange-
ments on the detection of radiosensitivity in cancer patients. Radiother. Oncol. 1997, 43,
189–195.
13. Stritzelberger, J.; Lainer, J.; Gollwitzer, S.; Graf, W.; Jost, T.; Lang, J.D.; Mueller, T.M.;
Schwab, S.; Fietkau, R.; Hamer, H.M.; Distel, L. Ex vivo radiosensitivity is increased in
non-cancer patients taking valproate. BMC Neurol. 2020, 20, 390.
14. Schuster, B.; Ellmann, A.; Mayo, T.; Auer, J.; Haas, M.; Hecht, M.; Fietkau, R.; Distel,
L.V. Rate of individuals with clearly increased radiosensitivity rise with age both in
healthy individuals and in cancer patients. BMC Geriatr. 2018, 18, 105.
15. Gryc, T.; Putz, F.; Goerig, N.; Ziegler, S.; Fietkau, R.; Distel, L.V.; Schuster, B. Idelalisib
may have the potential to increase radiotherapy side effects. Radiat. Oncol. 2017, 12,
109.
16. Rave-Fränk, M.; Virsik-Kopp, P.; Pradier, O.; Nitsche, M.; Grunefeld, S.; Schmidberger,
H. In vitro response of human dermal fbroblasts to X-irradiation: Relationship between
radiation-induced clonogenic cell death, chromosome aberrations and markers of prolif-
erative senescence or differentiation. Int. J. Radiat. Biol. 2001, 77, 1163–1174.
17. Barrios, L.; Miro, R.; Caballin, M.R.; Fuster, C.; Guedea, F.; Subias, A.; Egozcue, J.
Cytogenetic effects of radiotherapy. Breakpoint distribution in induced chromosome
aberrations. Cancer Genet. Cytogenet. 1989, 41, 61–70.
18. Cigarran, S.; Barrios, L.; Barquinero, J.F.; Caballin, M.R.; Ribas, M.; Egozcue, J.
Relationship between the DNA content of human chromosomes and their involvement in
radiation-induced structural aberrations, analysed by painting. Int. J. Radiat. Biol. 1998,
74, 449–455.
FISH—Detection of Individual Radio Sensitivity 169

19. Greulich, K.M.; Kreja, L.; Heinze, B.; Rhein, A.P.; Weier, H.G.; Bruckner, M.; Fuchs,
P.; Molls, M. Rapid detection of radiation-induced chromosomal aberrations in lympho-
cytes and hematopoietic progenitor cells by mFISH. Mutat. Res. 2000, 452, 73–81.
20. Liehr, T. Basics and literature on multicolor fuorescence in situ hybridization applica-
tion. http://cs-tl.de/DB/TC/mFISH/5-wcp-oth.html#2 [accessed on 01/12/2022].
21. Speicher, M.R.; Gwyn Ballard, S.; Ward, D.C. Karyotyping human chromosomes by
combinatorial multi-fuor FISH. Nat. Genet. 1996, 12, 368–375.
22. Schröck, E.; du Manoir, S.; Veldman, T.; Schoell, B.; Wienberg, J.; Ferguson-Smith,
M.A.; Ning, Y.; Ledbetter, D.H.; Bar-Am, I.; Soenksen, D.; Garini, Y.; Ried, T. Multicolor
spectral karyotyping of human chromosomes. Science. 1996, 273, 494–497.
23. Anderson, R.M.; Stevens, D.L.; Goodhead, D.T. M-FISH analysis shows that complex
chromosome aberrations induced by {alpha}-particle tracks are cumulative products of
localized rearrangements. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 12167–12172.
24. Savage, J.R.K.; Simpson, P.J. FISH “painting” pattern resulting from complex exchanges.
Mutat. Res. 1994, 312, 51–60.
25. Liehr, T. Molecular cytogenetics in the era of chromosomics and cytogenomic
approaches. Front. Genet. 2021, 12, 720507.
26. Liehr, T.; Heller, A.; Starke, H.; Rubtsov, N.; Trifonov, V.; Mrasek, K.; Weise, A.;
Kuechler, A.; Claussen, U. Microdissection based high resolution multicolor banding for
all 24 human chromosomes. Int. J. Mol. Med. 2002, 9, 335–339.
27. Johannes, C.; Chudoba, I.; Obe, G. Analysis of X-ray-induced aberrations in human
chromosome 5 using high-resolution multicolour banding FISH (mBAND). Chromosom.
Res. 1999, 7, 625–633.
28. Hande, M.P.; Azizova, T.V.; Geard, C.R.; Burak, L.E.; Mitchell, C.R.; Khokhryakov,
V.F.; Vasilenko, E.K.; Brenner, D.J. Past exposure to densely ionizing radiation leaves a
unique permanent signature in the genome. Am. J. Hum. Genet. 2003, 72, 1162–1170.
29. ISCN (2020). An International System for Human Cytogenomic Nomenclature;
McGowan-Jordan, J.; Hastings R.J.; Moore, S., Eds. S Karger, Basel; 1995.
 15 FISH—Detection of CNVs
Tigran Harutyunyan, Anzhela Sargsyan
and Rouben Aroutiounian

CONTENTS
Introduction............................................................................................................ 171
Samples/Tissues/Materials..................................................................................... 173
Human Whole Blood Culture ....................................................................... 174
Slides with Metaphase Chromosomes .......................................................... 174
Pod-FISH Probes .......................................................................................... 174
Pretreatment and Postfxation ....................................................................... 174
Pod-FISH and Washing................................................................................. 175
Description of Methods.......................................................................................... 175
Human Whole Blood Cultivation ................................................................. 175
Preparation of Metaphase Spreads................................................................ 175
Homemade Pod-FISH Probes....................................................................... 176
Slide Pretreatment......................................................................................... 176
Pod-FISH Procedure..................................................................................... 176
Washing and Counterstaining of Slides ........................................................ 177
Yields ..................................................................................................................... 177
Analysis of CNVs Using ImageJ Program ................................................... 177
Conclusions............................................................................................................ 178
Acknowledgments.................................................................................................. 179
References.............................................................................................................. 179

INTRODUCTION
DNA copy number variations (CNVs) occur in the genome due to spontaneous and
induced gains, losses, and/or insertions of 50 bp (as repeats) to several Mb long DNA
segments leading to copy number changes of a particular DNA sequence within the
chromosomes.[1] Thus, CNVs are structural changes within chromosomes and are
distinguished from whole chromosome gains and losses which are a separate class
of cytogenetic alterations (i.e. aneuploidy). When the frequency of a given CNV in
a population is greater than 1%, it is referred to as a copy number polymorphism.
Mounting evidence demonstrates the importance of de novo CNVs as a source of
genomic variation and in development of pathogenic conditions. Therefore, the
identifcation of CNVs in human chromosomes is of crucial task from clinical and
research perspectives. However, their detection remains challenging.

DOI: 10.1201/9781003223658-15 171

172 Cytogenetics and Molecular Cytogenetics

Zarrei et al.[2] constructed a high-resolution CNV map of the human genome that
includes 2,057,368 variants (195,084 gains and 1,862,284 losses). It was shown that
CNVs have unequal frequency among chromosomes. The highest frequencies of
gains were observed in chromosomes 22 and Y, followed by chromosomes 16, 9, and
15; however, the highest proportions of variable sequences with losses were detected
in chromosomes 19, 22, and Y. In addition, higher proportions of CNVs were iden-
tifed in pericentromeric and subtelomeric regions of all chromosomes. Moreover,
approximately for 100 genes it has been shown that they may be completely deleted
without apparent phenotypic consequences.[2] Overall, it was shown that CNVs con-
tribute to 4.8–9.7% of the variability in the human genome.
There are several databases for CNVs of which the “Database of Genomic
Variants” is the largest host for data of structural variations in healthy subjects from
worldwide populations.[3] The recently developed database CNVIntegrate is hosting
data from both healthy persons and cancer patients, and enables comparisons of
CNV frequencies within multiple ethnic populations.[4]
Based on differences in breakpoint positions during genomic rearrangements,
CNVs are classifed as recurrent and non-recurrent. Recurrent CNVs typically
occur via non-allelic homologous recombination between region-specifc low-copy
repeats with >95% nucleotide sequence identity.[5] Therefore, recurrent CNVs have
the same DNA sequence at the rearrangement breakpoints and similar size in unre-
lated individuals. In contrast, non-recurrent CNVs are thought to originate due to
inappropriate repair of accidental DNA double-strand breaks in proliferating cells
via microhomology-mediated pathways. Thus, non-recurrent CNVs have unique
sequences at the breakpoints in each individual.[6] Localization of recurrent and non-
recurrent CNVs in the genome is of high importance for distinguishing benign and
pathogenic variants, as well as for the identifcation of environmental factors that
potentially are capable of inducing CNVs.
Mounting evidence demonstrates the role of CNVs in various pathological condi-
tions including severe intellectual disability, autism, schizophrenia, cardiovascular
diseases, and cancer.[7–10] However, clinical interpretation of CNVs is still a challeng-
ing task. Fortin et al.[11] identifed CNVs in patients suffering from inherited epilepsy
with febrile seizures plus (GEFS+) syndrome. Deletions in 15q11.2, 19p13.3, and
22q11.2 and duplication in 10q11.22 were detected in four patients from 12 fami-
lies. Nevertheless, the clinical signifcance of these CNVs is diffcult to determine
considering the small cohort and the absence of strong evidence demonstrating the
causative impact of the identifed CNVs. Here intrachromosomal interactions may be
suggested as an underlying cause, as recently shown to play a role in 2q37-deletion
syndrome.[12]
Very large (> 500 kb) and rare (<1%) CNVs are more likely to be detected in
pathological conditions than small and common ones. Nevertheless, the number
of genes within the CNV, their dosage sensitivity, and functions (e.g. protein-
coding or non-coding) are likely the determinants of the clinical signifcance of
CNVs.[13]
Recently, the American College of Medical Genetics and Genomics and the
Clinical Genome Resource have developed guideline for the classifcation and quan-
titative interpretation of constitutional CNVs (inherited and de novo). Thus, there
FISH—Detection of CNVs 173

are fve main categories for postnatal CNVs according to their fnal point values of
evidence scoring metrics: pathogenic (≥0.99), likely pathogenic (0.90–0.98), variant
of uncertain signifcance (−0.89–0.89), likely benign (−0.90 and −0.98), and benign
(≤−0.99).[14] The authors developed a publicly available CNV classifcation calculator
(http://cnvcalc.clinicalgenome.org/cnvcalc/) to facilitate the use of this semi-quanti-
tative system. This tool allows applying points for individual evidence categories for
a given CNV and automatically determines the CNV category. However, the authors
emphasize that these standards cannot be implemented for the acquired CNVs in
neoplasia while CNVs are nearly ubiquitous in cancers. Therefore, identifcation of
clinically relevant CNVs and analysis of their frequency are important issues in can-
cer diagnostics.
Accumulating evidence demonstrates the potential of the environmental muta-
gens and carcinogens to induce de novo CNVs. In particular, DNA damage and/
or DNA replication stress-inducing agents, such as ionizing radiation, asbes-
tos, benzo(a)pyrene, and hydroxyurea are known to cause non-recurrent CNVs in
humans and animal cells.[1] Considering the mechanistic differences between recur-
rent and non-recurrent CNVs it can be assumed that different groups of environmen-
tal mutagens are likely to induce a specifc type of CNVs. Therefore, currently used
genetic endpoints of mutagenicity testing should also include CNVs as a marker of
mutagenic impact. However, the methods of the identifcation of CNVs and their loci
that should be analyzed in the standard genotoxicity testing are frontiers of modern
mutagenesis and clinical genetics.
Currently, fuorescence in situ hybridization (FISH) is the most commonly
used diagnostic tool and the gold standard for the detection of CNVs of differ-
ent genes of clinical importance. Namely, in patients with invasive breast cancer,
FISH analysis for HER2/NEU1 typically demonstrates the clear presence or lack
of HER2/NEU1 amplifcation.[15] However, CNVs in homologous chromosomes
can be used to distinguish otherwise cytogenetically similar homologs from each
other. Moreover, the application of BAC (bacterial artifcial chromosomes) probes
specifc for CNV loci and allows identifcation of the parental origin of the homol-
ogous chromosomes in the offspring. Accordingly, parental-origin-determination-
FISH (pod-FISH) analysis was developed.[16] Analysis of variations in fuorescence
intensities between treated and untreated groups enables the detection of altera-
tions in CNV loci. In afatoxin B1 treated human peripheral blood lymphocytes,
application of pod-FISH revealed losses in 8p21.2 and 15q11.2 chromosome loci,[17]
while in lymphocytes irradiated with AREAL laser-driven electron accelerator
(CANDLE, Armenia), gains were identifed in 1p31.1, 7q11.22, 9q21.3, 10q21.1,
and 16q23.1 chromosome loci.[18]
In this chapter, the semi-automatic quantitative analysis of CNVs by pod-FISH is
described, which can be used for CNV-studies of clinical importance and the inves-
tigation of alterations induced by mutagens in by pod-FISH accessible CNV loci.

SAMPLES/TISSUES/MATERIALS
Pod-FISH allows the detection of CNVs on a single-cell level in any cells; how-
ever, whole blood lymphocytes are most suitable for the analysis of spontaneous or
174 Cytogenetics and Molecular Cytogenetics

induced CNVs, as not only interphases but also metaphases can be evaluated. Slides
for the pod-FISH analysis can be prepared from heparinized whole blood or blood
collected in EDTA containing vacutainers.

HUMAN WHOLE BLOOD CULTURE

• RPMI-1640 medium with L-glutamine (Cat. No. 11875093, Gibco™)
• Fetal bovine serum (Cat. No. F2442, Sigma-Aldrich)
• Penicillin-Streptomycin (Cat. No. P4333, Sigma-Aldrich)
• Phytohemagglutinin (Cat. No. M5030-BC, Merck)

SLIDES WITH METAPHASE CHROMOSOMES

• Colcemid solution (Cat. No. 10295892001, Sigma-Aldrich)
• Potassium chloride (Cat. No. P9541, Sigma-Aldrich)
• Hypotonic solution of KCl: 0.075 M freshly prepared KCl solution used at
37°C
• Methanol 100 % (Cat. No. 34860, Sigma-Aldrich)
• Glacial acetic acid (Cat. No. 1000631011, Sigma-Aldrich)
• Carnoy’s fxative: methanol/glacial acetic acid freshly prepared mix in 3:1
(v/v) ratio and used at −20°C

POD-FISH PROBES
• BACs for the specifc CNV loci[16]
• Nick translation kit (Cat. No. 11745808910, Roche)
• EDTA 0.5 M (Cat. No. 324506, Sigma-Aldrich)
• Ethanol 100 % (Cat. No. 459836, Sigma-Aldrich)
• tRNA (Cat. No. 10109541001, Sigma-Aldrich)
• Sodium acetate buffer solution 3 M, pH 5.2 (Cat. No. S7899, Sigma-Aldrich)
• Dextran sulfate buffer: 2 g + 10 ml 50 % deionized formamide/2×SSC/50
mM phosphate buffer for 3 h, 70°C. Store at −20°C

PRETREATMENT AND POSTFIXATION

• Ethanol solutions: 70 %, 90 % and 100 %
• PBS (Cat. No. 806544, Sigma-Aldrich)
• Hydrochloric acid solution 0.2 M (Cat. No. 13–1720, Sigma-Aldrich)
• Pepsin (Cat. No. P7012, Sigma-Aldrich)
• Pepsin stock solution: 1 g pepsin + 50 ml double-distilled water. Aliquot in
0.5 ml, store at −20°C
• Pepsin pretreatment solution: 5 ml of 0.2 M HCl + 95 ml of distilled water
at 37°C
• Postfx solution: 5 ml 2 % paraformaldehyde + 4.5 ml PBS + 0.5 ml MgCl2
(1 M)
FISH—Detection of CNVs 175

POD-FISH AND WASHING

• COT DNA (Cat. No. 11581074001, Sigma-Aldrich)
• Fluoroshield with DAPI (Cat. No. F6057, Sigma-Aldrich)
• 20×SSC (Cat. No. S6639, Sigma-Aldrich)
• Formamide (Cat. No. F9037, Sigma-Aldrich)
• Tween 20 (Cat. No. P9416, Sigma-Aldrich)
• Denaturation buffer: 70 ml formamide + 10 ml 20×SSC + 20 ml distilled water
• 0.4×SSC solution: 10 ml 20×SSC + 490 ml distilled water
• 4×SSC/Tween solution: 100 ml 20×SSC + 400 ml distilled water + 0.25 ml
Tween 20

DESCRIPTION OF METHODS
Although pod-FISH is applicable for both interphase nuclei and metaphase chro-
mosomes, we recommend analyzing CNV signals in metaphase chromosomes,
which decreases the background noise and provides better resolution for the detec-
tion of small changes in fuorescence intensities of FISH signals. For the analysis of
radiation-induced CNVs, human whole blood should be exposed to radiation imme-
diately after sampling. For the analysis of chemical mutagen-induced CNVs, whole
blood should be cultured for 24 h before exposure.

HUMAN WHOLE BLOOD CULTIVATION

1. Add 1 ml of human whole blood to 9 ml RPMI-1640 medium, containing 10%
fetal bovine serum, 1% penicillin/streptomycin, and 10μg/ml phytohemagglu-
tinin-L, and incubate at 37°C. The total cultivation period should be 72h.
2. After 24 or 48 h growth, add mutagen (e.g. afatoxin B1) to the culture and
incubate at 37°C.

PREPARATION OF METAPHASE SPREADS

1. 1.5 h before harvesting (at 70.5 h of cultivation), add 0.1 ml colcemid to
the culture and incubate at 37°C to block the cell cycle in the metaphase.
2. Transfer the culture into a 15 ml centrifuge tube.
3. Centrifuge at room temperature for 7 min at 1500 rpm. Discard the super-
natant except for about 0.5 mL remaining above the cell pellet, and gently
resuspend the pellet.
4. Add 10 ml of prewarmed (37°C) 0.075 M hypotonic solution of KCl, and
incubate for 15 min at 37°C.
5. Repeat step 3.
6. Quickly add 5 ml of Carnoy’s fxative (−20°C) and vortex immediately to
avoid clumps. Add another 5 ml of Carnoy’s fxative (−20°C), and incu-
bate for 10 min at room temperature.
7. Repeat step 3.
176 Cytogenetics and Molecular Cytogenetics

8. Resuspend the pellet in 5 ml of Carnoy’s fxative (−20°C), and repeat

step 3.
9. Repeat step 8 twice.
10. Add 0.5 ml of Carnoy’s fxative (−20°C), and store the suspension at
−20°C until use.
11. Depending on the density of the suspension, drop (20–50 µl) 2–3 drops
onto the precooled (4°C) clean and humid slides, and let the slide dry on
the warming plate at 51°C.
12. Keep the slides at room temperature for short-term storage, while slides
for long-term storage should be kept at −20°C.

HOMEMADE POD-FISH PROBES

1. The BAC DNA for pod-FISH can be labeled using the corresponding nick
translation kit. In particular, nick translation mix (Cat. No. 11745808910,
Roche) is designed for direct fuorophore-labeling of FISH probes: dilute
1 µg BAC DNA in double-distilled water to get the fnal volume of 12 µl,
and add 4 µl of 5× fuorophore-labeling mix (contains fuorophore-labeled
dUTP) and 4 µl of nick translation mix. The labeled fragments usually have
a 200–500 nucleotide length.
2. Mix the solution using the 20 µl pipette tip and incubate at 15°C for 90 min.
3. Terminate the reaction with 1 µl of 0.5 M EDTA, mix vigorously, spin
down, and incubate at 65°C for 10 min.
4. Precipitate the labeled probe with 10 µl of tRNA + 5 µl sodium acetate +
100 µl 100 % ethanol at −20°C during the overnight.
5. Centrifuge the mixture at 15,000 rpm at 4°C for 15 min.
6. Discard the supernatant and dry the pellet using a speed vac.
7. Dilute the pellet in 20 µl of dextran sulfate buffer, and mix at 65°C for 5 min.
8. Keep the probe at −20°C until use.

SLIDE PRETREATMENT
1. Dehydrate slides in an ethanol series (70 %, 90 %, 100 %, 3 min each) and
air-dry at room temperature.
2. Add 0.5 ml of pepsin to 100 ml of pepsin pretreatment solution, and put
slides for 3–5 min in a Coplin jar at 37°C in the water bath.
3. Wash slides in the PBS for 5 min at room temperature.
4. Add 100 µl of postfx solution, and cover with coverslips for 10 min at room
temperature.
5. Remove coverslips; repeat step 3 and step 1.

POD-FISH PROCEDURE
1. Mix 1 µl of the probe with 7 µl dextran sulfate in the Eppendorf tube con-
taining COT DNA. The optimal concentration of the COT DNA should be
determined experimentally.
FISH—Detection of CNVs 177

2. Prewarm the mixture at 45°C for 20 min.

3. Add 100 µl of denaturation buffer to each slide, cover with coverslip and
incubate on a warming plate at 75°C for 3 min, and in parallel begin the
denaturation of the probe at 75°C.
4. Remove the coverslip and put slides in a 70 % ethanol at −20°C for 3 min.
5. Dehydrate slides in ethanol series of 90 % and 100 % at room temperature
for 3 min each and air-dry.
6. Add 8 µl of the probe onto half of the slide and cover with 24×24 coverslip
and seal with rubber cement.
7. Place the slides in the humid chamber and incubate at 37°C overnight.

WASHING AND COUNTERSTAINING OF SLIDES

1. Remove the rubber cement and coverslips, and put the slides in the Coplin
jar flled with 0.4×SSC at 65°C for 5 min in the water bath.
2. Put the slides in the new Coplin jar with 4×SSC/Tween on the shaker at
room temperature for 5 min.
3. Wash the slides in the PBS for 5 min, then dehydrate in ethanol series (70
%, 90 %, 100 %, 3 min each), and air-dry at room temperature.
4. Add 30 µl of fuoroshield containing DAPI for counterstaining, cover with
24×60 coverslip, and incubate at 4°C for at least 20 min.
5. Analyze the results under a fuorescent microscope.
6. For the analysis of spontaneous CNVs by pod-FISH, at least 20 metaphase
spreads should be investigated. However, for the analysis of mutagen-
induced CNVs, we recommend capturing at least 50 pairs of CNV sig-
nals of homologous chromosomes from each experimental point. This will
increase the statistical power of the analysis and will decrease the back-
ground noise (and/or false-positive variations).

YIELDS
ANALYSIS OF CNVS USING IMAGEJ PROGRAM
1. The fuorescence intensities of CNV signals can be measured using e.g.
the publically available ImageJ program.[19]
2. Capture CNV signals for each fuorochrome and separately save in “.TIF”
image.
3. Activate ImageJ and upload each image in the program by pressing
“Ctrl+O” and selecting images for evaluation.
4. Zoom in the area containing CNV signals by holding “Ctrl” and scrolling
the mouse (Figure 15.1A).
5. Select CNV signals using the “Freehand selection” tool (Figure 15.1B).
6. To obtain a numerical value of the CNV signal, select “Analyze” from the
command bar and select “Measure”, or simply press “Ctrl+M” after the
signal selection step. A “Results table” will occur with numerical values
of mean fuorescence intensity or mean area.
178 Cytogenetics and Molecular Cytogenetics

FIGURE  15.1 Example of the analysis of CNVs using pod-FISH. (A) CNV signals in
14q11.2 chromosome loci (Spectrum Green) are detected by pod-FISH. (B) In the ImageJ
program, CNV signals are selected using the “Freehand selection tool”, and the fuorescence
measurement values are presented in the “Results table”.

7. Repeat steps 5–7 for the second signal from a homologous chromosome.
Thus, the frst pair of signals will be measured, and so on (Figure 15.1B).
8. After measuring signals in the selected group of CNVs (e.g. control), go to
the “File” in the “Results table” and select “Save as” or press “Ctrl+S”.
9. After measuring signals in the control and the treatment groups, statistical
analysis can be performed to compare signal intensities within groups and
between groups.
10. This approach will allow the detection of even those CNVs that are not
detectable by fuorescence microscopy. In addition, the bias by the subjec-
tivity of the researcher visually analyzing the difference between CNV
signal intensities will signifcantly decrease.

CONCLUSIONS
Spontaneous CNVs are an important source of genomic variations and polymor-
phisms. However, de novo CNVs can have pathogenic consequences such as cancer
or neurological disorders. Moreover, induction of DNA strand breaks or inhibition
of DNA replication with environmental mutagens can also induce de novo CNVs
in human and animal cells. Therefore, we recommend including CNVs as a novel
genetic endpoint in mutagenicity testing of environmental factors. Alterations in
molecular-cytogenetically visible CNVs can be measured with the application of
the ImageJ program. Further studies with the application of standard mutagens are
required to establish threshold values of fuorescence intensities of CNVs, as well as
to determine CNV loci that can be useful for the determination of recurrent or non-
recurrent CNV-inducing agents.
FISH—Detection of CNVs 179

ACKNOWLEDGMENTS
This work was supported by the RA MESCS and BMBF (project #AG-01/20), and
RA MESCS (project #21AG-1F068).

REFERENCES
1. Hovhannisyan, G.; Harutyunyan, T.; Aroutiounian, R.; Liehr, T. DNA Copy number
variations as markers of mutagenic impact. Int. J. Mol. Sci. 2019, 20, 4723.
2. Zarrei, M.; MacDonald, J.R.; Merico, D.; Scherer, S.W. A copy number variation map of
the human genome. Nat. Rev. Genet. 2015, 16, 172–183.
3. MacDonald, J.R.; Ziman, R.; Yuen, R.K.; Feuk, L.; Scherer, S.W. The database of
genomic variants: A curated collection of structural variation in the human genome.
Nucleic Acids Res. 2014, 42, D986–D992.
4. Chattopadhyay, A.; Teoh, Z.H.; Wu, C.Y.; Juang, J.J.; Lai, L.C.; Tsai, M.H.; Wu, C.H.;
Lu, T.P.; Chuang, E.Y. CNVIntegrate: The frst multi-ethnic database for identifying
copy number variations associated with cancer. Database (Oxford). 2021, 2021, baab044.
5. Smajlagić, D.; Lavrichenko, K.; Berland, S.; Helgeland, Ø.; Knudsen, G.P.; Vaudel, M.;
Haavik, J.; Knappskog, P.M.; Njølstad, P.R.; Houge, G.; Johansson, S. Population preva-
lence and inheritance pattern of recurrent CNVs associated with neurodevelopmental
disorders in 12,252 newborns and their parents. Eur. J. Hum. Genet. 2021, 29, 205–215.
6. Conover, H.N.; Argueso, J.L. Contrasting mechanisms of de novo copy number muta-
genesis suggest the existence of different classes of environmental copy number muta-
gens. Environ. Mol. Mutagen. 2016, 57, 3–9.
7. Lew, A.R.; Kellermayer, T.R.; Sule, B.P.; Szigeti, K. Copy number variations in adult-
onset neuropsychiatric diseases. Curr. Genomics. 2018, 19, 420–430.
8. Velinov, M. Genomic copy number variations in the autism clinic: Work in progress.
Front. Cell Neurosci. 2019, 13, 57.
9. Prestes, P.R.; Maier, M.C.; Charchar, F.J. DNA copy number variations: Do these big
mutations have a big effect on cardiovascular risk? Int. J. Cardiol. 2020, 298, 116–117.
10. Brezina, S.; Feigl, M.; Gumpenberger, T.; Staudinger, R.; Baierl, A.; Gsur, A. Genome-
wide association study of germline copy number variations reveals an association with
prostate cancer aggressiveness. Mutagenesis. 2020, 35, 283–290.
11. Fortin, O.; Vincelette, C.; Chénier, S.; Ghais, A.; Shevell, M.I.; Simard-Tremblay, E.;
Myers, K.A. Copy number variation in genetic epilepsy with febrile seizures plus. Eur.
J. Paediatr. Neurol. 2020, 27, 111–115.
12. Maass, P.G.; Weise, A.; Rittscher, K.; Lichtenwald, J.; Barutcu, A.R.; Liehr, T.; Aydin,
A.; Wefeld-Neuenfeld, Y.; Pölsler, L.; Tinschert, S.; Rinn, J.L.; Luft, F.C.; Bähring,
S. Reorganization of inter-chromosomal interactions in the 2q37-deletion syndrome.
EMBO J. 2018, 37, e96257.
13. Rice, A.M.; McLysaght, A. Dosage sensitivity is a major determinant of human copy
number variant pathogenicity. Nat. Commun. 2017, 8, 14366.
14. Riggs, E.R.; Andersen, E.F.; Cherry, A.M.; Kantarci, S.; Kearney, H.; Patel, A.; Raca,
G.; Ritter, D.I.; South, S.T.; Thorland, E.C.; Pineda-Alvarez, D.; Aradhya, S.; Martin,
C.L. Technical standards for the interpretation and reporting of constitutional copy-num-
ber variants: A joint consensus recommendation of the American College of Medical
Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet
Med. 2020, 22, 245–257.
180 Cytogenetics and Molecular Cytogenetics

15. Liu, Y.; Wu, S.; Shi, X.; Mao, F.; Zeng, X. Cancer with a HER2 IHC2+ and FISH HER2/
CEP17 ratio ≥2.0 and an average HER2 gene copy number <4.0 per tumor cell: HER2
mRNA overexpression is a rare event. Front. Oncol. 2020, 10, 985.
16. Weise, A.; Gross, M.; Mrasek, K.; Mkrtchyan, H.; Horsthemke, B.; Jonsrud, C.; Von
Eggeling, F.; Hinreiner, S.; Witthuhn, V.; Claussen, U.; Liehr, T. Parental-origin-
determination fuorescence in situ hybridization distinguishes homologous human chro-
mosomes on a single-cell level. Int. J. Mol. Med. 2008, 21, 189–200.
17. Harutyunyan, T.; Hovhannisyan, G.; Babayan, N.; Othman, M.A.; Liehr, T.; Aroutiounian,
R. Infuence of afatoxin B1 on copy number variants in human leukocytes in vitro. Mol.
Cytogenet. 2015, 8, 25.
18. Harutyunyan, T.; Hovhannisyan, G.; Sargsyan, A.; Grigoryan, B.; Al-Rikabi, A.H.;
Weise, A.; Liehr, T.; Aroutiounian, R. Analysis of copy number variations induced by
ultrashort electron beam radiation in human leukocytes in vitro. Mol. Cytogenet. 2019,
12, 18.
19. Iourov, I.Y. Quantitative fuorescence in situ hybridization (QFISH). In: Methods in
Molecular Biology: Cancer Cytogenetics Methods and Protocols; Wan, T.S.K., Ed.
Springer, New York, 2017, pp. 143–149.
 16 FISH—Interphase
Applications
Including Detection
of Chromosome
Instability (CIN)
Ivan Y. Iourov, Svetlana G. Vorsanova
and Yuri B. Yurov

CONTENTS
Introduction ............................................................................................................ 181
Interphase FISH and CIN....................................................................................... 182
Interphase Chromosome-Specifc Multicolor Banding ......................................... 184
Diagnostic Issues ................................................................................................... 184
Conclusions ............................................................................................................ 184
Acknowledgments.................................................................................................. 185
References .............................................................................................................. 185

INTRODUCTION
Fluorescence in situ hybridization (FISH) is a technological basis of interphase
molecular cytogenetics. Interphase FISH is found useful for a wide spectrum of
applications from molecular cytogenetic diagnosis to interphase chromosome biol-
ogy.[1–3] Since a kind of a decrease in interest in interphase cytogenetics is observed
in the postgenomic era,[4, 5] a brief overview of interphase FISH applications seems
to be required.
Molecular cytogenetic analysis is an integral part of the research and medical care
in clinical/medical genetics, reproduction, oncology, neurology, psychiatry, pediatrics.
The signifcance of FISH applications in biomedicine has been consistently reported.[2, 3]
Interphase FISH techniques are currently suggested to be an important technological
element of research in somatic cell genetics/genomics, aging, single-cell biology, chro-
mosome/chromatin biology (i.e. genome organization in interphase).[1, 5]
A more specifc application of FISH is the analysis of chromosome instability
(CIN) in interphase nuclei. The growing importance of CIN analysis in current bio-
medicine results from observations on CIN involvement in genetic intercellular/
DOI: 10.1201/9781003223658-16 181
182 Cytogenetics and Molecular Cytogenetics

interindividual diversity and a variety of pathogenic processes including cancer-

ization, neurodegeneration, and tissue dysfunction.[6–9] Accordingly, addressing
technological issues of FISH analysis of CIN in interphase nuclei may represent an
intriguing part of a description of interphase FISH and related techniques. The pres-
ent chapter describes interphase FISH applications considering the opportunities in
analysis of chromosome abnormalities and instability in interphase nuclei and the
place it deserves in current biomedicine.

INTERPHASE FISH AND CIN

Interphase FISH is generally defned as a set of molecular cytogenetic techniques for
visualization analysis of genomic loci by means of DNA probes. FISH effciency and
resolution are determined by DNA probe properties. Such probes may be composed
of repetitive (e.g. centromeric and telomeric DNA probes), euchromatic (site-spe-
cifc DNA probes), and microdissected DNA (whole-chromosome painting probes
and DNA probes for multicolor banding or partial painting of chromosomes). As in
any kind of FISH-based assays, interphase FISH protocols are essentially composed
of cell suspension preparation, denaturation of sample/probe DNAs, hybridization,
and microscopic evaluation (visual and/or digital) of the results.[10, 11] Since inter-
phase FISH allows visual analysis of DNAs at any cell cycle stage at molecular reso-
lutions, it has become a method of choice for genome analyses at the chromosomal/
subchromosomal level in post-mitotic cells or large mitotic cellular populations.[12, 13]
Moreover, interphase FISH protocols are applicable for studying genome organiza-
tion in single cells.[3, 14, 15]
In the postgenomic era, interphase FISH is an effcient technological platform
for developing assays to analyze CIN and (re-)arrangements of chromosomal loci in
interphase nuclei. Figure 16.1 depicts different interphase FISH assays for studying
CIN in interphase (to show the possibilities of interphase FISH assays for studying
CIN in interphase nuclei, we provided data on the fetal human brain, inasmuch as
this tissue is affected by natural CIN).[16] Thus, FISH analysis with centromeric, chro-
mosome-enumeration, or chromosome-specifc DNA probes is used for detecting
numerical chromosome abnormalities (aneuploidy and polyploidy) (Figure 16.1A–
C), which are probably the most common types of abnormalities resulting in the
occurrence and propagation of CIN.[6, 7, 9, 17] FISH assays with site-specifc probes
for the visualization of genomic loci, usually containing DNA of specifc genes,
are generally applied for identifying structural chromosome imbalances. This type
of interphase FISH techniques is consistently used for specifc rearrangements of
chromosomes in cancers. The ability of this method to map altered genomic loci
provides an opportunity to analyze CIN affecting specifc chromosomal regions in
interphase.[11, 18–20] Several phenomena of chromosomal arrangement in interphase
nuclei may affect the performance of an interphase FISH assay.[1, 3] S phase DNA
replication generally results in a double FISH signal. These observations may be
used for studying DNA replication in interphase and require researcher’s experience
to differ replicative signals from signals showing a chromosome rearrangement.[21]
Associations or pairing of interphase (homologous) chromosomes, which is com-
mon in post-mitotic human cells, may affect the interpretation of FISH results. To
FISH—Interphase Applications and CIN 183

FIGURE  16.1 Interphase molecular cytogenetic analysis CIN in the fetal human brain.
Interphase FISH with chromosome-enumeration DNA probes:
(A) two nuclei characterized by additional chromosomes Y and X and a normal nucleus;
(B) a nucleus with monosomy of chromosome 15 and a normal nucleus;
(C) a nucleus with monosomy of chromosome 18 and a normal nucleus.
(D to G) interphase chromosome-specifc MCB: nuclei with monosomy, disomy, trisomy,
and G-banding ideograms with MCB color-code labeling of a chromosome (from left to
right), (D)—chromosome 9, (E)—chromosome 16, and (F)—chromosome 18. (G) inter-
phase QFISH: (1) a nucleus with two signals for chromosomes 18 (relative intensities: 2058
and 1772 pixels), (2) a nucleus with one paired signal mimics monosomy of chromosome 18
(relative intensity: 4012 pixels), (3) a nucleus with two signals for chromosomes 15 (relative
intensities: 1562 and 1622 pixels), (4) a nucleus with one signal showing monosomy of chro-
mosome 15 (relative intensity: 1678 pixels).
Source: From Yurov et al.,[16] an open-access article distributed under the terms of the Creative Commons
Attribution License.
184 Cytogenetics and Molecular Cytogenetics

solve this problem, quantitative FISH may be applied (Figure 16.1G). This technique
offers an opportunity to differ between chromosomal associations and chromosome
loss (aneuploidy/monosomy).[22–24]

INTERPHASE CHROMOSOME-SPECIFIC MULTICOLOR BANDING

A combination of microdissected DNA probes producing multicolor pseudo-band-
ing of chromosomes by FISH is the basis of multicolor banding (MCB).[25, 26] Using
chromosome-specifc MCB probes, the technique has been adopted for analysis of
interphase chromosomes. The adaptation is the method of interphase chromosome-
specifc MCB (ICS-MCB), a technique allowing the analysis of interphase chromo-
somes in their integrity at molecular resolutions (Figure 16.1D–F).[27, 28] ICS-MCB
has been repeatedly reported to be an effective technique for analysis of nuclear
genome organization at the chromosomal level and CIN in interphase nuclei.[29–34]
Indeed, ICS-MCB is actually a unique technique allowing the visualization of a
structurally rearranged chromosome in interphase.[32]

DIAGNOSTIC ISSUES
In the diagnostic context, the availability of single-cell molecular cytogenetic analy-
sis via visualization is a striking advantage offered by interphase FISH. Additional
advantage offered by FISH are the highest possible cell scoring potential by the
visualization of chromosomal changes in interphase nuclei. Since chromosomal
imbalances and CIN cause a wide spectrum of human morbid conditions, inter-
phase FISH-based methods are to be recognized as a valuable part of molecular
diagnosis.[1–3] It is noteworthy that a diagnosis is referred to as a knowledge about
molecular and/or cellular mechanism of a disease. Consequently, interphase FISH-
based techniques could be an important part of a diagnostic research (diagnosis
and monitoring) of CIN-mediated diseases. Interphase FISH-based study of chro-
mosomal changes may be an addition to pathway-based analysis of genome varia-
tions to model the functional consequences of chromosome imbalances and CIN.
Chromosomal biomarkers (specifc chromosome imbalances or reproducible pat-
terns of CIN) detectable by interphase FISH are to be addressed by systems biology
(bioinformatic) methodology for determining causes and consequences of CIN and
for developing algorithms of unraveling disease mechanisms.[8, 34–37] Finally, com-
bining immunohistochemical detection of proteins and interphase FISH (Immuno-
FISH) offers an opportunity to analyze interphase chromosomes (interphase CIN)
in nuclei of specifc cell types. This is found useful to detect chromosome instability
in post-mitotic tissues and in cancer cells.[31, 32, 38]

CONCLUSIONS
In the postgenomic era, collecting genomic data often lacks chromosomal context.
As a result, such important genetic phenomena/processes as chromosomal abnor-
malities and CIN are still poorly understood.[5, 39] Interphase FISH-based technolo-
gies may help to gain further understanding of causes and consequences of CIN and
FISH—Interphase Applications and CIN 185

chromosomal rearrangements. Using achievements and developments in genomics

and molecular biology, this molecular cytogenetic platform is able to provide new
opportunities in basic and diagnostic research. Mosaic and non-mosaic chromosome
imbalances and CIN are important targets of research in a signifcant number of
biomedical areas. Interphase FISH-based techniques allow the detection and moni-
toring of chromosomal changes in large cellular populations at molecular resolu-
tions and at all cell cycle stages. Therefore, interphase molecular cytogenetics using
FISH-based approaches represent an important part of studying genetics/genomics,
cellular and molecular basis of intercellular or interindividual diversity and dis-
eases. Certainly, interphase FISH should be integrated in sophisticated algorithms
including whole-genome scanning methods (optionally, single-cell whole-genome
analysis), techniques for visualization of nucleic acid molecules at single-cell level
and molecular resolutions, and bioinformatic/systems biology analysis for pathway-
based prioritization of chromosomal/genomic variations.

ACKNOWLEDGMENTS
The chapter is dedicated to Dr. Ilia V Soloviev. Interphase FISH studies in authors’
labs are supported by the Government Assignment of the Russian Ministry of
Science and Higher Education, Assignment no. AAAA-A19–119040490101–6, and
by the Government Assignment of the Russian Ministry of Health, Assignment no.
121031000238–1.

REFERENCES
1. Vorsanova, S.G.; Yurov, Y.B.; Iourov, I.Y. Human interphase chromosomes: A review of
available molecular cytogenetic technologies. Mol. Cytogenet. 2010, 3, 1.
2. Liehr, T. Fluorescence In Situ Hybridization (FISH): Application Guide. Springer,
Berlin; 2017.
3. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Human Interphase Chromosomes: Biomedical
Aspects. 2nd Edition. Springer, Berlin; 2020.
4. Liehr, T. From human cytogenetics to human chromosomics. Int. J. Mol. Sci. 2019, 20,
826.
5. Iourov, I.Y.; Yurov, Y.B.; Vorsanova, S.G. Chromosome-centric look at the genome. In:
Human Interphase Chromosomes: Biomedical Aspects; Iourov, I.Y.; Vorsanova, S.G.;
Yurov, Y.B., Eds. Springer, Berlin, 2020, pp. 157–170.
6. Heng, H.H.; Bremer, S.W.; Stevens, J.B.; Horne, S.D.; Liu, G.; Abdallah, B.Y.; Ye, K.J.;
Ye, C.J. Chromosomal instability (CIN): What it is and why it is crucial to cancer evolu-
tion. Cancer Metastasis Rev. 2013, 32, 325–340.
7. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B.; Kutsev, S.I. Ontogenetic and pathogenetic
views on somatic chromosomal mosaicism. Genes. 2019, 10, 379.
8. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B.; Zelenova, M.A.; Kurinnaia, O.S.; Vasin, K.S.;
Kutsev, S.I. The cytogenomic “theory of everything”: Chromohelkosis may underlie
chromosomal instability and mosaicism in disease and aging. Int. J. Mol. Sci. 2020, 21,
8328.
9. Ye, C.J.; Sharpe, Z.; Heng, H.H. Origins and consequences of chromosomal instability:
From cellular adaptation to genome chaos-mediated system survival. Genes. 2020, 11,
1162.
186 Cytogenetics and Molecular Cytogenetics

10. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Recent patents on molecular cytogenetics.
Recent Pat. DNA Gene Seq. 2008, 2, 6–15.
11. Liehr, T.; Othman, M.A.; Rittscher, K.; Alhourani, E. The current state of molecular
cytogenetics in cancer diagnosis. Expert Rev. Mol. Diagn. 2015, 15, 517–526.
12. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. Single cell genomics of the brain: Focus on
neuronal diversity and neuropsychiatric diseases. Curr. Genomics. 2012, 13, 477–488.
13. Bakker, B.; van den Bos, H.; Lansdorp, P.M.; Foijer, F. How to count chromosomes in a
cell: An overview of current and novel technologies. Bioessays. 2015, 37, 570–577.
14. Manvelyan, M.; Hunstig, F.; Bhatt, S.; Mrasek, K.; Pellestor, F.; Weise, A.; Simonyan, I.;
Aroutiounian, R.; Liehr, T. Chromosome distribution in human sperm: A 3D multicolor
banding-study. Mol. Cytogenet. 2008, 1, 25.
15. McClelland, S.E. Single-cell approaches to understand genome organisation throughout
the cell cycle. Essays Biochem. 2019, 63, 209–216.
16. Yurov, Y.B.; Iourov, I.Y.; Vorsanova, S.G.; Liehr, T.; Kolotii, A.D.; Kutsev, S.I.; Pellestor,
F.; Beresheva, A.K.; Demidova, I.A.; Kravets, V.S.; Monakhov, V.V.; Soloviev, I.V.
Aneuploidy and confned chromosomal mosaicism in the developing human brain. PLoS
One. 2007, 2, e558.
17. Iourov, I.Y.; Yurov, Y.B.; Vorsanova, S.G.; Kutsev, S.I. Chromosome instability, aging
and brain diseases. Cells. 2021, 10, 1256.
18. Liehr, T.; Starke, H.; Weise, A.; Lehrer, H.; Claussen, U. Multicolor FISH probe sets and
their applications. Histol. Histopathol. 2004, 19, 229–237.
19. Devadhasan, J.P.; Kim, S.; An, J. Fish-on-a-chip: A sensitive detection microfuidic sys-
tem for Alzheimer’s disease. J. Biomed. Sci. 2011, 18, 33.
20. Hu, Q.; Maurais, E.G.; Ly, P. Cellular and genomic approaches for exploring structural
chromosomal rearrangements. Chromosome Res. 2020, 28, 19–30.
21. Vorsanova, S.G.; Yurov, Y.B.; Kolotii, A.D.; Soloviev, I.V. FISH analysis of replication
and transcription of chromosome X loci: New approach for genetic analysis of Rett syn-
drome. Brain Dev. 2001, 23, S191–S195.
22. Iourov, I.Y.; Soloviev, I.V.; Vorsanova, S.G.; Monakhov, V.V.; Yurov, Y.B. An approach
for quantitative assessment of fuorescence in situ hybridization (FISH) signals for
applied human molecular cytogenetics. J. Histochem. Cytochem. 2005, 53, 401–408.
23. Vorsanova, S.G.; Iourov, I.Y.; Beresheva, A.K.; Demidova, I.A.; Monakhov, V.V.;
Kravets, V.S.; Bartseva, O.B.; Goyko, E.A.; Soloviev, I.V.; Yurov, Y.B. Non-disjunction
of chromosome 21, alphoid DNA variation, and sociogenetic features of Down syn-
drome. Tsitol. Genet. 2005, 39, 30–36.
24. Iourov, I.Y. Quantitative fuorescence in situ hybridization (QFISH). Methods Mol. Biol.
2017, 1541, 143–149.
25. Liehr, T.; Heller, A.; Starke, H.; Rubtsov, N.; Trifonov, V.; Mrasek, K.; Weise, A.;
Kuechler, A.; Claussen, U. Microdissection based high resolution multicolor banding for
all 24 human chromosomes. Int. J. Mol. Med. 2002, 9, 335–339.
26. Liehr, T.; Othman, M.A.; Rittscher, K. Multicolor karyotyping and fuorescence in situ
hybridization-banding (MCB/mBAND). Methods Mol. Biol. 2017, 1541, 181–187.
27. Iourov, I.Y.; Liehr, T.; Vorsanova, S.G.; Kolotii, A.D.; Yurov, Y.B. Visualization of inter-
phase chromosomes in postmitotic cells of the human brain by multicolour banding
(MCB). Chromosome Res. 2006, 14, 223–229.
28. Iourov, I.Y.; Liehr, T.; Vorsanova, S.G.; Yurov, Y.B. Interphase chromosome-specifc
multicolor banding (ICS-MCB): A new tool for analysis of interphase chromosomes in
their integrity. Biomol. Eng. 2007, 24, 415–417.
29. Lemke, J.; Claussen, J.; Michel, S.; Chudoba, I.; Mühlig, P.; Westermann, M.; Sperling,
K.; Rubtsov, N.; Grummt, U.W.; Ullmann, P.; Kromeyer-Hauschild, K.; Liehr, T.;
FISH—Interphase Applications and CIN 187

Claussen, U. The DNA-based structure of human chromosome 5 in interphase. Am. J.

Hum. Genet. 2002, 71, 1051–1059.
30. Yurov, Y.B.; Iourov, I.Y.; Vorsanova, S.G.; Demidova, I.A.; Kravetz, V.S.; Beresheva,
A.K.; Kolotii, A.D.; Monakchov, V.V.; Uranova, N.A.; Vostrikov, V.M.; Soloviev, I.V.;
Liehr, T. The schizophrenia brain exhibits low-level aneuploidy involving chromo-
some 1. Schizophr. Res. 2008, 98, 139–147.
31. Iourov, I.Y.; Vorsanova, S.G.; Liehr, T.; Yurov, Y.B. Aneuploidy in the normal,
Alzheimer’s disease and ataxia-telangiectasia brain: Differential expression and patho-
logical meaning. Neurobiol. Dis. 2009, 34, 212–220.
32. Iourov, I.Y.; Vorsanova, S.G.; Liehr, T.; Kolotii, A.D.; Yurov, Y.B. Increased chromo-
some instability dramatically disrupts neural genome integrity and mediates cerebellar
degeneration in the ataxia-telangiectasia brain. Hum. Mol. Genet. 2009, 18, 2656–2669.
33. Yurov, Y.B.; Vorsanova, S.G.; Liehr, T.; Kolotii, A.D.; Iourov, I.Y. X chromosome aneu-
ploidy in the Alzheimer’s disease brain. Mol. Cytogenet. 2014, 7, 20.
34. Iourov, I.Y.; Vorsanova, S.G.; Kurinnaia, O.S.; Zelenova, M.A.; Vasin, K.S.; Yurov, Y.B.
Causes and consequences of genome instability in psychiatric and neurodegenerative
diseases. Mol. Biol. 2021, 55, 37–46.
35. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. In silico molecular cytogenetics: A bioinfor-
matic approach to prioritization of candidate genes and copy number variations for basic
and clinical genome research. Mol. Cytogenet. 2014, 7, 98.
36. Liehr, T. Cytogenetically visible copy number variations (CG-CNVs) in banding and
molecular cytogenetics of human; about heteromorphisms and euchromatic variants.
Mol. Cytogenet. 2016, 9, 5.
37. Iourov, I.Y.; Vorsanova, S.G.; Yurov, Y.B. The variome concept: Focus on CNVariome.
Mol. Cytogenet. 2019, 12, 52.
38. Lv, Y.; Mu, N.; Ma, C.; Jiang, R.; Wu, Q.; Li, J.; Wang, B.; Sun, L. Detection value of
tumor cells in cerebrospinal fuid in the diagnosis of meningeal metastasis from lung
cancer by immuno-FISH technology. Oncol. Lett. 2016, 12, 5080–5084.
39. Heng, H.H. New data collection priority: Focusing on genome-based bioinformation.
Res. Results Biomed. 2020, 6, 5–8.
 17 FISH—Determination
of Telomere Length
(Q-FISH/CO-FISH)
Gordana Joksić, Jelena Filipović Tričković
and Ivana Joksić

CONTENTS
Introduction............................................................................................................ 189
Samples/Tissues ..................................................................................................... 191
Description Q-FISH Method.................................................................................. 191
Reagents........................................................................................................ 192
Procedure ...................................................................................................... 192
Description CO-FISH Method............................................................................... 194
Reagents........................................................................................................ 195
Procedure ...................................................................................................... 195
Yields ..................................................................................................................... 196
Q-FISH for Telomere Length ....................................................................... 196
Q-FISH for Telomere Fragility ..................................................................... 196
Co-FISH for Detection of Telomeric Sisterchromatid Exchanges
(T-SCEs) ........................................................................................... 196
Conclusions............................................................................................................ 198
References.............................................................................................................. 198

INTRODUCTION
Telomeres are specialized nucleoprotein structures at the ends of linear chromo-
somes. They consist of a shelterin protein complex and a long assembly of hexameric
repeats (TTAGGG) oriented in 5′ to 3′ direction, ending as a single strand 3′ over-
hang. This 3′ overhang invades 5′ double-stranded telomeric duplex and is housed
deep inside by clipping with shelterin heteroduplex POT1/TTP1. Shelterin sub-
units TRF1 and TRF2 (= telomere binding factors 1 and 2) directly bind the double
stranded TTAGGG, while TIN2 (= TERF1 interacting nuclear factor) links TPP1/
POT1 (= tripeptidyl peptidase 1/protection of telomeres 1) heterodimer and stabilizes
its association with chromosome ends. Shelterin assembly creates a telomere-specifc
structure T-loop (Figure 17.1) that caps the chromosomes and protects their termini
from being recognized as breakpoints by DNA repair mechanisms.[1]

DOI: 10.1201/9781003223658-17 189

190 Cytogenetics and Molecular Cytogenetics

FIGURE 17.1 Telomere T-loop formation.

By possessing at least two active domains in each subunit, shelterins directly

inhibit homologous recombination (HR) and non-homologous end-joining
(NHEJ).[2,  3] For inhibition of other DNA repair pathways, they use accessory pro-
teins that already act in genome maintenance.[4] Telomeres are transcribed into a
class of long noncoding RNA called telomeric repeat-containing RNAs (TERRA),[5]
that actively participates in the regulation of telomere homeostasis. TERRAs are
involved in the formation of telomeric heterochromatin and act as negative regula-
tors of telomerase.[6, 7] Telomeres shorten through each round of replication, and their
length is one of the most important indicators of their function. Telomere shortening
is a part of tumor suppressor mechanisms, whereas telomere length homeostasis
acts as a mitotic clock regulating cellular life-span.[8] Telomere loss can be reversed
by telomerase (in tissues where telomerase is normally active) or by DNA poly-
merase (in somatic cells), forming Okazaki fragments. Reassembling of 3′ overhang
to appropriate length enables correct T-loop formation and adequate telomere func-
tion. If telomeric 3′ overhangs are not regenerated, structural and functional integrity
of T-loop is altered, consequently leading to telomere attrition. Telomere shortening
to a defnite critical length (around 3−5 kb in human cells) leads cells in senes-
cence and additional shortening induces apoptosis.[9] In shortened telomeres under
critical length, the T-loop formation is altered, causing telomere fragility that can
be identifed in metaphase chromosomes by using quantitative fuorescence in situ
hybridization (Q-FISH). Fragile telomeres are seen as multiple or highly extended
telomeric signals at individual chromatid ends, split or lacking the corresponding
signals.[10] Loss of TRF1 is main cause of telomere fragility because their replication
is altered,[11] but telomere dysfunction can be caused by changes in other components
of shelterin complex, structure of telomeric DNA, structure of TERRA, helicases,
or nuclear lamina.[12] Telomere repeats are guanine-rich, which makes them very
susceptible to oxidative damages; therefore telomere dysfunction may be initiated by
oxidative stress shortening.[13] Oxidative lesions of guanine are highly unstable pre-
mutagenic lesions with adenine binding affnity causing transversions, mutations,
thus enhancing telomere fragility.[14] Loss of telomere length maintenance leads to
FISH—Determination of Telomere Length (Q-FISH/CO-FISH) 191

genome instability and is acknowledged as typical feature of carcinogenesis.[15] Most

of the tumor cells upregulate telomerase activity to facilitate telomere elongation.
Initially, assessment of telomere length was done by Southern-blot-based
techniques that gave estimate of average telomere length in population of cells.
Development of Q-FISH enabled measurement of individual chromosome telomere
length in single cells with the resolution of 200 base pairs, assessment of distribution
of telomere length (p-arms vs. q-arms), ratio of shortest/longest telomeres, and telo-
mere fragility. The method utilizes peptide nucleic acid PNA probes that are resistant
to degradation by nucleases and peptidases and show highly specifc hybridization
with DNA.[16] Staining effciency is almost 100%. Quantifcation of telomere length
has been standardized using centromeric PNA probe for chromosome 2 and mea-
surement tools for ISIS software (MetaSystems, Altlussheim, Germany) where cen-
tromere fuorescence is set to a value of 100%.[17] The telomere length is quantifed
from digital images of metaphase spreads and is expressed as telomere/centromere
(T/C) ratio. The rate of fragile telomeres is expressed as the ratio between the num-
ber of fragile telomeres and total number of analyzed metaphases.
Chromosome orientation FISH method (CO-FISH) is used to identify the direc-
tion of a synthetized DNA chains, or particular DNA sequence. Method is used for
identifcation of chromosome inversions associated with isochromosome formation,
pericentric inversions, rate of sister chromatid exchanges at telomeres (T-SCE),[18] or
centromeres (C-SCE).[19] Both variants of CO-FISH contribute to better understand-
ing molecular mechanisms leading to genomic instability.

SAMPLES/TISSUES
Telomere length can be determined in human and other vertebrate cells and should
be done in metaphases. The method is particularly convenient for species that
contain interstitial telomeric sites in their genome, as well as for species that have
ultra-long and heterogeneous telomeres, such as mice. A slide-based method can be
applied to all cells that can be cultivated (primary cells as lymphocytes, fbroblast,
and cell lines).

DESCRIPTION Q-FISH METHOD

Principle of the procedures are as follows: The target DNA (fxed on the slide sur-
face) and Cy3-conjugated PNA probes (telomeric + centromeric) are denatured
together at 80°C for 5 minutes under a coverslip. Hybridization takes place from
30 minutes up to 2 hours at room temperature (RT) in a moist chamber. After
post-hybridization washes and dehydration in ethanol series slides are stained with
DAPI (= 4′,6-diamidino-2-phenylindole)-antifade and covered by glass coverslips.
Analysis is performed using a high-power oil objective of fuorescent microscope
equipped with image capture and software for telomere measurement. The following
flters (excitation/emission) are recommended: 360/340 for the DAPI counterstain
and 550/640 for the Cy3 signals. Positive results are seen as red spots at the end of
each chromosomal arm, and centromere of chromosome 2.
192 Cytogenetics and Molecular Cytogenetics

A standard cell biological and molecular equipment (heating plate, incubator,

microcentrifuge, water bath for washing steps (with adjustable temperature), micro-
pipettes, tips, glass Hellendahl jars (100 ml), glass slides, glass coverslips for fuores-
cence microscopy, fuorescent microscope equipped with flters for Cy3 and DAPI,
digital imaging system and software for telomere length measurement (ISIS soft-
ware (MetaSystems, Altlussheim, Germany)) are necessary to perform Q-FISH.

REAGENTS
• 1×TBS (Tris-Buffered Saline, pH 7.5)—500 ml per experiment
• Pre-treatment stock solution: Dilute 2,000× concentrated proteinase K in
1×PBS
• Pre-treatment solution (working solution): mix 40 µl pre-treatment stock
solution with 80 ml TBS buffer at RT. Prepare fresh for each experiment.
• Telomere and centromere PNA probes ready to use probe (Cy3 conjugated
PNA probes in hybridization solution = 70% formamide) available on
request from manufacturer (DAKO/Agilent)
• Rinse solution ×1—Dilute 50× stock from DAKO/Agilent in pure water and
store at RT
• Wash solution ×1—Dilute 50× stock from DAKO/Agilent in pure water and
warm to 65°C ~2 h before experiment; solution might be milky, but it does
not infuence the results.
• Pure water (deionized or double-distilled water)
• 37% formaldehyde (e.g. Merck)
• Formaldehyde in TBS (working solution): add 8 ml of 37% formaldehyde to
72 ml 1×TBS buffer. Do not use more than four weeks.
• Cold ethanol series; 70%, 85%, 96% (e.g. 4°C in refrigerator)
• Mounting solution: Antifade containing DAPI as a counterstain. To pre-
serve signal intensity, the best is to use Vectashield (VectorLabs) supple-
mented with 0.1 µg/ml of DAPI.

PROCEDURE
1. Prepare metaphase spreads on a glass microscope slides employing stan-
dard cytogenetic procedure. Slide quality is the most important factor for
good hybridization results. Cells should be well fxed and well spread.
2. All following incubations are at RT if not otherwise specifed.
3. Heat working solution of Wash solution to 65oC in a water bath and
pre-worm heating incubator (e.g. Plate Shaker Thermostat PST-100 HL,
Biosan, Riga, Latvia, LV-1067) on 80oC.
4. Prepare six glass Hellendahl jars (100 ml), and fll in ~80 ml each of (i)
TBS, (ii) Formaldehyde in TBS (working solution), (iii) TBS, (iv) Pre-
treatment solution (working solution), (v) TBS, (vi) TBS
5. Immerse slides in TBS (i) for 2 min
6. Transfer and incubate slides in jar (ii) for 2 min exactly
7. Wash slides in TBS (i) and then in TBS (iii) 5 min, each
8. Incubate slides in jar (iv) 10 min
FISH—Determination of Telomere Length (Q-FISH/CO-FISH) 193

9. Wash slides in TBS (v) and then in TBS (vi) 5 min, each
10. Immerse slides in cold ethanol series (70%, 85% and 96%) 3×2 min
11. Airdry slides in a vertical position
12. Add 10 µl of PNA probes/Cy3 to carefully chosen area on the slide
13. Cover the area with a 18×18 mm coverslip
14. Place the slides in the pre-heated incubator to 80°C for 5 min
15. Place the slides in moist chamber (in the dark) at RT for 2 hours
16. Briefy immerse slides in Rinse solution ×1 to remove coverslip
17. Immerse slides in the pre-heated Wash solution ×1 for 5 min at 65°C
18. Repeat steps 11 and 12
19. Apply 2×10 µl of mounting solution onto each slide and put 24×50 mm
coverslip. For the counterstain color to develop, leave the slides 15 min
in the dark. Slides are ready for microscopy. Since a variety of micro-
scopes and image acquisitions systems are used in different laboratories,
the manufacturer manual for use should be followed. Figure 17.2 presents
captured metaphase for telomere length analysis.
20. For analysis of telomere length, using measurement tool for ISIS software
(MetaSystems, Altlussheim, Germany) with centromere fuorescence of
chromosome 2 set to value of 100% is described. Upon capturing, con-
trast and threshold of signals are adjusted; using option of inverse DAPI
staining chromosomes are arranged in karyogram form. Afterwards, the
karyogram is reverted to fuorescence mode. The telomere measurement
software displays two horizontal lines overlaid to each chromosome that
defne the centromere signal on chromosome 2 (red lines) and telomere
signals on p and q arms (green lines) (Figure 17.3).

FIGURE  17.2 Captured metaphase hybridized with Cy3-labeled PNA probe for measure-
ment of telomere length.
194 Cytogenetics and Molecular Cytogenetics

FIGURE  17.3 Fluorescence intensity of centromere signal is set at value of 100%. Each
chromosome arm is bordered with green software line.

DESCRIPTION CO-FISH METHOD

CO-FISH: CO-FISH involve labeling of S-phase synthetized DNA with BrdU and
BrdC during replication. After preparation, and slide making, newly synthetized
DNA chain is removed using Hoechst/UV nicking and enzymatic digestion of newly
synthetized DNA by exonuclease III. After this treatment, only the parental DNA
strands will be detectable. Depending of the cell type, and type of experiment, BrdU
and BrdC should be present one or two rounds of replication. After one round of
labeling, two telomeric signals at each chromosome ends will be visible, whereas
labeling with for two rounds of replication will display only one telomeric signal
per chromosome ends. Accordingly, the FISH-procedure is similar to Q-FISH—
however, to get CO-FISH results, a special sample preparation and handling is
necessary and described next, as outlined frst in the following: Label the S-phase
synthetized DNA with BrdU and BrdC during replication. Split cells in 3:1 ratio
either onto a T25 (= 25 ml culture fask) or T75 (= 75 ml culture fask) fasks. Wait
for 45 minutes until cells are settled in or attached onto the surface. Make BrdU/
BrdC mixture in a ratio 3:1 to achieve a fnal concentration of 10 µM of Brdu/BrdC.
Add colcemid (10 µl/ml) or Colchicine (25 mg/ml) 6–7 hours prior to the 24 hours’
deadline. Cells that pass two rounds of replication will display one telomeric signal
FISH—Determination of Telomere Length (Q-FISH/CO-FISH) 195

per chromosome ends. Activity of homologous replication attempting to extend telo-

meres will be seen as sister-chromatid exchanges; i.e. both chromatid ends will dis-
play the telomeric signal.

REAGENTS
• BrdU/BrdC: Take 15 µl of BrdU and 5 µl of BrdC from stock (50mM conc.)
to make a 3:1 ratio, mix well and add 1 µl of the mixture into 5 ml of T25
fask. N.B.: for a T75 add 3 µl of the mixture to make a fnal concentration
of 10 µM of Brdu/BrdC.
• Exonuclease III (e.g Thermofsher): Take 1.5 µl of stock (200 U/µl) and mix
it with 100 µl of buffer to make a fnal concentration of (3 U/µl)
• 70% formamide: 70ml formamide + 10 ml 20×SSC + 20 ml of ddH2O to
make a fnal volume of 100 ml
• Hoechst 33258: Take 50 µl of stock (0.5 µg/ml) and mix it with 50 ml of
ddH2O in a glass Hellendahl jar.
• Phosphate buffered saline: 1×PBS (e.g. Merck)
• 2×SSC: Dilute 20×SSC (commercially available, e.g. Merck) with ddH2O

PROCEDURE
1. Apply metaphase spread protocol and make slides and “age” them for few
days under room temperature.
2. Put the slides in glass Hellendahl jar comprising working solution of
Hoechst 33258 for 15 minutes at RT.
3. Remove and mount the slide with 2×SSC, and put the glass coverslip over
the each slide. The thin layer of 2×SSC between slide and glass coverslip
should be present during exposure to 365 nm UV light. Expose slides for
30 minutes.
4. Briefy wash the slides with 1×PBS by dipping to remove coverslips.
5. Add 20 µl of Exonuclease III (3 U/µl) onto each slide, and cover them with
paraflm for 10 minutes.
6. Wash the slide in 1×PBS by dipping into a glass Hellendahl jar.
7. Dehydrate the slide by adding 1 ml of 100% ethanol onto slide surface for
1 minute.
8. Airdry the slide.
9. Add 20 µl/slide of PNA probe (Cy3 or FITC- labelled)—and leave it to
hybridize for two minutes at 70ºC in the pre-heated incubator. Keep in wet
and dark containment for two hours.
10. Wash in 70% formamaide for 15 min twice.
11. Wash in 1×PBS for 10 minutes.
12. Dehydrate by serial dehydration (70%, 90% and 100%) with ethanol for
5 minutes.
13. Add 20 µl of DAPI Vectashield onto each slide, and put on 24x50mm
coverslip. For the counterstain color to develop, leave the slides 15 min in
the dark. Slides are ready for microscopy.
196 Cytogenetics and Molecular Cytogenetics

FIGURE 17.4 A) T-SCE staining using CO-FISH. Red arrows indicate for sister chromatid
exchanges at telomeres. B) Metaphase stained using CO-FISH without T-SCE.

14. Since a variety of microscopes and image acquisitions systems are used
in different laboratories, the manufacturer manual for use should be fol-
lowed. Upon capturing, contrast and threshold of signals are adjusted.
Cells that pass two rounds of replication will display one telomeric signal
per chromosome ends. Telomeric sisterchromatid exchanges (T-SCEs) are
seen as two signals on each chromatid (Figure 17.4).

YIELDS
Q-FISH FOR TELOMERE LENGTH
Employing measurement tools, intensity of each telomere signals results are dis-
played graphically and numerically. Results of measurement are expressed as T/C
ratio, i.e. percentage vs. centromere signal for each chromosome arm (Figure 17.5).

Q-FISH FOR TELOMERE FRAGILITY

Fragile telomeres are identifed as duplicated or extended signals on chromatid
arm, missing the signals or fusions (Figure 17.6). Rate of fragile telomeres (fragil-
ity index) is calculated as ratio between the numbers of fragile telomeres/number of
metaphases.

CO-FISH FOR DETECTION OF TELOMERIC SISTERCHROMATID EXCHANGES (T-SCES)

The incidence of T-SCE is expressed as number of chromosome displaying two sig-
nal at each chromosome ends per cell.
FISH—Determination of Telomere Length (Q-FISH/CO-FISH) 197

FIGURE 17.5 Telomere length for each chromosome arm expressed as T/C ratio.

FIGURE 17.6 Telomere fragility: split signals—red arrows; lack of signals—purple arrows;

telomere chromatid fusions—yellow arrow.
198 Cytogenetics and Molecular Cytogenetics

CONCLUSIONS
Telomeres are complex nucleoprotein structures whose integrity determines the sta-
bility of the genome. Many DNA damage repair proteins are located on telomeres.
They are activated upon early DNA damage or directly participate in its repair.
Telomeres shortening during each cell division, having the function of biological
“clock”, leads to elimination of cells with critically shortened telomeres. Rich in
guanine, telomeres are susceptible to guanine oxidative damage. While oxidized
bases are supplemented, break-induced repair can make disruption and elongation of
telomeres inducing genomic instability, which is known as a hallmark of tumor cells.
Molecular biology methods in combination with molecular cytogenetic methods will
certainly in the near future clarify the mechanisms by which telomeres maintain
genomic stability. This will certainly be important for the prevention and treatment
of many diseases.

REFERENCES
1. Blackburn, E.H.; Greider, C.W.; Jack, W.; Szostak, J.W. The nobel prize in physiology or
medicine 2009. Nobel Found. 2009, 1, 10–105.
2. de Lange, T. How telomeres solve the end-protection problem. Science. 2009, 326,
948–952.
3. de Lange, T. Shelterin-mediated telomere protection. Annu. Rev. Genet. 2018, 52,
223–247.
4. Sfeir, A.; de Lange, T. Removal of shelterin reveals the telomere end-protection problem.
Science 2012, 336, 593–597.
5. Deng, Z.; Wang, Z.; Stong, N.; Plasschaert, R.; Moczan, A.; Chen, H.-S.; Hu, S.;
Wikramasinghe, P.; Davuluri, R.V.; Bartolomei, M.S.; Riethman, H.; Lieberman, P.M.
A role for CTCF and cohesin in subtelomere chromatin organization, TERRA transcrip-
tion, and telomere end protection. EMBO J. 2012; 31, 4165–4178.
6. Bettin, N.; Pegorar, C.O.; Cusanelli, E. The emerging roles of TERRA in telomere main-
tenance and genome stability. Cells. 2019, 8, 246.
7. Muraki, K.; Nyhan, K.; Han, L.; Murnane, J. Mechanisms of telomere loss and their
consequences for chromosome instability. Front. Oncol. 2012, 2, 135.
8. Hayfick, L. Living forever and dying in attempt. Exp. Gerontol. 2003, 38, 1231–1241.
9. Tomáška, Ľ.; Cesare, A.J.; AlTurki, T.M.; Griffth, J.D. Twenty years of t-loops: A case
study for the importance of collaboration in molecular biology. DNA Repair (Amst).
2020, 94, 102901.
10. Liu, H.; Xie, Y.; Zhang, Z.; Mao, P.; Liu, J.; Ma, W.; Zhao, Y. Telomerric recombina-
tion induced by DNA damage results in telomere extension and length heterogeneity.
Neoplasia. 2018, 20, 905–916.
11. Martínez, P.; Thanasoula, M.; Muñoz, P.; Liao, C.; Tejera, A.; McNees, C.; McNees, C.;
Flores, J.M.; Fernández-Capetillo, O.; Tarsounas, M.; Blasco, M.A. Increased telomere
fragility and fusions resulting from TRF1 defciency lead to degenerative pathologies
and increased cancer in mice. Genes Dev. 2009, 23, 2060–2075.
12. Kychygina, A.; Dall’Osto, M.; Joshua, A.M.; Cadoret, J.C.; Piras, V.; Picket, H.A.;
Crabbe, L. Progerin impairs 3D genome organization and induces fragile telomeres by
limiting the dNTP pools. Sci. Rep. 2012, 11, 13195.
13. von Zglinicki, T. Oxidative stress shortens telomeres. Trends Biochem. Sci. 2002, 27,
339–344.
FISH—Determination of Telomere Length (Q-FISH/CO-FISH) 199

14. Barnes, R.P.; Fouquerel, E.; Opresko, P.L. The impact of oxidative DNA damage and
stress on telomere homeostasis. Mech. Ageing Dev. 2019, 177, 37–45.
15. Blackburn, E.H. Switching and signaling at the telomere. Cell. 2001, 106, 661–673.
16. Nielsen, P.E.; Egholm, M.; Berg, R.H.; Buchardt, O. Sequence-selective recognition of
DNA by strand displacement with a thymine-substituted polyamide. Science. 1991, 254,
1497–1500.
17. Perner, S.; Brüderlein, S.; Hasel, C.; Waibel, I.; Holdenried, A.; Ciloglu, N.; Chopurian,
H.; Nielsen, K.V.; Plesch, A.; Högel, J.; Möller, P. Quantifying telomere lengths of human
individual chromosome arms by centromere-calibrated fuorescence in situ hybridiza-
tion and digital imaging. Am. J. Pathol. 2003, 163, 1751–1756.
18. Bailey, S.M.; Williams, E.S.; Cornforth, M.N.; Goodwin, E.H. Chromosome orientation
fuorescence in situ hybridization or strand-specifc FISH. Methods Mol. Biol. 2010, 659,
173–183.
19. Giunta, S. Centromere chromosome orientation fuorescent in situ hybridization (Cen-
CO-FISH) detects sister chromatid exchange at the centromere in human cells. Bio.
Protoc. 2018, 8, e2792.
 18 FISH—in Three
Dimensions—3D-FISH
Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 201
Samples/Tissues ..................................................................................................... 202
Description of Methods.......................................................................................... 202
How to Proceed ............................................................................................. 202
Yields ..................................................................................................................... 203
Conclusions ............................................................................................................ 203
References .............................................................................................................. 203

INTRODUCTION
Molecular cytogenetics, particularly fuorescence in situ hybridization (FISH), can
be applied as one of nowadays three major approaches to study the nuclear architec-
ture (= 3D-FISH)[1]; the two other ways to access this problem are based on life-cell
imaging,[2] and long read sequencing,[3] and are not further covered here. However,
all studies applying such methods with the goal to unravel the peculiarities of inter-
phases can be summarized as nucleomics.[1]
Besides high-resolution laser scanning microscopes, it is also possible to use stan-
dard fuorescence microscopes for 3D-FISH studies. Even though such studies can
be performed in normally prepared, fattened nuclei,[4] there is a simple approach to
retain original shape and size of interphases before, during and after FISH-procedure,
as well as during microscopic evaluation, called suspension-FISH (S-FISH).[5]
As summarized elsewhere,[1] nucleomics can be traced back to 1885, when Carl
Rabl suggested that interphase chromosomes are threadlike but separated into indi-
vidual chromosomes.[6] Thus, he already suggested “chromosome territories”, a
wording shaped in 1909 by Theodor Boveri, and Eduard Strasburger at about the
same time.[1] In the 1950s, the inactive X-chromosome in females has been char-
acterized as a stainable Barr-body[7] within the nucleus. And since 1985, there was
evidence that chromosome territories and/or subdomains of them are able to change
shapes and structures during time in cell differentiation.[8] In the last decade fnally,
the next generation and long-read sequencing approaches could identify the topologi-
cally associating domains (TADs) and show that genes do interact in cis and trans
on DNA-level.[4, 9, 10]

DOI: 10.1201/9781003223658-18 201

202 Cytogenetics and Molecular Cytogenetics

According to data obtained during the last decades, in the interphase nucleus the
DNA is protein-covered, and there is never-ending transcription, de- and refolding,
and repair going on.[11] Even though the nucleus is imagined nowadays as fuent,
DNA-compartments of chromosomes, being subunits of the nucleus, are never really
separated,[10 –12] which surprisingly also includes a connection of maternal and pater-
nal genomes via spindles and spindle-pole.[1, 13] Besides, chromosomes are arranged
in a nucleus normally according to size and gene-density; an exception has yet been
seen only in rod cells in retina of nocturnal mammals, where an “inverted” arrange-
ment is needed to support night vision.[1, 14, 15]
Here the S-FISH approach is outlined.[5]

SAMPLES/TISSUES
Any kind of tissue can be used for 3D-FISH-anaylses, as long as intact interphase
nuclei can be accessed in it. However, to keep interphase cells in their original spher-
ical shape, to the best of our knowledge only the S-FISH approach has been reported
yet. For this technique, only cells fxed in Carnoy’s fxative from standard cytoge-
netic preparations are suited.[5]

DESCRIPTION OF METHODS
S-FISH was frst reported in 2002 and was e.g. successfully applied for 3D-FISH
studies of human sperm,[14] or in studied of great ape cells using human probes,[16]
or others like positioning-characterization of small supernumerary marker chro-
mosomes, (derivative) X-chromosomes, or chromosomes involved in chromosomal
rearrangements in leukemia cases.[17]

HOW TO PROCEED
A) Transfer prepared interphase cells from Carnoy’s fxative into hybridiza‑
tion buffer:
1. Centrifuge ~50–100 µl of a concentrated cell suspension diluted in Car-
noy’s fxative (1,500 rpm/4°C/10 min).
2. Wash in 500 μl methanol, incubate for 2 min and centrifuge as in step 1.
3. Wash in 500 μl 0.9% NaCl, incubate for 2 min and centrifuge as in step 1.
4. Incubate in 500 µl pepsin solution (950 μl of distilled water + 50 μl 0.2
N HCl + 5 μl pepsin stock solution) at 37°C for 5 min, and centrifuge as
in step 1.
5. Incubate in 500 μl 0.9% NaCl solution for 2 min at room temperature
(RT), and repeat step 1. Leave 50 μl suspension in the tube.
6. Denature at 95°C/5 min, pellet the cells by centrifugation for 10 min at
1,500 rpm, and repeat step 1. Leave 20 μl suspension in the tube.
7. Prepare a 3× concentrated probe (compared to normal FISH-experiment) in
25 µl hybridization buffer; if necessary add 5–50 μg of COT1 DNA, dena-
ture at 95°C for 5 min, and (if needed) prehybridize at 37°C for 30–60 min.
8. Now add this pre-hybridized probe (step 7) to suspension from step 6.
FISH—in Three Dimensions 203

B) S‑FISH procedure and evaluation:

1. Put vial with cells and hybridization buffer from step A/8 for 12–16 h
(overnight) on 37°C.
C) S‑FISH postwash
1. Add for washing 500 μl 0.4×SSC/68°C/2 min, and repeat step A/1.
2. Add for further washing 500 μl 4×SSC/RT/2 min, and repeat step A/1.
3. Add 150 μl DAPI solution (2 μl DAPI stock solution in 2 ml Vectashield
Antifade) at RT/10 min. Then wash in 500 μl/0.9% NaCl (RT), and
repeat step A/1.
4. Resuspend in 0.5% DAPI Vectashield gel (melted 250 mg agarose per 25
ml 0.9% NaCl at 600 W in a microwave = 1%; 2 ml of this to 2 ml of Vecta-
shield Antifade); transfer at once to a 15 μl well-slide; add a coverslip.
5. A fuorescence microscope being able to acquire Z-stacks is necessary;
Cell-P software (from Olympus) may be used for three-dimensional
analysis of the results.

YIELDS
By including data from electron microscopy not much considered up to now, Joan-
Ramon Daban could do the following statement recently:

Experimental evidence indicates that the chromatin flament is self-organized into a

multilayer planar structure that is densely stacked in metaphase and unstacked in inter-
phase. This chromatin organization is unexpected, but it is shown that diverse supra-
molecular assemblies are multilayered. The mechanical strength of planar chromatin
protects the genome integrity, even when double-strand breaks are produced. Here, it
is hypothesized that the chromatin flament in the loops and topologically associating
domains is folded within the thin layers of the multilaminar chromosomes. It is also
proposed that multilayer chromatin has two states: inactive when layers are stacked
and active when layers are unstacked. Importantly, the well-defned topology of planar
chromatin may facilitate DNA replication without entanglements and DNA repair by
homologous recombination.[18]

CONCLUSIONS
Based on data from nucleomics, some basic puzzle stones of chromosome and
interphase structure seem to be solved. Still, this just opens manifold doors of new
research directions, specifcally in clinical genetics and tumor genetics. Yet there are
just few such examples,[19–21] but more are expected to come.

REFERENCES
1. Liehr, T. Nuclear architecture. In: Cytogenomics; Liehr, T., Ed. Academic Press, New
York, 2021, pp. 297–305.
2. Potlapalli, B.P.; Schubert, V.; Metje-Sprink, J.; Liehr, T.; Houben, A. Application of Tris-
HCl allows the specifc labeling of regularly prepared chromosomes by CRISPR-FISH.
Cytogenett. Genome Res. 2020, 160, 156–165.
204 Cytogenetics and Molecular Cytogenetics

3. Eagen, K.P. Principles of chromosome architecture revealed by Hi-C. Trends Biochem.

Sci. 2018, 43, 469–478.
4. Maass, P.G.; Weise, A.; Rittscher, K.; Lichtenwald, J.; Barutcu, A.R.; Liehr, T.; Aydin,
A.; Wefeld-Neuenfeld, Y.; Pölsler, L.; Tinschert, S.; Rinn, J.L.; Luft, F.C.; Bähring,
S. Reorganization of inter-chromosomal interactions in the 2q37-deletion syndrome.
EMBO J. 2018, 37, e96257.
5. Steinhaeuser, U.; Starke, H.; Nietzel, A.; Lindenau, J.; Ullmann, P.; Claussen, U.; Liehr,
T. Suspension (S)-FISH, a new technique for interphase nuclei. J. Histochem. Cytochem.
2002, 50, 1697–1698.
6. Rabl, C. Über Zelltheilung. Morphologisches Jahrbuch. Gegenbaur, C., Ed. 1885, 10,
214–330.
7. Barr, M.L.; Bertram, L.F.; Lindsay, H.A. The morphology of the nerve cell nucleus,
according to sex. Anat Rec. 1950, 107, 283–297.
8. Blobel, G. Gene gating: A hypothesis. P.N.A.S. U.S.A. 1985, 82, 8527–8529.
9. Roy, S.S.; Mukherjee, A.K.; Chowdhury, S. Insights about genome function from spatial
organization of the genome. Hum. Genomics. 2018, 12, 8.
10. Melo, U.S.; Schöpfin, R.; Acuna-Hidalgo, R.; Mensah, M.A.; Fischer-Zirnsak, B.;
Holtgrewe, M.; Klever, M.K.; Türkmen, S.; Heinrich, V.; Pluym, I.D.; Matoso, E.; de
Sousa, S.B.; Louro, P.; Hülsemann, W.; Cohen, M.; Dufke, A.; Latos-Bieleńska, A.;
Vingron, M.; Kalscheuer, V.; Quintero-Rivera, F.; Spielmann, M.; Mundlos, S. Hi-C
identifes complex genomic rearrangements and TAD-shuffing in developmental dis-
eases. Am. J. Hum. Genet. 2020, 106, 872–884.
11. Cremer, T.; Cremer, M.; Cremer, C. Der Zellkern-eine Stadt in der Zelle: Teil 1:
Chromosomenterritorien und Chromatindomänen. Biol in uns. Zeit. 2016, 46, 290–299.
12. Lemke, J.; Claussen, J.; Michel, S.; Chudoba, I.; Mühlig, P.; Westermann, M.; Sperling,
K.; Rubtsov, N.; Grummt, U. W.; Ullmann, P.; Kromeyer-Hauschild, K.; Liehr, T.;
Claussen, U. The DNA-based structure of human chromosome 5 in interphase. Am. J.
Hum. Genet. 2002, 71, 1051–1059.
13. Weise, A.; Bhatt, S.; Piaszinski, K.; Kosyakova, N.; Fan, X.; Altendorf-Hofmann, A.;
Tanomtong, A.; Chaveerach, A.; De Cioff, M.B.; De Oliveira, E.; Walther, J.U.; Liehr, T.;
Chaudhuri, J. P. Chromosomes in a genome-wise order: Evidence for metaphase archi-
tecture. Mol Cytogenet. 2016, 9, 36.
14. Manvelyan, M.; Hunstig, F.; Bhatt, S.; Mrasek, K.; Pellestor, F.; Weise, A.; Simonyan, I.;
Aroutiounian, R.; Liehr, T. Chromosome distribution in human sperm: A 3D multicolor
banding-study. Mol Cytogenet. 2008, 1, 25.
15. Solovei, I.; Kreysing, M.; Lanctôt, C.; Kösem, S.; Peichl, L.; Cremer, T.; Guck, J.; Joffe,
B. Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolu-
tion. Cell. 2009, 137, 356–368.
16. Manvelyan, M.; Hunstig, F.; Mrasek, K.; Bhatt, S.; Pellestor, F.; Weise, A.; Liehr, T.
Position of chromosomes 18, 19, 21 and 22 in 3D-preserved interphase nuclei of human
and gorilla and white hand gibbon. Mol. Cytogenet. 2008, 1, 9.
17. Liehr, T. Chromosome architecture studied by high-resolution FISH banding in three-
dimensionally preserved human interphase nuclei. In: Human Interphase Chromosomes,
Biomedical Aspects; Iourov, I.; Vorsanova, S.; Yurov, Y., Eds. Springer, Berlin, 2020,
pp. 147–155.
18. Daban, J.R. Supramolecular multilayer organization of chromosomes: Possible func-
tional roles of planar chromatin in gene expression and DNA replication and repair.
FEBS Let. 2020, 594, 395–411.
FISH—in Three Dimensions 205

19. Manvelyan, M.; Kempf, P.; Weise, A.; Mrasek, K.; Heller, A.; Lier, A.; Höffken, K.;
Fricke, H.J.; Sayer, H.G.; Liehr, T.; Mkrtcyhan, H. Preferred co-localization of chromo-
some 8 and 21 in myeloid bone marrow cells detected by three dimensional molecular
cytogenetics. Int. J. Mol. Med. 2009, 24, 335–341.
20. Klonisch, T.; Wark, L.; Hombach-Klonisch, S.; Mai, S. Nuclear imaging in three dimen-
sions: A unique tool in cancer research. Ann. Anat. 2010, 192, 292–301.
21. Timme, S.; Schmitt, E.; Stein, S.; Schwarz-Finsterle, J.; Wagner, J.; Walch, A.; Werner,
M.; Hausmann, M.; Wiech, T. Nuclear position and shape deformation of chromosome 8
territories in pancreatic ductal adenocarcinoma. Analyt. Cell. Pathol. 2011, 34, 21–33.
 19 FISH—On Fibers
Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 207
Samples/Tissues ..................................................................................................... 207
Description of Methods ................................................................................ 208
Yields ..................................................................................................................... 209
Conclusions ............................................................................................................ 209
References .............................................................................................................. 209

INTRODUCTION
Fluorescence in situ hybridization (FISH) on stretched or highly extended chromo-
some fbers is known as Fiber-FISH,[1] or molecular combing.[2] In Figure 19.1, it is
visualized that FISH has a principal resolution from a few dozen to hundred base pairs
(in molecular combing), a few ten- to a few hundred-thousand base pairs (in Fiber-
FISH), a few hundred-thousand base pairs to mega base pairs in interphase-FISH
and mega base pair to chromosome size when accessing metaphases in FISH. Both
high-resolution approaches of FISH—Fiber-FISH,[1] and molecular combing[2]—were
described in the 1990s, and later on used in some specialized labs.[3–5] According to a
PubMed search,[6] molecular combing was applied in research at about the same rates
between 2001 and 2021 (Figure 19.2); on the other hand Fiber-FISH applications in
human samples had a kind of hype between 1994 and 2000 and afterwards declined
(Figure 19.2). In non-human-oriented research, Fiber-FISH has been used in about
the same rates since 1994 (Figure 19.2). While molecular combing has recently been
rediscovered and even commercialized,[7] for Fiber-FISH such a renaissance has not
happened yet.
As Fiber-FISH, also sometimes called nuclear chromatin release, neither needs
sophisticated equipment nor is complicated to perform, here the protocol for this
approach shall be brought back to molecular cytogeneticists’ minds.

SAMPLES/TISSUES
Each cell suspension prepared in Carnoy’s fxative (methanol/acetic acid = 3:1) is
suited as starting material to produce stretched DNA-fbers suited for Fiber-FISH.

DOI: 10.1201/9781003223658-19 207

208 Cytogenetics and Molecular Cytogenetics

FIGURE  19.1 Schematic depiction of resolution of FISH approaches compared to karyo-

typing and chromosome micro array. The approximate resolutions accessible by metaphase-
FISH, interphase-FISH, Fiber-FISH and molecular combing are shown.

FIGURE  19.2 A Pubmed search[6] revealed the number of research papers published
between 1994 and 2021 using molecular combing or Fiber-FISH (broken down in human and
non-human oriented studies).

DESCRIPTION OF METHODS
The Fiber-FISH approach is a modifcation from Fidlerová et  al.,[3] and was pre-
viously published elsewhere as a modifed version.[8] Cytogenetically worked up
material in Carnoy’s fxative (methanol/acetic acid = 3:1), previously being stored at
−20°C for up to several years, may be used.
FISH—On Fibers 209

1. Centrifuge the tube with Carnoy’s fxative (methanol/acetic acid = 3:1),

and cell pellet at 1,000 rpm for 5 min at room temperature (RT), and
replace the supernatant by fresh Carnoy’s fxative and mix the suspension
by inversion of the closed cup.
2. Adjust the number of cells to ~1×106 cells per ml by using a Fuchs-
Rosenthal hemocytometer under a phase contrast microscope.
3. Scratch a ~1.5 cm circle in a clean and fat-free slide using a diamond pen-
cil; and immerse slide in distilled water at RT.
4. Take the prepared slide out of water and clean below and outside the circle
by a tissue.
5. Place ~20 µl of suspension from step 2 within the circle, and let dry for
only 20 sec.
6. Transfer slide immediately into 1×PBS at RT for 30 sec to 1 min, accord-
ing to the suspension.
7. Remove slide from 1×PBS, clean quickly below and outside the circle by
a tissue, and add within the circle 100 µl of 0.5 M NaOH/30% ethanol for
30 sec; this step shall disrupt the nuclear shape and integrity. Do never
allow fuid to leave the region of the marked circle.
8. Add now 100 ml fresh methanol and let air dry at RT—optimally under a
hood.
9. Check dry slide(s) under phase contrast microscope—at this step nuclei
should have a ‘disrupted’ shape and a kind of compact short tail with
DNA-fbers should be visible.
10. Incubate slides at 60°C overnight and store until use in FISH at −20°C.
11. Do standard FISH, but avoid any kind of protein-degrading pretreatment.

YIELDS
The author of this paper used Fiber-FISH to clearly map the CMT1A-REP elements
being ~1.4 Mb apart from each other and the in-between localized PMP22 gene.[5, 8]
Alterations of regions being in range of 100,000 to 1,000,000 base pairs can be opti-
mally accessed by Fiber-FISH using locus-specifc probes.

CONCLUSIONS
In case of necessity to study chromosomal changes at a resolution not accessible any
more by interphase-FISH and being too large to be visualized in molecular comb-
ing, Fiber-FISH is the optimal alternative to correspondingly prepare cytogenetic
material.

REFERENCES
1. Heng, H.H.; Squire, J.; Tsui, L.C. High-resolution mapping of mammalian genes
by in situ hybridization to free chromatin. Proc. Natl. Acad. Sci. U. S. A. 1992, 89,
9509–9513.
210 Cytogenetics and Molecular Cytogenetics

2. Bensimon, A.; Simon, A.; Chiffaudel, A.; Croquette, V.; Heslot, F.; Bensimon, D.
Alignment and sensitive detection of DNA by a moving interface. Science. 1994, 265,
2096–2098.
3. Fidlerová, H.; Senger, G.; Kost, M.; Sanseau, P.; Sheer, D. Two simple procedures for
releasing chromatin from routinely fxed cells for fuorescence in situ hybridization.
Cytogenet. Cell Genet. 1994, 65, 203–205.
4. Heiskanen, M.; Peltonen, L.; Palotif, A. Visual mapping by high resolution FISH. Trends
Genet. 1996, 12, 379–382.
5. Rautenstrauss, B.; Fuchs, C.; Liehr, T.; Grehl, H.; Murakami, T.; Lupski, J.R. Visualization
of the CMT1A duplication and HNPP deletion by FISH on stretched chromosome fbers.
J. Peripher. Nerv. Syst. 1997, 2, 319–322.
6. Pubmed. https://pubmed.ncbi.nlm.nih.gov [accessed on 03/2022].
7. Bisht, P.; Avarello, M.D.M. Molecular combing solutions to characterize replication kinet-
ics and genome rearrangements. In: Cytogenomics; Liehr, T., Ed. Academic Press, New
York, 2021, pp. 47–72.
8. Fuchs, C.; Liehr, T.; Rautenstrauss, B. High-resolution FISH of stretched chromosome
fbers. Trends Genet. 1997, 13, 287.
20 FISH—and Single-Cell
Gel Electrophoresis
Assay (Comet Assay)
Galina Hovhannisyan, Tigran Harutyunyan
and Rouben Aroutiounian

CONTENTS
Introduction............................................................................................................ 212
Comet-FISH.................................................................................................. 212
Main Achievements of Comet-FISH Application......................................... 213
Comet-FISH in Studies for Gene Damage and Repair In Vitro........ 213
Comet-FISH in Studies of Chromosomes and Specifc
Chromosomal Regions In Vitro ......................................... 214
Comet-FISH In Vitro with Padlock and Strand-Specifc Probes ...... 214
Comet-FISH for In Vivo Studies....................................................... 214
Samples/Tissues ..................................................................................................... 215
Description of Methods.......................................................................................... 216
Materials ....................................................................................................... 216
Equipment and Supplies (CA and Comet-FISH).............................. 216
Reagents and Solutions (CA)............................................................ 216
Reagents and Solutions (FISH) ........................................................ 216
Method.......................................................................................................... 216
Cell Samples Preparation.................................................................. 216
Comet Assay ..................................................................................... 217
Comet-FISH...................................................................................... 217
Evaluation ......................................................................................... 217
Yields ..................................................................................................................... 218
Important Notes ............................................................................................ 219
Conclusions............................................................................................................ 219
Acknowledgments.................................................................................................. 219
References.............................................................................................................. 219

DOI: 10.1201/9781003223658-20 211

212 Cytogenetics and Molecular Cytogenetics

INTRODUCTION
The Comet Assay (= CA, also called single cell gel electrophoresis) was developed
in the 1980s as a relatively simple and fast way of detecting DNA damage and
repair at the level of individual cells.[1, 2] The main steps of the CA include fxa-
tion of cell suspension with agarose onto glass microscope slides, lysis of cells to
disrupt membranes and remove histones, and electrophoresis. Negatively charged,
fragmented DNA migrates out of the nucleus in the electric feld more rapidly than
intact DNA, forming a structure resembling the tail of a comet, whereas undam-
aged DNA forms the head of the comet.[3] Images of comets stained with fuores-
cent dyes are analyzed under a microscope using image analysis software. The
percentage of DNA in the tail refects the level of DNA damage in individual
cells.[4] The range of detection is between a few hundred DNA breaks per cell and
a few thousand, encompassing levels of damage that can be repaired and tolerated
by human cells.[5]
CA can be conducted under neutral or alkaline electrophoresis conditions. The
neutral version of CA allows the detection of double-strand breaks. Currently, the
most commonly used is the alkaline CA version, which detects a wider range of dam-
age, including single- and double-strand breaks and alkali-labile sites.[3] The alkaline
CA also identifes DNA lesions that are converted into strand breaks with various
lesion-specifc enzymes.[6] Up to now, twelve enzymes have been used; however, only
the bacterial formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III
(EndoIII), which induce breaks at sites of oxidized purines and pyrimidines, respec-
tively, are used extensively.[6, 7]
The ability to detect DNA damage, induced by genotoxic agents at subtoxic,
physiologically relevant exposures,[8] has contributed to the widespread use of the
CA in environmental biomonitoring and in vitro and in vivo genotoxicity testing in
animals,[9–11] plants,[12] and humans.[5, 13–15]

COMET-FISH
The alkaline CA was combined with fuorescence in situ hybridization (FISH)
to detect DNA damage and repair of specifc DNA sequences. According to Glei
et al.,[16]

the nature of the measured Comet-FISH endpoint precludes us from stating basically
that damage and repair are occurring within the specifc gene, it is at least possible to
evaluate whether the damage and repair are occurring within the vicinity of the gene
of interest.

The FISH protocol has been adapted to the experimental limitations of CA and
was frst applied to comet preparations to determine the spatial distribution of telo-
meres, centromeres, and segments of the O6-methylguanine–DNA methyltransfer-
ase (MGMT) gene in human lymphocytes.[17] To avoid damaging of agarose layer
with cells, the thermal denaturation of DNA in standard FISH was replaced with a
chemical denaturation in Comet-FISH.[17] Protocols of the Comet-FISH technique
FISH—and Comet Assay 213

and their modifcations have been developed for different DNA probes application to
a wide spectrum of biological material.[18–27] Several reviews provide technical and
theoretical aspects of Comet-FISH, limitations as well as advantages of the assay
and examples of its applications.[8, 28–32]

MAIN ACHIEVEMENTS OF COMET-FISH APPLICATION

Comet-FISH studies were carried out using DNA probes for genes, whole chromo-
somes, centromeric and telomeric regions, and other genomic loci, mainly to solve
questions in in vitro, and also in exceptionally for in vivo case studies in human,
animals, and plants. There are also limited examples for application padlock and
strand-specifc FISH probes on comets.

Comet-FISH in Studies for Gene Damage and Repair In Vitro

Among the various genes studied by Comet-FISH, the TP53 gene is the most often
accessed one, because of its important role in the etiology of many tumors and its
association with genome stability.[33] It was shown that the repair rates of TP53 gene
region in gamma-irradiated bladder cancer cell lines RT4 and RT112,[34] and mito-
mycin C-treated RT4 cells,[35] were higher compared to overall genomic DNA. In
addition, TP53 gene region is more rapidly repaired than the hTERT gene region in
gamma-irradiated RT4 and RT112 cells and in normal fbroblasts.[36]
Differences in the baseline levels of genetic instability of TP53 and HER‑2/NEU1
gene loci in the breast cancer cell lines MCF-7, MDA-MB-468, and CRL-2336 rela-
tive to the normal cell lines and preferential repair of TP53 in gamma-irradiated
normal and malignant cells were found.[37] Application of FISH probes for 5′ and 3′
regions of dihydrofolate reductase (DHFR) and MGMT genes in CHO cells, and the
human TP53 gene confrmed faster TP53 gene repair compared with total DNA.[38]
Damage of TP53 and c‑Myc loci was revealed in human lymphocytes treated with
pesticides terbuthylazine and carbofuran[39]; however, no signifcant effects were
observed in lymphocytes, exposed to insecticides chlorpyrifos, imidacloprid, and
α-cypermethrin.[40] The repair of the TP53 and telomerase reverse transcriptase
(hTERT) gene regions was more effcient in TK1+ compared to TK1- clones of the
γ-irradiated Raji lymphoblastoid cell line.[41] It was shown that products of oxidative
stress, such as hydrogen peroxide (H2O2), trans-2-hexenal, and 4-hydroxy-2-nonenal
(HNE), cause DNA damage in genes APC, KRAS, and TP53, relevant for human
colon cancer.[42]
There was no signifcant difference between primary mucosa cells of patients
with carcinoma and controls in susceptibility of the epidermal growth factor recep-
tor (EGFR) gene to benzo[a]pyrene-diolepoxide (BPDE), a major representative of
tobacco-associated carcinogens despite the fact that this gene involved in several
epithelial malignancies,[43] and no increase in BPDE-induced damage to the cyclin
D1 gene, which is of particular importance in in head and neck carcinogenesis.[44]
Inhibitory effect of grapefruit juice on the genotoxicity induced by hydrogen perox-
ide was found in human lymphocytes, based on concentration/time-dependent return
of intact TP53 signals to the head of comets.[45]
214 Cytogenetics and Molecular Cytogenetics

Comet-FISH in Studies of Chromosomes and Specifc

Chromosomal Regions In Vitro
Comet-FISH also appears to be a useful approach for detecting and comparing dam-
age to specifc chromosomal regions of signifcance in leukemogenesis. DNA breaks
at both 5q31 and 11q23 chromosomal regions were identifed in TK6 lymphoblastoid
cells exposed to melphalan, etoposide, or the benzene metabolite, and hydroquinone,
at various concentrations.[46] Signifcantly higher damage levels in chromosomes 3,
5, and 8 compared with chromosome 1 was found in primary mucosa cells harvested
during surgery from patients with and without carcinoma, treated with BPDE.[47]
Results of Comet-FISH showed no difference between the sensitivity of chromo-
some 16 and 13 to mitomycin C and radiation in lymphocytes of Fanconi anemia
(FA) patients, although the FA-A gene is located on chromosome 16.[48]
Telomere fragility was compared in normal human leukocytes and tumor cell
lines treated with cytostatic agents. It was shown that telomeres in CHO and CCRF-
CEM cells were about 2–3 times more sensitive towards BLM than global DNA,
while in HT1080 telomeres were less fragile than total DNA.[49–51]

Comet-FISH In Vitro with Padlock and Strand-Specifc Probes

In addition to conventionally labelled probes to particular DNA sequences, Comet-
FISH was also carried out using padlock probes.[21] According to Shaposhnikov
et al.,[52, 53] ends of padlock probes are designed complementary to adjacent sequences
in the target DNA, so that on hybridization the two ends are juxtaposed and can
readily be ligated to form a circle that is topologically linked to the target DNA.
By amplifying circularized padlock probes through rolling-circle amplifcation in
situ, specifcally reacted probes are copied and then detected by hybridization with
fuorescently labelled complementary DNA molecules. In comet preparations with
padlock probes Alu sequences in HeLa cells,[52, 53] and gene-specifc repair of three
genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum
group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) were ana-
lyzed in human lymphocytes.[54]
CA was also combined with strand-specifc FISH probes, which enables dis-
tinguishing of DNA damage and repair in the transcribed and the non-transcribed
strand.[26] Applying this approach, it has been demonstrated that 8-oxoguanine is
preferentially repaired in the transcribed strand of the ataxia-telangiesctasia mutated
(ATM) gene in UV-irradiated human fbroblasts.[55]

Comet-FISH for In Vivo Studies

Comet-FISH has also been successfully applied to assess genetic instability in vivo.
Analysis of Ret, Abl1 (cAbl), and Trp53 gene fragmentations on comet slides in
peripheral blood cells from irradiated C57BL/6 and CBA/J mice made it possible
to distinguish the effects of all doses as an indicator of DNA damage.[56] Prolonged
exposure to possible carcinogens (phenylhydrazine, ethylene oxide, dichlorometh-
ane, and 1,2-dichloroethane) in pharmaceutical industry workers delays DNA repair
and affects structural integrity of TP53.[57]
FISH—and Comet Assay 215

The possibility of using Comet-FISH in Pacifc oyster Crassostrea gigas was

confrmed using genotoxicant hydrogen peroxide model. The DNA damage was
detected in nucleolar organizer regions (NORs) in oyster’s hemocytes. As bivalves
are widely-used for monitoring environmental pollution in aquatic ecosystems, the
application of Comet-FISH to these organisms can provide additional information on
the nature of the genomic regions affected by genotoxicants.[58]
In plant research, Comet-FISH with DNA probes, detecting FokI element-
containing heterochromatin, NORs, or telomeres was frst applied to Vicia faba.
It was found that after treatment with specifc endonucleases, the distribution of
genomic loci in comet heads and tails refects the expected distribution of the endo-
nuclease-induced breaks.[59] Subsequently, DNA damage induced by maleic hydra-
zide in Crepis capillaris was localized in the genome using FISH with 5S and 25S
rDNA probes.[60] FISH with 5S and 25S rDNA and telomeric/centromeric probes was
applied to comets induced by maleic hydrazide, N-nitroso-N-methylurea (MNU),
and γ-rays in barley.[61] The DNA damage involving the 25S rDNA sequences was
analyzed in maleic hydrazide-treated Crepis capillaris with B chromosomes.[62]
Comet-FISH with the 45S rDNA and telomeric probes was performed in Vitis vinif‑
era L. treated with Cu2+.[63] The data obtained suggests that Comet-FISH in plants
may be a useful tool for environmental monitoring assessment.

SAMPLES/TISSUES
A major advantage of CA is that cells from various tissues of a wide variety of
eukaryotic organisms can be studied.[3, 10, 11, 26, 63, 64] CA slides are typically prepared
from fresh tissue; however, frozen tissue samples can also be applied[65]; in pre-
frozen cells, the background DNA level does not increase and the outcome of the
assay remains unchanged.[66, 67]
In principle, FISH can be applied to any comet slides. To date, Comet-FISH stud-
ies have been done on primary cells, including human,[17, 38–40, 45, 48–50, 54, 57] and mouse
blood,[56] human oropharyngeal mucosa cells,[43, 44, 47] human colon cells from surgi-
cal tissue,[42] and skin fbroblasts from Xeroderma pigmentosum patients.[55]
Furthermore, Comet-FISH studies have been carried out on cell lines, includ-
ing RT4 and RT112 (bladder carcinoma),[34–36] CHO (Chinese hamster ovary),[38, 51]
MCF-7, MDA-MB468, and CRL2336 (breast cancer), GM1310B (lymphoblastoma)
and AG11134 (normal mammary epithelial),[37] HT1080 (human fbrosarcoma) and
CCRF-CEM (human T lymphoblastoma),[51] TK6 (human lymphoblastoma),[46]
GM38 (normal fbroblast) and CSA and CSB (Cockayne syndrome fbroblast),[36] Raji
(human B lymphoblastoma),[41] and HeLa (human breast cancer cells).[52, 53] Moreover,
Comet-FISH was applied to the hemocytes of the Pacifc oyster Crassostrea gigas,[58]
and plant tissues, including seeds,[61] roots,[59] and leaves.[60, 62, 63]
The following protocol provides a detailed description of a standard Comet-FISH
experiment suitable for human blood cells and cell lines. Blood samples can be col-
lected and directly used or cultivated according to the experimental design. Cell
lines should be cultivated and harvested according to standard procedures.
216 Cytogenetics and Molecular Cytogenetics

DESCRIPTION OF METHODS
MATERIALS
Equipment and Supplies (CA and Comet-FISH)
• Glass slides.
• Glass cover slips.
• Incubator (37°C).
• Water bath
• Staining jars.
• Moist chamber.
• Electrophoresis tank (horizontal) and power supply (500 mA, 25 V).
• Fluorescence microscope.
• Image analysis system (e.g., Comet Assay IV, Perceptive Instruments,
Suffolk, UK).

Reagents and Solutions (CA)

• Normal melting point agarose (NMP agarose): 1% in H2O.
• Low melting point agarose (LMP agarose): 0.9% in PBS
• Phosphate buffered saline (PBS).
• Lysis solution: 10 mM Tris-(hydroxymethyl)-aminomethane, pH 7.5, 100
mM Na2EDTA, 2.5 M NaCl, 1% Triton X-100, pH 10, stored at 4°C.
• Electrophoresis solution: 1 mM Na2EDTA, 300 mM NaOH, pH 13.1, stored
at 4°C.
• Neutralization buffer: 0.4 M Tris-HCl, pH 7.5, 0.08 M Tris base, pH 7.2.
• SYBR-Green solution: (1:10,000) (30 μl per slide).

Reagents and Solutions (FISH)

• Telomere PNA FISH Kit/Cy3, or other DNA probes.
• Ethanol 75, 80, 95, and 100%.
• Rinse solution (included in the Telomere PNA FISH kit/Cy3).
• Hybridization buffer (included in the Telomere PNA FISH kit/Cy3). For
other DNA probes, follow the manufacturer’s instructions.
• Post-hybridization washing solution (included in the PNA FISH Kit/Cy3).
For other DNA probes, follow the manufacturer’s instructions.
• Phosphate-buffered detergent (PBD): 94 mM Na2HPO4(2 H2O), 6 mM
Na2HPO4/1 H2O, 0.06% Triton X-100.
• SYBR Green.
• Antifade.
• Counterstaining solution: 1 μl Sybr Green stock solution, 500 μl water, and
500 μl antifade; store in the dark at—20°C in 500 μl aliquots.

METHOD
Cell Samples Preparation
1. Collect human blood samples, use heparin as an anticoagulant. Use any cell
line, depending on the design of the experiment.
FISH—and Comet Assay 217

2. Cultivate the blood cells or cell lines according to standard procedure.

3. Treat cells with test compounds according to experiment design.
4. Get a suspension with concentration of about 1–2×106 cells/ml.

Comet Assay
1. Cover the glass with a layer of 1% normal melting point agarose by dipping
it in a vertical staining jar with melted agarose solution.
2. Dry slides at 37°C about 24 h in cell culture incubator to solidify agarose.
3. Drop 100 µl of cell/low–melting point agarose suspension (containing 10
µl of whole blood or cell line culture with 90 µl 0.9% low melting point
agarose in 1× phosphate buffered saline = PBS) onto microscope slide.
4. Cover slides with cover slips to allow a homogeneous distribution of mix-
ture of cells with low melting point agaroses on microscope slide.
5. Cool slides for 10 min at 4°C to solidify agarose.
6. Remove cover slips and immerse slides in cold lysis solution for 60 min at 4°C.
7. Place slides into an electrophoresis chamber containing cold (4°C) electro-
phoresis solution for 20 min.
8. Connect the electrophoresis tank to the power supply, and perform electro-
phoresis at 1.25 V/cm and 300 mA for 20 min at 4°C.
9. Remove slides from the electrophoresis tank, and wash them once for 20
min in neutralization buffer at room temperature (RT).

Comet-FISH
1. Dehydrate slides in absolute ethanol for at least three days.
2. Rehydrate slides in double-distilled H2O for 15 min.
3. Denature DNA in 0.5 M NaOH for 25 min at RT.
4. Dehydrate slides in an ethanol series (70, 80, and 95%, each for 5 min).
5. Dry slides at RT.
6. Denature telomere PNA probes by preheating to 80°C in water bath for 3
min.
7. Pipette 10 µl denatured probe to an area of approximately 20×20 mm,
cover with coverslip of appropriate size, and seal with rubber cement.
8. Incubate overnight at 37°C in a humid box.
9. Wash slides in prewarmed post-hybridization washing solution (from the
PNA FISH Kit/Cy3) at 65°C in water bath, without agitation for 2.5 min,
and cool the slides immediately in cold 1× phosphate-buffered detergent
(PBD).
10. Follow the supplier’s instructions for denaturation, hybridization, and
post-hybridization washing when using other DNA probes.
11. Counterstain the slides with SYBR Green including 50% antifade (30 μl
per slide), cover with a coverslip 24×60mm, and evaluate the results under
fuorescence microscope.

Evaluation
1. Apply comet analysis software (e.g. Comet Assay IV analysis system,
Perceptive Instruments, Suffolk, UK) for comet images.
218 Cytogenetics and Molecular Cytogenetics

2. Analyze visually the number of FISH signals per comet and the distribution
of FISH signals between head and tail for Comet-FISH images.

YIELDS
Comet-FISH allows the analysis of damage and repair at the level of the overall
DNA, and the localization of damage in specifc loci of the genome on the same
preparations (Figure  20.1). Methodological differences between Comet-FISH and
FISH are aimed at preserving the gel layer, and practice shows that this goal is quite
achievable. The following outputs are expected to be achieved:

1. Assessment of overall DNA damage. This requires an assessment of

%DNA in comet tail.
2. Assessment of loci-specifc DNA damage. The DNA locus can be con-
sidered intact if FISH signal is located in the head of a comet or damaged
if it is located in the tail of a comet. Quantifcation of damage in specifc
domains can be expressed as the percent of spots in heads and tails.
3. Assessment of overall DNA repair. This requires an assessment of
decrease of %DNA in tail over time.
4. Assessment of loci-specifc DNA repair. Reverse shift of fuorescent sig-
nals from the comet tail to the head over time corresponds to the repair of
a given DNA region. Quantifcation of repair in specifc domains can be
expressed as the percent of spots in heads and tails.
5. Comparison of damage and repair of specifc loci and whole DNA.
Comet-FISH allows to compare the rates of damage and repair of specifc
loci and whole DNA since both of these events can be tracked simultane-
ously in the same cell; thus, it is possible that individual loci of genome or
chromosomes can be damaged more or less frequent than the entire DNA.

FIGURE  20.1 The illustration shows the green-colored nucleus of a bleomycin-treated

human leukocyte in the form of a comet, which is obtained after the migration of damaged
DNA to the anode during electrophoresis. Red dots on the comet’s “head” and “tail” represent
fuorescently colored telomeres.
FISH—and Comet Assay 219

IMPORTANT NOTES
• The FISH signal is able to migrate to the comet’s tail if the break occurs
not only within a specifc loci of the genome, but also in the vicinity of the
area of interest.[16]
• Certain genes or parts of genes or other loci of the genome remain in the
head of comet even when there is a DNA break nearby, and it is likely that
this localization within the head refects the presence nearby of scaffold- or
matrix-associated areas.[16]
• Cells immersed in gel retain their minimally deformed three-dimensional
structure. However, the location of cells at different depths in the gel sug-
gests special attention when analyzing images,[28] and even does not exclude
the possibility that some signals immersed deep in the gel may be invisible.

CONCLUSIONS
FISH allows detecting genomic regions of interest immediately on comet prepara-
tions. Application of FISH on comets is based on replacing the thermal denaturation
of DNA with an alkaline one to preserve the structure of the gel in which the cells
are embedded. In that way, global genomic and sequence-specifc DNA damage and
repair of cells from almost any tissue can be comparatively investigated.
Comet-FISH studies were carried out using DNA probes for whole chromosomes,
specifc chromosome regions, centromeres, telomeres, genes, and any other genome
loci. Various examples of the application of the method on human, animal, and plant
cells confrmed sensitivity and the usefulness of this approach in genotoxicity testing
and biomonitoring.

ACKNOWLEDGMENTS
This work was supported by the RA MESCS and BMBF (project #AG-01/20), and
RA MESCS (project #21AG-1F068).

REFERENCES
1. Ostling, O.; Johanson, K.J. Microelectrophoretic study of radiation-induced DNA dam-
ages in individual mammalian cells. Biochem Biophys Res Commun. 1984, 123(1),
291–298.
2. Singh, N.P.; McCoy, M.T.; Tice, R.R.; Schneider, E.L. A simple technique for quan-
titation of low levels of DNA damage in individual cells. Exp Cell Res. 1988, 175(1),
184–191.
3. Cordelli, E.; Bignami, M.; Pacchierotti, F. Comet assay: A versatile but complex tool in
genotoxicity testing. Toxicol Res (Camb). 2021, 10(1), 68–78.
4. Karbaschi, M.; Ji, Y.; Abdulwahed, A.M.S.; Alohaly, A.; Bedoya, J.F.; Burke, S.L.;
Boulos, T.M.; Tempest, H.G.; Cooke, M.S. Evaluation of the major steps in the conven-
tional protocol for the alkaline comet assay. Int J Mol Sci. 2019, 20(23), 6072.
5. Azqueta, A.; Muruzabal, D.; Boutet-Robinet, E.; Milic, M.; Dusinska, M.; Brunborg, G.;
Møller, P.; Collins, A.R. Technical recommendations to perform the alkaline standard
220 Cytogenetics and Molecular Cytogenetics

and enzyme-modifed comet assay in human biomonitoring studies. Mutat Res Genet
Toxicol Environ Mutagen. 2019, 43, 24–32.
6. Muruzabal, D.; Collins, A.; Azqueta, A. The enzyme-modifed comet assay: Past, pres-
ent and future. Food Chem Toxicol. 2021, 147, 111865.
7. Muruzabal, D.; Sanz-Serrano, J.; Sauvaigo, S.; Treillard, B.; Olsen, A.K.; López de
Cerain, A.; Vettorazzi, A.; Azqueta, A. Validation of the in vitro comet assay for DNA
cross-links and altered bases detection. Arch Toxicol. 2021, 95(8), 2825–2838.
8. Spivak, G.; Cox, R.A.; Hanawalt, P.C. New applications of the Comet assay: Comet-
FISH and transcription-coupled DNA repair. Mutat Res. 2009, 681(1), 44–50.
9. de Lapuente, J.; Lourenço, J.; Mendo, S.A.; Borràs, M.; Martins, M.G.; Costa, P.M.;
Pacheco, M. The Comet assay and its applications in the feld of ecotoxicology: A mature
tool that continues to expand its perspectives. Front Genet. 2015, 4(6), 180.
10. Gajski, G.; Žegura, B.; Ladeira, C.; Pourrut, B.; Del Bo’, C.; Novak, M.; Sramkova, M.;
Milić, M.; Gutzkow, K.B.; Costa, S.; Dusinska, M.; Brunborg, G.; Collins, A. The comet
assay in animal models: From bugs to whales: (Part 1 Invertebrates). Mutat Res. 2019,
779, 82–113.
11. Gajski, G.; Žegura, B.; Ladeira, C.; Novak, M.; Sramkova, M.; Pourrut, B.; Del Bo’, C.;
Milić, M.; Gutzkow, K.B.; Costa, S.; Dusinska, M.; Brunborg, G.; Collins, A. The comet
assay in animal models: From bugs to whales: (Part 2 Vertebrates). Mutat Res Rev Mutat
Res. 2019, 781, 130–164.
12. Pietrini, F.; Iannilli, V.; Passatore, L.; Carloni, S.; Sciacca, G.; Cerasa, M.; Zacchini, M.
Ecotoxicological and genotoxic effects of dimethyl phthalate (DMP) on Lemna minor
L. and Spirodela polyrhiza (L.) Schleid. plants under a short-term laboratory assay. Sci
Total Environ. 2022, 1;806(Pt 4), 150972.
13. Azqueta, A.; Ladeira, C.; Giovannelli, L.; Boutet-Robinet, E.; Bonassi, S.; Neri, M.;
Gajski, G.; Duthie, S.; Del Bo’, C.; Riso, P.; Koppen, G.; Basaran, N.; Collins, A.; Møller,
P. Application of the comet assay in human biomonitoring: An hCOMET perspective.
Mutat Res Rev Mutat Res. 2020, 783, 108288.
14. Bonassi, S.; Ceppi, M.; Møller, P.; Azqueta, A.; Milić, M.; Neri, M.; Brunborg, G.;
Godschalk, R.; Koppen, G.; Langie, S.A.S.; Teixeira, J.P.; Bruzzone, M.; Da Silva,
J.; Benedetti, D.; Cavallo, D.; Ursini, C.L.; Giovannelli, L.; Moretti, S.; Riso, P.; Del
Bo’, C.; Russo, P.; Dobrzyńska, M.; Goroshinskaya, I.A.; Surikova, E.I.; Staruchova,
M.; Barančokova, M.; Volkovova, K.; Kažimirova, A.; Smolkova, B.; Laffon, B.;
Valdiglesias, V.; Pastor, S.; Marcos, R.; Hernández, A.; Gajski, G.; Spremo-Potparević,
B.; Živković, L.; Boutet-Robinet, E.; Perdry, H.; Lebailly, P.; Perez, C.L.; Basaran, N.;
Nemeth, Z.; Safar, A.; Dusinska, M.; Collins, A. hCOMET project. DNA damage in
circulating leukocytes measured with the comet assay may predict the risk of death. Sci
Rep. 2021, 11(1), 16793.
15. Milić, M.; Ceppi, M.; Bruzzone, M.; Azqueta, A.; Brunborg, G.; Godschalk, R.; Koppen,
G.; Langie, S.; Møller, P.; Teixeira, J.P.; Alija, A.; Anderson, D.; Andrade, V.; Andreoli,
C.; Asllani, F.; Bangkoglu, E.E.; Barančoková, M.; Basaran, N.; Boutet-Robinet, E.;
Buschini, A.; Cavallo, D.; Costa Pereira, C.; Costa, C.; Costa, S.; Da Silva, J.; Del Boˊ, C.;
Dimitrijević Srećković, V.; Djelić, N.; Dobrzyńska, M.; Duračková, Z.; Dvořáková, M.;
Gajski, G.; Galati, S.; García Lima, O.; Giovannelli, L.; Goroshinskaya, I.A.; Grindel,
A.; Gutzkow, K.B.; Hernández, A.; Hernández, C.; Holven, K.B.; Ibero-Baraibar,
I.; Ottestad, I.; Kadioglu, E.; Kažimirová, A.; Kuznetsova, E, Ladeira, C, Laffon, B,
Lamonaca, P, Lebailly, P, Louro, H, Mandina Cardoso, T, Marcon, F.; Marcos, R.;
Moretti, M.; Moretti, S.; Najafzadeh, M.; Nemeth, Z.; Neri, M.; Novotna, B.; Orlow, I.;
Paduchova, Z.; Pastor, S.; Perdry, H.; Spremo-Potparević, B.; Ramadhani, D.; Riso, P.;
Rohr, P.; Rojas, E.; Rossner, P.; Safar, A.; Sardas, S.; Silva, M.J.; Sirota, N.; Smolkova,
FISH—and Comet Assay 221

B.; Staruchova, M.; Stetina, R.; Stopper, H.; Surikova, E.I.; Ulven, S.M.; Ursini, C.L.;
Valdiglesias, V.; Valverde, M.; Vodicka, P.; Volkovova, K.; Wagner, K.H.; Živković, L.;
Dušinská, M.; Collins, A.R.; Bonassi, S. The hCOMET project: International database
comparison of results with the comet assay in human biomonitoring. Baseline frequency
of DNA damage and effect of main confounders. Mutat Res Rev Mutat Res. 2021, 787,
108371.
16. Glei, M.; Hovhannisyan, G.; Pool-Zobel, B.L. Use of Comet-FISH in the study of DNA
damage and repair: Review. Mutat Res. 2009, 681(1), 33–43.
17. Santos, S.J.; Singh, N.P.; Natarajan, A.T. Fluorescence in situ hybridization with comets.
Exp Cell Res. 1997, 232(2), 407–411.
18. Rapp, A.; Hausmann, M.; Greulich, K.O. The comet-FISH technique: A tool for detec-
tion of specifc DNA damage and repair. Methods Mol Biol. 2005, 291, 107–119.
19. Spivak, G. The Comet-FISH assay for the analysis of DNA damage and repair. Methods
Mol Biol. 2010, 659, 129–145.
20. Shaposhnikov, S.; Thomsen, P.D.; Collins, A.R. Combining fuorescent in situ hybridiza-
tion with the comet assay for targeted examination of DNA damage and repair. Methods
Mol Biol. 2011, 682, 115–132.
21. Henriksson, S.; Nilsson, M. Padlock probes and rolling circle amplifcation for detection
of repeats and single-copy genes in the single-cell comet assay. Methods Mol Biol. 2012,
853, 95–103.
22. Schlörmann, W.; Glei, M. Detection of DNA damage by comet fuorescence in situ
hybridization. Methods Mol Biol. 2012, 920, 91–100.
23. Laubenthal, J.; Anderson, D. Fluorescence in situ hybridization on electrophoresed cells
to detect sequence specifc DNA damage. Methods Mol Biol. 2013, 1054, 219–235.
24. Glei, M.; Schlörmann, W. Analysis of DNA damage and repair by comet fuorescence in
situ hybridization (Comet-FISH). Methods Mol Biol. 2014, 1094, 39–48.
25. Shaposhnikov, S.; El Yamani, N.; Collins, A.R. Fluorescent in situ hybridization on com-
ets: FISH comet. Methods Mol Biol. 2015, 1288, 363–373.
26. Mondal, M.; Guo, J. Comet-FISH for ultrasensitive strand-specifc detection of DNA
damage in single cells. Methods Enzymol. 2017, 591, 83–95.
27. Hovhannisyan, G.; Aroutiounian, R. Comet-FISH In: Fluorescence In Situ Hybridization
(FISH), Application Guide; Liehr, T., Ed. 2nd Edition. Springer, Berlin, 2017,
pp. 373–378.
28. Baugh, E.H.; Ke, H.; Levine, A.J.; Bonneau, R.A.; Chan, C.S. Why are there hotspot
mutations in the TP53 gene in human cancers? Cell Death Differ. 2018, 25(1),
154–160.
29. McKenna, D.J.; Rajab, N.F.; McKeown, S.R.; McKerr, G.; McKelvey-Martin, V.J. Use
of the comet-FISH assay to demonstrate repair of the TP53 gene region in two human
bladder carcinoma cell lines. Radiat Res. 2003, 159(1), 49–56.
30. McKenna, D.J.; Gallus, M.; McKeown, S.R.; Downes, C.S.; McKelvey-Martin, V.J.
Modifcation of the alkaline Comet assay to allow simultaneous evaluation of mitomycin
C-induced DNA cross-link damage and repair of specifc DNA sequences in RT4 cells.
DNA Repair (Amst). 2003, 2(8), 879–890.
31. McKenna, D.J.; Doherty, B.A.; Downes, C.S.; McKeown, S.R.; McKelvey-Martin, V.J.
Use of the comet-FISH assay to compare DNA damage and repair in p53 and hTERT
genes following ionizing radiation. PLoS One. 2012, 7(11), e49364.
32. Shaposhnikov, S.; Frengen, E.; Collins, A.R. Increasing the resolution of the comet assay
using fuorescent in situ hybridization: A review. Mutagenesis. 2009, 24(5), 383–389.
33. Glei, M.; Hovhannisyan, G.; Pool-Zobel, B.L. Use of Comet-FISH in the study of DNA
damage and repair: Review. Mutat Res. 2009, 681(1), 33–43.
222 Cytogenetics and Molecular Cytogenetics

34. Hovhannisyan, G.G. Fluorescence in situ hybridization in combination with the comet
assay and micronucleus test in genetic toxicology. Mol Cytogenet. 2010, 3, 17.
35. Azqueta, A.; Collins, A.R. The essential comet assay: A comprehensive guide to measur-
ing DNA damage and repair. Arch Toxicol. 2013, 87(6), 949–968.
36. Spivak, G. New developments in comet-FISH. Mutagenesis. 2015, 30(1), 5–9.
37. Kumaravel, T.S.; Bristow, R.G. Detection of genetic instability at HER-2/neu and p53
loci in breast cancer cells sing Comet-FISH. Breast Cancer Res Treat. 2005, 91(1), 89–93.
38. Horváthová, E.; Dusinská, M.; Shaposhnikov, S.; Collins, A.R. DNA damage and repair
measured in different genomic regions using the comet assay with fuorescent in situ
hybridization. Mutagenesis. 2004, 19(4), 269–276.
39. Mladinic, M.; Zeljezic, D.; Shaposhnikov, S.A.; Collins, A.R. The use of FISH-comet
to detect c-Myc and TP 53 damage in extended-term lymphocyte cultures treated with
terbuthylazine and carbofuran. Toxicol Lett. 2012, 211(1), 62–69.
40. Zeljezic, D.; Vinkovic, B.; Kasuba, V.; Kopjar, N.; Milic, M.; Mladinic, M. The effect of
insecticides chlorpyrifos, α-cypermethrin and imidacloprid on primary DNA damage,
TP 53 and c-Myc structural integrity by comet-FISH assay. Chemosphere. 2017, 182,
332–338.
41. McAllister, K.A.; Yasseen, A.A.; McKerr, G.; Downes, C.S.; McKelvey-Martin, V.J.
FISH comets show that the salvage enzyme TK1 contributes to gene-specifc DNA
repair. Front Genet. 2014, 5, 233.
42. Glei, M.; Schaeferhenrich, A.; Claussen, U.; Kuechler, A.; Liehr, T.; Weise, A.; Marian,
B.; Sendt, W.; Pool-Zobel, B.L. Comet fuorescence in situ hybridization analysis for
oxidative stress-induced DNA damage in colon cancer relevant genes. Toxicol Sci. 2007,
96(2), 279–284.
43. Reiter, M.; Welz, C.; Baumeister, P.; Schwenk-Zieger, S.; Harréus, U. Mutagen sensitivity
and DNA repair of the EGFR gene in oropharyngeal cancer. Oral Oncol. 2010, 46(7),
519–524.
44. Reiter, M.; Baumeister, P.; Hartmann, M.; Schwenk-Zieger, S.; Harréus, U.
Chemoprevention by celecoxib and mutagen sensitivity of cyclin d1 in patients with oro-
pharyngeal carcinoma. In Vivo. 2014, 28(1), 49–53.
45. Razo-Aguilera, G.; Baez-Reyes, R.; Alvarez-González, I.; Paniagua-Pérez, R.; Madrigal-
Bujaidar, E. Inhibitory effect of grapefruit juice on the genotoxicity induced by hydrogen
peroxide in human lymphocytes. Food Chem Toxicol. 2011, 49(11), 2947–2953.
46. Escobar, P.A.; Smith, M.T.; Vasishta, A.; Hubbard, A.E.; Zhang, L. Leukaemia-specifc
chromosome damage detected by comet with fuorescence in situ hybridization (comet-
FISH). Mutagenesis. 2007, 22(5), 321–327.
47. Harréus, U.A.; Kleinsasser, N.H.; Zieger, S.; Wallner, B.; Reiter, M.; Schuller, P.;
Berghaus, A. Sensitivity to DNA-damage induction and chromosomal alterations in
mucosa cells from patients with and without cancer of the oropharynx detected by a
combination of Comet assay and fuorescence in situ hybridization. Mutat Res. 2004,
563(2), 131–138.
48. Mohseni Meybodi, A.; Mozdarani, H. DNA damage in leukocytes from Fanconi ane-
mia (FA) patients and heterozygotes induced by mitomycin C and ionizing radiation as
assessed by the comet and comet-FISH assay. Iran Biomed J. 2009, 13(1), 1–8.
49. Arutyunyan, R.; Gebhart, E.; Hovhannisyan, G.; Greulich, K.O.; Rapp, A. Comet-FISH
using peptide nucleic acid probes detects telomeric repeats in DNA damaged by bleomy-
cin and mitomycin C proportional to general DNA damage. Mutagenesis. 2004, 19(5),
403–408.
50. Arutyunyan, R.; Rapp, A.; Greulich, K.O.; Hovhannisyan, G.; Haroutiunian, S.; Gebhart,
E. Fragility of telomeres after bleomycin and cisplatin combined treatment measured in
human leukocytes with the Comet-FISH technique. Exp Oncol. 2005, 27(1), 38–42.
FISH—and Comet Assay 223

51. Hovhannisyan, G.; Rapp, A.; Arutyunyan, R.; Greulich, K.O.; Gebhart, E. Comet-assay
in combination with PNA-FISH detects mutagen-induced DNA damage and specifc
repeat sequences in the damaged DNA of transformed cells. Int J Mol Med. 2005, 15(3),
437–442.
52. Shaposhnikov, S.; Azqueta, A.; Henriksson, S.; Meier, S.; Gaivão, I.; Huskisson, N.H.;
Smart, A.; Brunborg, G.; Nilsson, M.; Collins, A.R. Twelve-gel slide format optimised
for comet assay and fuorescent in situ hybridisation. Toxicol Lett. 2010, 195(1), 31–34.
53. Shaposhnikov, S.; Larsson, C.; Henriksson, S.; Collins, A.; Nilsson, M. Detection of
Alu sequences and mtDNA in comets using padlock probes. Mutagenesis. 2006, 21(4),
243–247.
54. Henriksson, S.; Shaposhnikov, S.; Nilsson, M.; Collins, A. Study of gene-specifc DNA
repair in the comet assay with padlock probes and rolling circle amplifcation. Toxicol
Lett. 2011, 202(2), 142–147.
55. Guo, J.; Hanawalt, P.C.; Spivak, G. Comet-FISH with strand-specifc probes reveals
transcription-coupled repair of 8-oxoGuanine in human cells. Nucleic Acids Res. 2013,
41(16), 7700–7712.
56. Amendola, R.; Basso, E.; Pacifci, P.G.; Piras, E.; Giovanetti, A.; Volpato, C.; Romeo, G.
Ret, Abl1 (cAbl) and Trp53 gene fragmentations in comet-FISH assay act as in vivo bio-
markers of radiation exposure in C57BL/6 and CBA/J mice. Radiat Res. 2006, 165(5),
553–5561.
57. Zeljezic, D.; Mladinic, M.; Kopjar, N.; Radulovic, A.H. Evaluation of genome damage
in subjects occupationally exposed to possible carcinogens. Toxicol Ind Health. 2016,
32(9), 1570–1580.
58. Pérez-García, C.; Rouxel, J.; Akcha, F. Development of a comet-FISH assay for the
detection of DNA damage in hemocytes of Crassostrea gigas. Aquat Toxicol. 2015, 161,
189–195.
59. Menke, M.; Angelis, K.J.; Schubert, I. Detection of specifc DNA lesions by a combina-
tion of comet assay and FISH in plants. Environ Mol Mutagen. 2000, 35(2), 132–138.
60. Kwasniewska, J.; Grabowska, M.; Kwasniewski, M.; Kolano, B. Comet-FISH with rDNA
probes for the analysis of mutagen-induced DNA damage in plant cells. Environ Mol
Mutagen. 2012, 53(5), 369–375.
61. Kwasniewska, J.; Kwasniewski, M. Comet-FISH for the evaluation of plant DNA damage
after mutagenic treatments. J Appl Genet. 2013, 54(4), 407–415.
62. Kwasniewska, J.; Mikolajczyk, A. Infuence of the presence of B chromosomes on DNA
damage in Crepis capillaris. PLoS One. 2014, 9(1), e87337.
63. Castro, C.; Carvalho, A.; Gaivão, I.; Lima-Brito, J. Evaluation of copper-induced DNA
damage in Vitis vinifera L. using Comet-FISH. Environ Sci Pollut Res Int. 2021, 28(6),
6600–6610.
64. Russo, C.; Acito, M.; Fatigoni, C.; Villarini, M.; Moretti, M. B-Comet Assay (Comet
Assay on buccal cells) for the evaluation of primary DNA damage in human biomonitor-
ing studies. Int J Environ Res Public Health. 2020, 17(24), 9234.
65. Hobbs, C.A.; Recio, L.; Winters, J.; Witt, K.L. Use of frozen tissue in the Comet Assay
for the evaluation of DNA damage. J Vis Exp. 2020, 157.
66. Pant, K.; Springer, S.; Bruce, S.; Lawlor, T.; Hewitt, N.; Aardema, M.J. Vehicle and posi-
tive control values from the in vivo rodent comet assay and biomonitoring studies using
human lymphocytes: Historical database and infuence of technical aspects. Environ
Mol Mutagen. 2014, 55(8), 633–642.
67. Ladeira, C.; Koppen, G.; Scavone, F.; Giovannelli, L. The comet assay for human bio-
monitoring: Effect of cryopreservation on DNA damage in different blood cell prepara-
tions. Mutat Res Genet Toxicol Environ Mutagen. 2019, 843, 11–17.
 21 Molecular Karyotyping
Ilda Patrícia Ribeiro, Luís Miguel Pires,
Susana Isabel Ferreira, Mariana Val,
Joana Barbosa Melo and Isabel Marques Carreira

CONTENTS
Introduction............................................................................................................ 225
The aCGH Technology and Data Interpretation .................................................... 226
Different Platforms: Past and at Present ................................................................ 229
Advantages and Limitations................................................................................... 232
Applications ........................................................................................................... 232
Molecular Karyotyping Approach in Human Genetic Diagnostics.............. 233
Molecular Karyotyping Approach in Prenatal Diagnostics .......................... 233
Molecular Karyotyping Approach in Cancer Research ................................ 233
Clinical Cases......................................................................................................... 233
Clinical Case 1.............................................................................................. 234
Clinical Case 2.............................................................................................. 236
Conclusion for Clinical Cases 1 and 2.......................................................... 237
Clinical Cases 3 and 4................................................................................... 238
Clinical Case 3.............................................................................................. 238
Clinical Case 4.............................................................................................. 240
Clinical Cases 5 and 6................................................................................... 243
Clinical Case 5.................................................................................. 243
Clinical Case 6.................................................................................. 243
The Impact of Molecular Karyotyping in Clinical Practice................................... 246
Conclusions............................................................................................................ 246
References.............................................................................................................. 247

INTRODUCTION
Nowadays, several diseases are linked to copy-number variants (CNVs), and various
genome-wide methods can be used for its detection. However, it is important to high-
light that the presence of CNVs by itself does not mean an abnormal or pathogenic
phenotype. The clinical relevance of CNVs depends on its functional impact, having
a high probability of a phenotypic effect if the region of imbalance maps critical
gene/s or an important regulatory region.
The most commonly used method for CNVs detection has been array-based
comparative genomic hybridization (aCGH). This technique was frst described
in 1997 by Solinas Toldo,[1] and its frst applications were published by the groups

DOI: 10.1201/9781003223658-21 225

226 Cytogenetics and Molecular Cytogenetics

of Pinkel, Snidjers and Buckley in the following years.[2–4] Initially, this technique
was introduced as matrix-CGH, and later coined as array-CGH (aCGH).[3] In 2005,
Vermeesch and colleagues proposed to call this technology as molecular karyo-
typing.[5] Currently, the use of aCGH technology is massifed worldwide either at
diagnostic or investigation point of view. Many reports were published concerning
chromosome imbalances associated with cancer as well as in constitutional cytoge-
netics, with postnatal and prenatal applications. The aCGH technology use whole
genome probes immobilized on glass slides (a microarray) that allow, due to the
deep resolution, the diagnosis of smaller genomic imbalances, being the method
of choice to small genomic imbalances identifcation with a wide range of clinical
applications. It was estimated that the introduction of aCGH technique in the clinical
practice allowed increasing the detection of apparently pathogenic genomic imbal-
ances in as much as 20% of cases that have had normal karyotyping tests.[6]

THE aCGH TECHNOLOGY AND DATA INTERPRETATION

Initially, the CGH technique was developed using metaphase chromosomes for anal-
ysis of chromosomal imbalances. This technique is based on the co-hybridization of
two samples of genomic DNA (test and reference), fuorescently labeled and mixed in
a 1:1 ratio, to normal metaphase chromosome spreads on a glass slide.[7] The ratios of
fuorescence intensities for each chromosome or chromosome segment show its rela-
tive copy number in the test genome compared with the control/reference genome.[8]
So, the result of hybridization is detected by the analysis of the intensities of the two
different fuorochromes, being the regions of gain or loss of DNA sequences, such as
deletions, duplications, or amplifcations, detected due to the fuctuations in the inten-
sities ratio of the two fuorochromes along the target chromosomes.[7] Besides, CGH
technique can be used to scan an entire genome for imbalances; its resolution (like
cytogenetic methods) is limited to alterations of approximately 5–10 Mb.[9] Between
1992 and 2000, this chromosome-based CGH approach was employed to human
genetics, more specially in the solid tumors feld.[10] Presently, CGH technique is still
used for comparison of closely related species in molecular cytogenetic studies and
to identify cryptic sex chromosomes within a species.[11, 12] Nevertheless, metaphase
spreads were replaced by arrays of DNA fragments such as BAC (bacterial artifcial
chromosomes) or PAC (P1 artifcial chromosome) clones and more recently by oli-
gonucleotides and SNP (single-nucleotide polymorphism) probes, being this modi-
fed version of the CGH test, named aCGH. In the aCGH technique, thousands to
millions of different ‘spots’ comprising several identical, immobilized single strand
DNA (ssDNA) probes complementary to a part of the genome are printed on a glass
slide, allowing much higher resolution and consequently the identifcation of sub-
microscopic aberrations, in comparison with CGH analysis and conventional cyto-
genetics.[13] In the aCGH technique, equal amounts of test and reference genomic
DNAs are differentially labeled with fuorochromes (usually Cyanine 5—red and
Cyanine 3—green, respectively), followed by co-hybridization onto a microarray,
a glass slide containing the DNA targets for whole genome. Since the DNAs have
been denatured, the single strands when applied to the glass slide hybridize with
the arrayed single-strand probes, by Watson-Crick base pairing.[14] Unbound labeled
Molecular Karyotyping 227

DNA is washed off the slide. The reference sample should be gender-matched with
the sample in study. After the hybridization, the slides are scanned into TIFF image
fles by a microarray scanner, which uses laser light to measure the relative intensity
of emission, usually at green (Cy3—for control) and red (Cy5—for biological sam-
ple in study) wavelengths being all targets’ intensities measured, and the image fles
are quantifed in a specifc feature extraction software. The green to red fuorescence
log2 ratio is calculated for each spot. The copy number alterations are calculated by
differences in fuorescence intensities along any given spot(s), enabling the detection
of gains and losses throughout the genome with high resolution. Therefore, equal
amount of green and red fuorescence for a specifc spot signifes that this region of
the genome to which that spot is mapped harbors an equal copy-number in the tested
and control samples. In the other hand, a ratio favoring red fuorescence implies
an excess of that genomic segment in the test genome (i.e., a copy-number gain)
in comparison with the control, whereas a green signal shows a copy number loss
(Figure 21.1).
The principle of aCGH technique can be demonstrated with the following sim-
ple example (Figure  21.2A). DNA from a male with 9p trisomy and a partial 9q
(9q13-q31.2) trisomy, labeled in red, is co-hybridized with DNA from a normal male
(46,XY) labeled in green against an array slide. At each target sequence of the array
slide without copy number variants, a balanced, yellow color is observed. Since at
chromosome 9 (specifcally 9p and 9q13-q31.2) there is more DNA labeled with red
due to trisomy 9, this means that in the competitive hybridization higher quantity of
‘DNA red’ will be linked in comparison with DNA labelled with green (from con-
trol) for this chromosome, and consequently a gain of chromosome 9 material will
be visualized after this experiment (represented in a positive scale, by a right devia-
tion of the blue color in the Figure 21.2A).
A genotype–phenotype correlation is crucial to interpret the CNVs detected. Not
all CNVs are associated with unfavorable clinical phenotypes, being in some cases
classifed as benign—without impact in the phenotype. In other cases, CNVs are
linked to a spectrum of clinical phenotypes that may range from benign to pathologi-
cal, including a very challenging class of CNVs named VUS (variant of unknown/
uncertain clinical signifcance). Therefore, CNVs could be phenotypically benign,
because of phenotypic differences created by evolution, or could be responsible for
disease or for disease susceptibility. Additionally, the CNVs could be categorized
as germ line (constitutional), where every cell of the body is altered, or somatic,
where only a subgroup of cells in the organism harbors the alteration, such as in
cancer or in mosaics. The interpretation of the results and consequently the assess-
ment of the pathogenicity of a CNV could be challenging and needs to consider the
size and location of the copy number alteration, breakpoint information, whether it
has occurred de novo, and gene content, namely whether the region contains genes
of known importance. Determining whether a specifc CNV is present in affected
(or unaffected) relatives can also be important as well as verify in databases if other
unrelated but affected individuals with similar clinical phenotypes are described,
namely the use of CNV databases such as DECIPHER (www.deciphergenomics.org/)
and if those CNVs are also reported among healthy individuals, using CNV data-
bases such as the Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home).
 228
Cytogenetics and Molecular Cytogenetics
FIGURE 21.1 aCGH results of chromosome 16 from three different samples, using CytoGenomics software for analysis.
A) Chromosome view.
B) Gene view, presenting the gene content in this chromosome region. Sample 1 presents a deletion of 541 kb at 16p11.2, involving 30 genes arr[GRCh37]
16p11.2(29656684_30197341)x1; Sample 2 without CNVs at 16p11.2; and Sample 3 presents a duplication of 544 kb at 16p11.2, involving 34 genes
arr[GRCh37] 16p11.2(29652999_30197341)x3.
Molecular Karyotyping 229

FIGURE  21.2 A) Chromosome view and respective gene content of a 9p trisomy and a
partial 9q trisomy. Results obtained using aCGH technique and analyzed with Cytogenomics
software. In orange are represented the OMIM Morbid Map genes, in blue the OMIM genes
and in grey the RefSeq genes. Trisomy is represented as a positive scale. B) Plot of chro-
mosome 22 displaying a gain at 22q11.21, detected by aCGH technique and analyzed with
Cytogenomics software. The duplicated region is shown in the UCSC Genome Browser
(https://genome.ucsc.edu/) with some selected tracks to assist in the genotype–phenotype
correlation and consequently in the clinical relevance determination. Example of these tracks
are chromosome band, RefSeq genes, OMIM genes, DECIPHER CNVs, ClinGen CNVs and
DGV.

There are also several public databases and sources such as PubMed (https://
pubmed.ncbi.nlm.nih.gov/), OMIM (www.omim.org/) and human genome browsers
(UCSC—https://genome.ucsc.edu/, Ensembl—www.ensembl.org/index.html) that
provide useful tracks of genome-oriented data (Figure 21.2B).[15]

DIFFERENT PLATFORMS: PAST AND AT PRESENT

The aCGH technology is based in the deposit and immobilization of small
amounts of single-strand DNA (ssDNA), known as probes, on a solid support, such
230 Cytogenetics and Molecular Cytogenetics

as a glass slide, in an ordered way. The frst-generation aCGH approach included

probes derived from bacterial artifcial chromosomes (BACs) or complementary
DNA (cDNA)–cloned genomic segments. Nevertheless, the BAC-based arrays
present some constraints, namely the diffculty in the calculation of CNV sizes
and boundaries as well as the fact that its production is prone to errors.[16] The
progress of aCGH platforms and the reference human genome sequencing allowed
to design oligonucleotide probes with higher resolution. The size of the probes is
very variable; for example, BAC-based arrays use large-insert genomic clones of
100–150  kb, while oligonucleotide-based arrays use probes of ~60-mer.[17] The
resolution of aCGH varies according to the size of the genomic fragments and their
density—the genomic distance between DNA probes.[5] For example, a microar-
ray design with probes for whole genome that are 1 Mb apart will be unable to
identify copy number variations of small sequence.[14] So, the resolution of the
array increases directly with the decrease of the size of the nucleic acid probes
and the reduction of the distance (more contiguous) between the targets on the
microarray. The higher resolution allows more accurate identifcation of chromo-
somal breakpoints and gene alterations. Nowadays, the great majority of the com-
mercial and academic laboratories are using oligonucleotide- or single-nucleotide
polymorphism (SNP)–based arrays.[17] The advantages of oligonucleotide-based
arrays consist of more fexibility in terms of probe selection, which enable higher
probe density and customization of array content. Arrays designed specifcally for
copy-number analysis use longer oligonucleotide probes that offer a better signal-
to-noise ratios in comparison to SNP-platforms, having however platforms with
SNP-detecting arrays that include a mixture of SNP and copy-number probes to
address this issue.[17] It is important to stress that single oligonucleotide probes do
not provide accurate determination of copy number, there being required multiple
consecutive probes with the same copy-number change to correctly establish a gain
or a loss.[17] Usually, at least three consecutive probes with abnormal log2 ratios
are needed to identify a copy number alteration. However, the number of con-
secutive probes required could vary between arrays of oligonucleotide probes and
SNP probes. There are different platforms of oligonucleotide-based microarray,
which means that depending on the selected platform, the number of oligonucle-
otide probes used for a whole-genome single-sample analysis can vary, and con-
sequently the density of probes and the resolution are also different (Figure 21.3).
The more recent array-based method on the market is SNP array genotyping, also
recognized as SNP chips. The SNP platforms perform allelic discrimination to
interrogate polymorphisms at specifc positions in the genome, being its great
advantage the capability of also identify long stretches of homozygosity, which
might represent uniparental disomy (UPD) or consanguinity not suspected in the
clinical history.[17] The SNP array genotype data can be downstream evaluated
based on the B-allele frequency (BAF), which can reveal, when the BAF deviates
from the trimodal expected distribution (AA = 0, AB = 0.5, and BB = 1), allelic
imbalances, namely CNVs, regions of absence of heterozygosity (AOH) or unipa-
rental disomy (UPD), all detected within the same assay.[16, 18] It is also important
to stress that SNP-based aCGH can only detect quite long stretches of isodisomy,
and this approach misses the UPD based on heterodisomy.[19]
 Molecular Karyotyping
FIGURE 21.3 Representation of the number of oligonucleotide probes present at chromosome 7 in two different platforms, 180K and 60K of Agilent
oligonucleotide based-microarray, using Agilent Genomic Workbench v6.5 software.

231
232 Cytogenetics and Molecular Cytogenetics

ADVANTAGES AND LIMITATIONS

In a single assay, aCGH allows to simultaneously identify genome-wide copy num-
ber alterations with high resolution. This technique presents several benefts and
constraints. Among the advantages of aCGH technique, we can highlight:

• high-resolution whole genomic view for all euchromatic regions of genomes,

• ability to simultaneously detect aneuploidies, deletions, duplications, and/or
amplifcations of any locus represented on an array,
• detection of submicroscopic chromosomal abnormalities,
• only needing DNA (or RNA for gene expression microarrays), so not need-
ing any living or dividing cells, which enable the use of both fresh and
some archival DNA, including uncultured specimens and DNA obtained
from postmortem samples, such as some formalin fxed paraffn embedded
(FFPE) tissues,
• short turnaround time usually not exceeding few days and more cost-effec-
tive than multiple fuoresence in situ hybridization (FISH) assays,

Considering the limitations of aCGH, we can highlight:

• only detecting unbalanced chromosomal aberrations,

• alterations in polyploidy is not depictable (except using SNP array), since
this technique relies on normalization of the intensity ratios,
• mosaic with low expression rate is usually, in regular routine settings, not
detected. Cells with a chromosomal imbalance need to be present in at
least 20% of the studied tissue, otherwise the aberrant cell clone may be
missed,[20]
• small indels (insertion or deletion of bases) are not detected, in areas that
are not so well covered by probes,
• without information available about centromeric-regions and heterochro-
matic regions, which corresponding to about 10% of the human genome,[21]
• being unable to defne the direction of inserted sequences within the
genomic context (i.e., direct vs inverted orientation).[16]

APPLICATIONS
The application of aCGH into the clinical practice has introduced a paradigm shift
in the diagnostic workup and accelerated the identifcation of the molecular basis of
several genetic diseases.[22] Initially, CGH was developed as a research tool for the
investigation of CNVs in cancer. However, nowadays, aCGH technique is mainly
used as a routine diagnostic tool, being the frst-tier clinical diagnostic test for
patients with intellectual disability, congenital anomalies and autistic spectrum dis-
order, and more recently has also been applied for the detection of genomic imbal-
ances in prenatal genetic diagnosis. Since CNVs are common in the genome, the
clinical signifcance and the genotype-phenotype correlation of aCGH results can be
Molecular Karyotyping 233

challenging, parental analysis and the use of several internet-based databases being
pivotal. The application of aCGH technique in three main areas will be discussed:

MOLECULAR K ARYOTYPING APPROACH IN HUMAN GENETIC DIAGNOSTICS

Array CGH has been applied to high-resolution analysis of constitutional abnormali-
ties. The most important beneft of molecular karyotyping in human genetics is its
capability to detect submicroscopic CNVs correlated to clinical phenotype in chil-
dren and adults with normal GTG-banding based karyotype. This high-resolution
technique allows the identifcation of novel disease-causing CNVs that can affect
individual genes, exons, and regulatory regions. Therefore, besides the detection of
CNVs for well-known genomic disorders, aCGH has also revealed new genomic
disorders and disease-causing genes.

MOLECULAR K ARYOTYPING APPROACH IN PRENATAL DIAGNOSTICS

Several studies have also showed the feasibility of performing aCGH for prenatal
diagnosis using a wide range of sample types. In 2006, Rickman et  al. analyzed
30 native prenatal samples by aCGH and demonstrated its potential for aneuploidy
detection in DNA isolated from 1 ml of native amniotic fuid.[23] In this study, 29/30
samples were accurately diagnosed, except for a case of triploidy. Since aCGH tech-
nique allows the chromosomal aneuploidy and submicroscopic imbalances detection
in whole genome with higher resolution, it is recommended as a frst-tier approach
in prenatal diagnosis for detection of CNVs in fetuses with structural anomalies
observed by ultrasound.[24, 25] So, aCGH has been progressively used in prenatal
diagnosis with high clinical impact in the clarifcation of the karyotype fndings
signifcance and also in the diagnosis of not identifable imbalances using only
karyotyping.[26]

MOLECULAR K ARYOTYPING APPROACH IN CANCER RESEARCH

The aCGH technology was frst developed as a research tool for the investigation of
genomic alterations in cancer and has proven valuable in the identifcation of DNA
copy number signatures and profles for different types of tumors. This technique
has been applied to a large number of cancer studies with reproducible results, some
examples being head and neck cancer,[27, 28] basal cell carcinomas,[29] cholangiocar-
cinoma,[30] multiple myeloma,[31] bladder cancer,[32, 33] acute myeloid leukemia,[34, 35]
among others.

CLINICAL CASES
Selected clinical cases will be presented as example of aCGH applications in post-
natal human genetic diagnosis, in prenatal diagnosis and in cancer characterization.
All cases were analyzed by aCGH technique using Agilent SurePrint G3 Human
Genome microarray 180 K (Agilent Technologies, Santa Clara, CA, USA).
234 Cytogenetics and Molecular Cytogenetics

For all clinical post and prenatal cases, three questions are to be answered:

i) How to interpret this result? How to classify this CNV? What is the most
probable mechanism of origin of the alteration?
ii) What is the origin of this CNV?
iii) What can we offer to this couple? What is the future strategy for the family?

Clinical cases 1 and 2 are two examples of aCGH application in postnatal human
genetic diagnosis (using DNA extracted from peripheral blood), with different
outcomes.

CLINICAL CASE 1
A 11-year-old boy with renal insuffciency, retinopathy and ataxia was referred for
aCGH that identifed a 105 kb homozygous deletion at chromosome 2, arr[GRCh37]
2q13(110862477_110967912)x0 (Figure 21.4A).

i) How to interpret this result? How to classify this CNV? What is the most
probable mechanism of origin of the alteration?

There are several CNVs described in this region at DECIPHER database. Two genes,
MALL and NPHP1, are mapped in this region. NPHP1 gene is present in OMIM
Morbid Map and is associated with nephronophthisis 1, juvenile, an autosomal reces-
sive cystic kidney disease that leads to renal failure. Almost 15% of nephronophthi-
sis patients present extrarenal symptoms affecting other organs, such as eyes, liver,
bones and central nervous system.[36] Around two thirds of the patients with familial
juvenile nephronophthisis have a homozygous deletion at the 2q13.[37]
This homozygous deletion seems to explain the clinical phenotype of the boy. In
order to establish the most probable mechanism of origin of the alteration, the aCGH
study of the progenitors were recommended.

ii) What is the origin of this CNV?

Both progenitors, not relatives, present 105kb heterozygous deletion, arr[GRCh37]

2q13(110862477_110967912)x1 (Figure 21.4B). The parents do not exhibit any clini-
cal phenotype. Thus, deletion of one copy is not harmful for the parents. Although
inherited, the 2q13 deletion is clearly pathogenic for the child, because it is in
homozygosity.

iii) What can we offer to this couple? What is the future strategy for the
family?

This couple received genetic counselling, where the implication of the molecular
fndings was explained, and prenatal diagnosis was offered for future pregnancies.
Later on, this couple had a new pregnancy and requested an invasive prenatal diag-
nosis. DNA from amniotic fuid was analyzed by aCGH technique, and it was verifed
that the fetus has inherited the deletion from one of the parents (Figure 21.4C). Thus,
it is expected that the fetus did not present any phenotype like the carrier progenitors.
FIGURE  21.4 A) Chromosomal view and corresponding gene content of a 2q13 homo-
zygous deletion identifed in the 11-year-old boy. Results obtained using aCGH technique
and analyzed using Agilent Genomic Workbench v6.5 software. The results are according
to Human Genome build 19 and include imbalances with at least three consecutive probes
with abnormal log2 ratios. Deletion is represented in a negative scale. B) Chromosomal view
and respective gene content of the 2q13 deletion identifed in the mother and the father of
the 11-year-old boy. Results obtained using aCGH technique and analyzed using Agilent
Genomic Workbench v6.5 software. The results are according to Human Genome build 19
and include imbalances with at least three consecutive probes with abnormal log2 ratios.
Deletion is represented in a negative scale. C) Chromosomal view and respective gene content
of the 2q13 deletion identifed in a sample of amniotic fuid during pregnancy of this couple
that have a heterozygous deletion in arr[GRCh37] 2q13(110862477_110967912)x1 and a son
that inherited from both parents this deletion. Results obtained using aCGH technique and
analyzed with Cytogenomics software. Deletion is represented in a negative scale.
236 Cytogenetics and Molecular Cytogenetics

CLINICAL CASE 2
A newborn with several congenital anomalies, such as axial hypotonia, retrog-
nathism, fetal growth restriction, high palate, and hypogonadism was referred
for aCGH that identifed two genomic anomalies (Figure  21.5): arr[GRCh37]
10p12.1(26182512_26815179)x3,15q11.2q13.1(22765628_28940098)x4. The mother
has a brother with global psychomotor developmental delay and a son with hyperac-
tivity and global psychomotor developmental delay.

FIGURE 21.5 Chromosomal view and respective gene content of

A) 10p12.1 duplication and
B) 15q11.2q13.1 triplication.
Molecular Karyotyping 237

Results obtained using aCGH technique and analyzed with CytoGenomics soft-
ware. Gain of genetic material is represented in a positive scale.

i) How to interpret this result? How to classify this CNV? What is the most
probable mechanism of origin of the alteration?
• duplication of 632 Kb at 10p12.1 includes three genes described at OMIM
database (OMIM ID: 606808-MYO3A, 138275‑GAD2, 609036-APB‑
B1IP). The MYO3A gene is described at OMIM Morbid Map and related
to autosomal recessive deafness. CNVs in duplication are not reported
for this region in healthy individuals at Database of Genomic Variants.
In the DECIPHER database, there is one patient with cognitive defcit
that has a similar duplication (ID: 300716) with unknown origin, which
was classifed as likely pathogenic. However, this reported patient also
presented another genomic alteration, a 657Kb deletion at 1p36.23 of
unknown origin, also classifed as likely pathogenic. At the ClinGen data-
base, there are bigger CNVs in duplication than those of our case, which
were classifed either as benign or pathogenic or even likely pathogenic.
• triplication of 6.1Mb at 15q11.2q13.1 includes several genes, 24 reported
at OMIM database and 10 at OMIM Morbid Map database (OMIM ID:
608145-NIPA1, 603856-MKRN3, 605283-MAGEL2, 602117-NDN,
182279-SNRPN, 601623-UBE3A, 137192-GABRB3, 137142-GABRA5,
611409-OCA2, 605837-HERC2). In the literature, triplication of
15q11.2q13.1 is related with hypotonia, motor delay, intellectual disabil-
ity and autism.[38] These disabilities ft with the clinical phenotype of our
patient.
ii) What is the origin of this CNV?

Both parents were also analyzed by aCGH. It was possible to conclude that the
10p12.1 duplication of our patient was inherited from the mother (phenotypi-
cally normal) and was classifed, considering the present knowledge as a CNV
of unknown clinical signifcance. The tetrasomy of 15q11.2q13.1 is de novo and
classifed as pathogenic. arr[GRCh37]10p12.1(26182512_26815179)×3mat,15q11
.2q13.1(22765628_28940098)×4 dn.

iii) What can we offer to this couple? What is the future strategy for the
family?

This couple was referred to genetic counselling. Monitoring and testing future preg-
nancies were recommended.

CONCLUSION FOR CLINICAL CASES 1 AND 2

These two clinical cases show the challenges in the interpretation and classifcation
of CNVs, where the detailed clinical characteristics of the patients, the analysis of
specifc databases and the progenitors are pivotal to perform the genotype-phenotype
correlations. Sometimes the current knowledge is yet scarce to infer without doubt
238 Cytogenetics and Molecular Cytogenetics

about the clinical signifcance of some CNVs, which is even more challenging in
prenatal cases. It is important, whenever possible to revisit some of these VUS, spe-
cifcally when a new pregnancy occurs in that family.

CLINICAL CASES 3 AND 4

Clinical cases 3 and 4 are two examples of aCGH application in prenatal diagnosis,
with different outcomes.

CLINICAL CASE 3
A 29 weeks and 5 days’ pregnancy, showed by ultrasound a fetus with cleft lip, cleft
palate and defect in the ventricular septum. Amniotic fuid was collected and conven-
tional cytogenetic and aCGH techniques requested. Both techniques identifed two
genomic anomalies in this female fetus: 46,XX,rec(4)dup(4p)inv(4)(p15.33q31.3)
a r r[GRCh37]4p16.3p1533(45882 _11887210)×3,4q313q35.2(155125113_
190469337)×1.

i) How to interpret this result? How to classify this CNV? What is the most
probable mechanism of origin of the alteration?

The fetus 1 presented (Figure 21.6A):

• a 11.8 Mb 4pter duplication that harbors several genes, including 71 reported

at OMIM database and 21 reported at OMIM Morbid map database;
• a 35.3 Mb 4qter deletion that harbors several genes, including 73 reported at
OMIM database and 19 reported at OMIM Morbid map database.

The imbalances were also observed in conventional cytogenetics analysis of fetus 1

(Figure 21.6C), allowing a correlation with the ultrasound abnormalities seen on the
fetus. They are also suggestive of a recombinant that could have originated from a
balanced structural rearrangement in one of the progenitors. Therefore, karyotyping
study of both parents was requested.

ii) What is the origin of this CNV?

Regarding the most probable biological mechanism, it can be concluded that the
male progenitor is a carrier of a pericentric inversion of chromosome 4 (Figure 21.6C
and 21.6D). Thus, from the conventional cytogenetic analysis of the fetus and the
progenitors, it was demonstrated that the fetus inherited a recombinant chromosome
4 resulting from the crossing over in the pericentric inversion loop of the paternal
inverted chromosome 4 during spermatogenesis.

Fetus 1–46,XX,rec(4)dup(4p)inv(4)(p15.33q31.3)pat. arr[GRCh37]4p16.3p

1533(45882_11887210)×3,4q313q35.2(155125113_190469337)×1
Father: 46,XY,inv(4)(p15.33q31.3)
Molecular Karyotyping 239

FIGURE  21.6 A) Chromosome view of the 4pter duplication and 4qter deletion identi-
fed in a sample of amniotic fuid (Fetus 1) and B) of the 4pter deletion and 4qter duplica-
tion identifed in fetus 2 and 3. Results obtained using aCGH technique and analyzed with
Cytogenomics software. Duplication is represented in a positive scale and deletion in a nega-
tive scale. C) Pair of chromosomes 4 obtained from representative G-banded metaphases of
the amniotic cells from the fetus D) and of the peripheral blood from the father.
240 Cytogenetics and Molecular Cytogenetics

These two genomic imbalances identifed in the fetus seem to be in the origin of the
ultrasound malformations detected and consequently are correlated to the clinical
phenotype.

iii) What can we offer to this couple? What is the future strategy for the
family?

This couple was offered genetic counselling where it was well explained the conse-
quence of this pericentric inversion in this phenotypically normal father for future
pregnancies and the need of family studies (parents, brothers, sisters and other
family members at risk). To all carriers, invasive prenatal diagnosis should be
offered.
This couple had two more pregnancies. Trophoblastic cells from fetuses 2 and
3 were collected and aCGH revealed two genomic imbalances in mirror compared
with fetus 1: 4p16.3p15.33 deletion and 4q31.3q35.2 duplication.
Fetuses 2 and 3 had similar alterations but different from fetus 1 (Figure 21.6B)
that resulted from the meiotic recombination in the spermatogenesis of the male
progenitor.
Fetus 2 and 3: 46,XX,rec(4)dup(4q)inv(4)(p15.33q31.3)pat—arr[GRCh37]
4p16.3p15.33(72447_1,887210)x1,4q31.3q35.2(155125113_190976439)x3.
Although in fetus 2 and 3 the genomic imbalances in chromosome 4 are in oppo-
site way to that observed in fetus 1 (mirror, i.e., fetus 1 presented 4pter duplication
and fetus 2 and 3 deletion and fetus 1 presented 4qter duplication and fetus 2 and 3
a deletion), the mechanism of origin is the same: inheritance of a recombinant chro-
mosome 4 as a result of a paternal pericentric inversion in chromosome 4.
Deletions in the 4p16.3 region observed in the fetus are the main cause of Wolf-
Hirschhorn syndrome, which presents as clinical manifestations growth delay, sei-
zures, intellectual disability and distinct craniofacial features. In prenatal, 4pter
deletion/4qter duplication were already reported with clinical manifestations like
our patient.[39]
This case shows the clinical implications for the offspring of a balanced structural
rearrangement in one of the progenitors and the need to offer to this couple prenatal
diagnosis in future gestations and genetic counselling to explain the clinical implica-
tions of these genetic fndings, the risk of recurrence and the need to offer family
studies to other relatives.

CLINICAL CASE 4
Cells cultured from amniotic fuid of a fetus were analyzed by conventional cytoge-
netics, being the clinical indication for the invasive prenatal diagnosis a positive bio-
chemical screening. The G-banded metaphases of the amniotic cells revealed a male
fetus with two structural alterations: a paracentric inversion in chromosome 3 and a
reciprocal translocation between chromosomes 2 and 5 (Figure 21.7A). 46,XY,t(2;5)
(q33;q22)dn,inv(3)(p26.2p23).

i) How to interpret this result? How to classify this CNV? What is the most
probable mechanism of origin of the alteration?
Molecular Karyotyping 241

FIGURE  21.7 A) Representation of the pair of chromosomes 3, 2 and 5 obtained from

representative G-banded metaphases of the amniotic cells from the fetus. The arrows show
the structural imbalances observed: a paracentric inversion in chromosome 3 and a recipro-
cal translocation between the long arms of chromosomes 2 and 5. B) Results of FISH tech-
nique using a whole chromosomic painting (WCP) probe for chromosome 3 (WCP3, Cambio)
labelled with SpectrumOrange and a subtelomeric probe for 3p26.3 (D3S4559, TotelVysion,
Vysis) labelled with SpectrumGreen. C) Chromosome view and respective gene content of
the 3q22.2q22.3 and 5p13.2 deletion identifed in the DNA from amniotic cells of the fetus
(red arrows). Results obtained using aCGH technique and analyzed using Agilent Genomic
Workbench v6.5 software. The blue arrows represent the chromosome bands involved in the
structural rearrangements identifed by conventional cytogenetics and without genomic imbal-
ances in the region observed by aCGH. The results are according to Human Genome build
19 and include imbalances with at least three consecutive probes with abnormal log2 ratios.
Deletion is represented in a negative scale.
242 Cytogenetics and Molecular Cytogenetics

In order to ascertain the origin of these structural alterations, both parents were also
analyzed by conventional cytogenetics, and it was possible to infer that these altera-
tions of the fetus were de novo.
In order to further characterize the inversion, molecular cytogenetics (FISH tech-
nique) (Figure 21.7B) was also performed in the amniotic cells from the fetus using
a whole chromosome painting (WCP) probe for chromosome 3 (WCP3, Cambio)
labelled with SpectrumOrange that showed a continuum pattern in the chromosome
3; e.g. the inversion in the chromosome 3 did not change the pattern observed in this
or any chromosome with this probe. A subtelomeric probe for 3p26.3 (D3S4559,
TotelVysion, Vysis) labelled with SpectrumGreen was needed to confrm that this
terminal region in the short arm of the chromosome 3 (3p26.3) was not involved in
the inversion.
With these two techniques we can concluded that the fetus carries two structural
chromosomal alterations that are de novo and seem to be balanced. Being de novo,
the risk for the fetus of intellectual disability would be 6–10%.
In order to ascertain whether there were micro imbalances aCGH technique
was performed and revealed losses of genetic material in two of the chromosomes
involved (chromosomes 3 and 5) in the structural rearrangements observed by
conventional cytogenetics arr[GRCh37]3q22.2q22.3(135247918_136673227)×1
,5p13.2(33825339_35133937)×1 (Figure 21.7C):

• Deletion of 1.5 Mb at 3q22.2q22.3, where are mapped seven genes, being

six described at OMIM database (OMIM ID: 604944-PPP2R3A, 614802-
MSL2, 232050-PCCB, 604358-STAG1, 600508-NCK1, 605621-IL20RB)
and 1 at OMIM Morbid Map (OMIM ID: 232050-PCCB).
• Deletion of 1.3 Mb at 5p13.2, where are mapped thirteen genes, being nine
described at OMIM database (OMIM ID: 606184-ADAMTS12, 609445-
RXFP3, 606202-SLC45A2, 604489-AMACR, 612045-C1ATNF3, 606586-
RAI14, 603153-RAD1, 612471-AGXT2, 176716-PRLR) and 2 at OMIM
Morbid Map (OMIM ID: 606202-SLC45A2, 604489-AMACR).

ii) What is the origin of this CNV?

DNA from the peripheral blood of the progenitors were also analyzed by aCGH and
did not reveal these imbalances (data not shown), which were therefore classifed as
de novo in the fetus.

iii) What can we offer to this couple? What is the future strategy for the
family?

This clinical case is a clear example of the importance of complementary techniques

like conventional cytogenetics and aCGH to achieve a correct risk of recurrence
for the offspring as well as to identify the mechanisms of origin of the chromo-
some imbalances and its accurate breakpoints. The conventional cytogenetics in this
case reveals a double structural chromosome rearrangement de novo and apparently
balanced with the inherent risks for the fetus. With complementarity of the aCGH
Molecular Karyotyping 243

technique, we can conclude that besides these structural rearrangements detected by

conventional cytogenetics, two additional imbalances in two of these chromosomes,
but at different locations were present, which increase the risk of intellectual disabil-
ity and developmental disorders in the carrier/fetus. All these results were explained
to the couple in order to decide about the pregnancy in course. Monitoring and test-
ing future pregnancies were recommended.

CLINICAL CASES 5 AND 6

Clinical cases 5 and 6 are two examples of aCGH application in cancer genetic char-
acterization in order to detect the genetic changes associated with cancer develop-
ment and progression. In cancer research, this technology due to the study of whole
genome allows to identify new chromosomal regions and genes related to different
cancer types as well as to perform the correlation of the identifed genomic profles
with tumor stage, risk factors, disease evolution, response to treatment and patients’
survival.

Clinical Case 5
DNA from tumor tissue of a 47 years-old woman diagnosed with foor of the mouth
tumor, stage I, human papilloma virus (HPV) negative and with tobacco consump-
tion, was analyzed by aCGH. This whole genome approach revealed imbalances
in several chromosomes, such as, chromosomes 1, 2, 3, 7 and 9, where there are
mapped several genes with key roles for the carcinogenesis process (Figure 21.8).
We observed that tumor cells of this patient displayed numerous rearrangements on
several chromosomes simultaneously, while showing a small number of chromo-
somes without or with few aberrations. The interpretation of the large-scale genomic
profling of cancer samples has been challenging due to the intra- and inter-tumor
heterogeneity displayed by solid tumors. The aCGH technique has helped in the
identifcation and translation to clinical practice, of specifc diagnostic and prognos-
tic cancer biomarkers, which are of utmost importance in order to improve patient’s
treatment selection, prophylactic screening strategies and ultimately the patients
quality of life.

Clinical Case 6
A primary cell culture established from a surgical tongue tumor resected sample was
characterized by aCGH and banding cytogenetics. This primary culture presented
a huge karyotypic heterogeneity with different numerical and structural chromo-
somal abnormalities identifed in the analyzed metaphases (Figure 21.9A). Results
from aCGH helped to establish the specifc chromosomal bands involved in copy
number gains and losses, assisting in the delimitation of the size of the imbalances
and the establishment of specifc breakpoints as well as to identify additional small
copy number gains and losses not detected by conventional cytogenetics due to their
reduced size. With the integration of these two techniques, we can observe aneuploi-
dies and also to infer and characterize some structural rearrangements that are in the
origin of the CNVs and aneuploidy (Figure 21.9 B–C).
 244
Cytogenetics and Molecular Cytogenetics
FIGURE 21.8 Chromosome view of chromosomes 1, 2, 3, 7 and 9 displaying several CNVs (gains and losses). The results were obtained through the
analysis of DNA from a foor of the mouth tumor using aCGH technique and Agilent Genomic Workbench v6.5 software. The results are according to
Human Genome build 19 (GRCh37) and include imbalances with at least three consecutive probes with abnormal log2 ratios. Deletion is represented in
a negative scale and amplifcation in a positive scale.
Molecular Karyotyping 245

FIGURE  21.9 A) Karyogram of a representative G-banded metaphase for a primary cell

culture established from a tongue tumor. B) Chromosome view of chromosomes 20 and
1 displaying whole chromosome gain detected by aCGH technique and analyzed through
Agilent Genomic Workbench v6.5 software. Gains are represented in a positive scale.
C) Representation of the chromosomes 20 and 1 obtained from a representative G-banded
metaphase of the primary cell culture, showing different structural rearrangements that cul-
minated in copy number gain of these chromosomes.
246 Cytogenetics and Molecular Cytogenetics

THE IMPACT OF MOLECULAR KARYOTYPING

IN CLINICAL PRACTICE
Molecular karyotyping is a strong tool for the detection of submicroscopic chro-
mosomal anomalies in clinical practice. The implementation of aCGH technique
in postnatal and prenatal studies seems to increase the detection rate of chromo-
somal imbalances in comparison of conventional karyotyping. Several large-scale
studies reveal that aCGH has a 10%–20% detection rate of chromosomal abnor-
malities in children with mental retardation/developmental delay with or without
congenital anomalies, only 3%–5% of these genomic alterations being detectable
by other techniques, such as conventional cytogenetics.[14] In 2013, Wapner and
colleagues showed in a large multicenter NICHD sponsored study the clinical
utility of aCGH in prenatal diagnosis.[40] In this study, aCGH presents, in preg-
nancies with fetal structural anomalies and a normal karyotype, an incremental
diagnostic yield of about 6% in comparison to karyotype detection rate.[40] In
miscarriages, the use of aCGH is limited to few studies; nevertheless, there seems
to be an increased detection rate of chromosomal abnormalities in about 10% of
the cases with normal karyotype and nearly 50% in samples with culture failure
when karyotyping was impossible.[41] aCGH technique has some important advan-
tages for diagnosing human diseases, namely the higher resolution and the shorter
turnaround time due to not need cell culture, being a useful tool to identify new
disease-causing genes, which has allowed to expand the knowledge of the etiol-
ogy of several genetic disorders. Additionally, SNP-based microarrays are also
able to diagnosis uniparental disomy, improving the diagnostic capabilities in the
pre- and postnatal settings.

CONCLUSIONS
Molecular karyotyping is a powerful tool to identify with high resolution chromo-
somal and gene alterations for diagnostic and prognostic purposes. It is important to
stress that aCGH cannot detect all genetic anomalies and that a normal aCGH result
does not reduce the risk for other genetic conditions or even, in prenatal context, for
birth defects, related to balanced rearrangements or single gene mutations, which are
the detection limitations of this technique. Therefore, the results of molecular karyo-
typing are frequently complementary to those obtained with conventional karyotyp-
ing and Next Generation Sequencing (NGS) mutation studies.
The implementation of aCGH technique in the post and prenatal feld has been a
signifcant impact on the diagnosis of human constitutional diseases, since allowed
to detect genomic copy number alterations undetectable by other cytogenetic and
molecular technologies. This technique allows not only identifying numerous
well-characterized genetic syndromes but also to fnd new genomic disorders and
disease-causing genes, improving the knowledge of human genetic disorders and
consequently the capability to diagnosis and treat the patients. The implementation
of this technique in the clinical practice can help therapeutic selection and novel
molecular targets identifcation, improving precision medicine in a wide range of
human genetic disorders and cancer.
Molecular Karyotyping 247

REFERENCES
1. Solinas-Toldo, S.; Lampel, S.; Stilgenbauer, S.; Nickolenko, J.; Benner, A.; Dohner, H.;
Cremer, T.; Lichter, P.; Matrix-based comparative genomic hybridization: Biochips to
screen for genomic imbalances. Genes Chromosomes Cancer. 1997, 20, 399–407.
2. Snijders, A.M.; Nowak, N.; Segraves, R.; Blackwood, S.; Brown, N.; Conroy, J.;
Hamilton, G.; Hindle, A.K.; Huey, B.; Kimura, K.; Law, S.; Myambo, K.; Palmer, J.;
Ylstra, B.; Yue, J.P.; Gray, J.W.; Jain, A.N.; Pinkel, D.; Albertson, D.G. Assembly of
microarrays for genome-wide measurement of DNA copy number. Nat. Genet. 2001, 29,
263–264.
3. Pinkel, D.; Segraves, R.; Sudar, D.; Clark, S.; Poole, I.; Kowbel, D.; Collins, C.; Kuo,
W.L.; Chen, C.; Zhai, Y.; Dairkee, S.H.; Ljung, B.M.; Gray, J.W.; Albertson, D.G. High
resolution analysis of DNA copy number variation using comparative genomic hybrid-
ization to microarrays. Nat. Genet. 1998, 20, 207–211.
4. Buckley, P.G.; Mantripragada, K.K.; Benetkiewicz, M.; Tapia-Paez, I.; Diaz De Stahl, T.;
Rosenquist, M.; Ali, H.; Jarbo, C.; De Bustos, C.; Hirvela, C.; Sinder Wilen, B.; Fransson,
I.; Thyr, C.; Johnsson, B.I.; Bruder, C.E.; Menzel, U.; Hergersberg, M.; Mandahl, N.;
Blennow, E.; Wedell, A.; Beare, D.M.; Collins, J.E.; Dunham, I.; Albertson, D.; Pinkel,
D.; Bastian, B.C.; Faruqi, A.F.; Lasken, R.S.; Ichimura, K.; Collins, V.P.; Dumanski, J.P.
A full-coverage, high-resolution human chromosome 22 genomic microarray for clinical
and research applications. Hum. Mol. Genet. 2002, 11, 3221–3229.
5. Vermeesch, J.R.; Melotte, C.; Froyen, G.; Van Vooren, S.; Dutta, B.; Maas, N.; Vermeulen,
S.; Menten, B.; Speleman, F.; De Moor, B.; Van Hummelen, P.; Marynen, P.; Fryns,
J.P.; Devriendt, K. Molecular karyotyping: Array CGH quality criteria for constitutional
genetic diagnosis. J. Histochem. Cytochem. 2005, 53, 413–422.
6. Lee, C.; Hyland, C.; Lee, A.S.; Hislop, S.; Ihm, C. Copy number variation and human
health. In: Genomic and Personalized Medicineed; Willard, P.D.H.F.; Ginsburg, G.S.,
Eds. Academic Press, New York, 2009, pp. 108–119.
7. Kallioniemi, A.; Kallioniemi, O.P.; Sudar, D.; Rutovitz, D.; Gray, J.W.; Waldman, F.;
Pinkel, D. Comparative genomic hybridization for molecular cytogenetic analysis of
solid tumors. Science. 1992, 258, 818–821.
8. du Manoir, S.; Speicher, M.R.; Joos, S.; Schröck, E.; Popp, S.; Döhner, H.; Kovacs, G.;
Robert-Nicoud, M.; Lichter, P.; Cremer, T. Detection of complete and partial chromo-
some gains and losses by comparative genomic in situ hybridization. Hum. Genet. 1993,
90, 590–610.
9. Lichter, P.; Joos, S.; Bentz, M.; Lampel, S. Comparative genomic hybridization: Uses and
limitations. Semin Hematol. 2000, 37, 348–357.
10. Gebhart, E.; Liehr, T. Patterns of genomic imbalances in human solid tumors (Review).
Int J Oncol 2000, 16, 383–399.
11. Spangenberg, V.; Kolomiets, O.; Stepanyan, I.; Galoyan, E.; de Bello Cioff, M.;
Martynova, E.; Martirosyan, I.; Grishaeva, T.; Danielyan, F.; Al-Rikabi, A.; Liehr,
T.; Arakelyan, M. Evolution of the parthenogenetic rock lizard hybrid karyotype:
Robertsonian translocation between two maternal chromosomes in Darevskia ros-
tombekowi. Chromosoma. 2020, 129, 275–283.
12. Deon, G.A.; Glugoski, L.; Vicari, M.R.; Nogaroto, V.; Sassi, F.M.C.; Cioff, M.B.;
Liehr, T.; Bertollo, L.A.C.; Moreira-Filho, O. Highly rearranged karyotypes and mul-
tiple sex chromosome systems in armored catfshes from the genus Harttia (Teleostei.;
Siluriformes). Genes. 2020, 11, 1366.
13. Szczaluba, K.; Demkow, U. Array comparative genomic hybridization and genomic
sequencing in the diagnostics of the causes of congenital anomalies. J. Appl. Genet.
2017, 58, 185–198.
248 Cytogenetics and Molecular Cytogenetics

14. Theisen, A. Microarray-based comparative genomic hybridization (aCGH). Nature

Educ. 2008, 1, 45.
15. de Leeuw, N.; Dijkhuizen, T.; Hehir-Kwa, J.Y.; Carter, N.P.; Feuk, L.; Firth, H.V.; Kuhn,
R.M.; Ledbetter, D.H.; Martin, C.L.; van Ravenswaaij-Arts, C.M.A.; Scherer, S.W.;
Shams, S.; Van Vooren, S.; Sijmons, R.; Swertz, M.; Hastings, R. Diagnostic interpreta-
tion of array data using public databases and internet sources. Hum. Mutat. 2012, 33,
930–940.
16. Zepeda Mendoza, C.J.; Gonzaga-Jauregui, C. Genomic disorders in the genomics era.
In: Genomics of Rare Diseases; Gonzaga-Jauregui, C.; Lupski, J.R., Eds. Academic
Press, New York, 2021, pp. 35–59.
17. Miller, D.T.; Adam, M.P.; Aradhya, S.; Biesecker, L.G.; Brothman, A.R.; Carter,
N.P.; Church, D.M.; Crolla, J.A.; Eichler, E.E.; Epstein, C.J.; Faucett, W.A.; Feuk, L.;
Friedman, J.M.; Hamosh, A.; Jackson, L.; Kaminsky, E.B.; Kok, K.; Krantz, I.D.; Kuhn,
R.M.; Lee, C.; Ostell, J.M.; Rosenberg, C.; Scherer, S.W.; Spinner, N.B.; Stavropoulos,
D.J.; Tepperberg, J.H.; Thorland, E.C.; Vermeesch, J.R.; Waggoner, D.J.; Watson, M.S.;
Martin, C.L.; Ledbetter, D.H. Consensus statement: Chromosomal microarray is a frst-
tier clinical diagnostic test for individuals with developmental disabilities or congenital
anomalies. Am. J. Hum. Genet. 2010, 86, 749–764.
18. Brady, P.D.; Vermeesch, J.R. Genomic microarrays: A technology overview. Prenat.
Diagn. 2012, 32, 336–343.
19. Liehr, T. Cases with uniparental disomy; 2022. http://cs-tl.de/DB/CA/UPD/0-Start.html.
20. Weise, A.; Liehr, T. Molecular karyotyping. In: Cytogenomics; Liehr, T., Ed. Academic
Press, New York, 2021, pp. 73–85.
21. Liehr, T. Repetitive elements, heteromorphisms, and copy number variants. In:
Cytogenomics; Liehr, T., Ed. Academic Press, New York, 2021, pp. 373–388.
22. Cheung, S.W.; Bi, W. Novel applications of array comparative genomic hybridization in
molecular diagnostics. Exp. Rev. Mol. Diagn. 2018, 18, 531–542.
23. Rickman, L.; Fiegler, H.; Shaw-Smith, C.; Nash, R.; Cirigliano, V.; Voglino, G.; Ng,
B.L.; Scott, C.; Whittaker, J.; Adinolf, M.; Carter, N.P.; Bobrow, M. Prenatal detection
of unbalanced chromosomal rearrangements by array CGH. J. Med. Genet. 2006, 43,
353–361.
24. Genetics ACoOaGCo. Committee opinion No. 581: The use of chromosomal microarray
analysis in prenatal diagnosis. Obstet. Gynecol. 2013, 122, 1374–1377.
25. Society for Maternal-Fetal Medicine Electronic address pso; Dugoff, L.; Norton, M.E.;
Kuller, J.A. The use of chromosomal microarray for prenatal diagnosis. Am. J. Obstet.
Gynecol. 2016, 2015, B2–B9.
26. Cheung, S.W.; Pursley, A.N. Comparative genomic hybridization in the study of human
disease; 2011, Wiley. https://onlinelibrary.wiley.com/doi/epdf/10.1002/9780470015902.
a0005955.pub2.
27. Ribeiro, I.P.; Caramelo, F.; Esteves, L.; Menoita, J.; Marques, F.; Barroso, L.; Migueis,
J.; Melo, J.B.; Carreira, I.M. Genomic predictive model for recurrence and metastasis
development in head and neck squamous cell carcinoma patients. Scient. Rep. 2017, 7,
13897.
28. Ribeiro, I.P.; Esteves, L.; Santos, A.; Barroso, L.; Marques, F.; Caramelo, F.; Melo, J.B.;
Carreira, I.M. A seven-gene signature to predict the prognosis of oral squamous cell
carcinoma. Oncogene. 2021, 40, 3859–3869.
29. Cardoso, J.C.; Ribeiro, I.P.; Caramelo, F.; Tellechea, O.; Barbosa de Melo, J.; Marques
Carreira, I. Basal cell carcinomas of the scalp after radiotherapy for tinea capitis in
childhood: A genetic and epigenetic study with comparison with basal cell carcinomas
evolving in chronically sun-exposed areas. Exp. Dermatol. 2021, 30, 1126–1134.
Molecular Karyotyping 249

30. Tavares, I.; Martins, R.; Ribeiro, I.P.; Esteves, L.; Caramelo, F.; Abrantes, A.M.; Neves,
R.; Caetano-Oliveira, R.; Botelho, M.F.; Barbosa de Melo, J.; Diogo, D.; Tralhao, J.G.;
Carreira, I.M. Development of a genomic predictive model for cholangiocarcinoma
using copy number alteration data. J. Clin. Patho. 2022, 75, 274–278.
31. Couto Oliveira, A.; Ribeiro, I.P.; Pires, L.M.; Goncalves, A.C.; Paiva, A.; Geraldes, C.;
Roque, A.; Sarmento-Ribeiro, A.B.; Barbosa de Melo, J.; Carreira, I.M. Genomic char-
acterisation of multiple myeloma: Study of a Portuguese cohort. J. Clin Pathol, 2022, 75,
422–425.
32. Pinto-Leite, R.; Carreira, I.; Melo, J.; Ferreira, S.I.; Ribeiro, I.; Ferreira, J.; Filipe, M.;
Bernardo, C.; Arantes-Rodrigues, R.; Oliveira, P.; Santos, L. Genomic characterization
of three urinary bladder cancer cell lines: Understanding genomic types of urinary blad-
der cancer. Tumour Biol. 2014, 35, 4599–4617.
33. Spasova, V.; Mladenov, B.; Rangelov, S.; Hammoudeh, Z.; Nesheva, D.; Serbezov, D.;
Staneva, R.; Hadjidekova, S.; Ganev, M.; Balabanski, L.; Vazharova, R.; Slavov, C.;
Toncheva, D.; Antonova, O. Clinical impact of copy number variation changes in blad-
der cancer samples. Exp. Ther. Med. 2021, 22, 901.
34. Capela de Matos, R.R.; Ney Garcia, D.R.; Othman, M.A.K.; Moura Ferreira, G.; Melo,
J.B.; Carreira, I.M.; Meyer, C.; Marschalek, R.; Costa, E.S.; Land, M.G.P.; Liehr, T.;
Ribeiro RC.; Silva, M.L.M. A new complex karyotype involving a KMT2A-r variant
three-way translocation in a rare clinical presentation of a pediatric patient with acute
myeloid leukemia. Cytogenet. Genome Res. 2019, 157, 213–219.
35. Ronaghy, A.; Yang, R.K.; Khoury, J.D.; Kanagal-Shamanna, R. Clinical applications of
chromosomal microarray testing in myeloid malignancies. Curr. Hematol. Malign. Rep.
2020, 15, 194–202.
36. Wolf, M.T.F. Nephronophthisis and related syndromes. Curr. Opin. Pediatr. 2015, 27,
201–211.
37. Ala-Mello, S.; Koskimies, O.; Rapola, J.; Kääriäinen, H. Nephronophthisis in Finland:
Epidemiology and comparison of genetically classifed subgroups. Europ. J. Hum.
Genet. 1999, 7, 205–211.
38. Lu, Y.; Liang, Y.; Ning, S.; Deng, G.; Xie, Y.; Song, J.; Zuo, N.; Feng, C.; Qin, Y. Rare
partial trisomy and tetrasomy of 15q11-q13 associated with developmental delay and
autism spectrum disorder. Mol. Cytogenet. 2020, 13, 21.
39. Bi, W.; Cheung, S.-W.; Breman, A.M.; Bacino, C.A. 4p16.3 microdeletions and microdu-
plications detected by chromosomal microarray analysis: New insights into mechanisms
and critical regions. Am. J. Med. Genet. A. 2016, 170, 2540–2550.
40. Wapner, R.J.; Martin, C.L.; Levy, B.; Ballif, B.C.; Eng, C.M.; Zachary, J.M.; Savage,
M.; Platt, L.D.; Saltzman, D.; Grobman, W.A.; Klugman, S.; Scholl, T.; Simpson, J.L.;
McCall, K.; Aggarwal, V.S.; Bunke, B.; Nahum, O.; Patel, A.; Lamb, A.N.; Thom, E.A.;
Beaudet, A.L.; Ledbetter, D.H.; Shaffer, L.G.; Jackson, L. Chromosomal microarray ver-
sus karyotyping for prenatal diagnosis. N. Engl. J. Med. 2012, 367, 2175–2184.
41. Gao, J.; Liu, C.; Yao, F.; Hao, N.; Zhou, J.; Zhou, Q.; Zhang, L.; Liu, X.; Bian, X.; Liu, J.
Array-based comparative genomic hybridization is more informative than conventional
karyotyping and fuorescence in situ hybridization in the analysis of frst-trimester spon-
taneous abortion. Mol. Cytogenet. 2012, 5, 33.
22 FISH—Mitochondrial
DNA
Tigran Harutyunyan

CONTENTS
Introduction............................................................................................................ 251
Samples/Tissues/Materials..................................................................................... 254
Human Whole Blood Culture ....................................................................... 254
Slides with Metaphase Chromosomes .......................................................... 254
FISH Probe for mtDNA................................................................................ 254
Pretreatment and Postfxation ....................................................................... 255
mtDNA-FISH and Washing .......................................................................... 255
Description of Methods.......................................................................................... 255
Human Whole Blood Cultivation ................................................................. 255
Preparation of Metaphase Spreads................................................................ 256
Homemade mtDNA-FISH Probes ................................................................ 256
Slide Pretreatment......................................................................................... 257
mtDNA-FISH Procedure .............................................................................. 257
Washing and Counterstaining of Slides ........................................................ 258
Yields ..................................................................................................................... 258
Analysis of mtDNA Insertions in Metaphase Chromosomes....................... 258
Conclusions............................................................................................................ 258
Acknowledgments.................................................................................................. 259
References.............................................................................................................. 259

INTRODUCTION
Mitochondria are the powerhouses of cells, generating over 90% of energy in the
form of adenosine triphosphate (ATP) required for the normal cell functioning.[1]
Moreover, mitochondria are involved in cell cycle regulation and intrinsic apopto-
sis. In addition, each mitochondrion contains up to several thousand copies of its
own DNA molecule. In human cells, the mitogenome consists of a circular molecule
called mitochondrial chromosome, or better mitochondrial DNA (mtDNA), that har-
bors 16,569 bp and 37 genes, including 13 proteins (for the OXPHOS system), 22
tRNAs, and 2 rRNAs. In addition, at least eight mitochondrial-derived peptides have
been identifed which are encoded by 12S and 16S rRNA genes.[2]
Recent data indicates horizontal transfer of mitochondria as a mechanism of inter-
cellular signaling in normal and pathological conditions.[3] In addition, numtogenesis,

DOI: 10.1201/9781003223658-22 251

252 Cytogenetics and Molecular Cytogenetics

a de novo insertion of mtDNA in the nuclear genome, was observed at high fre-
quency in cancer cell, which can also result in genetic diseases.[4] It was shown that
environmental mutagens can increase the frequency of de novo mtDNA insertions
in human chromosomes after exposure to DNA double-strand break (DSB) induc-
ing agents.[5] Also, Lutz-Bonengel et  al.[6] and Wei et  al.[7] demonstrated the pres-
ence of amplifed NUMTs (nuclear mtDNA sequences) or mega-NUMTs in nuclear
genome of healthy individuals, resembling paternal mtDNA. In addition, implica-
tion of mtDNA in the innate immunity and COVID-19 pathogenesis was recently
shown.[8] Therefore, availability of tools for identifcation of mitochondrial exchange
and integration of mtDNA in the nuclear genome has clinical, forensic, phylogenetic
and research importance.
Sequencing of eukaryotic genomes revealed the presence of NUMTs in nuclear
DNA (nDNA) which occurred by insertion of corresponding sequences during evo-
lution, with variant frequencies within different species.[9] In the human genome,
over 1,000 NUMTs have been identifed, with the sizes ranging from 39 to 16,106
bp and encompassing ~400 kb of DNA stretches. Interestingly, the number of these
insertions in chromosomes positively correlate with the relative chromosome length
indicating random insertion of mtDNA. However, there is also evidence that inser-
tion loci of NUMTs in (human) genome are non-random, since enrichment with
repetitive elements in 2 kb of both fanking regions were identifed.[9] In recent years,
it was also shown that NUMTs might cause bias in the interpretation of nDNA
sequencing data due to high homology with mtDNA sequences. In particular, bipa-
rental inheritance of mtDNA was suggested after sequencing and analysis for het-
eroplasmy in the offspring of three heteroplasmic fathers, which indicated for the
presence of maternally and paternally inherited mtDNA haplotypes.[10] However,
the authors did not completely rule out NUMT contamination of the sequencing
data. Recently, the results of Lutz-Bonengel et al.[6] have challenged this hypothesis
by demonstrating the presence of heteroplasmy in the offspring carrying paternal
U4c1 mitogenome haplogroup along with maternal V haplogroup. Sequencing stud-
ies revealed the presence of maternal V mitotype only in cells devoid of nucleus (e.g.
thrombocytes and hair shafts), while cells depleted from mtDNA had only U4c1
mitotype. Fluorescence in situ hybridization (FISH) analysis confrmed the inser-
tion of mtDNA in 14q31 chromosome loci, proving the presence of mega-NUMT in
nDNA.[6] Thus, NUMTs should be considered in phylogenetic, medical genetic, and
forensic studies to avoid false-positive results.
While the biological role of inherited NUMTs is not determined, the occurrence
of de novo NUMTs and mtDNA insertions in the nDNA, defned as numtogenesis,
can be observed in pathological conditions, too.[4] The analysis of whole-genome
sequencing data from over 2,600 cancers demonstrated elevation in frequencies of
somatically acquired mtDNA insertions being highest in HER2+ breast cancer cells
and squamous cell lung cancers.[11] Elevation of de novo NUMTs was also observed
in colorectal adenocarcinoma genomes, which contained up to 4.2-fold higher
NUMTs compared to blood-derived normal genomes.[12]
Furthermore, human diseases caused by de novo insertions of mtDNA in
nDNA are described, although in a limited number of cases. Such insertions
were identifed in single patients with severe plasma factor VII defciency,[13]
FISH—Mitochondrial DNA 253

Usher syndrome type 1C,[14] Pallister-Hall syndrome,[15] mucolipidosis-IV,[16] lis-

sencephaly,[17] and X-linked hyper-IgM syndrome combined with immunodef-
ciency syndrome.[18] Thus, identifcation of mtDNA insertions in nDNA is of
diagnostic importance.
Recently detected was horizontal exchange of mitochondria between different
cells defned as “momiome” (mobile functions of mitochondria and the mitochon-
drial genome).[19] Thus, randomly exchanged mitochondria can be defned as per-
mutational mitochondria. Permutational mitochondria mainly occur via tunneling
nanotubes (TNTs), extracellular vesicles, and Cx43 gap junctions which can be
observed between normal and injured/transformed cells.[3] In the co-culture sys-
tem of human vascular smooth muscle cells (VSMCs) and mesenchymal stem cells
(MSCs), the shuttling of mitochondria from VSMCs signifcantly elevated the pro-
liferation of MSCs,[20] while the enhanced mitochondrial transfer from MSCs to epi-
thelial cells can rescue epithelial injury.[21]
Interestingly, the presence of a particular cell type in the tumor microenvironment
is important for the horizontal transfer of mitochondria. In the co-culture of bone
marrow stromal cells, endothelial cells, and MCF7 cells, mitochondria transfer was
observed only from endothelial cells to MCF7 cells.[22] It was shown that horizon-
tal mitochondrial transfer in the tumor microenvironment can restore the OXPHOS
and support the survival and chemoresistance in cancer cells.[23] Thus, inhibition of
permutational mitochondria occurrence in cancer cells may be a promising future
approach for anticancer therapy.
The potential of the environmental mutagens to induce translocation of the mtDNA
in the nuclear genome, defned as numtomutagenesis, has been demonstrated. First
studies in yeast showed that numtomutagenesis can occur in Saccharomyces cerevi‑
siae after induction of DSBs using I-SceI nuclease.[24] Ionizing radiation can induce
numtomutagenesis in chicken (Gallus gallus) genome after X-ray-irradiation of
eggs.[25] In our own studies, the numtomutagenic activity of DSB-inducing antican-
cer drug doxorubicin was analyzed in metaphase chromosomes of human periph-
eral blood lymphocytes. FISH analysis allowed to identify the number of mtDNA
insertions in each chromosome, which positively correlated with the frequency of
doxorubicin-induced micronuclei (marker of chromosome damage).[5] Sequencing of
the mtDNA insertion junctions in cancer cells demonstrated microhomologies sug-
gesting the involvement of the non-homologous end joining repair mechanism in the
insertion of the mtDNA fragments in nDNA.[26] Thus, DSB-inducing environmental
factors can also have numtomutagenic activity, which should be considered as a new
marker in mutagenicity testing.
Mutations of mtDNA can result in mitochondrial dysfunction and mtDNA deple-
tion syndromes with a broad spectrum of clinically heterogeneous symptoms due
to abnormal energy production by OXPHOS.[27] In addition, mtDNA copy num-
ber variations were detected in cancer cells and in patients infected with Human
Immunodefciency Virus 1 (HIV-1).[28, 29]
Circulating cell-free mtDNA which occurs due to intracellular mitochondrial
damage, is an early indicator of severe illness and mortality from COVID-19.[30]
Thus, rapid detection of mtDNA quantitative changes, as well as integration of
mtDNA in nDNA and permutational mitochondria, has clinical benefts.
254 Cytogenetics and Molecular Cytogenetics

FISH is a gold standard of the molecular cytogenetic diagnostics enabling quan-

titative and qualitative analysis with application of specifc probes for the target
sequences of variable size. Janes et  al.[31] analyzed depletion of mtDNA in fbro-
blasts treated with 2′,3′-dideoxycytidine, a standard medication for the treatment of
patients with HIV-1, using FISH; and Lucic et al.[32] demonstrated the applicability of
FISH for the detection of HIV-1 in cells infected with the virus in vitro.
In tumor cells of breast cancer patient the insertion of mtDNA in chromosome 10
was identifed using fber FISH technique.[26] Caro et al.[33] demonstrated the accu-
mulation of mtDNA fragments in nDNA with age in rat tissues. FISH analysis con-
frmed the presence of mtDNA in centromeric regions in 20 out of 40 chromosomes,
while Koo et al.[34] developed fber FISH for the detection of NUMTs in MCF-12A/
MDA-MB-231 normal epithelial cells.
In this chapter, FISH analysis of mtDNA insertions in metaphase chromosomes
of whole blood lymphocytes is described. However, this protocol can be also useful
for the analysis of permutational mitochondria and alterations of mtDNA copy num-
bers under pathological conditions.

SAMPLES/TISSUES/MATERIALS
FISH enables the detection of mtDNA on a single-cell level in any cells. Slides for
FISH analysis of numtomutagenesis can be prepared from both heparinized whole
blood or blood collected in EDTA containing vacutainers. Nevertheless, if meta-
phase chromosomes should be used in the analysis the heparinized blood is more
suitable in contrast to EDTA.

HUMAN WHOLE BLOOD CULTURE

• RPMI-1640 medium with L-glutamine (Cat. No. 11875093, Gibco™)
• Fetal bovine serum (Cat. No. F2442, Sigma-Aldrich)
• Penicillin-Streptomycin solution (Cat. No. P4333, Sigma-Aldrich)
• Phytohemagglutinin (Cat. No. M5030-BC, MERCK)

SLIDES WITH METAPHASE CHROMOSOMES

• Colcemid solution (Cat. No. 10295892001, Sigma-Aldrich)
• Potassium chloride (Cat. No. P9541, Sigma-Aldrich)
• Hypotonic solution of KCl: 0.075 M freshly prepared KCl solution used at
37°C
• Methanol 100 % (Cat. No. 34860, Sigma-Aldrich)
• Glacial acetic acid (Cat. No. 1000631011, Sigma-Aldrich)
• Carnoy’s fxative: methanol/glacial acetic acid freshly prepared mix in 3:1
(v/v) ratio and used at −20°C

FISH PROBE FOR MTDNA

• mtDNA
• Primers for mtDNA amplifcation and labeling
FISH—Mitochondrial DNA 255

• EDTA 0.5 M (Cat. No. 324506, Sigma-Aldrich)

• Ethanol 100 % (Cat. No. 459836, Sigma-Aldrich)
• tRNA (Cat. No. 10109541001, Sigma-Aldrich)
• Sodium acetate buffer solution 3 M, pH 5.2 (Cat. No. S7899, Sigma-Aldrich)
• Dextran sulfate buffer: 2 g + 10 ml 50 % deionized formamide/2×SSC/50
mM phosphate buffer for 3 h, 70°C. Store at −20°C.

PRETREATMENT AND POSTFIXATION

• Ethanol solutions: 70 %, 90 %, and 100 %
• PBS (Cat. No. 806544, Sigma-Aldrich)
• Hydrochloric acid solution 0.2 M (Cat. No. 13–1720, Sigma-Aldrich)
• Pepsin (Cat. No. P7012, Sigma-Aldrich)
• Pepsin stock solution: 1 g pepsin + 50 ml double-distilled water. Aliquot in
0.5 ml, store at −20°C
• Pepsin pretreatment solution: 5 ml of 0.2 M HCl + 95 ml of distilled water
at 37°C
• Postfx solution: 5 ml 2 % paraformaldehyde + 4.5 ml PBS + 0.5 ml MgCl2
(1 M)

MTDNA-FISH AND WASHING

• mtDNA FISH probe
• COT DNA (Cat. No. 11581074001, Sigma-Aldrich)
• Fluoroshield with DAPI (Cat. No. F6057, Sigma-Aldrich)
• 20×SSC (Cat. No. S6639, Sigma-Aldrich)
• Formamide (Cat. No. F9037, Sigma-Aldrich)
• Tween 20 (Cat. No. P9416, Sigma-Aldrich)
• Denaturation buffer: 70 ml formamide + 10 ml 20×SSC + 20 ml distilled
water
• 0.4×SSC solution: 10 ml 20×SSC + 490 ml distilled water
• 4×SSC/Tween solution: 100 ml 20×SSC + 400 ml distilled water + 0.25 ml
Tween 20

DESCRIPTION OF METHODS
Although mtDNA-FISH is applicable for both interphase nuclei and metaphase chro-
mosomes, the analysis of insertions of mtDNA in nDNA using metaphase chromo-
somes is recommended. This approach permits the identifcation of chromosomes
with mtDNA insertion(s) and insertion loci, enabling the analysis of the distribution of
mtDNA insertions in the genome. For the analysis of numtomutagenesis, human whole
blood should be exposed to the mutagen and cultivated under standard conditions.

HUMAN WHOLE BLOOD CULTIVATION

1. Add 1 ml of human whole blood to 9 ml RPMI-1640 medium, contain-
ing 10% fetal bovine serum, 1% penicillin/streptomycin, and 10 μg/ml
256 Cytogenetics and Molecular Cytogenetics

phytohemagglutinin-L, and incubate at 37°C. The total cultivation period

should be 72 h.
2. After 24 or 48 h growth add mutagen (e.g. doxorubicin) to the culture and
incubate at 37°C.

PREPARATION OF METAPHASE SPREADS

1. 1.5 h before harvesting (at 70.5 h of cultivation), add 0.1 ml colcemid to the
culture and incubate at 37°C to block the cell cycle in the metaphase.
2. Transfer the culture into a 15 ml centrifuge tube.
3. Centrifuge at room temperature for 7 min at 1500 rpm. Discard the super-
natant except for about 0.5 ml remaining above the cell pellet and gently
resuspend the pellet.
4. Add 10 ml of prewarmed (37°C) 0.075 M hypotonic solution of KCl, and
incubate for 15 min at 37°C.
5. Repeat step 3.
6. Quickly add 5 ml of Carnoy’s fxative (−20°C), and vortex immediately to
avoid clumps. Add another 5 ml of Carnoy’s fxative (−20°C), and incubate
for 10 min at room temperature.
7. Repeat step 3.
8. Resuspend the pellet in 5 ml of Carnoy’s fxative (−20°C), and repeat step 3.
9. Repeat step 8 twice.
10. Add 0.5 ml of Carnoy’s fxative (−20°C), and store the suspension at −20°C
until use.
11. Depending on the density of the suspension drop (20–50 µl) 2–3 drops onto
the precooled (4°C) clean and humid slides, and let the slide dry on the
warming plate at 51°C.
12. Keep the slides at room temperature for short-term storage, while slides for
long-term storage should be kept at −20°C.

HOMEMADE MTDNA-FISH PROBES

1. The mtDNA for FISH can be amplifed using specifc pair of primers (for-
ward: 5′-GAGCCGGAGCACCCTATGT-3′, reverse: 5′-GGGGAACG
TGTGGGCTATTT-3′) in the following PCR reaction mixture of 50 µl:
• 5 µl of Advantage 2 PCR Buffer (Takara, Kyoto, Japan),
• 4 µl of dNTP Mix, 5 pM/µl of each primer (1 µl each),
• 1 µl of Advantage 2 Titanium Taq Polymerase Mix (Takara),
• 6 µl of mtDNA (~50 pg)
• 30 µl of H2O.
2. The following PCR conditions for amplifcation of the mtDNA insert can be
set: denaturation at 95°C for 2 min, followed by 32 cycles of 95°C for 15 s, 61°C
for 30 s, 72°C for 1 min 30 s, then 72°C for 10 min and termination at 4°C.
FISH—Mitochondrial DNA 257

3. Labeling can be performed using nine pairs of primers specifc for

mtDNA amplifcation,[35] with the PCR conditions mentioned previously.
The optional reaction mixture is as follows:
• 2 µl of buffer,
• 2 µl of Label-mix (Atto488 NT Labeling Kit, Jena Bioscience, Jena,
Germany),
• 2 µl of 25 mM MgCl2,
• 0.12 µl of AmpliTaq DNA Polymerase,
• 2 µl of fuorochromes,
• 12.08 µl of H2O.
4. The specifcity of the obtained probe can be confrmed using positive con-
trol with mtDNA insertion in chromosomes or using cells with undamaged
mitochondria. In addition, a negative control (non-human chromosomes
or mitochondria) is recommended to be used.
5. Mix the solution using the 20 µl pipette tip, and incubate at 15°C for 90
min.
6. Terminate the reaction with 1 µl of 0.5 M EDTA, mix vigorously, spin
down and incubate at 65°C for 10 min.
7. Precipitate the labeled probe with 10 µl of tRNA + 5 µl sodium acetate +
100 µl 100 % ethanol at −20°C during the overnight.
8. Centrifuge the mixture at 15,000 rpm at 4°C for 15 min.
9. Discard the supernatant and dry the pellet using a speed vac.
10. Dilute the pellet in 20 µl of dextran sulfate buffer, and mix at 65°C for 5
min.
11. Keep the probe at −20°C until use.

SLIDE PRETREATMENT
1. Dehydrate slides in an ethanol series (70 %, 90 %, 100 %, 3 min each) and
air-dry at room temperature.
2. Add 0.5 ml of pepsin to 100 ml of pepsin pretreatment solution, and put
slides for 3–5 min in a Coplin jar at 37°C in the water bath.
3. Wash slides in the PBS for 5 min at room temperature.
4. Add 100 µl of postfx solution and cover with coverslips for 10 min at room
temperature.
5. Remove coverslips; repeat step 3 and step 1.

MTDNA-FISH PROCEDURE
1. Mix 1 µl of the probe with 7 µl dextran sulfate in the Eppendorf tube con-
taining COT DNA. The optimal concentration of the COT DNA should be
determined experimentally.
2. Prewarm the mixture at 45°C for 20 min.
258 Cytogenetics and Molecular Cytogenetics

3. Add 100 µl of denaturation buffer to each slide, cover with coverslip, and
incubate on a warming plate at 81°C for 3 min and in parallel begin the
denaturation of the probe at 75°C in PCR machine.
4. Remove the coverslip and put slides in a 70 % ethanol at −20°C for 3 min.
5. Dehydrate slides in ethanol series of 90 % and 100 % at room temperature
for 3 min each and air-dry.
6. Add 8 µl of the probe onto half of the slide, and cover with 24×24 coverslip
and seal with rubber cement.
7. Place the slides in the humid chamber and incubate at 37°C overnight.

WASHING AND COUNTERSTAINING OF SLIDES

1. Remove the rubber cement and coverslips, and put the slides in the Coplin
jar flled with prewarmed 0.4×SSC at 65°C for 5 min in the water bath.
2. Put the slides in the new Coplin jar with 4×SSC/Tween on the shaker at
room temperature for 5 min.
3. Wash the slides in the PBS for 5 min, then dehydrate in ethanol series
(70 %, 90 %, 100 %, 3 min each), and air-dry at room temperature.
4. Add 30 µl of fuoroshield containing DAPI for counterstaining, cover with
24×60 mm coverslip, and incubate at 4°C for at least 20 min.
5. Analyze the results under a fuorescent microscope.

YIELDS
ANALYSIS OF MTDNA INSERTIONS IN METAPHASE CHROMOSOMES
1. For the detection of mtDNA insertions in metaphase chromosomes by
FISH, at least 1,800 metaphase spreads are recommended to be analyzed.
2. Capture mtDNA signals using fuorescent microscope quipped with 100×
objective and the camera with high sensitivity.
3. Statistical analysis can be performed using t-test for the comparison of
mtDNA insertion frequencies between untreated and treated variants.
4. Construction of the map with mtDNA insertions in each chromosome
according to Harutyunyan et  al.[5] is recommended for the description of
the distribution of mtDNA insertions in the genome.
5. For evaluation of the quantitative changes in mitochondrial network,
the application of free-to-use program ImageJ (developed at the NIH) is
recommended.

CONCLUSIONS
Functional mitochondria are important players of intracellular and intercellular sig-
naling. However, exchange of mitochondria between cells, mutations of mtDNA, or
translocation of mtDNA in the nuclear genome can have deleterious effects for the
organism. Moreover, DSB-inducing environmental agents are capable to increase
numtomutagenic events in the cells. Thus, methods for rapid detection of aberrant
FISH—Mitochondrial DNA 259

FIGURE 22.1 Insertion of mtDNA in chromosome 14 identifed by mtDNA-FISH; the same

patient as presented in [5] was studied here.

behavior of mitochondria or mtDNA will have diagnostic importance in many path-

ological conditions, including cancer, viral infections, and mtDNA disorders.
FISH analysis of mtDNA (Figure 22.1) enables quantitative and qualitative analy-
sis of permutational mitochondria, numtogenesis, and numtomutagenesis in human
cells. Further studies are required for identifcation of molecular pathways regulating
mtDNA escape, transfer, and insertion in human chromosomes.

ACKNOWLEDGMENTS
This work was supported by the RA MESCS and BMBF (project #AG-01/20), and
RA MESCS (project #21AG-1F068).

REFERENCES
1. Yan, C.; Duanmu, X.; Zeng, L.; Liu, B.; Song, Z. Mitochondrial DNA: Distribution,
mutations, and elimination. Cells. 2019, 8(4), 379.
260 Cytogenetics and Molecular Cytogenetics

2. Merry, T.L.; Chan, A.; Woodhead, J.; Reynolds, J.C.; Kumagai, H.; Kim, S.J.; Lee, C.
Mitochondrial-derived peptides in energy metabolism. Am J Physiol Endocrinol Metab.
2020, 319(4), E659–E666.
3. Valenti, D.; Vacca, R.A.; Moro, L.; Atlante, A. Mitochondria can cross cell boundaries:
An overview of the biological relevance, pathophysiological implications and therapeutic
perspectives of intercellular mitochondrial transfer. Int J Mol Sci. 2021, 22(15), 8312.
4. Singh, K.K.; Choudhury, A.R.; Tiwari, H.K. Numtogenesis as a mechanism for develop-
ment of cancer. Semin Cancer Biol. 2017, 47, 101–109.
5. Harutyunyan, T.; Al-Rikabi, A.; Sargsyan, A.; Hovhannisyan, G.; Aroutiounian, R.;
Liehr, T. Doxorubicin-induced translocation of mtDNA into the nuclear genome of
human lymphocytes detected using a molecular-cytogenetic approach. Int J Mol Sci.
2020, 21(20), 7690.
6. Lutz-Bonengel, S.; Niederstätter, H.; Naue, J.; Koziel, R.; Yang, F.; Sänger, T.; Huber, G.;
Berger, C.; Pfugradt, R.; Strobl, C.; et al. Evidence for multi-copy Mega-NUMTs in the
human genome. Nucleic Acids Research. 2021, 49(3), 1517–1531.
7. Wei, W.; Pagnamenta, A.T.; Gleadall, N.; Sanchis-Juan, A.; Stephens, J.; Broxholme, J.;
Tuna, S.; Odhams, C.A.; Genomics England Research Consortium; NIHR BioResource;
et  al. Nuclear-mitochondrial DNA segments resemble paternally inherited mitochon-
drial DNA in humans. Nat Commun. 2020, 11(1), 1740.
8. Singh, K.K.; Chaubey, G.; Chen, J.Y.; Suravajhala, P. Decoding SARS-CoV-2 hijack-
ing of host mitochondria in COVID-19 pathogenesis. Am J Physiol Cell Physiol. 2020,
319(2), C258–C267.
9. Calabrese, F.M.; Balacco, D.L.; Preste, R.; Diroma, M.A.; Forino, R.; Ventura, M.;
Attimonelli, M. NumtS colonization in mammalian genomes. Sci Rep. 2017, 7(1), 16357.
10. Luo, S.; Valencia, C.A.; Zhang, J.; Lee, N.C.; Slone, J.; Gui, B.; Wang, X.; Li, Z.; Dell,
S.; Brown, J.; et al. Biparental inheritance of mitochondrial DNA in humans. Proc Natl
Acad Sci U S A. 2018, 115(51), 13039–13044.
11. Yuan, Y.; Ju, Y.S.; Kim, Y.; Li, J.; Wang, Y.; Yoon, C.J.; Yang, Y.; Martincorena, I.;
Creighton, C.J.; Weinstein, J.N.; et  al. Comprehensive molecular characterization of
mitochondrial genomes in human cancers. Nat Genet. 2020, 52(3), 342–352.
12. Srinivasainagendra, V.; Sandel, M.W.; Singh, B.; Sundaresan, A.; Mooga, V.P.; Bajpai,
P.; Tiwari, H. K.; Singh, K.K. Migration of mitochondrial DNA in the nuclear genome of
colorectal adenocarcinoma. Genome Med. 2017, 9(1), 31.
13. Borensztajn, K.; Chafa, O.; Alhenc-Gelas, M.; Salha, S.; Reghis, A.; Fischer, A.M.;
Tapon-Bretaudière, J. Characterization of two novel splice site mutations in human fac-
tor VII gene causing severe plasma factor VII defciency and bleeding diathesis. Br J
Haematol. 2002, 117(1), 168–171.
14. Ahmed, Z.M.; Smith, T.N.; Riazuddin, S.; Makishima, T.; Ghosh, M.; Bokhari, S.;
Menon, P.S.; Deshmukh, D.; Griffth, A.J.; Riazuddin, S.; et al. Nonsyndromic recessive
deafness DFNB18 and Usher syndrome type IC are allelic mutations of USHIC. Hum
Genet. 2002, 110(6), 527–531.
15. Turner, C.; Killoran, C.; Thomas, N.S.; Rosenberg, M.; Chuzhanova, N.A;, Johnston,
J.; Kemel, Y.; Cooper, D.N.; Biesecker, L.G. Human genetic disease caused by de novo
mitochondrial-nuclear DNA transfer. Hum Genet. 2003, 112(3), 303–309.
16. Goldin, E.; Stahl, S.; Cooney, A.M.; Kaneski, C.R.; Gupta, S.; Brady, R.O.; Ellis, J.R.;
Schiffmann, R. Transfer of a mitochondrial DNA fragment to MCOLN1 causes an
inherited case of mucolipidosis IV. Hum Mutat. 2004, 24(6), 460–465.
17. Millar, D.S.; Tysoe, C.; Lazarou, L.P.; Pilz, D.T.; Mohammed, S.; Anderson, K.;
Chuzhanova, N.; Cooper, D.N.; Butler, R. An isolated case of lissencephaly caused by
FISH—Mitochondrial DNA 261

the insertion of a mitochondrial genome-derived DNA sequence into the 5′ untranslated

region of the PAFAH1B1 (LIS1) gene. Hum Genomics. 2010, 4(6), 384–393.
18. Li, X.; Xu, D.; Cheng, B.; Zhou, Y.; Chen, Z.; Wang, Y. Mitochondrial DNA insert into
CD40 ligand gene-associated X-linked hyper-IgM syndrome. Mol Genet Genomic Med.
2021, 9(5), e1646.
19. Singh, B.; Modica-Napolitano, J.S.; Singh, K. Defning the momiome: Promiscuous
information transfer by mobile mitochondria and the mitochondrial genome. Semin
Cancer Biol. 2017, 47, 1–17.
20. Vallabhaneni, K.C.; Haller, H.; Dumler, I. Vascular smooth muscle cells initiate prolif-
eration of mesenchymal stem cells by mitochondrial transfer via tunneling nanotubes.
Stem Cells Dev. 2012, 21(17), 3104–3113.
21. Ahmad, T.; Mukherjee, S.; Pattnaik, B.; Kumar, M.; Singh, S.; Kumar, M.; Rehman, R.;
Tiwari, B.K.; Jha, K.A.; Barhanpurkar, A.P.; et al. Miro1 regulates intercellular mito-
chondrial transport & enhances mesenchymal stem cell rescue effcacy. EMBO J. 2014,
33(9), 994–1010.
22. Pasquier, J.; Guerrouahen, B.S.; Al Thawadi, H.; Ghiabi, P.; Maleki, M.; Abu-Kaoud, N.;
Jacob, A.; Mirshahi, M.; Galas, L.; Rafi, S.; et al. Preferential transfer of mitochondria
from endothelial to cancer cells through tunneling nanotubes modulates chemoresis-
tance. J Transl Med. 2013, 11, 94.
23. Sahinbegovic, H.; Jelinek, T.; Hrdinka, M.; Bago, J. R.; Turi, M.; Sevcikova, T.; Kurtovic-
Kozaric, A.; Hajek, R.; Simicek, M. Intercellular mitochondrial transfer in the tumor
microenvironment. Cancers (Basel). 2020, 12(7), 1787.
24. Ricchetti, M.; Fairhead, C.; Dujon, B. Mitochondrial DNA repairs double-strand breaks
in yeast chromosomes. Nature. 1999, 402(6757), 96–100.
25. Abdullaev, S.A.; Fomenko, L.A.; Kuznetsova, E.A.; Gaziev, A.I. Experimental detection
of integration of mtDNA in the nuclear genome induced by ionizing radiation. Radiats
Biol Radioecol. 2013, 53(4), 380–388.
26. Ju, Y.S.; Tubio, J.M.; Mifsud, W.; Fu, B.; Davies, H.R.; Ramakrishna, M.; Li, Y.; Yates,
L.; Gundem, G.; Tarpey, P.S.; et al. Frequent somatic transfer of mitochondrial DNA into
the nuclear genome of human cancer cells. Genome Res. 2015, 25(6), 814–824.
27. Basel, D. Mitochondrial DNA Depletion Syndromes. Clin Perinatol. 2020, 47(1),
123–141.
28. Reznik, E.; Miller, M.L.; Şenbabaoğlu, Y.; Riaz, N.; Sarungbam, J.; Tickoo, S.K.;
Al-Ahmadie, H.A.; Lee, W.; Seshan, V.E.; Hakimi, A.A.; et al. Mitochondrial DNA copy
number variation across human cancers. Elife. 2016, 5, e10769.
29. Schank, M.; Zhao, J.; Moorman, J.P.; Yao, Z. Q. The impact of HIV- and ART-induced
mitochondrial dysfunction in cellular senescence and aging. Cells. 2021, 10(1), 174.
30. Scozzi, D.; Cano, M.; Ma, L.; Zhou, D.; Zhu, J.H.; O’Halloran, J.A.; Goss, C.; Rauseo,
A.M.; Liu, Z.; Sahu, S.K.; et al. Circulating mitochondrial DNA is an early indicator of
severe illness and mortality from COVID-19. JCI insight. 2021, 6(4), e143299.
31. Janes, M.S.; Hanson, B.J.; Hill, D.M.; Buller, G.M.; Agnew, J.Y.; Sherwood, S.W.; Cox,
W.G.; Yamagata, K.; Capaldi, R.A. Rapid analysis of mitochondrial DNA depletion by
fuorescence in situ hybridization and immunocytochemistry: Potential strategies for
HIV therapeutic monitoring. J Histochem Cytochem. 2004, 52(8), 1011–1018.
32. Lucic, B.; Wegner, J.; Stanic, M.; Jost, K.L.; Lusic, M. 3D immuno-DNA fuorescence in
situ hybridization (FISH) for detection of HIV-1 and cellular genes in primary CD4+ T
cells. Methods Mol Biol. 2021, 2157, 239–249.
33. Caro, P.; Gómez, J.; Arduini, A.; González-Sánchez, M.; González-García, M.; Borrás,
C.; Viña, J.; Puertas, M.J.; Sastre, J.; Barja, G. Mitochondrial DNA sequences are present
262 Cytogenetics and Molecular Cytogenetics

inside nuclear DNA in rat tissues and increase with age. Mitochondrion. 2010, 10(5),
479–486.
34. Koo, D.H.; Singh, B.; Jiang, J.; Friebe, B.; Gill, B.S.; Chastain, P.D.; Manne, U.; Tiwari,
H.K.; Singh, K.K. Single molecule mtDNA fber FISH for analyzing numtogenesis. Anal
Biochem. 2018, 552, 45–49.
35. Ramos, A.; Santos, C.; Barbena, E.; Mateiu, L.; Alvarez, L.; Nogués, R.; Aluja, M.P.
Validated primer set that prevents nuclear DNA sequences of mitochondrial origin
co-amplifcation: A revision based on the New Human Genome Reference Sequence
(GRCh37). Electrophoresis. 2011, 32(6–7), 782–783.
23 FISH—in Birds
Rafael Kretschmer, Michelly da Silva dos Santos,
Ivanete de Oliveira Furo, Edivaldo Herculano
Correa de Oliveira and Marcelo de Bello Cioff

CONTENTS
Introduction............................................................................................................ 263
Material .................................................................................................................. 264
FISH Probe Generation and Labeling........................................................... 265
Slide Pretreatment......................................................................................... 265
FISH Procedure ............................................................................................ 266
Methods.................................................................................................................. 266
Slide Preparation: Dropping the Cell Suspension ........................................ 266
FISH Probes.................................................................................................. 267
Commercially Available Probes........................................................ 267
Homemade Probes ............................................................................ 267
Probes Obtained by Flow Sorting................................................................. 270
Probe Labeling.............................................................................................. 272
Nick Translation................................................................................ 272
DOP-PCR Labeling .......................................................................... 272
Precipitation of DNA Probe.......................................................................... 273
Fluorescence In Situ Hybridization (FISH).................................................. 273
FISH with rDNA and Telomeric Sequences ..................................... 274
Slide Pretreatment with Pepsin..................................................................... 274
Chromosome Painting................................................................................... 274
FISH with Microsatellite Sequences ............................................................ 275
Important Notes for Proceeding FISH.......................................................... 276
Acknowledgments.................................................................................................. 276
References.............................................................................................................. 276

INTRODUCTION
Birds are the most diverse biological group among tetrapods, with more than
10,900 existing species.[1] Molecular and morphological studies support the division
of living birds into three monophyletic groups: Palaeognathae, Galloanserae, and
Neoaves.[2] Due to this wide variation, birds have been used as model species in
numerous biological studies, focusing on ecology, behavior, genetics, and cytogenet-
ics, and others.[3–6]

DOI: 10.1201/9781003223658-23 263

264 Cytogenetics and Molecular Cytogenetics

Cytogenetics is a useful tool in bird cytotaxonomy, especially for groups with a great
chromosome variability. Most of the karyological data in birds are based on classical
cytogenetic techniques, involving conventional staining and chromosomal banding
methods, such as C-banding, (used to detect constitutive heterochromatin), G-banding
(to identify regions rich in adenine and thymine (AT), and cytosine and guanine (CG)),
and AgNOR silver staining method applied to reveal nucleolar organizer regions.[7–9]
Despite its signifcant contribution to the characterization of karyotypes and provision
of basic genomic information used in cytotaxonomy, banding resolution stands as a
limitation in the identifcation of chromosome homology and comparative studies.
In this sense, the emergence of the fuorescence in situ hybridization (FISH) technique
allowed an advance towards more accurate cytogenetic studies, enabling the identifca-
tion and localization of DNA sequences or specifc regions along the chromosomes.[10]
Therefore, FISH helped to overcome limitations of comparative chromosomal studies
based on banding methods, especially in birds, with typically high 2n and many micro-
chromosomes (i.e. punctiform elements not easily distinguishable from each other).[6]
Chromosome painting data are available for 117 species, which represent less than
1% of all known bird species.[11] So far, whole chromosome painting probes (WCP)
from only six different bird species have been used in avian comparative cytoge-
netics: chicken (Gallus gallus—GGA, Galliformes, 2n=78), stone-curlew (Burhinus
oedicnemus—BOE, Charadriiformes, 2n=42), white hawk (Leucopternis albicollis—
LAL, Falconiformes, 2n= 68), griffon vulture (Gyps fulvus—GFU, Falconiformes,
2n=66), eared dove (Zenaida auriculata—ZAU, Columbiformes, 2n=76), and monk
parakeet (Myiopsitta monachus—MMO, Psittaciformes, 2n=48).[12–17] Among them,
G. gallus probes stand out as the most used ones due to the economic importance
of chickens, its well-known genome, and by having a karyotype very similar to the
putative avian ancestral one.[6] Overall, comparative chromosome studies using WCP
in birds have detected numerous inter- and intrachromosomal rearrangements, even
in species with apparently conserved karyotypes.[6, 11, 18]
In addition to WCP, the development of probes using specifc DNA sequences for
FISH has allowed cytogenetic mapping of different repetitive sequences in distinct
birds species, such as telomeric sequences, 18S/28SrDNA, 5S rDNA, and microsat-
ellites, establishing chromosomal landmarks and providing information concerning
different aspects such as karyotype organization and evolution and, besides, allow-
ing insights on origin and differentiation of sex chromosomes.[19–26]
For all these reasons, FISH has become an indispensable tool for cytogenomic
investigations in birds, and in this chapter, we present the current FISH protocol used
in our laboratories. We also include details about the isolation and preparation of the
most commonly used probes in avian cytogenetics. In addition, we highlight some
steps related to the quality of avian chromosome preparations, which is a crucial
condition for successful hybridizations.

MATERIAL
Besides the standard cell biological and molecular cytogenetic equipment, including
standard reagents (e.g., colchicine, methanol, acid acetic, ethanol, among others),
specialized items required are listed next.
FISH—in Birds 265

FISH PROBE GENERATION AND LABELING

• AmpliTaq® DNA Polymerase, Stoffel Fragment (Cat. No.: N8080038,
Applied Biosystems, USA)
• Biotin 14-dATP (Cat. No.: 19524016, Invitrogen, USA)
• Biotin16 NT Labeling Kit (Cat. No.: PP-310S-BIO16, Jena Bioscience,
Germany)
• Cy5-dUTP (Cat. No.: 25005446, GE Healthcare Life Science, USA)
• Deionized formamide (Cat. No.: 1 09684 2500, Merck, Germany; aliquot
and store at +4oC (See Note 1)
• Deoxinucleotides 100mM (Cat. No.: DNTP100–1KT, Sigma, USA)
• Dextran sodium sulfate (Cat. No.: D8906–10G, Sigma, USA)
• Digoxigenin NT Labeling Kit (Cat. No.: PP-310S-DIGX, Jena Bioscience,
Germany)
• Digoxigenin-11-dUTP (Cat. No.: 11093088910, Roche Diagnostics,
Switzerland)
• dNTP label mix: 2mM dATP/dCTP/dGTP, 1mM dTTP)
• DOP primer (5′- CCG ACT CGA GNN NNN NAT GTG G -3′)
• Double-distilled water (ddH20) = Aqua ad iniectabilia (Cat. No.: 2351544,
Braun, Germany; aliquot and store at −20°C)
• EDTA 0.5 M (e.g. Merck, Germany; store at −20°C).
• Hybridization buffer: Dissolve 2g dextran sodium sulfate in 10 ml 50%
deionized formamide/2×SSC/50 mM phosphate buffer for 3 h at 70°C;
adjusted pH to 7 with phosphate buffer; hydrochloric acid destabilizes buf-
fer solution. Aliquot and store at −20°C.
• Nick translation mix (Cat. No.: 11 745 808 910, Roche Diagnostics,
Switzerland)
• Ribonucleic acid transfer—tRNA (Cat. No.: R8508, Sigma, USA)
• Sodium acetate solution (3 M, pH 5.2; e.g. Merck, Germany; store at
−20°C)
• Spectrum-Orange dUTP (Cat. No.: 6J9415, Abbott, USA)
• Spectrum-Green dUTP (Cat. No.: 6J9410, Abbott, USA)
• TexasRed-dUTP (Cat. No.: C-7631, Molecular Probes, USA)
• TexasRed-dUTP/Spectrum-Orange-dUTP/Spectrum-Green-dUTP/
Streptavidin-Cy3/Streptavidin-Cy5 and Avidin-FITC working solutions:
Reconstitute 1 mg with 1.0 ml of double-distilled water (ddH20), dispense
into suitable aliquots and freeze

SLIDE PRETREATMENT
• 1×PBS (phosphate-buffered saline—Cat. No.: L1825, Biochrom, Germany;
store at room temperature)
• Pepsin stock solution (Cat. No.: P-7012, Sigma, USA)
• Pepsin solution (0.005%): 99 µl H2O, 10µl HCl and 2.5µl Pepsin (20 mg/ml)
• RNase A (Cat. No.: R4642, Sigma, USA)
• RNase solution (10µg/ml): 1.5 µl RNase A (10 mg/ml) and 1.5 ml 2×SSC.
266 Cytogenetics and Molecular Cytogenetics

FISH PROCEDURE
• Avidin-FITC (Cat. No.: A2901, Sigma, USA)
• Anti-digoxigenin fuorescein (Cat. No.: 11207741910, Roche Diagnostics,
Switzerland)
• Anti-digoxigenin rhodamin (Cat. No.: 11207750910, Roche Diagnostics,
Switzerland)
• Denaturation buffer: 70% (v/v) deionized formamide (See Note 1), 20% (v/v)
fltered double distilled water, 10% (v/v) 20×SSC; make fresh as required
• Detection solution 1: anti-biotin-labeled probes using a layer of Cy3-a or
Cy5-avidin (1:1000)
• Detection solution 2: anti-digoxigenin-labeled probes using monoclonal
anti-digoxin antibody produced in mice (1:500)
• Detection solution 3: FITC-labeled probes were detected with a layer of
rabbit anti-FITC (1:200) followed by a layer of goatanti-rabbit-FITC anti-
bodies (1:100)
• Formamide solution: 2×SSC/50% deionized formamide, pH 7.0 (See
Note 1)
• Hybridization buffer: Dissolve 2g of dextran sodium sulfate in 10 ml of 50%
formamide/2×SSC/50 mM phosphate buffer for 1 hour at 70°C. Aliquot
and stock at −20°C
• 1×PBS(phosphate-buffered saline—Cat. No.: L1825, Biochrom, Germany;
store at room temperature).
• Phosphate buffer: prepare 0.5 M Na2HPO4 and 0.5 M NaH2PO4, mix these
two solutions (1:1) to get pH 7.0, and then aliquot and store at −20°C
• 20×SSC = saline sodium citrate (Cat. No.: 15557–036; Invitrogen, USA;
store at room temperature); set up 1× and 2× SSC before use
• Streptavidin-Cy5 (Cat. No.: 25800881, GE Healthcare Life Sciences, USA)
• Streptavidin-Cy3 (Cat. No.: S6402, Sigma, USA)
• Tween 20 = polyoxyethylene-sorbitan monolaurate (Cat. No.: 10670–1000,
Sigma, Germany, store at room temperature)
• Vectashield Mounting Medium with DAPI FR/10 ml (Cat. No.: H-1200,
Vector, USA)
• Washing buffer (4×SCCT): 4×SSC, 0.05% Tween 20; make fresh as
required.

METHODS
SLIDE PREPARATION: DROPPING THE CELL SUSPENSION
To obtain chromosome preparations from birds demands biological samples; hence,
it is important to notice that such procedures usually require previous approval by
an Animal Experimentation Ethics Committee and permission to sample the ani-
mals, either for in situ or ex situ studies in birds. Avian mitotic chromosomes can
be directly obtained from samples of different tissues, including kidney, liver, bulb
FISH—in Birds 267

feather, and bone marrow,[27] as well as from embryos/eggs.[28] However, chromo-

somes can also be obtained from lymphocyte cultures,[29] and the most used pro-
tocol is culture of fbroblast cells.[24] The general protocol for mitotic chromosome
preparation consists of treatment with colchicine, hypotonic solution, and fxation in
methanol and acid acetic (3:1).
The quality of chromosomal preparations, including i) high amount of metaphase
plates, ii) correct spreading of the chromosomes, and iii) good chromosomal mor-
phologies, is important for successful FISH experiments. If not accurately done,
cytoplasm and other cellular materials may remain on the metaphase plates, block-
ing the proper probe hybridization and evaluable results. Therefore, the chromo-
somes obtained from fbroblast culture usually provides the best preparations.
To obtain high-quality metaphase spreads, some conditions such as humidity and
temperature should be considered while dropping the mitotic cells on the slides:

1. Wash and thoroughly clean a glass slide, and prepare a fresh fxative solu-
tion (3:1 methanol: acetic acid).
2. Place a coupling jar with distillate water in a microwave to heat. Hold the
slide over boiling water to create a flm of condensation. Alternatively,
place the slide under a rack in a water bath at 55°C.
3. Pipette about 20 μl of the cell suspension on the slide.
4. Pipette immediately 20 μl fxative on the slide.
5. Dry the preparation directly on the air.

FISH PROBES
Commercially Available Probes
We have used two kinds of commercially available probes for FISH in bird species:

1. Microsatellite probes: here labeled oligonucleotides containing mono-, di-,

and tri- microsatellites have been used. These sequences can be ordered as
directly labeled with Cy3 or Cy5 at 5′ terminal during synthesis (Sigma,
St. Louis, MO, USA). Among the most used microsatellites are (CA)15,
(CAC)10, (CAG)10, (CGG)10, (GA)15, (GAA)10, and (GAG)10.
2. Telomeric probes: Telomeric (TTAGGG)n repeats can be detected by
FISH using a Telomere PNA FISH Kit/FITC (Cat. No.: K5325, DAKO) or
Telomere PNA FISH Kit/Cy3 (Cat. No.: K5326, DAKO). Telomeric probes
can also be generated by PCR (please see next).

Homemade Probes
Several methods are available to obtain probes for FISH experiments. Usually, the
probes for FISH experiments in birds can be obtained through PCR amplifcation
(rDNA and telomeric) or isolated by fow cytometry followed by PCR amplifca-
tion (whole chromosome probes). Next, we list the methods for obtaining the most
used probes in our laboratories.
268 Cytogenetics and Molecular Cytogenetics

• rDNAs: The distribution of the 45S gene has been widely investigated in
different groups of birds, being a useful marker for evolutionary studies.[26]
These genes are highly conserved and organized in multigene families con-
sisting of many copies, in which the 45S rDNA encodes for 18S, 5.8S, and
28S rRNAs.[30] The 5S ribosomal gene, which encodes the 5S rRNA′,[30] has
been investigated only in a few species.
1. For sequence amplifcation, mix the reagents listed next in a 0.6 ml tube
(DNAse free).
2. Perform amplifcation in a thermocycler according to the program
described next.
3. Check 2 μl of amplifcation product on a 1% agarose gel with appro-
priate size markers. The product should yield a visible smear between
~1,400bp.
4. Store the amplifed DNA at −20°C.
• 18S-rDNA directed PCR amplifcation can be obtained using 18SF
(5′CCGAGGACCTCACTAAACCA-3′) and 18SR (5′-CCGCTTTGG
TGACTCTTGAT-3′) as primers to generate a fragment of approximately
1,400 base pairs. The primers were developed from the fsh Hoplias mala‑
baricus,[31] and due to the high sequence conservation, it has been applied
successfully in several birds species.[26] Here, we amplifed the 18S rDNA
gene using the Emu (Dromaius novaehollandiae) DNA as a template. It is
known that the Emu has this multigene family in a pair of microchromo-
somes,[32] and we confrmed this result (Figure 23.1 A). Reagents and details
for the reaction are shown in Table 23.1.
• 5S-rDNA directed PCR: The probes were obtained from the DNA
of the fsh Leporinus obtusidens by PCR using the primers 5SF
(5′-TACGCCCGATCTCGTCCGATC-3′) and 5SR (5′-CAGGCTGGTAT
GGCCGTAAGC-3′), according to.[33] These fragments of approximately
200 bp were obtained and successfully applied in FISH experiments in

FIGURE  23.1 18S (A, green) and 5S (B, red) rDNA distribution in the Emu (Dromaius
novaehollandiae).
FISH—in Birds 269

TABLE 23.1
a) How to Prepare 25 μl Reaction Master Mix (per Sample) for 18S-rDNA
Reagent Amount Final Concentration
Genomic DNA 25 ng -
Buffer 10× 2.5 µl 1×
MgCl2 (50 mM) 0.75 µl 1.5 mM
Primer reverse (10 mM) 0.5 µl 0.2 mM
Primer forward (10 mM) 0.5 µl 0.2 mM
dNTP Mix (10mM each) 3.1 µl 2.5mM each (dATP, dCTP, dGTP, dTTP)
Adjust with ddH2O 16.45 µl -
Taq polymerase 5U/µl 0.2 µl 1.0 U

b) Thermocycler Program to Produce 18S-rDNA Probes

Number of Cycles Reaction Temperature, Time
1 Initial denaturation 94°C—5′
Denaturation 95°C—1′00′′
35 cycles Annealing 60°C—1′00′′
Extension 72°C—1′30′′
1 Final extension 72°C—7′00′′
- Hold 4º ∞

birds.[34] Reagents and details for the reaction are shown in Tables 23.1 and
23.2. As an example, here we amplifed the 5S rDNA gene using the Emu
DNA as a template. This gene was found in a small macrochromosome,
probably the ninth pair in the Emu (Figure 23.1 B).
1. For the sequence amplifcation, mix the reagents listed next in a 0.6 ml
tube (DNAse free).
2. Perform amplifcation in a thermocycler according to the program
described next.
3. Check 2 μl of amplifcation product on a 1% agarose gel with appropriate
size markers. The product should yield a visible smear between ~200bp.
4. Store the amplifed DNA at −20°C.
• Telomere directed PCR amplifcation can be obtained using Primer: F
(5′ TTAGGG-3′)5 e R (5′ CCCTAA-3′)5.[35] In this case, the amplifcation
reaction can be placed with (Table  23.3) or without DNA as a template
(Table 23.4).
1. For the sequence amplifcation, mix the reagents listed next in a 0.6 ml
tube (DNAse free).
2. Perform amplifcation in a thermocycler according to the program
described next (Table 23.2).
3. Check 5 μl of amplifcation product on a 1% agarose gel with appropriate
size markers. The product size should be up to 25,000bp or 25Kb.
4. Store the amplifed DNA at −20°C.
270 Cytogenetics and Molecular Cytogenetics

TABLE 23.2
Thermocycler Program to Produce 5S-rDNA Probes Are Shown
Number of cycles Reaction Temperature, Time
1 Initial denaturation 94°C—5′
Denaturation 94°C—45′′
35 cycles Annealing 59°C—45′′
Extension 72°C—1′00′′
1 Final extension 72°C—10′
- Hold 4º∞
For reagents for this reaction, see Table 23.1.

TABLE 23.3
a) Reagents for the Telomere Directed PCR Amplifcation Reaction with
DNA (50 μl Reaction Master Mix per Sample)
Reagent Amount Final Concentration
Genomic DNA Variable 1–100 ng DNA
Buffer 10× 5.0 µl 1×
MgCl2 (50 mM) 2.0 µl 2.0 mM
Primer 18SR (10 mM) 1.5 µl 0.3 mM
Primer 18SF (10 mM) 1.5 µl 0.3 mM
2.5 mM each (dATP, dCTP, dGTP)
dNTP Mix 3.7 µl
1.25 mM (dTTP)
Adjust with ddH2O 33.9 µl -
Dye 1.0 µl -
Taq polymerase 5U/µl 0.4 µl 1.0 U

b) Thermocycler Program to Produce Telomeric DNA Probes

Number of cycles Reaction Temperature, Time
1 Initial denaturation 95°C—10′
Denaturation 94°C—45′′
34 cycles Annealing 50°C—1′00′′
Extension 68°C—1′00′′
1 Final extension 68°C—7′
- Hold 12º∞

PROBES OBTAINED BY FLOW SORTING

Whole chromosome painting probes from birds have been generated by fow-sorting.
For this, chromosome suspensions can be prepared according to standard protocol,[36]
sorted on a dual laser cell sorter (FAC-Star Plus, Becton Dickinson) as described
in Yang et al. (1999),[37] and amplifed by degenerate oligonucleotide-primes PCR
(= DOP-PCR).[36, 38] An example is highlighted in Figure 23.2.
FISH—in Birds 271

TABLE 23.4
a) Reagents for the Telomere Directed PCR Amplifcation Reaction without
DNA (50 μl Reaction Master Mix per Sample)
Reagent Amount Final Concentration
Buffer 10× 5.0 µl 1×
MgCl2 (50 mM) 2.5µl 2.5 mM
Primer 18SR (10 mM) 0.5 µl 0.1 mM
Primer 18SF (10 mM) 0.5 µl 0.1 mM
2.5mM each (dATP, dCTP, dGTP)
dNTP Mix 3.7 µl
1.25 mM (dTTP)
Adjust with ddH2O 36.8 µl -
Dye 0.6 µl -
Taq polymerase 5U/µl 0.4 µl 1.0 U

b) Thermocycler Program to Produce Telomeric DNA Probes

Number of Cycles Reaction Temperature, Time
Denaturation 94°C—1′00′′
10 cycles Annealing 55°C—0′30′′
Extension 72°C—1′00′′
Denaturation 94°C—1′00′′
30 cycles Annealing 60°C—0′30′′
Extension 72°C—1′00′′
- Hold 4º∞

FIGURE 23.2 Examples of FISH experiments with fow sorted whole chromosome probes
derived from Gallus gallus (GGA) (A) and Leucopternis albicollis (LAL) (B) onto white-
tipped dove Leptotila verreauxi (LVE). The probes were labeled with biotin and detected
with Cy3-streptavidin. Both signals are on LVE 1.
272 Cytogenetics and Molecular Cytogenetics

PROBE LABELING
Probe labeling is an essential step for a successful FISH experiment and usually,
labeling methods such as Nick translation and DOP-PCR are effective. The Nick
translation procedure follows the manufacturer’s instructions and is appropriate
when the DNA sequence of interest was already isolated by fow sorting. In addi-
tion, DOP-PCR labeling is also applied after the fow sorting procedure to label
sequences of whole chromosomes.[36, 38] By these methods, probes can be directly or
indirectly labeled. In the frst case, nucleotides carrying a specifc fuorochrome are
incorporated into the probe-DNA. In the second case, nucleotides carrying haptens
(like biotin and digoxigenin) are incorporated, requiring a further detection step
with appropriate fuorochrome-conjugated antibodies to enable the visualization of
the hybridization sites.

Nick Translation
1. Add 1 µg of probe DNA to 4 µl biotin- or digoxigenin nick translation mix
and fll up to 20 µl with ddH2O; continue with step 3.
2. OR: add 1 µg of probe DNA to 4 µl of Nick translation mix and 4 µl of 5×
concentrated fuorophore labeling mixture* and fll up to 20 µl with ddH2O.
– *Fluorophore labeling mixture:
– 5 µl 2.5 mM dATP
– 5 µl 2.5 mM dCTP
– 5 µl 2.5 mM dGTP
– 3.4 µl 2.5 mM dTTP
– 4 µl of either 1mM of Cy5 dUTP/Spectrum-Green dUTP/Spectrum-
Orange dUTP/Texas Red-dUTP
– 27.6 µl ddH2O
3. Mix carefully using the tip of a 20 µl pipette, and incubate at 15°C for
90 min.
4. Stop the reaction by adding 2 µl of EDTA 0.5M (pH 8.0) and incubating at
65°C for 10 min.

DOP-PCR Labeling
DOP-PCR is particularly used for the amplifcation of chromosome painting
probes, which are usually generated by fow-sorted chromosomes. The amplifca-
tion can be obtained using the degenerate 6 MW primer sequence: CCGACTCGAG
NNNNNNATGTGG.

1. For a standard reaction for secondary DOP-PCR amplifcation, mix the

reagents listed in Table 23.5 in a 0.6 ml tube (DNAse free).
2. Perform the amplifcation in a thermocycler according to the program
described next.
3. Check 2 μl of amplifcation product on a 1% agarose gel with appropriate
size markers. The product should yield a visible smear between ~200bp or
1.5 kb.
4. Store the amplifed DNA at −20°C (up to several years).
FISH—in Birds 273

TABLE 23.5
a) Reagents for the DOP-PCR Amplifcation Reaction (25 μl Reaction Master
Mix per Sample)
Reagent Amount Final Concentration
Genomic DNA Variable 1–100 ng DNA
Buffer 10× 2.5 µl 1×
MgCl2 (50 mM) 1.25 µl 2.5 mM
6MW primer (20 mM) 2 µl 2 mM
dNTP Mix 2.5 µl 2.5mM each (dATP, dCTP, dGTP)
1.25 mM (dTTP)
Adjust with ddH2O 12.5 µl -
Dye 2,5 µl -
Taq polymerase 5U/µl 0.25 µl 1.25 U

b) Thermocycler Program to Do DOP-PCR

Number of Cycles Reaction Temperature, Time
1 Initial denaturation 94°C—5′
28 cycles Denaturation 94°C—1′30′′
Annealing 62°C—1′30′′
Extension 72°C—3′00′′
1 Final extension 72°C—8′00′′
- Hold 4º∞

PRECIPITATION OF DNA PROBE

The procedure is the same to precipitate the probes labeled by nick translation, DOP-
PCR, or general PCR amplifcation. First, per each µg of the probe, it is necessary to
add 2.5 vol of ethanol (100%) and 0.1 vol of sodium acetate (3 M, pH 5.2) to the fnal
volume of labeled probes.

1. Mix carefully and incubate at −80°C for 30 min or at −20°C for at least 2 h.
2. Centrifugate at 13,000 rpm for 20 min at −4°C, and remove the supernatant
with a micropipette without touching the pellet.
3. Dry the pellet in a vacuum centrifuge or let it at room temperature (RT)
until it dries.
4. Add 1 µl of ddH20, spin, and let at 37°C for 5 min to help dissolve.
5. Add 20 µl of the hybridization buffer, vortex it, and let it shake at 65°C
for 5 min. In the end, a fnal concentration of 50 ng/µl of labeled probe is
obtained.

FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

Chromosome painting follows three principal steps: DNA Precipitation, Slide pre-
treatment, and FISH. In the case of using rDNA or telomeric sequences as probes,
it is necessary to add an additional step with RNase before the slides pretreatment
274 Cytogenetics and Molecular Cytogenetics

with pepsin, to remove nuclear RNA and prevent nonspecifc hybridization between
probes and single-strand sequences.

FISH with rDNA and Telomeric Sequences

1. Add 200 µl of the RNase A solution to slide and incubate at 37°C for 25 min
(1 µl of the RNase 10mg/ml solution in 500 µl of the 2×SSC).
2. Followed by treatment with pepsin (protocol described next).
3. Follow the steps for FISH described next.

SLIDE PRETREATMENT WITH PEPSIN

The pretreatment step of the slides is essential to achieve high-quality hybridization
signals, especially in chromosomes obtained directed from bone marrow, which usu-
ally contains cytoplasm or other cellular residuals.

1. Add 100 µl of a pepsin solution 0.01% on the slides, cover with a 22×50 mm
coverslip, and keep at 37°C for 5 min.
2. Remove the coverslip and incubate the slides in a coupling jar containing
2×SSC for 5 min at RT.
3. Repeat three times step 2.
4. Dehydrate the slides in ethanol series (70, 90%) for 2 min in each and 100%
for 4 min (RT).
5. Air-dry and incubate the slides at 60°C for 1 h or 37°C overnight.

CHROMOSOME PAINTING
1. Incubate the slides in a formamide solution at 72°C, 1 min and 20 seconds,
for DNA chromosomal denaturation.
2. Transfer the slides to a coupling jar flled with cold 70% ethanol (−20°C;
2 min) to conserve target DNA as single strands. Proceed slides into the
ethanol series (90 and 100%, RT, 2 min each) and air-dry.
3. Denature the hybridization mix (containing 20 µl of the hybridization buf-
fer and 100 ng of the labeled probe) in a thermocycler at 75°C for 10 min.
(this step can be done while ethanol series is performed).
4. Add 20 µl of the hybridization mix to the slides, cover with a 22×50 mm
coverslip, and incubate at 37°C for 72 h in a darkened moist chamber.
5. Remove the coverslip and wash the slides twice in 50% formamide and
twice in 2×SSC at a temperature ranging from 40 to 44°C, depending on
the applied probes and the phylogenetic distance between the species inves-
tigated (Note 3).
6. Add 100 µl of the appropriated detection solution (see Section 2.3), to each
slide, cover with a 22×50 mm coverslip and incubate at 37°C for 1h in a
darkened moist chamber. Detection steps 6 are necessary if the probes were
indirectly labeled. In the case of directly labeled probes, proceed to step 7.
FISH—in Birds 275

7. Remove the coverslip and wash 5 min for 3 times in 4×SSC/Tween = wash-
ing buffer (RT), under agitation (shaker).
8. Add 20 µl of Vectashield Mounting Medium with DAPI on each slide, cover
with a 22×50 mm coverslip, and press gently. The slides are ready to be
analyzed in a fuorescence microscope. For long-term storage, slides can be
kept at 4°C to prolong the fuorescence intensity.

FISH WITH MICROSATELLITE SEQUENCES

Different from other tetrapods, the avian genomes have only a small amount of
repetitive DNAs (4–10%). However, the importance of these repetitive sequences
in sex chromosome evolution has been demonstrated in the last years.[22, 24] The fol-
lowing protocol has been used to map repetitive DNA in the chromosomes of birds.

1. Incubate the slides at 60°C for 1 hour.

2. Add 100 µl of RNAse Solution (1.5 µl RNAse in 100 µl 2×SSC) per slide
(using a 24×60 mm coverslip). Incubate at 37°C for 1h 30 min in a moist
chamber.
3. Remove the coverslip and incubate the slides in a coupling jar containing
2×SSC for 5 min at RT in a shaker.
4. Add 3 µl of pepsin (stock solution 1.0g in 50ml of water) into previously
heated Water + HCl solution. Mix and homogenize the solution. Add
100µl on the slides for pepsin treatment for 10 minutes at 37°C in a moist
chamber.
5. Incubate the slides in a coupling jar containing 2×SSC for 5 min at RT in
a shaker.
6. Repeat step 5 twice.
7. Dehydrate the slides in ethanol series (70, 90, 100%) for 2 min in each
(RT).
8. Air-dry the slides.
9. Incubate the slides in a formamide solution 70% at 72°C, 1 min and 20 sec
for DNA chromosomal denaturation.
10. Repeat step 7.
11. Denature the hybridization mix (containing 11 µl of the hybridization buf-
fer and 100 ng of the labeled probe) in a thermocycler at 80°C for 10 min.
(This step can be done while the ethanol series is in progress).
12. Add the hybridization mix to the slides, cover with a 22×50 mm coverslip,
and incubate at 37°C for 16 h or overnight in a darkened moist chamber.
13. Remove the coverslip and wash the slides twice in 2×SSC and once in
1×SSC at RT, fve minutes each in a shaker.
14. Wash the slides once in 1×PBS for fve minutes in a shaker.
15. Repeat step 7.
16. Add 20 µl of Vectashield Mounting Medium with DAPI on each slide,
cover with a 22×50 mm coverslip, and press gently. The slides are ready
to be analyzed in a fuorescence microscope. For long-term storage, slides
can be kept at 4°C to prolong the fuorescence intensity.
276 Cytogenetics and Molecular Cytogenetics

IMPORTANT NOTES FOR PROCEEDING FISH

• Please, remember to discard the formamide solution as hazardous waste.
• If possible, try to use the formamide solution in a fume hood.
• Use low stringency temperatures (around 40°C) if you are hybridizing
chicken probes in a distant species, such as Passerines. Use higher strin-
gency temperatures (around 44°C) if you are hybridizing chicken probes in
closely related species, such as Galliformes and Anseriformes.

ACKNOWLEDGMENTS
We would like to thank the Brazilian funding agencies FAPESP (Fundação de Amparo
à Pesquisa do Estado de São Paulo, Process 2020/11669–2) and CNPq (Conselho
Nacional de Desenvolvimento Científco e Tecnológico, Process 307382/2019–2 and
302449/2018–3) for the fnancial support.

REFERENCES
1. Gill, F.; Donsker, D.; Rasmussen, P. (Eds.). IOC world bird list; 2021. www.worldbird-
names.org/new/ [accessed on 08/31/2021].
2. Hackett, S.J.; Kimball, R.T.; Reddy, S.; Bowie, R.C.; Braun, E.L.; Braun, M.J.;
Chojnowski, J.L.; Cox, W.A.; Han, K.L.; Harshman, J.; Huddleston, C.J.; Marks,
B.D.; Miglia, K.J.; Moore, W.S.; Sheldon, F.H.; Steadman, D.W.; Witt, C.C.; Yuri, T.
A phylogenomic study of birds reveals their evolutionary history. Science 2008, 320,
1763–1768.
3. Isaksson, C.; Rodewald, A.D.; Gil, D. Editorial: Behavioural and ecological conse-
quences of urban life in birds. Front. Ecol. Evol. 2018, 6, 50.
4. Jarvis, E.D.; Mirarab, S.; Aberer, A.J.; Li, B.; Houde, P.; Li, C.; Ho, S.Y.; Faircloth, B.C.;
Nabholz, B.; Howard, J.T.; Suh, A.; Weber, C.C.; da Fonseca, R.R.; Li, J.; Zhang, F.; Li,
H.; Zhou, L.; Narula, N.; Liu, L.; Ganapathy, G.; Boussau, B.; Bayzid, M.S.; Zavidovych,
V.; Subramanian, S.; Gabaldón, T.; Capella-Gutiérrez, S.; Huerta-Cepas, J.; Rekepalli,
B.; Munch, K.; Schierup, M.; Lindow, B.; Warren, W.C.; Ray, D.; Green, R.E.; Bruford,
M.W.; Zhan, X.; Dixon, A.; Li, S.; Li, N.; Huang, Y.; Derryberry, E.P.; Bertelsen, M.F.;
Sheldon, F.H.; Brumfeld, R.T.; Mello, C.V.; Lovell, P.V.; Wirthlin, M.; Schneider, M.P.;
Prosdocimi, F.; Samaniego, J.A.; Vargas Velazquez, A.M.; Alfaro-Núñez, A.; Campos,
P.F.; Petersen, B.; Sicheritz-Ponten, T.; Pas, A.; Bailey, T.; Scofeld, P.; Bunce, M.;
Lambert, D.M.; Zhou, Q.; Perelman, P.; Driskell, A.C.; Shapiro, B.; Xiong, Z.; Zeng, Y.;
Liu, S.; Li, Z.; Liu, B.; Wu, K.; Xiao, J.; Yinqi, X.; Zheng, Q.; Zhang, Y.; Yang, H.; Wang,
J.; Smeds, L.; Rheindt, F.E.; Braun, M.; Fjeldsa, J.; Orlando, L.; Barker, F.K.; Jønsson,
K.A.; Johnson, W.; Koepfi, K.P.; O’Brien, S.; Haussler, D.; Ryder, O.A.; Rahbek, C.;
Willerslev, E.; Graves, G.R.; Glenn, T.C.; McCormack, J.; Burt, D.; Ellegren, H.; Alström,
P.; Edwards, S.V.; Stamatakis, A.; Mindell, D.P.; Cracraft, J.; Braun, E.L.; Warnow, T.;
Jun, W.; Gilbert, M.T.; Zhang, G. Whole-genome analyses resolve early branches in the
tree of life of modern birds. Science 2014, 346, 1320–1331.
5. Prum, R.O.; Berv, J.S.; Dornburg, A.; Field, D.J.; Townsend, J.P.; Lemmon, E.M.;
Lemmon, A.R. A comprehensive phylogeny of birds (Aves) using targeted next-genera-
tion DNA sequencing. Nature 2015, 526, 569–577.
6. Kretschmer, R.; Ferguson-Smith, M.A.; de Oliveira, E.H.C. Karyotype evolution in
birds: From conventional staining to chromosome painting. Genes 2018, 9, 181.
FISH—in Birds 277

7. Howell, W.M.; Black, D.A. Controlled silver-staining of nucleolus organizer regions with
a protective colloidal developer: A 1-step method. Experientia 1980, 36, 1014–1015.
8. Seabright, M. A rapid banding technique for human chromosomes. Lancet. 1971, 2,
971–972.
9. Sumner, A.T. A simple technique for demonstrating centromeric heterochromatin. Exp.
Cell Res. 1972, 75, 304–306.
10. Pinkel, D.; Straume, T.; Gray, J.W. Cytogenetic analysis using quantitative, high-sensitiv-
ity, fuorescence hybridization. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 2934–2938.
11. Degrandi, T.M.; Barcellos, A.S.; Costa, A.L.; Garnero, A.D.V.; Hass, I.; Gunski, R.J.
Introducing the bird chromosome database: An overview of cytogenetic studies in birds.
Cytogenet. Genome Res. 2020, 160, 199–205.
12. Griffn, D.K.; Haberman, F.; Masabanda, J.; O’Brien, P.; Bagga, M.; Sazanov, A.;
Smith, J.; Burt, D.W.; Ferguson-Smith, M.; Wienberg, J. Micro- and macrochromosome
paints generated by fow cytometry and microdissection: Tools for mapping the chicken
genome. Cytogenet. Cell Genet. 1999, 87, 278–281.
13. Nie, W.; O’Brien, P.C.M.; Ng, B.L.; Fu, B.; Volobouev, V.; Carter, N.P.; Ferguson-Smith,
M.A.; Yang, F. Avian comparative genomics: 112 reciprocal chromosome painting
between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus,
Charadriiformes): An atypical species with low diploid number. Chromosome Res. 2009,
17, 99–113.
14. de Oliveira, E.H.C.; Tagliarini, M.M.; Rissino, J.D.; Rissino, J.D.; Pieczarka, J.C.;
Nagamachi, C.Y.; O’Brien, P.C.; Ferguson-Smith, M.A. Reciprocal chromosome painting
between white hawk (Leucopternis albicollis) and chicken reveals extensive fusions and
fssions during karyotype evolution of accipitridae (Aves, Falconiformes). Chromosome
Research. 2010, 18, 349–355.
15. Nie, W.; O’Brien, P.C.M.; Fu, B.; Wang, J.; Su, W.; He, K.; Bed’Hom, B.; Volobouev, V.;
Ferguson-Smith, M.A.; Dobigny, G.; Yang, F. Multidirectional chromosome painting
substantiates the occurrence of extensive genomic reshuffing within Accipitriformes.
BMC Evol. Biol. 2015, 15, 205.
16. Kretschmer, R.; Furo, I.O.; Gunski, R.J.; Del Valle Garnero, A.; Pereira, J.C.; O’Brien,
P.C.M.; Ferguson-Smith, M.A.; de Oliveira, E.H.C.; de Freitas, T.R.O. Comparative
chromosome painting in Columbidae (Columbiformes) reinforces divergence in Passerea
and Columbea. Chromosome Res. 2018, 26, 211–223.
17. Furo, I.O.; Kretschmer, R.; O’Brien, P.C.; Pereira, J.C.; Garnero, A.D.V.; Gunski, R.J.;
O’Connor, R.E.; Griffn, D.K.; Gomes, A.J.B.; Ferguson-Smith, M.A.; de Oliveira,
E.H.C. Chromosomal evolution in the phylogenetic context: A remarkable karyotype
reorganization in neotropical parrot myiopsitta monachus (Psittacidae). Front. Genet.
2020, 11, 721.
18. Griffn, D.K.; Robertson, L.B.; Tempest, H.G.; Skinner, B.M. The evolution of the avian
genome as revealed by comparative molecular cytogenetics. Cytogenet. Genome Res.
2007, 117, 64, 77.
19. dos Santos, M.S.; Kretschmer, R.; Silva, F.A.O.; Silva, F.A.; Ledesma, M.A.; O’Brien,
P.C.; Ferguson-Smith, M.A.; Del Valle Garnero, A.; de Oliveira, E.H.; Gunski, R.J.
Intrachromosomal rearrangements in two representatives of the genus Saltator
(Thraupidae, Passeriformes) and a case of polymorphism in Z chromosome. Genetica
2015, 143, 535–543.
20. dos Santos, M.S.; Kretschmer, R.; Frankl-Vilches, C.; Bakker, A.; Gahr, M.; O’Brien,
P.C.; Ferguson-Smith, M.A.; de Oliveira, E.H. Comparative cytogenetics between
two important sonbird, models: The zebra fnch and the canary. PLoS One 2017, 12,
e0170997.
278 Cytogenetics and Molecular Cytogenetics

21. dos Santos, M.S.; Furo, I.O.; Tagliarini, M.M.; Kretschmer, R.; O’Brien, P.C.M.;
Ferguson-Smith, M.A.; de Oliveira, E.H.C. The karyotype of the hoatzin (Opisthocomus
hoazin): A phylogenetic enigma of the neornithes. Cytogenet. Genome Res. 2018, 156,
158–164.
22. de Oliveira, T.D.; Kretschmer, R.; Bertocchi, N.A.; Degrandi, T.M.; de Oliveira, E.H.;
Cioff, M.B.; Garnero, A.D.; Gunski RJ. Genomic organization of repetitive DNA in
woodpeckers (Aves, Piciformes): Implications for karyotype and ZW sex chromosome
differentiation. PLoS One 2017, 12, e0169987.
23. Kretschmer, R.; de Oliveira, T.D.; Furo, I.O.; Oliveira Silva, F.A.; Gunski, R.J.; Del
Valle Garnero, A.; de Bello Cioff, M.; de Oliveira, E.H.C.; de Freitas, T.R.O. Repetitive
DNAs and shrink genomes: A chromosomal analysis in nine Columbidae species (Aves,
Columbiformes). Genet. Mol. Biol. 2018, 41, 1, 98–106.
24. Furo, I.O.; Kretschmer, R.; dos Santos, M.S.; de Lima Carvalho, C.A.; Gunski, R.J.;
O’Brien, P.C.M.; Ferguson-Smith, M.A.; Cioff, M.B.; de Oliveira, E.H.C. Chromosomal
mapping of repetitive DNAs in Myiopsitta monachus and Amazona aestiva
(Psittaciformes, Psittacidae: Psittaciformes), with emphasis on the sex chromosomes.
Cytogenet. Genome Res. 2017, 151, 151–160.
25. Barcellos, S.A.; Kretschmer, R.; Souza, M.S.; Costa, A.L.; Degrandi, T.M.; Dos Santos,
M.S.; de Oliveira, E.H.C.; Cioff, M.B.; Gunski, R.J.; Garnero, A.D.V. Karyotype evo-
lution and distinct evolutionary history of the W chromosomes in swallows (Aves,
Passeriformes). Cytogenet. Genome Res. 2019, 158 (2), 98–105.
26. Degrandi, T.M.; Gunski, R.J.; Garnero, A.D.V.; Oliveira, E.H.C.; Kretschmer, R.; Souza,
M.S.; Barcellos, S.A.; Hass, I. The distribution of 45S rDNA sites in bird chromosomes
suggests multiple evolutionary histories. Genet. Mol. Biol., 2020, 43, 2, e20180331.
27. Garnero, A.V.; Gunski, R.J. Comparative analysis of the karyotype of Nothura maculosa
and Rynchotus rufescens (Aves: Tinamidae). A case of chromosomal polymorphism.
The Nucleus, 2000, 43, 64–70.
28. Barcellos, S.A.; de Souza, M.S.; Tura, V.; Pereira, L.R.; Kretschmer, R.; Gunski, R.J.;
Del Valle Garnero, A. Direct chromosome preparation method in Avian embryos for
cytogenetic studies: Quick, easy and cheap. DNA 2022, 2, 22–29.
29. Moorhead, P.S.; Nowell, P.C.; Mellman, W.J.; Battips, D.M.; Hungerford, D.A.
Chromosome preparations of leukocytes cultured from human peripheral blood. Exp
Cell Res. 1960, 20, 613–616.
30. López-Flores, I.; Garrido-Ramos, M.A. The repetitive DNA content of eukaryotic
genomes. Genome Dyn, 2012, 7, 1–28.
31. Cioff, M.B.; Martins, C.; Centofante, L.; Jacobina, U.; Bertollo, L.A. Chromosomal vari-
ability among allopatric populations of Erythrinidae fsh Hoplias malabaricus: Mapping
of three classes of repetitive DNAs. Cytogenet. Genome Res. 2009, 125, 132–141.
32. Nishida-Umehara, C.; Tsuda, Y.; Ishijima, J.; Ishijima, J.; Ando, J.; Fujiwara, A.; Matsuda,
Y.; Griffn, D.K. The molecular basis of chromosome orthologies and sex chromosomal
differentiation in palaeognathous birds. Chromosome Res. 2007, 15, 721–734.
33. Martins, C.; Galleti, P.M. Chromosomal localization of 5S rDNA genes in Leporinus fsh
(Anostomidae, Characiformes). Chromosome Res. 1999, 7, 363–367.
34. Gunski, R.J.; Kretschmer, R.; de Souza, M.S.; de Oliveira Furo, I.; Barcellos, S.A.; Costa,
A.L.; Cioff, M.B.; de Oliveira, E.H.C.; Del Valle Garnero, A. Evolution of bird sex
chromosomes narrated by repetitive sequences: Unusual W chromosome enlargement in
Gallinula melanops (Aves: Gruiformes: Rallidae). Cytogenet. Genome Res. 2019, 158,
152–159.
35. Ijdo, J.W.; Wells, R.A.; Baldini, A.; Reeders, S.T. Improved telomere detection using a
telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acid Res. 1991, 19, 4780.
FISH—in Birds 279

36. Yang, F.; Carter, N.P.; Shi, L.; Ferguson-Smith, M.A. A comparative study of karyotypes
of muntjacs by chromosome painting. Chromosoma 1995, 103, 642–652.
37. Yang, F.; O’Brien, P.C.M.; Milne, B.S.; Graphodatsky, A.S.; Solanky, N.; Trifonov, V.;
Rens, W.; Sargan, D.; Ferguson-Smith, M.A. A complete comparative chromosome map
for the dog, red fox and human and its integration with canine genetic maps. Genomic.
1999, 62, 189–202.
38. Telenius, H.; Ponder, B.A.J.; Tunnacliffe, A.; Carter, N.P.; Behmel, A.; Ferguson-Smith,
M.A.; Nordenskjöld, M.; Pfragner, R.; Ponder, B.A. Cytogenetic analysis by chromosome
painting using DOP-PCR amplifed fow-sorted chromosomes. Genes Chromosomes
Cancer 1992, 4, 257–263.
 24 FISH—in Fish
Chromosomes
Francisco de M. C. Sassi, Gustavo A. Toma
and Marcelo de Bello Cioff

CONTENTS
Introduction............................................................................................................ 282
Material .................................................................................................................. 282
FISH Probe Labeling and Precipitation........................................................ 283
Via Polymerase Chain Reaction (PCR) ............................................ 283
Via Nick-Translation Kit................................................................... 283
Precipitation...................................................................................... 283
Slide Pre-FISH Treatment............................................................................. 283
FISH Workfow ............................................................................................. 284
If an Indirectly Labeled Probe Is Used ............................................. 284
Methods.................................................................................................................. 285
Samples......................................................................................................... 285
Cell Suspension and Slides Preparation ....................................................... 285
Probes ........................................................................................................... 285
Ribosomal DNA ........................................................................................... 286
Genomics ...................................................................................................... 286
Transposable Elements (TEs) ....................................................................... 287
Small Nuclear RNAs (snRNAs) ................................................................... 287
Microdissection............................................................................................. 287
Comparative Genomic Hybridization (CGH)............................................... 287
Probe Labeling.............................................................................................. 288
Multicolor FISH................................................................................ 288
Precipitation and Hybridization Mixture ...................................................... 288
FISH Probes...................................................................................... 288
CGH and WCP Probes...................................................................... 288
Fish-FISH Protocol....................................................................................... 289
Variations of FISH Protocol for WCP and CGH .............................. 292
Sequential FISH................................................................................ 293
Image Capture and Treatment....................................................................... 293
Acknowledgments.................................................................................................. 293
References.............................................................................................................. 293

DOI: 10.1201/9781003223658-24 281

282 Cytogenetics and Molecular Cytogenetics

INTRODUCTION
Representing more than half of the vertebrate species richness, fishes are a
very diverse group of organisms, with approximately 36,000 valid species.[1, 2, 3]
They include distinct groups such as bony (ray-finned fish, lungfish, coel-
acanth), cartilaginous (sharks, rays), and jawless fishes (hagfishes, lampreys).[3]
Their worldwide distribution, basal phylogenetic position among vertebrates,
and niche diversity make them valuable experimental models in many scien-
tific fields, such as ecological, evolutionary, and genetics.[3] Furthermore, with
the increased number of newly described sex chromosome systems (with the
coexistence of both ZZ/ZW- and XX/XY-derived systems), characterization of
inter- and intra-specific chromosomal rearrangements, and the implementation
of novel genomic research tools, fish cytogenetics has steadily grown over the
years. Many investigations on chromosomes have helped to solve taxonomic
issues, elucidate meiotic behaviors, and even provided information regarding
the evolution of repetitive DNA sequences, such as satellite DNAs and transpo-
sons.[4–6] In all these cases, chromosomal mapping of DNA by fluorescence in
situ hybridization (FISH) has been an indispensable tool, allowing a more refined
look into the genomic content of chromosomes, and providing a way to search
for DNA sequences that may have a relevant role in chromosomal differentia-
tion (e.g., the origin of B and sex chromosomes, identification of chromosomal
rearrangements, etc.).
Despite that frst applications of FISH occurred in end 1980s,[7] FISH in fshes
was frst applied only around 1993 using primarily rRNA genes and other tan-
dem repeats as probes.[8–11] These protocols were mostly similar to those used
in human molecular cytogenetics,[7, 12–14] with some differences in post-hybrid-
ization washes (especially in concentration of solutions and time of washes) and
other minor adaptations. However, with the improvement of hybridization probes
and rising numbers of FISH-derived techniques, such as genome in situ hybrid-
ization (GISH), comparative genomic hybridization (CGH), and whole chromo-
some painting (WCP), a new optimized FISH in fsh protocol was designed.[15]
This, instead, greatly differ from those previously used, as many changes were
proposed to have a more meticulous hybridization procedure, which could be
used in any fsh group.
Here we present an updated fsh-FISH protocol, in which we show a fgure-guided
protocol for the whole procedure and discuss some minor adaptations made over
the years, such as adjustments for a sequential FISH and problems that have usually
emerged using an overabundance of labeled probes.

MATERIAL
As usual, the standard cytogenetics equipment and reagents (centrifuge, pipettes,
colchicine, methanol, etc.) are obligatory, and specialized items are listed next,
including their working solutions.
FISH—in Fish Chromosomes 283

FISH PROBE LABELING AND PRECIPITATION

Via Polymerase Chain Reaction (PCR)
• AmpliTaq® DNA Polymerase, Stoffel Fragment (Cat. No.: N8080038,
Applied Biosystems, USA)
• Biotin 14-dATP (Cat. No.: 19524016, Invitrogen, USA)
• DNA template (~50 ng/µl)
• dNTP label mix (usually dUTP coupled with Cyanine 5, SpectrumGreen,
SpectrumOrange, TexasRed plus other non-labelled dNTPs)
• Double-distilled water (ddH2O) or Ultrapure Water
• MgCl2
• Primer (DOP-primer or reverse/forward primers fanking the interesting
section)
• SpectrumGreen dUTP (Cat. No.: 6J9410, Abbott, USA)
• SpectrumOrange dUTP (Cat. No.: 6J9415, Abbott, USA)
• TexasRed dUTP (Cat. No.: C-7631, Molecular Probes, USA)
Via Nick-Translation Kit
Commercially available label kits, usually composed of an enzyme mix (Polymerase +
DNAse I), a labeling buffer, the labeling mix (usually dATP, dCTP, dGTP, and dTTP
unlabelled + dUTP labeled with the selected fuorophore), PCR-grade water (for mount-
ing the reaction), and a stop buffer (usually EDTA 0.5 M). The most common ones are:

• Biotin16 NT Labeling Kit (Cat. No.: PP-310S-BIO16, Jena Bioscience, Germany)

• Biotin Nick Translation Mix (Cat. No.: 11745824910, Roche Diagnostics,
Switzerland).
• Digoxigenin NT Labeling Kit (Cat. No.: PP-310S-DIGX, Jena Bioscience,
Germany)
• DIG Nick Translation Mix (Cat. No.: 11745816910, Roche Diagnostics,
Switzerland).
• Nick translation mix (Cat. No.: 11 745 808 910, Roche Diagnostics,
Switzerland)
• Atto550 or Atto488 NT Labeling Kit (Cat. No.: PP-305S-550, Jena
Bioscience, Germany)
Precipitation
• Ethanol 100%
• Ribonucleic acid transfer—tRNA (Cat. No.: R8508, Sigma, USA)
• Sodium acetate solution (3 M, pH 5.2; e.g., Merck, Germany; store at −20°C)

SLIDE PRE-FISH TREATMENT

• 10×PBS (phosphate-buffered saline) stock for 1×PBS dilution: for 1l solution
use 75.8g of sodium chloride (NaCl), 9.93g of disodium phosphate (Na2HPO4),
4.14g of monosodium phosphate (NaH2PO4). Dissolve in 1l of distilled water
284 Cytogenetics and Molecular Cytogenetics

• 1×PBS (phosphate-buffered saline—Cat. No.: L1825, Biochrom, Germany;

store at room temperature = RT)
• Pepsin stock solution (Cat. No.: P-7012, Sigma, USA)
• Pepsin solution (0.005%): 99 µl H2O, 10µl HCl, and 2.5µl pepsin (20 mg/
ml)
• Post fxation solution (10 ml, 1% paraformaldehyde): mix 50 ml of 2% para-
formaldehyde with 45 ml of 1×PBS and 5 ml 1M MgCl2 (make fresh as
required).
• RNAse A (Cat. No.: R4642, Sigma, USA)
• RNase solution (10µg/ml): 1.5 µl RNase A (10 mg/ml) and 1.5 ml 2×SSC.
• Ethanol 100% stock for diluted solutions (70% and 85%)

FISH WORKFLOW
• 20×SSC (Saline Sodium Citrate) stock solution, store at 4oC. For 1×SSC
and 2×SSC dilutions, store at RT (Cat. No.: 15557–036; Invitrogen, USA)
or prepare: 1l solution using 175.3g of sodium chloride (NaCl) and 88.23g
of trisodium citrate dihydrate (Na3C6H507.2H2O). Dissolve in 1l of distilled
water.
• Deionized formamide (Cat. No.: 1 09684 2500, Merck, Germany; aliquot
and store at 4oC).
• Denaturation buffer: 70% (v/v) deionized formamide, 20% (v/v) fltered
double distilled water, 10% (v/v) 20×SSC; make fresh as required.
• Dextran sodium sulfate (Cat. No.: D8906–10G, Sigma, USA).
• Phosphate buffer: prepare 0.5 M Na2HPO4 and 0.5 M NaH2PO4, mix these
two solutions (1:1) to get pH 7.0, and then aliquot and store at −20°C.
• Hybridization buffer: Dissolve 2 g dextran sodium sulfate in 10 ml 50%
deionized formamide/2×SSC/50 mM phosphate buffer for 3 h at 70°C; pH
adjusted to 7 with phosphate buffer; hydrochloric acid destabilizes buffer
solution. Aliquot and store at −20°C.
• Tween 20 = polyoxyethylene-sorbitan monolaurate (Cat. No.: 10670–1000,
Sigma, Germany, store at RT).
• Washing buffer (4×SCCT): 4×SSC, 0.05% Tween 20; make fresh as
required.
• Vectashield Mounting Medium with DAPI FR/10ml (Cat. No.: H-1200,
Vector, USA).

If an Indirectly Labeled Probe Is Used

• Avidin-FITC (Cat. No.: A2901, Sigma, USA).
• Anti-digoxigenin fuorescein (Cat. No.: 11207741910, Roche Diagnostics,
Switzerland).
• Anti-digoxigenin rhodamine (Cat. No.: 11207750910, Roche Diagnostics,
Switzerland).
• Detection solution 1: 994 µl of 3% NFDM/4×SSC, 2 µl of avidin-FITC
working solution (1mg ml−1), and 5 µl of anti-digoxigenin rhodamine
(200µg ml−1).
FISH—in Fish Chromosomes 285

• Detection solution 2: 995 µl of 3% NFDM/4×SSC, 10 µl of streptavidin-

Cy3, and 5µl of anti-digoxigenin fuorescein (200 µg ml−1).
• Detection solution 3: 995 µl of 3% NFDM/4×SSC, 10 µl of streptavidin-
Cy5, and 5 µl of anti-digoxigenin fuorescein (200µg ml−1).
• Detection solution 4: 995 µl of 3% NFDM/4×SSC, 10 µl of streptavidin-
Cy5, and 5 µl of anti-digoxigenin rhodamine (200µg ml−1).
• NFDM = Nonfat dried milk powder (Cat. No.: 9999, Cell Signaling
Technology, USA).
• NFDM3%/4×SSC: 40 ml 20×SSC, 160 ml ddH20, and 5 g of NFDM.
Prepare the solution in constant agitation to dissolve the NFDM.
• Streptavidin-Cy5 (Cat. No.: 25800881, GE Healthcare Life Sciences, USA).
• Streptavidin-Cy3 (Cat. No.: S6402, Sigma, USA).

METHODS
Since fsh-FISH methods were recently revisited, here we provide a review and an
update on the technique. For the complete PCR cycles, conditions of amplifcation,
and probe labeling.[15]

SAMPLES
Mitotic chromosomes can be obtained by the classical air-drying method,[16, revisited in 17]
which consists of the application of colchicine to stop chromosomes in metaphase,
followed by hypotonization of cells and fxation in Carnoy 2 (methanol 3:1 glacial
acetic acid). The kidney is the preferred organ to obtain metaphase chromosomes,
due to its hematopoietic function in adult teleosts,[18] but good preparations were
already obtained from other organs such as the spleen.[19] Cell culture could also be
an alternative for mitotic chromosome obtainment, using a tissue fragment that usu-
ally is muscle or fn.[20] To obtain meiotic chromosomes of fshes, the most common
protocol follows the squash of the testes to liberate cells and proceed to hypotoniza-
tion in KCl 0.075 M, followed by fxation in Carnoy 2.[21]

CELL SUSPENSION AND SLIDES PREPARATION

To obtain good fsh results, chromosomes should be well spread (Figure 24.1). For
this, the cell suspension needs to be dropped (usually 10–20 µl) into a clean, heated,
and humid slide (50–60ºC in a heater plate, with some humid paper). Small droplets
of Carnoy 2 can also be immediately dropped over the drop with cell suspension,
helping to spread the metaphase plate.

PROBES
The most important step for the success of a FISH procedure is the establishment of
a probe of good quality. Such probe can be obtained by different methods, such as
target DNA amplifcation via PCR, microdissection, and whole-genome extraction.
Besides, commercially available probes (as microsatellite and telomeric sequences)
286 Cytogenetics and Molecular Cytogenetics

FIGURE 24.1 Examples of a good (a) and a bad (b) metaphase plate to analyze with FISH.
a) Mitotic chromosomes of Chalceus erythrurus (Teleostei, Characiformes) hybridized with
ribosomal DNA probes (a) 5S rDNA in green, and 18S rDNA in red.
b) Lebiasina bimaculata (Teleostei, Characiformes) chromosomes hybridized with GC
microsatellite.
Observe that in a) chromosomes are separated and well defned, while in b) they present some
overlapped regions and nuclei around (arrowheads), which may interfere with the hybridiza-
tion and the detection of hybridized regions.

can also be produced. We list next the methods for obtaining the most common
probes used in fsh cytogenetics.

RIBOSOMAL DNA
The chromosomal localization of ribosomal genes is an important tool for the com-
prehension of the evolutionary process on fshes.[22] These multigene families are
highly conserved and give origin to the ribosomal RNA, that actively participate in
protein synthesis. They are expressed by two multigene families: the 45S rDNA and
5S rDNA. In the 45S rDNA are codifed the 5.8S, the 18S, and the 28S, separated by
two internal transcribed spacers (ITS1 and ITS2), two external transcribed spacers
(ETS1 and ETS2), and a non-transcribed spacer (NTS). On other hand, 5S rDNA
is organized into a series of repetitions, separated by an NTS, also being found as
pseudogenes dispersed on chromosomes.[23] In fsh cytogenetics, the most common
probes of rDNA are 5S and 18S. For the obtainment of such probes, we use previ-
ously designed primers (18S from [24] and 5S from[25]) to isolate the sequences from
a targeted genome, which usually is from Hoplias malabaricus (Characiformes,
Erythrinidae), due to the previous existence of well-isolated libraries.

GENOMICS
More recently, genomics has become an important mechanism for probes obtain-
ment in fsh cytogenetics. Briefy, sequences from next-generation sequencing
FISH—in Fish Chromosomes 287

(NGS) are obtained, and bioinformatics pipelines are applied to isolate specifc
libraries, where primers can be designed for the targeted sequences. Then, these
primers are used in a PCR reaction to amplify the target sequence and thus be
used as a FISH probe after labeling. Satellite DNAs and TE probes are the most
common class of DNA used,[e.g. 26] but studies are still scarce when compared to the
high diversity of fshes, and consequently of the possibilities of this technique for
more DNA classes.

TRANSPOSABLE ELEMENTS (TES)

Another repetitive DNA class that represents an important target for fsh cytoge-
netics is TEs, since they represent a highly diverse pool of sequences that can be
associated with ribosomal DNAs and other DNA classes, helping them to spread
into the genome.[27–30] Transposons are class II TEs that have the biggest abundance
in fsh genomes,[30] and are commonly mapped in fsh chromosomes, such as the Rex
family.[e.g. 31, 32] They can be PCR-isolated using already known primers, especially
those isolated from Xiphophorus (Perciformes, Poeciliidae).[33]

SMALL NUCLEAR RNAS (SNRNAS)

In eukaryotic genomes, small RNAs have an important role in intron splicing
and other RNA processing, as the maturation of primary transcripts in mes-
senger RNA (mRNA).[34] Indeed, in fsh karyotypes, they present a variable
pattern of distribution, although its sequence remains conserved.[35] To obtain
such probes, it can be amplifed using the primers already designed for the U’s
snRNAs.[27, 36, 37]

MICRODISSECTION
For the whole-chromosome painting (WCP) procedure, single chromosomes (or spe-
cifc regions of them) can be directly isolated by microdissection, which involves the
use of a glass needle to scuff up out the chromosome from the slide, followed by its
amplifcation via PCR and its label with a fuorescent dUTP. The complete procedure
is reviewed in [38].

COMPARATIVE GENOMIC HYBRIDIZATION (CGH)

Another FISH-derived technique that is currently used in fsh cytogenetics is the
comparison of genomes by Comparative Genomic Hybridization (CGH). This tech-
nique is very resolutive, especially in the identifcation of sex chromosome sys-
tems,[39, 40] and to detect hidden variability among similar karyotypes.[41] Genomes
can be obtained from the same species, varying from sexes or populations, or differ-
ent species, according to the proposed objective. Total DNA is extracted and labeled
via Nick Translation. Unlabeled DNA is used as a blocker (usually C 0 t-1 DNA) and
is hybridized together with the genomic probe to impair the hybridization in highly
288 Cytogenetics and Molecular Cytogenetics

repetitive regions of chromosomes. Then, a specifc pattern of hybridization can be

obtained varying from the amount of blocker used. For intraspecifc comparisons,
we have great results with 10–15 µg of blocker, while for interspecifc experiments,
20–25 µg.

PROBE LABELING
The probe needs to be hapten- or fuorescent-labeled for FISH. For this purpose, the
isolated DNA can be directly labeled with a fuorescent molecule (e.g., Cy5, Atto
550, Atto 488) or be linked with haptens (e.g., biotin and digoxigenin), thus constitut-
ing indirect labeling. As previously described, two main techniques can be used for
this: a standard PCR reaction (using designed or degenerated primers) or via Nick
Translation (NT). While for the frst the labeled nucleotides are incorporated during
the amplicon syntheses, the second method is based on the incorporation of fuores-
cent/haptens-dNTPs in small aleatory gaps generated by a DNAse. When labeling
by NT, it’s very important to control the fnal size of fragments, which should vary
between 200 and 600 base pairs (bp). For this purpose, several commercially NT
reactions kits can be widely found.

Multicolor FISH
To hybridize different probes in a single experiment, these should be labeled with
distinct fuorophores. Usually, the combination of red (Atto550, Rhodamine, Cy3,
Spectrum Orange, etc.) and green (Atto488, FITC, Spectrum green, among others)
probes produces good and distinguishable patterns, since DAPI (blue) is the most
common counterstain used.

PRECIPITATION AND HYBRIDIZATION MIXTURE

FISH Probes
Probes need to be precipitated before the hybridization. For this, use a combination
of sodium acetate 3M, tRNA, and 100% ethanol in a proportion of 5:10:100 µl for
each 20 µl of the probe. Mix carefully and incubate at −20°C overnight or −80°C for
30 min. Centrifugate at 13,000 rpm for 20 min at −4°C and remove the supernatant,
leaving the pellet for dry at RT. This step should proceed with caution, given that a
super-dried pellet will be diffcult to dissolve in the hybridization buffer. Once dry,
resuspend the pellet in 20 µl of the hybridization buffer, using a vortex for better
results. The hybridization buffer can be prepared before the FISH procedure and
stored at −20 °C.

CGH and WCP Probes

For the FISH-derived techniques, the precipitation step needs to be differentiated.
For WCP and CGH procedures, precipitate a ‘blocker-DNA’ (usually C 0 t-1 DNA) to
avoid unspecifc background hybridization frst, and then mount in the same tube
the hybridization mixture with the hybridization buffer and the whole-chromosome
probes already labeled.
FISH—in Fish Chromosomes 289

FISH-FISH PROTOCOL
Day 1 (Figure 24.2):

1. Drop the chromosome preparation into clean glass slides;

2. Incubate the slides for 1h at 60°C in a glass Coplin jar;
3. Add 100 µl of RNAse solution (1.5µl RNAse in 100 µl 2×SSC) per slide
(using a 24×60 mm coverslip). Incubate at 37°C for 1h 30min in a humid
dark chamber;
4. In parallel put 1 ml of dd-H2O + 10 µl of HCl 0.1 M to heat to 37°C and
5. put a Coplin jar with the denaturation buffer (70% (v/v) deionized for-
mamide, 20% (v/v) fltered double distilled water, 10% (v/v) 20×SSC) to
heat in a water bath to 72°C;
6. After 1h 30min in RNAse solution, remove the coverslip and wash the
glass slide in 1×PBS for 5 min at RT on a shaker;
7. Add 3 µl of pepsin into previously heated 1 ml water + HCl solution
(step 4) and homogenize. Add 100 µl on the still ‘1×PBS—humid’ slide,
cover with 24×60mm coverslips, and incubate for 10 min at 37 °C in a
humid dark chamber;
8. Remove the coverslip and wash the glass slide in 1×PBS for 5 min on a shaker;
9. Quickly submerge the glass slide in distilled water;
10. Dehydrate the slides in ethanol 70%, 85%, and 100% for 2 min each, at
RT. Completely air-dry the slides;
11. Denature the slides in the previously heated at 72°C denaturation buffer
(step 5) for 3 min 15 sec;
12. Dehydrate the slides in −20°C cold ethanol 70% for 3 min, followed by
ethanol 85% and 100% for 2 min each, at RT. Completely air-dry the slides;
13. Denature the probes in a thermal cycler at 85°C for 10 min, followed by
cooling at 4°C;
14. Add 20 µl of the hybridization mixture (probes + hybridization buffer) to
the slides. Cover with 24×50 mm coverslips and incubate at 37°C over-
night in a dark humid chamber.

Day 2 for directly labeled probes (Figure 24.3a):

15. Heat a Coplin jar with 1×SSC to 65°C, remove the coverslip and wash the
slides for 5 min on a shaker;
16. Transfer the slides to the washing buffer (4×SSC/Tween solution) and
wash for 5 min in on shaker at RT;
17. Wash the slides in 1×PBS for 1 min at RT using a shaker;
18. Quickly submerge the glass slide in distilled water;
19. Dehydrate the slides in ethanol 70%, 85%, and 100% for 2 min each, at
RT. Completely air-dry the slides;
20. Add 20 µl of DAPI + antifade onto slides, cover with a 24×50 mm cover-
slip and stabilize the fuorophores by leaving the slides at 4°C for 30 min
before the analysis in a fuorescence microscope.
290 Cytogenetics and Molecular Cytogenetics

FIGURE  24.2 Illustrated fsh-FISH protocol of the frst day of the procedure. The steps
correspond to the ones described in the fsh-FISH protocol (Day 1).
FISH—in Fish Chromosomes 291

FIGURE 24.3 The second day of the illustrated fsh-FISH protocol, with variations accord-
ing to the label method used (a: directly labeled; b: indirectly labeled). The steps correspond
to the ones described in the fsh-FISH protocol (Day 2).
292 Cytogenetics and Molecular Cytogenetics

Day 2 for indirectly labeled probes (Figure 24.3b):

15. Heat a Coplin jar with 2×SSC at 42°C, remove the coverslip and wash the
slides for 5 min in a shaker;
16. Wash the slides in 1×SSC at RT for 5 min on a shaker;
17. Incubate the slides in 3% NFDM/4×SSC for 5 min at RT;
18. Apply 50 µl of the detection solution (will vary according to the combina-
tion of fuorophores used), cover with 24×50mm coverslips, and incubate
at 37 °C in a humid dark chamber for 1h;
19. Remove the coverslips, transfer the slides to the washing buffer (4×SSC/
Tween solution), and wash for 5 min on a shaker at RT;
20. Repeat the wash as done in step 19 twice;
21. Wash the slides in 1×PBS for 1 min at RT with a shaker;
22. Quickly submerge the glass slide in distilled water;
23. Dehydrate the slides in ethanol 70%, 85%, and 100% for 2 min each, at
RT. Completely air-dry the slides;
24. Add 20 µl of DAPI + antifade onto slides, cover with a 24×50mm cover-
slip and stabilize the fuorophores by leaving the slides at 4°C for 30 min
before the analysis in a fuorescence microscope.

Variations of FISH Protocol for WCP and CGH

For the FISH-basic protocol derived techniques, some steps of the previously men-
tioned protocol should be modifed. Given that the amount of probe is greater than
in classical FISH, modifcations in denaturation of probes and post-hybridization
washes must occur as follows:
Day 1 for WCP and CGH:
[. . .]

13. Denature the probes in a thermal cycler at 85°C for 10 min, followed by a
pre-annealing at 37°C for 30 min, followed by cooling at 4°C.
14. Add 20 µl of the hybridization mixture (probes + hybridization buffer) to
the slides. Cover with a 24 × 50mm coverslip, seal the borders of the cov-
erslip with rubber cement and incubate at 37°C overnight in a dark humid
chamber.

Day 2 for WCP and CGH:

15. Heat two Coplin jars with 1×SSC at 65°C, remove the coverslip and wash
the slides for 5 min on a shaker;
16. Repeat step 15 to washing the slides again;
17. Transfer the slides to the washing buffer (4×SSC/Tween solution) and
wash for 5 min on a shaker at RT;
18. Wash the slides in 1×PBS for 1 min at RT with a shaker;
19. Finish as described before
FISH—in Fish Chromosomes 293

Sequential FISH
Depending on the degree of chromosomal damage during the FISH technique, a
second round of hybridization can be realized. For this, the previous probe-chromo-
some assembly must be disassembled as described next:

1. Remove the coverslip and wash the slides in 1×SSC (65°C) for 5 min on a shaker;
2. Repeat step 1;
3. Transfer the slides to the washing buffer (4×SSC/Tween solution) and wash
for 5 min an a shaker at RT;
4. Repeat step 3;
5. Wash the slides in 1×PBS for 1 min at RT with a shaker;
6. Quickly submerge the glass slide in distilled water;
7. Dehydrate the slides in ethanol 70%, 85%, and 100% for 2 min each, at RT.
Completely air-dry the slides.

This should be enough to disassembly the previously applied FISH probes from
chromosomes and the FISH procedure should proceed from step 11 (denaturation of
slides). Be careful with the time of denaturation in corresponding buffer, since chro-
mosomes can be very fragile. This sequential FISH can be very helpful to identify
shared regions of distinct probes.

IMAGE CAPTURE AND TREATMENT

The image capture should be performed in a fuorescence microscope with the
respective flter combination corresponding to the light-wave excitation of probes.
After being captured, images from each flter should be exported separately and
overlapped in image software that allows the work in separated layers (such as GIMP
or Adobe Photoshop).

ACKNOWLEDGMENTS
The authors are thankful for the funding of Fundação de Amparo à Pesquisa do Estado
de São Paulo (Process numbers 2020/02681–9; 2018/14677–6 and 2020/11772–8).
Additionally, we would like to thank CNPq and the Humboldt Foundation for fnan-
cial support.

REFERENCES
1. Nelson, J.S.; Grande, T.C.; Wilson, M.V. Fishes of the World. John Wiley & Sons, New
York, 2016.
2. Salzburger, W. Understanding explosive diversifcation through cichlid fsh genomics.
Nat. Rev. Genet. 2018, 19, 705–717.
3. Fricke, R.; Eschmeyer, W.N.; Van der Laan, R. (Eds.). Eschmeyer’s catalog of fshes:
Genera, species, references. http://researcharchive.calacademy.org/research/ichthyol-
ogy/catalog/fshcatmain.asp [accessed on 01/06/2022].
294 Cytogenetics and Molecular Cytogenetics

4. Lehmann, R.; Kovařík, A.; Ocalewicz, K.; Kirtiklis, L.; Zuccolo, A.; Tegner, J.N.;
Wanzenböck, J.; Bernatchez, L.; Lamatsch, D.K.; R. Symonová. DNA transposon expan-
sion is associated with genome size increase in Mudminnows. Genome Biol. Evol. 2021,
13, evab228.
5. Santos, R.Z.; Calegari, R.M.; Silva, D.M.Z.A.; Ruiz-Ruano, F.J.; Melo, S.; Oliveira, C.;
Foresti, F.; Uliano-Silva, M.; Porto-Foresti, F.; Utsunomia, R. A long-term conserved
satellite DNA that remais unexpanded in several genomes of Characiformes fsh is
actively transcribed. Genome Biol. Evol. 2021, 13, evab002.
6. Yano, C.F.; Sember, A.; Kretschmer, R.; Bertollo, L.A.C.; Ezaz, T.; Hatanaka, T.;
Liehr, T.; Ráb, P.; Al-Rikabi, A.; Viana, P.F.; Feldberg, E.; Oliveira, E.A.; Toma, G.A.;
Cioff, M.B. Against the mainstream: Exceptional evolutionary stability of ZW sex
chromosomes across the fsh families Triportheidae and Gasteropelecidae (Teleostei:
Characiformes). Chromosome Res. 2021, 29, 1–26.
7. Pinkel, D.; Straume, T.; Gray, J.W. Cytogenetic analysis using quantitative, high sensitiv-
ity, fuorescence hybridization. Proc. Nat. Acad. Sci. 1986, 83, 2934–2938.
8. Pendás, A.M.; Morán, P.; Garcia-Vázquez, E. Ribosomal RNA genes are interspersed
throughout a heterochromatic chromosome arm in Atlantic salmon. Cytogenet. Genome
Res. 1993, 63, 128–130.
9. Pendás, A.M.; Morán, P.; García-Vázquez, E. Multi-chromosomal location of ribosomal
RNA genes and heterochromatin association in brown trout. Chromosome Res. 1993, 1,
63–67.
10. Pendás, A.M.; Moran, P.; Freije, J.P.; Garcia-Vazquez, E. Chromosomal mapping and
nucleotide sequence of two tandem repeats of Atlantic salmon 5S rDNA. Cytogenet.
Genome Res. 1994, 67, 31–36.
11. Phillips, R.B. Application of fuorescence in situ hybridization (FISH) to fsh genetics
and genome mapping. Marine Biotechnol. 2001, 3, S145–S152.
12. Wiegant, J.; Ried, T.; Nederlof, P.M.; Ploeg, M.V.D.; Tanke, H.J.; Raap, A.K. In situ
hybridisation with fuoresceinated DNA. Nucl. Acids Res. 1991, 19, 3237–3241.
13. Wiegant, J.; Galjart, N.J.; Raap, A.K.; d’Azzo, A. The gene encoding human protective
protein (PPGB) is on chromosome 20. Genomics 1991, 10(2), 345–349.
14. Freije, J.P.; Pendas, A.M.; Velasco, G.; Roca, A.; Abrahamson, M.; Lopez-Otin, C.
Localization of the human cystatin D gene (CST5) to chromosome 20p11. 21 by in situ
hybridization. Cytogenet. Genome Res. 1993, 62, 29–31.
15. Yano, C.F.; Bertollo, L.A.C.; Cioff M.B. Fish‑FISH: Molecular Cytogenetics in
Fish Species: In Fluorescence In Situ Hybridization (FISH). Springer, Berlin, 2017,
pp. 429–443.
16. Bertollo, L.A.C. Cytotaxonomic considerations on Hoplias lacerdae (Pisces,
Erythrinidae). Brazil. J. Genet. 1978, 1, 103–120.
17. Bertollo, L.A.C.; Cioff, M.B.; Moreira-Filho, O. Direct chromosome preparation
from freshwater teleost fshes. In: Fish Cytogenetic Techniques (Chondrichthyans and
Teleosts); Ozouf-Costaz, C.; Pisano, E.; Foresti, F.; Almeida Toledo, L.F., Eds., CRC
Press/Taylor & Francis Group, Oxon, 2015, pp. 21–26.
18. Zapata, A. Ultrastructural study of the teleost fsh kidney. Development. Comp. Immunol.
1979, 3, 55–65.
19. Oliveira, E.A.; Bertollo, L.A.C.; Rab, P.; Ezaz, T.; Yano, C.F.; Hatanaka, T.; Jegede, O.I.;
Tanomtong, A.; Liehr, T.; Sember, A.; Maruyama, S.R.; Feldberg, E.; Viana, P.F.; Cioff,
M.B. Cytogenetics, genomics and biodiversity of the South American and African
Arapaimidae fsh family (Teleostei, Osteoglossiformes). PloS One 2019, 14, e0214225.
20. Paim, F.G.; Maia, L.; Landim-Alvarenga, F.D.C.; Foresti, F.; Oliveira, C. New protocol
for cell culture to obtain mitotic chromosomes in fshes. Meth. Protoc. 2018, 1, 47.
FISH—in Fish Chromosomes 295

21. Traut, W.; Winking, H. Meiotic chromosomes and stages of sex chromosome evolution
in fsh: Zebrafsh, platyfsh and guppy. Chromosome Res. 2001, 9, 659–672.
22. Rebordinos, L.; Cross, I.; Merlo, A. High evolutionary dynamism in 5S rDNA of fsh:
State of the art. Cytogenet. Genome Res. 2013, 141, 103–113.
23. Lafontaine, D.L.; Tollervey, D. The function and synthesis of ribosomes. Nat. Rev. Mol.
Cell Biol. 2001, 2, 514–520.
24. Cioff, M.B.; Martins, C.; Centofante, L.; Jacobina, U.; Bertollo, L.A.C. Chromosomal vari-
ability among allopatric populations of Erythrinidae fsh Hoplias malabaricus: Mapping
of three classes of repetitive DNAs. Cytogenet. Genome Res. 2009, 125, 132–141.
25. Martins, C.; Galetti, P.M. Chromosomal localization of 5S rDNA genes in Leporinus fsh
(Anostomidae, Characiformes). Chromosome Res. 1999, 7, 363–367.
26. Crepaldi, C.; Martí, E.; Gonçalves, É.M.; Martí, D.A.; Parise-Maltempi, P.P. Genomic
differences between the sexes in a fsh species seen through satellite DNAs. Front.
Genet. 2021, 12, 728670.
27. Volff, J.N.; Schartl, M. Variability of genetic sex determination in poeciliid fshes.
Genetica. 2001, 111, 101–110.
28. Ferreira, D.C.; Porto-Foresti, F.; Oliveira, C.; Foresti, F. Transposable elements as a
potential source for understanding the fsh genome. Mobile Genet. Elements. 2011, 1,
112–117.
29. Chalopin, D.; Naville, M.; Plard, F.; Galiana, D.; Volff, J.N. Comparative analysis of
transposable elements highlights mobilome diversity and evolution in vertebrates.
Genome Biol. Evol. 2015, 7, 567–580.
30. Carducci, F.; Barucca, M.; Canapa, A.; Carotti, E.; Biscotti, M.A. Mobile elements in
ray-fnned fsh genomes. Life. 2020, 10, 221.
31. Ferreira, A.M.V.; Marajó, L.; Matoso, D.A.; Ribeiro, L.B.; Feldberg, E. Chromosomal
mapping of Rex retrotransposons in Tambaqui (Colossoma macropomum Cuvier, 1818)
exposed to three climate change scenarios. Cytogenet. Genome Res. 2019, 159, 39–47.
32. da Silva, F.A.; Guimarães, E.M.C.; Carvalho, N.D.; Ferreira, A.M.; Schneider, C.H.;
Carvalho-Zilse, G.A.; Feldberg, E.; Gross, M.C. Transposable DNA elements in
Amazonian fsh: From genome enlargement to genetic adaptation to stressful environ-
ments. Cytogenet. Genome Res. 2020, 160, 148–155.
33. Volff, J.N.; Körting, C.; Sweeney, K.; Schartl, M. The non-LTR retrotransposon Rex3
from the fsh Xiphophorus is widespread among teleosts. Mol. Biol. Evol. 1999, 16,
1427–1438.
34. Marz, M.; Kirsten, T.; Stadler, P.F. Evolution of spliceosomal snRNA genes in metazoan
animals. J. Mol. Evol. 2008, 67, 594–607.
35. Cabral-de-Mello, D.C.; Valente, G.T.; Nakajima, R.T.; Martins, C. Genomic organization
and comparative chromosome mapping of the U1 snRNA gene in cichlid fsh, with an
emphasis in Oreochromis niloticus. Chromosome Res. 2012, 20, 279–292.
36. Silva, D.M.; Utsunomia, R.; Pansonato-Alves, J.C.; Oliveira, C.; Foresti, F. Chromosomal
mapping of repetitive DNA sequences in fve species of Astyanax (Characiformes,
Characidae) reveals independent location of U1 and U2 snRNA sites and association of
U1 snRNA and 5S rDNA. Cytogenet. Genome Res. 2015, 146, 144–152.
37. Utsunomia, R.; Silva, D.M.Z.A.; Ruiz-Ruano, F.J.; Araya-Jaime, C.; Pansonato-Alves,
J.C.; Scacchetti, P.C.; Hashimoto, D.T.; Oliveira, C.; Trifonov, V.A.; Porto-Foresti, F.;
Camacho, J.P.M.; Foresti, F. Uncovering the ancestry of B chromosomes in Moenkhausia
sanctaeflomenae (Teleostei, Characidae). PLoS One. 2016, 11, e0150573.
38. Yang, F.; Trifonov, V.; Ng, B.L.; Kosyakova, N.; Carter, N.P. Generation of Paint
Probes from Flow‑Sorted and Microdissected Chromosomes: In Fluorescence In Situ
Hybridization (FISH). Springer, Berlin; 2017, pp. 63–79.
296 Cytogenetics and Molecular Cytogenetics

39. Sassi, F.M.C.; Deon, G.A.; Moreira-Filho, O.; Vicari, M.R.; Bertollo, L.A.C.; Liehr, T.;
de Oliveira, E.A.; Cioff, M.B. Multiple sex chromosomes and evolutionary relation-
ships in Amazonian catfshes: The outstanding model of the genus Harttia (Siluriformes:
Loricariidae). Genes. 2020, 11, 1179.
40. Sassi, F.M.C.; Moreira-Filho, O.; Deon, G.A.; Sember, A.; Bertollo, L.A.C.; Liehr, T.;
Oliveira, V.C.S.; Viana, P.F.; Feldberg, E.; Vicari, M.R.; Cioff, M.B. Adding new pieces
to the puzzle of karyotype evolution in Harttia (Siluriformes, Loricariidae): Investigation
of Amazonian species. Biology. 2021, 10, 922.
41. Sassi, F.M.C.; Perez, M.F.; Oliveira, V.C.S.; Deon, G.A.; de Souza, F.H.S.; Ferreira,
P.H.N.; de Oliveira, E.A.; Hatanaka, T.; Liehr, T.; Bertollo, L.A.C.; Cioff, M.B. High
genetic diversity despite conserved karyotype organization in the giant Trahiras from
genus Hoplias (Characiformes, Erythrinidae). Genes. 2021, 12, 252.
25 FISH—and the
Characterization of
Synaptonemal Complex
Victor Spangenberg

CONTENTS
Introduction ............................................................................................................ 297
Immuno-FISH Approach on Total Preparations of Synaptonemal Complexes ..... 298
The Logical Scheme of Immuno-FISH Is as Follows............................................ 299
DNA FISH Probes and Spread Preparation of Synaptonemal Complexes ............ 300
Spreading Technique .............................................................................................. 302
Materials ....................................................................................................... 302
Procedure ...................................................................................................... 302
Conclusions ............................................................................................................ 303
References .............................................................................................................. 304

INTRODUCTION
Meiotic prophase I is the key stage of meiosis and includes several sequential sub-
stages connected with synaptonemal complex (SC), which is the skeletal structure
of meiotic bivalents. Therefore, studies of SC-preparations are not focused on one
specifc stage. On the contrary, it is a complex analysis of dynamic changes in the
nuclei of germ cells of all prophase I stages of meiosis: leptotene, zygotene, pachy-
tene, diplotene and diakinesis. In biological objects without strict synchronization
in meiosis, it is usually possible to obtain all of the listed stages on one cytological
preparation. The stages of assembly and subsequent disassembly of SC are associ-
ated with numerous protein markers of DNA double-strand breaks (DSB) processing
(their formation and subsequent repair), meiotic recombination, chromatin remod-
eling and many others. Thus, the SC is a highly dynamic nucleoprotein structure.
The study of protein markers of meiosis using immunocytochemical (ICC) methods
makes SC-karyotyping an important extension to the analysis of metaphase mitotic
chromosomes.
There is a wide variety of methods for obtaining preparations of meiotic chro-
mosomes, i.e. total preparations of SCs. Most applicable methods for the analysis of
meiotic nuclei are spread techniques.[1, 2] The main feature of spreading approaches
is obtaining a fat (spread) preparation from a native three-dimensional nucleus.
Notably, spread preparation of SCs should not contain too many overlapped biva-
lents. Although such confgurations (overlapping) are unavoidable when studying

DOI: 10.1201/9781003223658-25 297

298 Cytogenetics and Molecular Cytogenetics

TABLE 25.1
Basic Morphological Criteria of Meiotic Prophase I Stages and Most-Used
Protein Markers—Synaptonemal Complex Associated Proteins
Stages of Meiotic Synaptonemal Complex Basic Protein Markers Used for
Prophase I Identifcation of Stages
Leptotene formation of axial elements of SPO11, RAD51, γH2AFX
chromosomes, intense DSB processing
and repair.
Early zygotene clustering of chromosome’s telomeres— SYCP3, SYCP1, RAD51,
formation of “chromosomal bouquet” γH2AFX
structure. Initiation of peritelomeric/
interstitial synapsis.
Late zygotene prolongation of chromosomal synapsis, SYCP3, SYCP1, RAD51,
declustering of telomere ends. γH2AFX
Pachytene complete assembly of synaptonemal SYCP3, SYCP1, MLH1,
complexes (meiotic bivalents). γH2AFX(on the XY-bivalent only)
Early diplotene initiation of homologous chromosomes SYCP3, SYCP1, MLH1
desynapsis—desynaptic “forks”, or
interstitial regions of desynapsis.
Late diplotene progression of desynapsis, elongation and SYCP3
fragmentation of axial elements,
synaptonemal complex disassemly.
Diakinesis complete removal of SYCP3 from SYCP3
chromosome axes, but remnants near
centromeres.

species with long chromosomes, following the spread protocols one can achieve
analyzable total preparations of SCs.
Methods for obtaining total preparations of SCs were developed for mammals,[1, 2]
widely used for reptiles,[3, 4] and also adapted for other groups, such as fshes,[5]
insects,[6] plants,[7] and fungi,[8] with some modifcations. Particular care should be
taken when going for preparations from meiocytes of organisms that have strong
enzymatic systems, for example fungi.[8] Ice-cold conditions or use of protease inhib-
itors are highly recommended, since during hypotonic shock and cell destruction,
enzymes can destroy proteins of SC. Interestingly, some authors recommend to use
protease inhibitors for mammalian cells as well.[9] The most useful protein markers
of SC (i.e. morphologi-cal criteria for determining the stages of meiotic prophase I)
are presented in Table 25.1.

IMMUNO-FISH APPROACH ON TOTAL PREPARATIONS

OF SYNAPTONEMAL COMPLEXES
Immuno-FISH is a method combining immunocytochemistry (ICC) with fuores-
cence in situ hybridization (FISH).[5, 10] However, the combination of ICC and FISH
FISH—and the Characterization of Synaptonemal Complex 299

methods applied to the same SC preparation imposes several limitations. They

address key methodological differences between ICC and FISH:

I. ICC methods require keeping proteins in the most native conformation so

that epitopes (both linear and 3D) are accessible by the antibodies used.
Thus, buffered fxative to maintain pH is highly recommended, and all pro-
cedures of making spread SC-preparation should be carried out in ice-cold
conditions.[10, 11] Using the buffered fxative allows to use ICC approaches
and detect even minor foci of specifc proteins involved in the processes
of homologous chromosomes synapsis, DSB repair, meiotic recombination,
transcription activity or silencing of chromatin. As stated before, meiocytes
of organisms with strong enzymatic systems (fungi,[5] plants,[10] maybe even
mammalian cells[9]) need ice-cold conditions or protease inhibitors during
hypotonic shock and cell destruction.
II. FISH methods include a step of DNA denaturation in 50–70% formamide
solution at a high temperature (temperature depends on the concentration
of formamide used). Therefore, proteins on SC-preparations can denature
during this step.

Nevertheless, in some cases, it is possible to use polyclonal and some monoclonal

antibodies even after FISH as an additional round of ICC.[12, 13] Apparently, heat
treatment does not destroy some of the epitopes (possibly the linear ones) and thus
helps to get more data from one SC-preparation. On the other hand, protocols for
unmasking protein epitopes in citrate buffer using high temperature and pressure are
well-known in histological and cytological practice.[14] Many proteins could be suc-
cessfully immunostained only after one round of unmasking. These facts gave rise
to experiments applying certain antibodies after FISH.
Immuno-FISH studies on mammalian and reptile preparations have shown
that some major proteins could be immunostained using stable fuorophores
(AlexaFluor488 or AlexaFluor555) retaining partial fuorescence even after FISH.
This fact must be taken into account when choosing spectral channels to avoid prob-
lems in data interpretation ICC and FISH on one spread preparation.[13, 15]

THE LOGICAL SCHEME OF IMMUNO-FISH IS AS FOLLOWS

1. ICC rounds (one or several)
2. Microscopy
3. FISH
4. Microscopy
5. Additional ICC round (if possible)
6. Fusion of ICC and FISH images.

After FISH, it is recommended to make images not only the optical channel
under study, but all those available on the microscope assembly. This is important
because chromatin becomes less contrasting after denaturation. The more opti-
cal channels are captured, the easier it is to colocalize the ICC and FISH images
300 Cytogenetics and Molecular Cytogenetics

using morphological structures as reference. The most convenient for this are
the SC axial structures (immunostaining of major SC protein SYCP3 is usually
detectable after FISH if stable antibody conjugates are used), or secondly, chro-
matin contrasted with 4′,6-diamidino-2-phenylindole (= DAPI) before and after
FISH.
Thus, immuno-FISH on SC-karyotypes allows to study chromatin changes (DNA
and proteins) in dynamics—during successive stages of meiotic prophase I. It is
important to know that meiotic bivalents are on average 10 times longer than meta-
phase mitotic chromosomes. Therefore, many tasks related to precise positioning of
DNA-loci can be more easily solved on SC spreads. On the other hand, important
discoveries on the elimination of part of the genomes also have been made using
immuno-FISH on SC-preparations: germline-restricted chromosome (GRC), elimi-
nated from somatic cells and spermatids,[16] or elimination of extra X-chromosome
from 47,XXY karyotype in human.[9]

DNA FISH PROBES AND SPREAD PREPARATION

OF SYNAPTONEMAL COMPLEXES
According to FISH probe’s type, the methodological section can be divided into two
basic parts:

1. FISH with probes for tandem repetitive DNA sequences (pericentromeric

satellite DNA, telomere repeats, rDNA).
2. FISH with probes to unique DNA loci (locus-specifc probes, whole chro-
mosome probes).

The main difference of FISH on preparations of SC spreads is signifcantly less DNA

compaction compared to metaphase mitotic chromosomes; therefore, the expected
FISH-signal intensity is lower. On the other hand, in pachytene bivalents there are
four copies of each DNA sequence located close to each other, and thus could pro-
vide stronger FISH-signal.[17] Figure 25.1 shows one of the hypothetical models for
the interaction of four chromatin loops (DNA locus) during the assembly of the SC
from leptotene to pachytene (Figure 25.1).
Short oligo-DNA probes (~18–40 bp) are mainly applicable to tandemly organized
repetitive DNA-loci on meiotic chromosomes, such as telomeres, or pericentromeric
satellites. To obtain a bright fuorescent signal, terminal fuorescent labeling of the 5′
end or both (5′ and 3′) ends is usually suffcient.[13] More expensive short PNA/LNA-
FISH probes for tandemly repeated DNA provide reliable results and very specifc
signal.[13, 19] This type of probes is recommended for poorly preserved preparations
or after several rounds of denaturation and washings.[15]
Locus-specifc DNA FISH-probes have been successfully used to study meio-
sis and made it possible to visualize true chromatin loops in the structure of
SCs (Figure  25.2).[12, 20–23] Probes derived from BAC clones containing dispersed
repetitive DNA or so-called repeat-free (RF) probes are available for studies of
human and mouse loci.[24, 25] Tyramide-FISH method has been successfully used
to detect sequences shorter than 1,000 bps for the frst time on mitotic metaphase
FISH—and the Characterization of Synaptonemal Complex 301

FIGURE  25.1 a) Model of interaction between chromatin loops in prophase I of meiosis.

1 and 2—sister chromatin loops of the homolog I; 3 and 4—sister chromatin loops of the
homolog II. AE—axial elements of the chromosomes, LE—lateral elements of the assembled
synaptonemal complex. b) Cross-section of the same model (from[18] with modifcations).

FIGURE  25.2 Immuno-FISH study of 160 Kb (yellow) and 90 Kb (violet) DNA loci on
chromosome 17 in human spermatocyte I, early diplotene.
a) 160 Kb probe (yellow) on the bivalent 17.
b) 160 Kb probe (yellow) visualizes chromatin loops on the bivalent 17.
c) Immunostaining of crossing over marker, MLH1 (red) on the bivalent 17 near 160 Kb
probe (yellow) Scale bar—5μm (from[18] with modifcations).
302 Cytogenetics and Molecular Cytogenetics

chromosomes.[26, 27] This approach also showed applicability when used on prepara-
tions of SCs to detect single-copy genes.[28]
In general, FISH techniques on preparations of SCs are similar to the main proto-
cols used on mitotic metaphase chromosomes. Particularly to those protocols where
signal amplifcation is used (biotin-avidin, tyramide detection systems). Minor dif-
ferences relate to less compaction, and hence greater accessibility of chromatin on
meiotic prophase I preparation. Therefore, for example, RNAse treatment can be
done in half the time compared to somatic metaphase plates.

SPREADING TECHNIQUE
MATERIALS
• Microscope slides: All preparations of primary spermatocyte’s of oocytes
spread nuclei (i.e. total preparations of SCs) should be done on slides with
an adhesive coating, such as poly-L-lysine. This is important for attachment
and preservation of chromatin during several rounds: ICC (immunostaining
and washing) and FISH (denaturation, annealing, washing).
• Fixative for spreads: 1% paraformaldehyde, pH 8.4 containing 0.15% Triton
X-100. Bi-distilled water with the addition of paraformaldehyde powder
must be heated to 60–65°C. Add 1–2 small drops (10–15 µl) of 1N NaOH
until a transparent solution is obtained (avoid boiling). Let the fxative cool,
flter using flter paper, add Triton X-100, and adjust to pH8.4 with 0.1M
sodium tetraborate.
• Hypotonic solution: 0.1 M sucrose (1.7g sucrose in 50ml bi-distilled water).
• Wash solution: 0.4% Photofo (Kodak) adjusted to pH 8.2–8.4 with borate
buffer. Can be stored as a 4% stock solution and diluted before use.

PROCEDURE
1. Isolate gonadal tissues and transfer to cold Eagle’s medium (without
L-Glutamine) or 1×PBS (pH 7.4) in a small Petri dish. Samples should be
isolated shortly before preparations. Alternatively, storage is allowed in
cold (+4°C) conditions in the Eagle medium (or similar) or in 1×PBS for
several hours. Avoid drying of tissues. Using two forceps, disaggregate
the tissue of the seminiferous tubules. In the case of ovarian tissue, cell
disaggregation can be carried out mechanically between glass slide and
coverslip (better to use concave microscope slide). Using a stereomicro-
scope, gently press the dissecting needle on the coverslip over the pressed
piece of tissue and collect the suspension with a pipette. Avoid drying out
and keep the temperature near 4°C using cold plate thermostat.
2. Transfer 50 μl of cell suspension from step 1 in a 1.5 ml reaction tube and
continue to disrupt aggregates using pipette with a 200 μl tip with one mm
shortened tip. Avoid formation of bubbles. Check a small amount of the
suspension in a light microscope for large spermatocytes and in case of
FISH—and the Characterization of Synaptonemal Complex 303

overabundance of spermatids and/or fat inclusions centrifuge for 10 min

at 400–500 g and remove the supernatant (for next steps use lower fraction
only).
3. Add slowly an equivalent amount (50 μl) of hypotonic solution (0.1 M
sucrose). Keep for 5–10 min at 4°C.
4. Repeat step 3 two times by adding 50 μl of 0.1 M sucrose every time. Keep
the vial on +4°C.
5. Immerse the microscope slide with poly-L-lysine coating in cold fxative
for spreading (1% paraformaldehyde, pH 8.4, containing 0.15 % Triton
X-100, see Materials). The wet area can vary (24×24 or 24×50 mm)
depending on the number of slides needed or suspension quality. Wipe off
the fxative from the lower side of slide.
6. Using pipette, accurately touch the 20–30 μl hanging drop of the cell
suspension with the surface of the cold fxative on the slide. It is better
to use a not very sharp pipette tip (200 μl); this allows forming a rounded
hanging drop of the cell suspension. Distribute the suspension well by
turning the slide in different directions. If the suspension contains a
small number of cells (oocytes), then 2–3 drops can be added per slide,
but in this case, use a large (24×50 mm) area of the fxative solution on
the slide.
7. Immediately after dropping, transfer the slides in a water bath at 4°C for
one hour.
8. Wash three times in cold 0.04% PhotoFlo solution (pH 8.4) for 20 seconds
each without agitation.
9. Place the slides to dry in a vertical position in a cold place under the air
fow for 0.5–1 hour.
10. Store the slides at −20°C before use.
11. Slides should be rehydrated in PBS for 1 min before immunostaining.

CONCLUSIONS
In general, the immuno-FISH method on preparations of meiotic chromosomes is
promising for working with a variety of biological objects due to the availability of
antibodies with a wide range of immune homology, as well as due to the accumula-
tion and analysis of genomic data. Thus, it is possible to study the key stage of the
life cycle of an organism using both, (i) a variety of meiosis associated protein mark-
ers, and (ii) DNA markers (whole chromosome painting, unique DNA loci, satellite
DNA, telomeric repeats, rDNA). In the near future, it is obvious that a large num-
ber of new protein markers associated with the SC in particular, and with meiosis
prophase I in general, will become available.[29] Therefore, when using immuno-
FISH-protocols, frst of all highest quality preparation of spread meiotic bivalents
is required, with preservation of the native form of protein markers. Second, FISH
protocols should take into account the lower chromatin density in meiosis compared
to the metaphase of mitosis. Overall, this means that methods of amplifying detected
signals are important.
304 Cytogenetics and Molecular Cytogenetics

REFERENCES
1. Navarro, J.; Vidal, F.; Guitart, M.; Egozcue, J. A method for the sequential study of syn-
aptonemal complexes by light and electron microscopy. Hum. Genet. 1981, 59, 419–421.
2. Peters, A.H.F.M.; Plug, A.W.; Van Vugt, M J.; De Boer, P. A drying-down technique
for the spreading of mammalian meiocytes from the male and female germline.
Chromosome Res. 1997, 5, 66–68.
3. Lisachov, A.; Poyarkov, N.; Pawangkhanant, P.; Borodin, P.; Srikulnath, K. New karyo-
type of Lygosoma bowringii (Günther, 1864) suggests cryptic diversity. Herpet. Notes.
2018, 11, 1083–1088.
4. Spangenberg, V.; Arakelyan, M.; Galoyan, E.; Martirosyan, I.; Bogomazova, A.;
Martynova, E.; de Bello Cioff, M.; Liehr, T.; Al-Rikabi, A.; Osipov, F.; Petrosyan, V.;
Kolomiets, O. Meiotic synapsis of homeologous chromosomes and mismatch repair
protein detection in the parthenogenetic rock lizard Darevskia unisexualis. Molecular
Reproduction and Development. 2021, 88, 119–127.
5. Araya-Jaime, C.; Serrano, É.A.; de Andrade Silva, D.M.Z.; Yamashita, M.; Iwai, T.;
Oliveira, C.; Foresti, F. Surface-spreading technique of meiotic cells and immunodetec-
tion of synaptonemal complex proteins in teleostean fshes. Mol. Cytogenet. 2015, 8,
1–6.
6. Viera, A.; Santos, J.L.; Parra, M.T.; Calvente, A.; Gómez, R.; de la Fuente, R.; Suja, J.Á.;
Page, J.; Rufas, J.S. Cohesin axis maturation and presence of RAD51 during frst meiotic
prophase in a true bug. Chromosoma. 2009, 118(5), 575–589.
7. Loidl, J.; Jones, G.H. Synaptonemal complex spreading in Allium. Chromosoma. 1986,
93, 420–428.
8. Mazheika, I.S.; Kolomiets, O.L. A method for preparation of synaptonemal complexes of
meiotic chromosomes from basidial protoplasts of the white button mushroom Agaricus
bisporus (Lange) Imbach. Russ J Genet. 2003, 39, 283–287.
9. Sciurano, R.B.; Luna Hisano, C.V.; Rahn, M.I.; Brugo Olmedo, S.; Rey Valzacchi, G.;
Coco, R.; Solari, A.J. Focal spermatogenesis originates in euploid germ cells in classical
Klinefelter patients. Hum. Reprod. 2009, 24, 2353–2360.
10. Sepsi, A.; Fábián, A.; Jäger, K.; Heslop-Harrison, J.S.; Schwarzacher, T. ImmunoFISH:
Simultaneous visualisation of proteins and DNA sequences gives insight into meiotic
processes in nuclei of grasses. Front Plant Sci. 2018, 9, 1193.
11. Kolomiets, O.L.; Lelekova, M.A.; Kashintsova, A.A.; Kurilo, L.F.; Bragina, E.E.;
Chernykh, V.B.; Gabliya, M.Y.; Vinogradov, I.V.; Vityazeva, I.I.; Bogolyubov, S.V.;
Spangenberg, V.E. Detection of human meiotic and spermatogenetic anomalies using
light, electron and fuorescence microscopy. Androl Genit Surgery. 2018, 19, 24–35.
12. Pigozzi, M.I. Localization of single-copy sequences on chicken synaptonemal complex
spreads using fuorescence in situ hybridization (FISH). Cytogenet Genome Res. 2007,
119, 105–112.
13. Spangenberg, V.; Losev, M.; Volkhin, I.; Smirnova, S.; Nikitin, P.; Kolomiets, O. DNA
Environment of centromeres and non-homologous chromosomes interactions in mouse.
Cells. 2021, 10, 3375.
14. Sciurano, R.B.; Solari, A.J. Ultrastructural and immunofuorescent methods for the
study of the XY body as a biomarker. In: Functional Analysis of DNA and Chromatin;
Humana Press, Totowa, NJ, 2014, pp. 137–149.
15. Spangenberg, V.; Arakelyan, M.; Galoyan, E.; Pankin, M.; Petrosyan, R.; Stepanyan,
I.; Grishaeva, T.; Danielyan, F.; Kolomiets, O. Extraordinary centromeres: Differences
FISH—and the Characterization of Synaptonemal Complex 305

in the meiotic chromosomes of two rock lizards species Darevskia portschinskii and
Darevskia raddei. Peer J. 2019, 7, e6360.
16. Torgasheva, A.A.; Malinovskaya, L.P.; Zadesenets, K.S.; Karamysheva, T.V.; Kizilova,
E.A.; Akberdina, E.A.; Pristyazhnyuk, I.E.; Shnaider, E.P.; Volodkina, V.A.; Saiftdinova,
A.F.; Galkina, S.A.; Larkin, D.M.; Rubtsov, N.B.; Borodin, P.M. Germline-restricted
chromosome (GRC) is widespread among songbirds. Proc. Natl. Acad. Sci. 2019, 116,
11845–11850.
17. Zadesenets, K.S.; Katokhin, A.V.; Mordvinov, V.A.; Rubtsov, N.B. Telomeric DNA in
chromosomes of fve opisthorchid species. Parasitol Internat. 2012, 61, 81–83.
18. Spangenberg, V. Dynamics of the structural organization of the chromosomes during
the meiotic prophase I in human and mouse. PhD thesis. NI Vavilov Institute of General
Genetics RAS (Moscow, 2013).
19. Lisachov, A.P.; Borodin, P.M. Microchromosome polymorphism in the sand lizard,
Lacerta agilis Linnaeus, 1758 (Reptilia, Squamata). Comparat. Cytogenet. 2016, 10, 387.
20. Beliveau, B.J.; Joyce, E.F.; Apostolopoulos, N.; Yilmaz, F.; Fonseka, C.Y.; McCole,
R.B.; Wu, C.T. Versatile design and synthesis platform for visualizing genomes with
Oligopaint FISH probes. Proc. Natl. Acad. Sci. 2012, 109, 21301–21306.
21. Froenicke, L.; Anderson, L.K.; Wienberg, J.; Ashley, T. Male mouse recombination maps
for each autosome identifed by chromosome painting. Am. J. Hum. Genet. 2002, 71,
1353–1368.
22. Bogdanov, Y.F.; Spangenberg, V.E.; Dadashev, S.Y.; Vityazeva, I.I.; Bogoliubov, S.V.;
Kolomiets, O.L. Morphological manifestation of unique DNA segments in human mei-
otic prophase I. Cell Tiss. Biol. 2012, 6, 407–411.
23. Grishaeva, T.M.; Spangenberg, V.E.; Kolomiets, O.L.; Dadashev, S.Y.; Bogdanov, Y.F.
Peculiarities of chromosome organization in meiosis. Cell Tiss. Biol. 2013, 7, 343–346.
24. Liehr, T. Commercial FISH probes. In: Fluorescence In Situ Hybridization (FISH);
Springer, Berlin, 2017, pp. 49–61.
25. Swennenhuis, J.F.; Foulk, B.; Coumans, F.A.; Terstappen, L.W. Construction of repeat-
free fuorescence in situ hybridization probes. Nucl. Acids Res. 2012, 40, e20.
26. Khrustaleva, L.I.; Kik, C. Localization of single-copy T-DNA insertion in transgenic
shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplifcation.
Plant J. 2001, 25, 699–707.
27. Kudryavtseva, N.; Ermolaev, A.; Karlov, G.; Kirov, I.; Shigyo, M.; Sato, S.; Khrustaleva,
L. A dual-color tyr-FISH method for visualizing genes/markers on plant chromosomes
to create integrated genetic and cytogenetic maps. Int J Mol Sci. 2021, 22, 5860.
28. Kirov, I.V.; Van Laere, K.; Khrustaleva, L.I. High resolution physical mapping of single
gene fragments on pachytene chromosome 4 and 7 of Rosa. BMC Genet. 2015, 16, 1–10.
29. da Cruz, I.; Rodríguez-Casuriaga, R.; Santiñaque, F.F.; Farías, J.; Curti, G.; Capoano,
C.A.; Geisinger, A. Transcriptome analysis of highly purifed mouse spermatogenic cell
populations: Gene expression signatures switch from meiotic-to postmeiotic-related pro-
cesses at pachytene stage. BMC Genomics. 2016, 17, 1–19.
 26 RNA-FISH—
on Lampbrush
Chromosomes:
Visualization of Individual
Transcription Units
Tatiana Kulikova and Alla Krasikova

CONTENTS
Introduction............................................................................................................ 308
Description of Methods.......................................................................................... 309
Chromosome Isolation and Pretreatment...................................................... 309
RNA Preservation ......................................................................................... 309
Chromosome Fixation .................................................................................. 309
Probe Labeling.............................................................................................. 310
RNA-FISH Procedure................................................................................... 310
RNA FISH Procedure for Double Stranded DNA-Probes
(PCR- or Nick-Translation Products) ............................................... 311
Hybridization Mixture and In Situ Hybridization ............................ 311
Post-Hybridization Washings............................................................ 311
Detection of Hapten Labeled Probes ................................................ 311
RNA FISH Procedure for Oligonucleotide Probes....................................... 312
Hybridization Mixture and In Situ Hybridization ............................ 312
Post-Hybridization Washings............................................................ 312
Detection of Hapten Labeled Oligonucleotide Probes ..................... 312
Microscopy and Image Analysis................................................................... 313
Control Experiments .............................................................................................. 313
Interpretation of Results......................................................................................... 315
Conclusions............................................................................................................ 315
Acknowledgements................................................................................................ 315
References.............................................................................................................. 315

DOI: 10.1201/9781003223658-26 307

308 Cytogenetics and Molecular Cytogenetics

INTRODUCTION
RNA directed fuorescence in situ hybridization (FISH) is an effective approach
to localize RNA sequences in situ, visualize gene transcripts, and estimate gene
expression in time and space.[1–3] In a typical interphase nucleus, transcribed genes,
together with nascent transcripts at the site of their synthesis, are visible as dot-
like foci.[4, 5] The only exception are several highly expressed genes with very
long introns that form loop-like structures with nascent transcripts in Drosophila
spermatocytes,[6] and in human somatic cells.[7] However, in fshes, amphibians,
reptiles, birds, and certain insects, hypertranscription during oogenesis leads to
decompactization of hundreds of transcribed genomic regions and transforma-
tion of the meiotic chromosomes into highly elongated lampbrush chromosomes
(LBCs).[8–11] Lateral loops of LBCs representing sites of ongoing transcription
serve as a model for exploration into nascent RNA synthesis and processing at
the cytological level.[9, 12] Single transcription units on the lateral loops of amphib-
ian and avian LBCs with a gradient of RNP matrix can be seen even by light
microscopy.[13–15]
First in situ hybridization experiments on LBCs of newt Notophthalmus viride‑
scens were performed with radioactively labeled cRNA probes detected by autora-
diography.[16] In further studies, radioactive 5S rDNA probes were hybridized with
non-denatured N. viridescens LBCs to reveal nascent RNA.[17] In these early DNA/
RNA in situ hybridization experiments nascent 5S RNA transcripts were revealed
on a set of lateral loops. In control preparations, RNase pretreatment eliminated
5S RNA transcripts from the ribonucleoprotein (RNP) matrix of these loops. By
hybridization of the radioactively labelled DNA-probe to nascent RNA transcripts
on LBCs of crested newt, Triturus cristatus carnifex, Varley and co-authors evi-
denced transcription of highly repetitive DNA during oogenesis.[18] Similarly, radio-
active probes to satellite I DNA hybridized to non-denatured LBCs of N. viridescens
without RNase digestion demonstrated intense labeling of lateral loops.[19] Next
FISH method was developed and employed to detect nascent RNA on the lateral
loops of amphibian and avian LBCs.[13, 20–23] Nowadays, RNA-FISH can be success-
fully applied to reveal newly synthesized pre-mRNA and non-coding RNA on LBC
lateral loops. For example, probes made by in vitro transcription and bacterial artif-
cial chromosome (BAC)–derived probes were effectively hybridized to transcription
units of intensively expressed protein-coding genes on the lateral loops of axolotl and
chicken LBCs.[15, 24]
Visualization of nascent gene transcripts on individual transcription units could
be accompanied by immunofuorescent staining as described elsewhere.[25] For
example, by RNA-FISH after immunofuorescent detection of the elongating form
of RNA polymerase II we demonstrated that short 41 bp repeats are transcribed on
the lateral loops of chicken LBCs by RNA polymerase II with hyperphosphorylated
C-terminal domain.[13] For other examples of RNA-FISH after LBC immunostain-
ing, see in references [22] and [26]. RNA-FISH can also be combined with the target-
ing of labeled protein components to the RNP matrix of LBC lateral loops. Besides,
FISH according to nascent RNA hybridization protocol (see later) can be performed
on LBC preparations after incorporation of RNA precursors such as bromouridine
RNA-FISH—on Lampbrush Chromosomes 309

triphosphate (BrUTP), giving an opportunity to estimate transcription rates for indi-

vidual genes.
In this chapter, we provide an effective RNA-FISH protocol that includes steps
essential to preserve RNA on LBC preparations; also necessary control experiments
to estimate gene expression are presented. The described methodology enables
simultaneous visualization of up to 10 transcribed single-copy genomic loci on the
same LBC.

DESCRIPTION OF METHODS
CHROMOSOME ISOLATION AND PRETREATMENT
Methods for obtaining preparations of LBCs and nuclear bodies from amphibian and
avian diplotene oocytes are described in detail elsewhere (for example in [27] and
[28], or on https://projects.exeter.ac.uk/lampbrush/protocols.htm). In these protocols,
oocyte nucleus content is spread within an isolation chamber mounted on a standard
microscope slide. After microdissection from the oocyte nucleus LBC spreads are
fxed in paraformaldehyde (PFA) in phosphate buffered saline (PBS) followed by
alcohol-based fxation. Depending on the antigen detected, other fxation protocols
may be used: without ethanol or with reduced time of fxation in PFA.
Permeabilization can be omitted, since preparations of native chromosomes iso-
lated from the oocyte nucleus by microdissection are used. The absence of permea-
bilization results in the preservation of native chromosome morphology.
Nascent RNA on LBC preparations exists in a complex with a number of RNA
binding proteins and other RNPs; therefore, pretreatment with proteinases is not
performed, as this may reduce the specifc hybridization signal. For the RNA FISH
protocol no ribonuclease pretreatment should be done to detect nascent RNA targets
on LBC spreads. However, certain control slides imply RNase digestion step (see
later in the section “Control Experiments”).

RNA PRESERVATION
To achieve optimal results, RNA preservation measures must be followed, from chro-
mosome isolation procedure to post-hybridization washings. Bench surfaces should
be treated with 5% H2O2 or commercial RNase inhibitors. Slides and chambers for
chromosome isolation, lab beakers and other glassware, as well as metal instruments
should be washed in 2% 7X-detergent, thoroughly rinsed in distilled water and dried
in a drying oven at 200°C or autoclaved. Deionized water used for buffers should
be autoclaved. Whole chromosome isolation procedure and slide handling should be
performed with sterile laboratory gloves.

CHROMOSOME FIXATION
1. Fix freshly isolated LBC preparations in 2% PFA in a PBS for 30 minutes
at room temperature.
2. Dehydrate in 50% ethanol and 70% ethanol for 5 minutes each.
310 Cytogenetics and Molecular Cytogenetics

3. Store overnight in 70% ethanol.

4. Mark chamber positions on the opposite side of the slide by a diamond
blade.
5. Remove the isolation chambers with a razor blade; avoid slide drying.
6. Dehydrate preparations in 96% ethanol.
7. Leave to dry on air.
8. Examine preparations in the microscope equipped with the phase contrast
or Nomarski differential interference contrast.

PROBE LABELING
A variety of probes can be used for RNA-FISH on LBC preparations including
PCR-derived DNA probes, probes obtained via nick-translation, cDNA probes bio-
tinylated by random-primed oligonucleotide labeling, RNA probes made by in vitro
transcription, and synthetic oligonucleotide or locked nucleic acid probes. Protocol
for labeling DNA probes for FISH on LBC preparations by (degenerated oligo-
nucleotide primed = DOP-) polymerase chain reaction (PCR) was given earlier in
[23]. Labeling of longer probes (for example isolated BAC clone DNA) by Nick-
translation can be performed as described in [29]. Oligonucleotide probes are usually
labeled by terminal deoxynucleotidyl transferase.[30, 31] Probes can be labeled directly
by fuorochrome-modifed nucleotides or indirectly by hapten-modifed nucleotides
further detected by a fuorochrome. The quality of all labeled probes can be verifed
by DNA-FISH on metaphase chromosome preparations.
To visualize multiple gene transcripts in the same experiment, a mixture of ~10
BAC clone-based probes can be applied on a single LBC set. If gene targets localize
in a relatively small genomic region (2–2.5 Mbp), BAC probes should be labeled with
different haptens and fuorochromes.

RNA-FISH PROCEDURE
RNA-FISH on LBC preparations is performed according to conventional protocol[32]
with modifcations.[20] Stringency of the hybridization can be adjusted by varying
the concentration of formamide, the incubation temperature during hybridization,
concentration of Na+ in the washing buffer, and the temperature of the washing buf-
fer. The duration of hybridization should not be greatly reduced since it may lead to
underrepresentation of specifc nascent RNA targets; however, in case of oligonucle-
otide probes, hybridization time can be reduced up to 4 hours. To detect nascent
transcripts on LBC lateral loops, amplifcation of the FISH signal is usually not
required due to the presence of multiple closely spaced RNA targets on a single
transcription unit.
Re-FISH according to RNA hybridization protocol can be performed on LBC
spreads after image acquisition. In this case, after coverslip removal, slides with
LBCs are washed in 4×SSC (saline-sodium citrate buffer) with 0.01% Tween 20 at
37°C. Then RNA FISH procedure is applied with another probe set. However, it
should be noted that RNA on the lateral loops could be partially degraded during
RNA-FISH—on Lampbrush Chromosomes 311

this procedure. Thus, Re-FISH protocol can be best applied to co-localize probes
which, as shown earlier, hybridize with nascent RNA.

RNA FISH PROCEDURE FOR DOUBLE STRANDED DNA-PROBES

(PCR- OR NICK-TRANSLATION PRODUCTS)
Hybridization Mixture and In Situ Hybridization
1. To prepare 20 μl of hybridization mixture mix 0.8 μg of labeled DNA-
probe/s with 50× excess of carrier nucleic acid (salmon sperm DNA or
yeast tRNA) and a competitor DNA (e.g. C0t DNA fractions), if needed;
add 5M NaCl (0.1 of the DNA mixture volume); add ice-cold 96% ethanol
(2.5 of the mixture volume). Freeze for several hours or overnight.
2. Precipitate the DNA by centrifugation at 13,000 rpm at +4°C for 15 min-
utes. Remove supernatant.
3. Wash the pellet with 200 μl ice-cold 70% ethanol followed by centrifuga-
tion at 13,000 rpm at +4°C for 10 minutes. Remove supernatant and dry
the DNA pellet on air.
4. Dissolve the DNA pellet in 10 μl 100% formamide, vortex thoroughly.
5. Add 10 μl of the mixture of 4×SSC (20×SSC: 3M NaCl, 0.3M sodium
citrate, pH 7) and 20% dextran sulfate (half of the fnal volume of hybrid-
ization mixture), vortex thoroughly and siege.
6. Denature hybridization mixture at 95°C for 5 minutes. If a competitive
DNA is added, it is necessary to adjust the pre-annealing conditions.
7. Place the tube on ice.
8. Drop 1 μl of hybridization mixture per each isolation chamber.
9. Cover all four isolation chambers with 18×18 mm coverslip. If individ-
ual hybridization mixtures are applied, each droplet can be covered with
round coverslip (8 mm) or quarter sliced 18×18 mm coverslip.
10. Seal the coverslips with rubber cement.
11. Place slides into a moist chamber at 37°C, overnight.

Post-Hybridization Washings
12. Gently remove rubber cement with the forceps.
13. Place slides into a Coplin jar or 50 ml beaker with 2×SSC for 5–10 min-
utes to rinse coverslips.
14. Wash slides through three changes of 0.2×SSC at 60°C in a water bass
with rocking.
15. Wash slides through two changes of 2×SSC at 45°C in a water bass with
rocking.

Detection of Hapten Labeled Probes

16. Put the slides into a moist chamber and apply 100 μl of blocking solution (1%
blocking reagent or 1% BSA in 4×SSC with 0.1% Tween 20). Immediately
cover the drops with squares of paraflm. Incubate at 37°C for 40 minutes.
312 Cytogenetics and Molecular Cytogenetics

17. Remove paraflm and excess of the blocking solution and apply 50–100 μl
of primary antibodies against a hapten or avidin/streptavidin conjugated
with a fuorochrome dissolved in blocking solution. Immediately cover the
drops with squares of paraflm. Incubate in a moist chamber at 37°C for
40 minutes.
18. Wash slides through three changes of 4×SSC with 0.01% Tween 20 at
42°C in a water bass with rocking.
19. Put slides into a moist chamber and apply 50–100 μl of secondary
antibodies or anti-avidin/streptavidin antibodies in blocking solution.
Immediately cover the drops with squares of paraflm. Incubate at 37°C
for 40 minutes.
20. Wash slides through three changes of 4×SSC with 0.01% Tween 20 at
42°C in a water bass with rocking.
21. If biotin is used, repeat steps 17 and 18.
22. Rinse slides with deionized water.
23. Dehydrate in increasing concentration of ethanol: 70% and 96%, for
5 minutes each.
24. Dry on air.
25. Apply 7 μl of antifade solution (65% glycerol, 0.01M TrisHCl, 2%
DABCO, pH 7.5) containing 1 μg/μl DAPI and mount 24×24 coverslips.

RNA FISH PROCEDURE FOR OLIGONUCLEOTIDE PROBES

Hybridization Mixture and In Situ Hybridization
1. Prepare the hybridization mixture containing 42% formamide, 10% dextran
sulfate, 2×SSC, 10–30 ng/μl oligonucleotide probe and 50× excess of car-
rier nucleic acid (salmon sperm DNA or yeast tRNA). Vortex thoroughly.
2. Cover all four isolation chambers with 18×18 coverslip. If individual hybrid-
ization mixtures are applied, each droplet can be covered with round 8 mm
or quarter sliced 18×18 coverslip.
3. Seal the coverslips with rubber cement.
4. Place slides into a moist chamber at room temperature, overnight.

Post-Hybridization Washings
5. Gently remove rubber cement with the forceps.
6. Place slides into a Coplin jar or 50 ml beaker with 2×SSC for 5–10 minutes
to rinse coverslips.
7. Wash slides through three changes of 2×SSC at 37°C in a water bass with
rocking.

Detection of Hapten Labeled Oligonucleotide Probes

Hapten labeled oligonucleotide probes are detected by the same procedure as double-
stranded DNA-probes (steps 16–24). Apply 7 μl of antifade solution (65% glycerol, 0.01M
TrisHCl, 2% DABCO, pH 7.5) containing 1 μg/μl DAPI and mount 24×24 coverslips.
RNA-FISH—on Lampbrush Chromosomes 313

MICROSCOPY AND IMAGE ANALYSIS

Conventional 2D fuorescence microscopy is used to examine LBC preparations
after FISH. Additionally, phase contrast or Nomarski differential interference con-
trast (DIC) can be used to obtain images of the chromosomes themselves. Generally,
no image processing is required when the transcripts are visualized on the lateral
loops of LBCs. Since the images of giant LBCs consist of fragments, appropriate
software is used to compile a whole chromosome image.
Because LBCs represent meiotic diplotene bivalents, four hybridization signals
for each single-copy genomic loci can be seen on each LBC set. Closely spaced
nascent RNA transcripts are visible as a polarized RNP matrix with a thin end cor-
responding to the start of transcription (Figure 26.1b–b′).

CONTROL EXPERIMENTS
A combination of control experiments is necessary to accurately estimate genuine
gene transcription on LBC spreads by RNA-FISH (Figure 26.1):

• DNA-RNA FISH with a positive control probe is applied to estimate RNA

preservation on LBC preparations. As a positive control, a probe to gene
loci previously demonstrated to be transcribed on LBC lateral loops can
be used.
• DNA-RNA FISH after Ribonuclease A (RNase A) pretreatment is applied
to check the specifcity of probe hybridization to RNA (Figure 26.1c–c′′).
Other specifc ribonucleases (for example RNase H, RNase R, S7 nuclease)
can be applied on separate LBC preparations to establish the structure of
targeted RNA.
• DNA-RNA FISH with sense single-stranded oligonucleotide probes can be
used as a negative control for single-copy genomic regions transcribed from
one strand.
• DNA-DNA FISH after RNase A pretreatment is performed on LBC prepa-
rations as a positive control for probe hybridization to the target sequence
(Figure 26.1d–d′′).
• DNA-RNA FISH procedure, but without labelled probe in the hybridiza-
tion mixture, followed by post-hybridization washings and probe detection
is used as a control for non-specifc binding of the detection system to the
RNP-matrix of LBC lateral loops.
• DNA-RNA FISH procedure without any probe followed by post-
hybridization washings but without probe detection is performed as a control
for autofuorescence.

Control experiments should be performed in parallel to the main DNA-RNA FISH

experiment on LBC preparations obtained on the same day and under the same con-
ditions. Images should be acquired with the same parameters as the images in the
main DNA-RNA FISH experiment.
FIGURE 26.1 a-d′′—Visualization of nascent RNA within individual transcription units of a
single-copy gene on chicken lampbrush chromosomes.
a—A genomic region of chicken chromosome 4 occupied by BAC-clone 49F17 and overlap-
ping with COL25A1 gene (snapshot of the Integrative Genomics Viewer, chicken genome
assembly version galGal5);
b–d′—FISH with BAC-clone 49F17 (CHORI-261 chicken BAC-library) DNA-probe (green) on
lampbrush chromosome (LBC) 4 with control experiments, chromosomes are counterstained
with DAPI (blue); fragments of individual homologous chromosomes (half-bivalents) with a
pair of lateral loops containing transcription units of COL25A1 gene are shown.
b–b′—RNA-FISH on native LBCs.
c–c′—RNA-FISH on LBCs pretreated with RNase A.
d–d′—DNA-FISH on denatured LBCs pretreated with RNase A.
b′–d′—fuorescent images merged with phase contrast. Scale bar—10 μm.
b′′–d′′—schematic drawings of the corresponding microphotographs. LBCs were imaged by
a monochrome CCD camera (1.3 MegaPixel resolution) using fuorescent microscope Leica
DM 4000 (Leica Microsystems).
RNA-FISH—on Lampbrush Chromosomes 315

INTERPRETATION OF RESULTS
When we interpret the results of an RNA-FISH experiment on LBC preparations,
we should take into consideration the quality of spreads, RNA preservation, results
of control experiments and genomic characteristics of the targeted region. Control
experiments are essential to reliably establish the transcriptional activity of genes
on LBCs by RNA-FISH, for example [20, 21]. It should be noted that different RNA
species could be partially and nonuniformly degraded leading to variability across
RNA FISH results.[33] Moreover, analysis of gene expression on LBC spreads can
depend on the transcriptional activity of a certain gene, i.e. the number of nascent
RNA transcripts associated with individual transcription units. In addition, nascent
transcripts of tandem repeats could be still detectable on those LBC preparations
on which single-gene transcripts are already undetectable due to RNA degradation.

CONCLUSIONS
Here we describe an optimized RNA-FISH protocol directed to effective visual-
ization of transcribed genes on LBC preparations. In contrast to the basic DNA/
(DNA+RNA-transcript) FISH protocol on LBC spreads,[23] critical steps necessary
to detect nascent gene transcripts on lampbrush lateral loops are emphasized. The
delineated protocol was successfully implemented to establish transcription of pro-
tein-coding and non-coding RNA genes on LBCs of birds,[24] and can be applied to
LBC preparations of other diverse model organisms.

ACKNOWLEDGEMENTS
The research was supported by the RSF (grant #19–74–20075). Microscopy was
performed in the research resource center “Molecular and cell technologies” of
St. Petersburg State University.

REFERENCES
1. Chartrand, P.; Bertrand, E.; Singer, R.H.; Long, R.M. Sensitive and high-resolution
detection of RNA in situ. Methods Enzymol. 2000, 318, 493–506.
2. Brown, J.M.; Buckle, V.J. Detection of nascent RNA transcripts by fuorescence in situ
hybridization. Methods Mol. Biol. 2010, 659, 33–50.
3. Long, X.; Colonell, J.; Wong, A.M.; Singer, R.H.; Lionnet, T. Quantitative mRNA imag-
ing throughout the entire Drosophila brain. Nat. Methods. 2017, 14, 703–706.
4. Femino, A.M.; Fay, F.S.; Fogarty, K.; Singer, R.H. Visualization of single RNA tran-
scripts in situ. Science. 1998, 280, 585–590.
5. Ferraro, T.; Lucas, T.; Clémot, M.; De Las Heras Chanes, J.; Desponds, J.; Coppey, M.;
Walczak, A.M.; Dostatni, N. New methods to image transcription in living fy embryos:
The insights so far, and the prospects. WIREs Dev. Biol. 2016, 5, 296–310.
6. Fingerhut, J.M.; Moran, J.V.; Yamashita, Y.M. Satellite DNA-containing gigantic introns
in a unique gene expression program during Drosophila spermatogenesis. PLoS Genet.
2019, 15, e1008028.
7. Leidescher, S.; Ribisel, J.; Ullrich, S.; Feodorova, Y.; Hildebrand, E.; Galitsyna, A.;
Bultmann, S.; Link, S.; Thanisch, K.; Mulholland, C.; Dekker, J.; Leonhardt, H.; Mirny,
316 Cytogenetics and Molecular Cytogenetics

L.; Solovei, I. Spatial organization of transcribed eukaryotic genes. Nat. Cell Biol. 2022,
24, 327–339.
8. Callan, H.G. Lampbrush chromosomes. Springer, Berlin; 1986.
9. Morgan, G.T. Lampbrush chromosomes and associated bodies: New insights into prin-
ciples of nuclear structure and function. Chromosome Res. 2002, 10, 177–200.
10. Gaginskaya, E.; Kulikova, T.; Krasikova, A. Avian lampbrush chromosomes: A pow-
erful tool for exploration of genome expression. Cytogenet. Genome Res. 2009, 124,
251–267.
11. Krasikova, A.; Kulikova, T. Lampbrush Chromosomes: Current Concepts and Research
Perspectives. St Petersburg University Publishing House, St Petersburg, 2019.
12. Morgan, G.T. Imaging the dynamics of transcription loops in living chromosomes.
Chromosoma. 2018, 127, 361–374.
13. Deryusheva, S.; Krasikova, A.; Kulikova, T.; Gaginskaya, E. Tandem 41-bp repeats in
chicken and Japanese quail genomes: FISH mapping and transcription analysis on lamp-
brush chromosomes. Chromosoma. 2007, 116, 519–530.
14. Kaufmann, R.; Cremer, C.; Gall, J. G. Superresolution imaging of transcription units on
newt lampbrush chromosomes. Chromosome Res. 2012, 20, 1009–1015.
15. Keinath, M.C.; Davidian, A.; Timoshevskiy, V.; Timoshevskaya, N.; Gall, J.G.
Characterization of axolotl lampbrush chromosomes by fuorescence in situ hybridiza-
tion and immunostaining. Exp. Cell Res. 2021, 401, 112523.
16. Barsacchi, G.; Gall, J.G. Chromosomal localization of repetitive DNA in newt, Triturus.
J Cell Biol. 1972, 54, 580–591.
17. Pukkila, P.J. Identifcation of the lampbrush chromosome loops which transcribe 5S
ribosomal RNA in Notophthalmus (Triturus) viridescens. Chromosoma. 1975, 53,
71–89.
18. Varley, J.M.; Macgregor, H.C.; Erba, H.P. Satellite DNA is transcribed on lampbrush
chromosomes. Nature. 1980, 283, 686–688.
19. Diaz, M.O.; Barsacchi-Pilone, G.; Mahon, K.A.; Gall, J.G. Transcripts from both strands
of a satellite DNA occur on lampbrush chromosome loops of the newt Notophthalmus.
Cell. 1981, 24, 649–659.
20. Weber, T.; Schmidt, E.; Scheer, U. Mapping of transcription units on Xenopus laevis
lampbrush chromosomes by in situ hybridization with biotin-labeled cDNA probes. Eur.
J. Cell Biol. 1989, 50, 144–153.
21. Solovei, I.; Gaginskaya, E.R.; Macgregor, H.C. The arrangement and transcription of
telomere DNA sequences at the ends of lampbrush chromosomes of birds. Chromosome
Res. 1994, 2, 460–470.
22. Sallacz, N.B.; Jantsch, M.F. Chromosomal storage of the RNA-editing enzyme ADAR1
in Xenopus oocytes. Mol. Biol. Cell. 2005, 16, 3377–3386.
23. Zlotina, A.; Krasikova, A. FISH in lampbrush chromosomes. In Fluorescence In Situ
Hybridization (FISH); Springer Protocols Handbooks; Liehr, T., Ed. Springer Berlin
Heidelberg, Berlin; 2017, pp. 445–457.
24. Kulikova, T.; Maslova, A.; Starshova, P.; Rodriguez, S.J.; Krasikova, A. Comparison of
the somatic TADs and lampbrush chromomere-loop complexes in transcriptionally active
prophase I oocytes. Chromosoma. 2022. https://doi.org/10.1007/s00412-022-00780-5.
25. Krasikova, A.V.; Kulikova, T.V. Distribution of heterochromatin markers in lampbrush
chromosomes in birds. Russ. J. Genet. 2017, 53, 1022–1029.
26. Kulikova, T.; Chervyakova, D.; Zlotina, A.; Krasikova, A.; Gaginskaya, E. Giant
poly(A)-rich RNP aggregates form at terminal regions of avian lampbrush chromo-
somes. Chromosoma. 2016, 125, 709–724.
RNA-FISH—on Lampbrush Chromosomes 317

27. Morgan, G.T. Working with oocyte nuclei: Cytological preparations of active chromatin
and nuclear bodies from amphibian germinal vesicles. Methods Mol. Biol. 2008, 463,
55–66.
28. Gall, J.G.; Nizami, Z.F. Isolation of giant lampbrush chromosomes from living oocytes
of frogs and salamanders. J. Vis. Exp. 2016, 118, 54103.
29. Green, M.R.; Sambrook, J. Labeling of DNA probes by nick translation. Cold Spring
Harb Protoc. 2020, 2020, prot100602.
30. Green, M.R.; Sambrook, J. Labeling the 3′ termini of oligonucleotides using terminal
deoxynucleotidyl transferase. Cold Spring Harb Protoc. 2021, 2021, prot100685.
31. Green, M.R.; Sambrook, J. Preparation of labeled DNA, RNA, and oligonucleotide
probes. Cold Spring Harb Protoc. 2022, 2022, pdb.top100578.
32. Young, A.P.; Jackson, D.J.; Wyeth, R.C. A technical review and guide to RNA fuores-
cence in situ hybridization. Peer J. 2020, 8, e8806.
33. Baudrimont, A.; Jaquet, V.; Wallerich, S.; Voegeli, S.; Becskei, A. Contribution of RNA
degradation to intrinsic and extrinsic noise in gene expression. Cell Rep. 2019, 26, 3752–
3761.e5.
 27 FISH—in Insect
Chromosomes
Vladimir E. Gokhman and Valentina G. Kuznetsova

CONTENTS
Introduction............................................................................................................ 319
Samples/Tissues ..................................................................................................... 320
Description of Methods.......................................................................................... 320
General Notes ............................................................................................... 320
FISH Protocol for Insect Chromosomes....................................................... 321
Chemicals (in Alphabetic Order) ...................................................... 322
General Procedure (Based on [24]) .................................................. 323
Alternative Procedure (Based on [17]) ............................................. 324
Use of C0t-1 DNA (Based on the Preceding Protocol,
Instead of Step 3)............................................................... 325
Yields ..................................................................................................................... 325
Physical Mapping of DNA Sequences.......................................................... 325
Repetitive Sequences ........................................................................ 325
Unique Sequences............................................................................. 326
Chromosome Painting (= Multicolor FISH)................................................. 327
Study of Various Properties of Particular Chromosomes ................. 327
Comparative Genomic Hybridization (CGH) and Genomic In Situ
Hybridization (GISH) ....................................................................... 328
Conclusions............................................................................................................ 328
Acknowledgement ................................................................................................. 329
References.............................................................................................................. 329

INTRODUCTION
Insects, i.e., the class Insecta within the subphylum Hexapoda, are an extremely
diverse and species-rich group of predominantly terrestrial animals. Recent esti-
mates suggest that there are about 5 to 15 million species of insects in the world
fauna, with more than a million already described.[1, 2] In fact, these creatures play an
essential role in many, if not all, terrestrial and freshwater ecosystems.[3] Given this
information, one can conclude that both the morphological variation and ecological
importance of insects are enormous.[1, 3] Moreover, this vast group is also substan-
tially diverse in terms of cytology and genetics, including chromosome morphol-
ogy and behavior.[4–12] For example, different types of the chromosome structure,

DOI: 10.1201/9781003223658-27 319

320 Cytogenetics and Molecular Cytogenetics

i.e., monocentric (including polytene) and holocentric (= holokinetic), are found in

various members of Insecta.[4, 13, 14] This cytogenetic diversity apparently needs to
be studied at the level of DNA sequences, chromosomes and entire genomes.[15, 16]
To perform this research, a wide array of modern techniques is used.[13, 14] Among
these methods, fuorescence in situ hybridization, or FISH, represents an essential
technique on which molecular cytogenetics of insects is currently based for the most
part.[17–20] With this method, fuorochrome-labeled DNA sequences (probes) are
usually hybridized to metaphase chromosomes or interphase nuclei, thus providing
important information necessary for physical mapping of these sequences.[18, 21,  22]
The present work is aimed to review the current state and future perspectives of
FISH in insect chromosomes.

SAMPLES/TISSUES
To obtain reliable results of FISH, this technique must be performed on a suff-
cient number of high-quality mitotic and/or meiotic divisions. In turn, appropriate
developmental stages and tissues of insects must be chosen to achieve this goal.
Specifcally, male testes often harbor numerous spermatocytes undergoing either
mitotic or meiotic divisions.[23, 24] Nevertheless, certain numbers of cell divisions
can be found in the midgut as well as in the developing ovaries.[23, 25–28] Both
adults and immature stages of insects can generally become a valuable source of
mitoses and meioses, depending on the taxon under study.[25–29] However, devel-
oping tissues of insect larvae (and, to a lesser extent, pupae) often contain cells of
varying ploidy, thus making the precise identifcation of the chromosome num-
ber substantially diffcult, if at all possible. Exceptions from this rule include
salivary glands of dipteran larvae, which contain giant polytene chromosomes,
and imaginal discs of many Holometabola,[26, 30] for example, cerebral ganglia of
hymenopteran and coleopteran prepupae.[7, 23] Embryos extracted from develop-
ing eggs or viviparous females also provide high-quality mitotic divisions.[25, 31]
In some special cases, e.g., in many members of the suborder Symphyta (= lower
Hymenoptera), unfertilized eggs can also be taken out of adult females and incu-
bated in a moist chamber to stimulate their development.[32] It must also be noted,
however, that a precise morphological identifcation of feld-collected immature
stages is often diffcult, especially in holometabolous insects, but use of addi-
tional characters (for example, molecular ones) could offer a possible solution to
this problem.

DESCRIPTION OF METHODS
GENERAL NOTES
At present, two main groups of methods are used to produce high-quality chromo-
some preparations of insects, i.e., squash and air-drying techniques.[13, 31] All these
methods are aimed to ensure preservation of the chromosome structure, good spread-
ing of chromosomes as well as their reliable attachment to the slide. Nevertheless,
squash technique achieves these goals by applying physical pressure to a piece of
FISH—in Insect Chromosomes 321

preliminarily fxed tissue, which is turned into a monolayer of cells on the slide. This
preparation can be subsequently converted into a permanent one using dry ice or liq-
uid nitrogen.[30, 33] On the other hand, air-drying technique implies initial hypotonic
treatment of the tissue; after that, it is macerated and turned into cell suspension,
which is then applied to a slide, fxed and air-dried.[34] Both methods have their own
advantages and drawbacks. Specifcally, squash technique can readily utilize mate-
rial preserved with e.g. Carnoy’s fxative, whereas air-drying one predominantly
deals with living tissues. On the other hand, the air-drying method usually ensures
better chromosome morphology, but it apparently needs additional post-fxation to
keep the chromosomes attached to the slide.
In addition to the conventional FISH, a few studies of insect chromosomes were
conducted using the so-called fber-FISH.[25, 35] With this technique, FISH is performed
on the stretched DNA fbers (= mechanically stretched chromosomes) obtained from
interphase nuclei destroyed by either heat or chemical shock.[36] Fiber-FISH can detect
DNA sequences with a substantially increased resolution of 1–400 Kb, as opposed to
1–3 Mb provided by regular FISH on metaphase chromosomes.[25, 37]
As noted earlier, probes of different types are used to perform FISH. Among
these probes, both repetitive and unique DNA sequences, together with the so-called
chromosome paints (see later) can be listed.[38] On the other hand, these sequences
are either directly synthesized or amplifed from the existing genomic material. In
turn, amplifcation of the probes is substantially facilitated using bacterial artif-
cial chromosomes (BACs).[38–41] Regardless of the technique used for the prepara-
tion of chromosomes and probes, the process of FISH includes certain essential
stages, which can be subdivided into pre-hybridization, hybridization per se, and
post-hybridization[18, 19]:

(1) preparation of chromosomes on the slide,

(2) labeling of DNA probe,
(3) denaturation of the probe,
(4) pre-treatment of the chromosome preparation,
(5) denaturation of DNA on the chromosome preparation,
(6) renaturation of DNA from the chromosomes and the probe (=DNA hybrid-
ization per se),
(7) detection of the DNA probe, and
(8) counterstaining, mounting and examination of the chromosome preparation.

FISH PROTOCOL FOR INSECT CHROMOSOMES

In general, a few standard FISH protocols for various organisms were published,
e.g. [21, 42]. These protocols include a number of modifcations of the probe label-
ing (nick translation and polymerase chain reaction (PCR), direct and indirect
labeling), probe denaturation, slide pretreatment and denaturation, hybridization in
a strict sense, detection of hybridization sites etc. The protocol presented next is
mainly based on the technique we routinely use in our lab.[24] This protocol produces
strong, specifc and reproducible signals for all insect orders we worked with, i.e.,
Odonata, Mantophasmatodea, Psocoptera, Hemiptera, Lepidoptera, Neuroptera and
322 Cytogenetics and Molecular Cytogenetics

Hymenoptera.[24, 28, 43–52] Nevertheless, we also provide an alternative protocol, which

was used to perform FISH on chromosomes of various insects.[17]

Chemicals (in Alphabetic Order)

• 1×Taq DNA polymerase (Ref. EP0404, Thermo Fisher Scientifc)
• 2-Propanol (Ref. I9516, Sigma-Aldrich)
• 10×PBS = phosphate-buffered saline: dilute 75.8 g NaCl, 9.93 g Na2HPO4
and 4.14 g NaH2PO4 in 1 l of Milli-Q water
• 20×SSC = saline sodium citrate: dilute 175.6 g NaCl and 88.2 g sodium
citrate in 1 l of Milli-Q water. If necessary, use HCl to reach pH 7.0. Set up
0.4×, 1× and 2× before use
• Anti-digoxigenin-fuorescein/rhodamine, Fab fragments (Ref. 11207741910/
11207750910, Roche)
• Antibody solution: 10 μl anti-digoxigenin-fuorescein/rhodamine (200 μl/
ml) and 990 μl Marvel solution, make fresh as required
• Avidin, fuorescein/rhodamine conjugate (Ref. A2662/A6378, Thermo
Fisher Scientifc)
• Biotin-11-dUTP (Ref. R0081, Thermo Fisher Scientifc)
• Bovine serum albumin (Ref. 05473, Sigma-Aldrich)
• Denaturation buffer: 0.7 volume formamide, 0.2 volume Milli-Q water and
0.1 volume 20×SSC; store at 4 °C
• Dextran sulphate (Ref. 67578, Sigma-Aldrich)
• DIG-Nick Translation Mix (Ref. 11745816910, Roche)
• dNTP Mix (Ref. R0191, Thermo Fisher Scientifc)
• dTTP (Ref. R0171, Thermo Fisher Scientifc)
• EDTA = ethylenediaminetetraacetic acid (Ref. 03690, Sigma-Aldrich)
• Ethanol 70, 95 and 100 %
• Fluoroshield™ with DAPI (Ref. F6057, Sigma-Aldrich)
• Formamide (Ref. F7503, Sigma-Aldrich)
• Hybridization buffer: dissolve 2 g dextran sulphate in 10 ml 50 % deionized
formamide/2×SSC/50 mM phosphate buffer for 3 h at 70 °C. Adjust pH to
7 with phosphate buffer. Aliquot and store at −20 °C
• Marvel solution: dilute 0.1 g milk powder Marvel in 2 ml washing buffer
• Milli-Q water (Merck)
• Pepsin solution: mix 10 μl 1 M HCl and 2.5 μl volume 20 ng/ml pepsin in
990 μl of Milli-Q water; make fresh as required
• Pepsin stock (Ref. P7012, Sigma-Aldrich)
• Phosphate buffer: dilute 20.214 g of Na2HPO4×7H2O and 3.394 g
NaH2PO4×H2O in 800 ml of Milli-Q water; store at 2–8 °C
• Post-fxation solution: mix 5 ml 2 % paraformaldehyde, 4.5 ml 1×PBS and
0.5 ml 1 M MgCl2; store at 4 °C
• ProLong™ Gold with DAPI (Ref. P36931, Thermo Fisher Scientifc)
• RNAse A solution: add 0.05 volume RNAse A at 10 mg/ml in 0.95 volume
2×SSC and mix well; make fresh as required
• RNAse A stock (Ref. 10109142001, Roche)
FISH—in Insect Chromosomes 323

• Salmon sperm DNA (Ref. D7656, Sigma-Aldrich)

• Sodium acetate solution (Ref. 71196, Sigma-Aldrich)
• Specifc primers, e.g., 18S_F (5′-GATCCTGCCAGTAGTCATATG-3′) and
18S_R (5′-GAGTCAAATTAAGCCGCAGG-3′)[24]; 5S_F (5′-AACGACC
ATACCACGCTGAA-3′) and 5S_R (5′-AAGCGGTCCCCCATCTAAGT)[23];
TTAGG_F (5′-AACCTAACCTAACCTAACCTAA-3′) and TTAGG_R
(5′-GGTTAGGTTAGGTTAGGTTAGG-3′) (for the canonical insect telo-
mere sequence)[24]
• Tween 20 = polyoxyethylene-sorbitan monolaurate (Ref. P9416, Sigma-
Aldrich)
• Washing buffer: 0.2 volume 20×SSC, 0.8 volume Milli-Q water and 0.05
volume Tween 20

General Procedure (Based on [24])

1. Make a chromosome preparation using either air-drying or squash
technique.
2. Isolate the DNA probe (either by direct synthesis, PCR or microdissection;
see earlier). Prepare 50 µl of PCR mixture containing 50–100 ng DNA
probe, 0.1 mM Biotin-11-dUTP, 0.1 mM dTTP, 0.2 mM other dNTPs (each),
2.5 mM MgCl2, 50 pmol specifc primers, 1 U 1×Taq DNA polymerase and
5 µl 10×Taq buffer. Perform PCR with the following parameters: denatur-
ation: 1 min at 94 °C, main stage: 30–35 cycles of 30 sec at 94 °C, anneal-
ing: 30 sec at 50–56 °C (temperature depends on the length and composition
of the primer), elongation: 0.5–2 min at 72 °C (temperature depends on the
fragment size; for example, 1 min is enough for reliable amplifcation of 1
kb fragment), and fnal extension: 5 min at 72 °C. Precipitate labeled DNA
with 5–10 volumes of 96–100 % ethanol at −20 °C and 0.1 volumes of 3 M
NaCl for 30–60 min at −80 °C or 12–24 h at −20 °C. Make a DNA pellet
by centrifugation at 15,000 rpm and 4 °C for 30 min. Wash labeled DNA
with 500–700 μl ice-cold 70 % ethanol by centrifugation at 15,000 rpm and
4 °C for 15–20 min, then air-dry at 37 °C. Resuspend the pellet in 20–30 μl
Milli-Q water. The pellet can be kept at −20 °C for a long time.
3. Put 100 μl RNAse A solution on slide and cover with coverslip, then incu-
bate in humid chamber at 37 °C for 1 h. Remove the coverslip and wash in
2×SSC for 5 min and air-dry. Add 50 μl 0.005 % pepsin solution and cover
with coverslip for 10 min at room temperature (= RT). Wash in 1×PBS for 5
min at RT. Incubate the slide in post-fxation solution in a Coplin jar for 10
min at RT. Wash for 5 min in 1×PBS. Dehydrate the slide in ethanol (70, 95
and 100 %, 3 min each) and air-dry.
4. Add 6.5–7 μl of denatured probe solution (about 100 ng of labeled probe,
50 % formamide, 2×SSC, 10 % (w/v) dextran sulfate, 1 % (w/v) Tween 20)
containing 10 μg salmon-sperm DNA on the slide, cover with coverslip and
seal with rubber cement. Incubate the slide on preheated hot plate at 75 °C
for 5 min.
5. Incubate the slide overnight at 37 °C in a humid chamber.
324 Cytogenetics and Molecular Cytogenetics

6. Wash the slide in 2×SSC for 3 min at 45 °C, then in 50 % formamide/2×SSC

for 10 min at 45 °C. Wash the slide twice in 2×SSC at 45 °C (10 min each),
then twice in 0.2×SSC at 45 °C (10 min each). Block the reaction in 1.5 %
(w/v) bovine serum albumin/4×SSC/0.1 % Tween 20 for 30 min at 37 °C in
a humid chamber. Detect the biotin-labeled probe with 5 μg/ml avidin-fu-
orescein/rhodamine in 1.5 % bovine serum albumin/4×SSC/0.1 % Tween
20 under coverslip. Remove the coverslip and put the slide in 4×SSC/0.02
% Tween 20 for 5 min on the shaker; repeat this step three times using new
portions of the solution. Rinse in Milli-Q water briefy, pass through the
ethanol series (70, 95 and 100 %) for 3 min each at RT, and air-dry. Apply
20 μl of ProLong™ Gold with DAPI and cover with coverslip. Examine
under the microscope.

Alternative Procedure (Based on [17])

1. Make a chromosome preparation using either air-drying or squash
technique.
2. Isolate the DNA probe (either by direct synthesis, PCR or microdissection,
see earlier). Put 3.5 μl of the probe solution (suggested concentration 50 ng/
μl) in 12.5 μl autoclaved Milli-Q water in a microtube, and add 4 μl of the
DIG-Nick Translation Mix; mix and spin briefy. Incubate for 90 min at
16 °C. Add 1 μl 0.5M EDTA and put for 10 min at 65 °C to stop the process.
Store at −20 °C.
3. Denature 2 μl of the labeled probe in 18 μl of hybridization buffer for 5 min
at 85 °C, then keep the solution on ice or at 4 °C before use.
4. Put 100 μl RNAse A solution on slide and cover with coverslip, then incu-
bate in humid chamber at 37 °C for 1 h. Remove the coverslip and wash in
2×SSC for 5 min and air-dry. Add 50 μl 0.005 % pepsin solution and cover
with coverslip for 10 min at RT. Wash in 1×PBS for 5 min at RT. Incubate
the slide in 100 μl post-fxation solution under the coverslip for 10 min at
RT. Remove the coverslip and wash for 5 min in 1×PBS. Dehydrate the slide
in ethanol (70, 95 and 100 %, 3 min each) and air-dry.
5. Put 100 μl of denaturation buffer on the slide and cover with coverslip, and
then denature on preheated hot plate at 73 °C for 3 min. Remove the cover-
slip and place the slide in 70 % ethanol at −20 °C for 3 min. Dehydrate the
slide in ethanol (70, 95 and 100 %, 3 min each) and air-dry.
6. Add 20 μl of denatured probe solution on the slide and cover with coverslip.
Incubate the slide overnight at 37 °C in a humid chamber.
7. Remove the coverslip and wash the slide in 100 ml 0.4×SSC at 65–68 °C
for 3–4 min in a Coplin jar in a water bath. Put the slide into 100 ml of
4×SSC/0.2 % Tween 20 for 5 min at RT in a shaker. Add 100 μl Marvel solu-
tion under coverslip at 37 °C for 10–15 min in a humid chamber. Remove
the coverslip and wash the slide in 4×SSC/0.2 % Tween 20 for 2 min at RT.
Detect the probe by incubation of the slide in 100 μl antibody solution for
20–35 min at 37 °C in a humid chamber.
8. Remove the coverslip and put the slide in 4×SSC/0.2 % Tween 20 for 5 min
on the shaker; repeat this step three times using new portions of the solution.
FISH—in Insect Chromosomes 325

Rinse in Milli-Q water briefy, pass through the ethanol series (70, 95 and
100 %) for 3 min each at RT, and air-dry. Apply 20 μl of Fluoroshield™
with DAPI and cover with coverslip. Examine under the microscope.

Use of C0t-1 DNA (Based on the Preceding Protocol, Instead of Step 3)

Precipitate the labeled probe and C0t-1 DNA together using a single volume of 2-pro-
panol and 0.1 volume of sodium acetate. For better results, the use of proportions
higher than 1:20 probe to C0t-1 DNA is recommended. Centrifuge at 14,000–15,000
rpm for 20 min at 4 °C, discard the supernatant and dry the DNA pellet at RT. Dilute
the pellet in 20 μl of hybridization buffer. Denature the probe solution for 5 min at
85 °C, and then perform pre-hybridization for 30 min at 37 °C. Keep the solution on
ice or at 4 °C before use.

YIELDS
PHYSICAL MAPPING OF DNA SEQUENCES
Repetitive Sequences
FISH in insect chromosomes is most often used for mapping repetitive DNA
sequences, in which it is especially effcient.[19] These sequences can be provisionally
subdivided into the so-called satellite DNA, which usually occurs in clusters,[53] and
transposable elements.[54] Among the satellite sequences, ribosomal DNAs (rDNAs)
are the most frequently used as probes.[20, 43, 51, 55–57] Eukaryotic rDNA is composed
of tandem repeats divided into large (45S) and small (5S) transcriptional units. In
turn, these units form characteristic clusters, which usually contain hundreds or even
thousands of copies.[58] The large unit contains genes encoding 18S, 5.8S and 28S
rRNAs, which are separated by internal transcribed spacers.[59, 60] The small unit
encodes multiple 5S rRNA genes separated by non-transcribed spacers. 5S rDNA
is mostly distributed independently of 45S rDNA throughout the genome, although
it often forms clusters of its own.[25, 35, 61–67] However, 45S and 5S rRNA genes are
interspersed in the coleopteran Lagria villosa (Fabricius).[68] Nevertheless, the main
rDNA clusters of insects remain the most studied, with 18S rDNA being the most
frequently used probe.[24, 46, 48, 52, 61, 63, 66, 67, 69–74, etc.] The number and localization of
the rDNA clusters vary among closely related forms,[33, 75–81] and, occasionally, even
among particular populations and individuals of the same species. In the latter case,
different sections of the 45S rDNA cistron can evolve more or less independently.[60]
Moreover, certain rDNA-like sequences sometimes spread over the whole genome
and can therefore be detected on every chromosome.[82]
In addition to rDNA, other repetitive sequences can also be studied using
FISH. For example, histones are basic proteins, which, together with DNA, form
the fundamental units of chromatin structure.[83] Histone-coding genes are usually
arranged in clusters, and these clusters also can be physically mapped with this
technique.[35, 61, 63, 64, 67, 74, 78, 84–86] In insects, clusters of these genes are often (but not
always) co-localized with those of 5S rDNA.[61–63, 87, 88] Histone-coding genes can be
arranged either in clusters of tandem repeats formed by sequences by all types (H1,
H2A, H2B, H3 and H4), spaced by non-coding DNA, as individual genes or their
326 Cytogenetics and Molecular Cytogenetics

random combinations. For example, these genes are grouped into quartets and quin-
tets in several Drosophila species.[89]
Microsatellites represent short tandemly repeated DNA sequences (usually about
two to six base pairs),[58, 90] which can be effciently mapped using FISH.[35, 66, 71, 77, 91, 92]
Although these sequences are predominantly associated with heterochromatic seg-
ments[53] (but see[93]), microsatellites can have diverse distribution patterns within
the given chromosome set. Specifcally, they can be either scattered along the whole
genome or restricted to certain chromosomes and/or their segments, being either
dispersed or united into clusters.[77, 93, 94]
Small nuclear DNAs (snDNAs) encode corresponding RNAs (snRNAs), which
play important roles in processing pre-messenger RNA (= splicing), telomere main-
tenance, and a few other processes.[95] Various types of these DNAs (e.g. U1 and U2
snDNA), which are organized in clusters, can also be mapped using FISH.[64–67, 71, 96–98]
Tandemly repeated centromeric sequences of satellite DNA[53] are located in the
pericentromeric regions of chromosomes.[99] FISH visualized specifc sequences of
that kind in certain grasshoppers, beetles and ants,[100–102] but microdissection exper-
iments also showed that centromeric sequences can vary not only between different
species, but between different chromosomes of the same species as well (see e.g.[103]).
Telomeric sequences are located in the terminal regions of chromosomes.[9] In
most insects, as well as in other arthropods, TTAGG is the most widespread and
apparently ancestral telomeric repeat.[48, 52, 77, 104–107] However, telomeric motifs of
many insect taxa are highly variable, being often substituted by other sequences with
either known or, more frequently, unknown structure.[9, 108, 109] In addition, telomeric
repeats are substituted by transposons in higher Diptera, and these transposons can
be detected using FISH as well (see later).[19]
Although mobile repetitive DNA sequences, i.e., transposable elements,[54] are
often not arranged in clusters, they also can be revealed in insect chromosomes using
FISH.[19, 25, 110 –113] Sometimes, whole fractions of repetitive DNA, e.g., C0t-1 DNA,
can be physically mapped to insect chromosomes as well.[35, 66, 88, 114] This analysis
can provide an insight on spreading of repetitive sequences among different chromo-
somes, especially within their heterochromatic regions.[65, 115]

Unique Sequences
In addition to repetitive DNA sequences, certain unique, single-copy genes also can
be physically mapped on insect chromosomes using FISH. Previously, regular FISH
could only reveal longer sequences.[32] Later on, sensitivity of FISH was substantially
enhanced using e.g. tyramide signal amplifcation (TSA-FISH).[41, 74, 116]
During the last decades, an important role of chromosomal inversions in main-
taining genetic diversity among and within different populations became obvi-
ous.[117] In particular, these inversions preserve certain gene complexes within the
same chromosome, i.e., the so-called supergenes,[118] by preventing crossing over.
A few cases of supergenes are found in insects, including ants, in which their pres-
ence is demonstrated using FISH.[119] In addition, recent research revealed that
genomes of many parasitoid Hymenoptera harbor symbiotic polydnaviruses (dou-
ble-stranded DNA viruses) that are integrated into insect chromosomes, and then
FISH—in Insect Chromosomes 327

excised from the genome and further injected into the host during oviposition.[120]
Although DNA sequences of these viruses were detected in chromosomes of a
particular parasitic wasp using non-fuorescence in situ hybridization,[121] similar
studies could be conducted using FISH. Moreover, FISH also demonstrated that
a number of physiologically important genes are sex-linked in most clades of the
moth family Tortricidae.[40]

CHROMOSOME PAINTING (= MULTICOLOR FISH)

Chromosome painting mainly differs from the previously mentioned procedure
in the nature and preparation of the probes. Specifcally, these probes can be pre-
pared either from entire chromosomes or from their particular sections.[16] To cap-
ture the corresponding material, the so-called microdissection is normally used.
For studying insect chromosomes, glass-needle,[103, 122–126] or laser microdissection is
employed.[127–129] When DNA is isolated from this material, it can be amplifed using
the so-called degenerate oligonucleotide primed polymerase chain reaction (DOP-
PCR). This reaction performs non-specifc amplifcation of DNA.[130] The amplifed
DNA then must be sheered e.g. using autoclavation, and labeled with a particular
fuorochrome.[126] However, insect chromosomes usually contain large amounts of
repetitive sequences, and therefore either blocking DNA (usually C0t-1 DNA),[17] or
digital techniques,[131] must be used to avoid unwanted cross-hybridization (see ear-
lier) or to detect the specifc signal using specialized software.
During the last decade, a new powerful technique of chromosome painting, i.e.,
the so-called Oligopaint technique, which visualizes parts of the genome using
Oligopaint FISH probes,[132] was introduced into the chromosomal study of insects.
This method implies use of fuorochrome-tagged custom-synthetized oligonucle-
otides (oligos), which specifcally highlight selected chromosomes. However, this
technique is based on the results of full genome sequencing, and therefore is cur-
rently available for just a few insect species (see e.g.[133]).

Study of Various Properties of Particular Chromosomes

In insects, chromosome painting is most often used to explore origin and evolu-
tion of particular chromosomes and their regions by studying their specifc
properties.[39, 123, 124, 127, 129, 134, 135, etc.] In particular, any chromosome and/or chro-
mosomal segment within a given karyotype can be reliably recognized using this
technique. In certain species with low chromosome numbers, as in the parasitic
wasp Nasonia vitripennis (Walker) with n = 5, a study of that kind has already
been conducted for every chromosome.[122] Among specifc elements of the karyo-
type, sex chromosomes, especially W chromosomes of butterfies and moths with
ZZ/ZW sex determination, as well as B chromosomes in Orthoptera are the most
studied.[39, 123–125, 127, 129, 135]
In addition to FISH with known centromeric repeats (see earlier), specifc
regions of certain insect chromosomes are also studied using microdissection.
These works include studying centromeres,[103] and heterochromatic segments.[134]
This research demonstrated that centromeric regions of different chromosomes
328 Cytogenetics and Molecular Cytogenetics

within a given karyotype could substantially differ in their DNA content. On

the contrary, the latter characteristic of heterochromatic segments in stingless
bees (Hymenoptera) appeared to be very similar across the whole chromosome
sets.[134]
To our best knowledge, the only study of a particular fusion/fssion with the so-
called whole chromosome painting (WCP), was conducted a few years ago on the
two cryptic species of parasitoids of the Lariophagus distinguendus (Förster) com-
plex with n = 5 and 6.[126] To identify target chromosomes, morphometric analysis of
chromosome sets of both species was performed.[34] This study showed that the only
acrocentric and a smaller metacentric in the chromosome set with n = 6 respectively
correspond to the shorter and longer arms of the largest metacentric chromosome in
the karyotype with n = 5. This assumption was confrmed using both microdissec-
tion and WCP.[126]
In particular, the technique of Oligopaint FISH probes (see earlier) provides a
novel opportunity to explore meiosis in the model organism, i.e., the domestic silk
moth, Bombyx mori (Linnaeus), at an unprecedented scale. Specifcally, a number
of interesting phenomena, e.g., asynchronous pairing of homologous chromosomes,
were observed for the frst time using this technique.[133]

COMPARATIVE GENOMIC HYBRIDIZATION (CGH) AND GENOMIC IN SITU

HYBRIDIZATION (GISH)
The CGH technique explores the number of copies of a particular DNA sequence
in the test sample vs. the reference one.[22] These samples most often represent total
genomic DNA, and the corresponding method is therefore called GISH. This tech-
nique can effectively highlight particular sex chromosomes in different groups of
insects (see [136] and references therein). For example, GISH identifed a particular
segment of the neo-X element as an original X chromosome in certain true bugs
(Hemiptera, Pyrrhocoridae).[128] CGH also recognized differentiated W chromo-
somes, which are enriched with female-specifc sequences and/or common repetitive
ones, in various members of the family Geometridae (Lepidoptera).[129, 137] Moreover,
this method highlighted chromosomes originating from different parent taxa in a
rare case of homoploid hybrid speciation in the genus Agrodiaetus (Lepidoptera,
Lycaenidae).[138]

CONCLUSIONS
The previously mentioned results demonstrate that FISH represents a powerful tool
used in the cytogenetic study of insects (Figure 27.1). In addition to physical mapping
of various DNA sequences, it can highlight different genomic and chromosomal
rearrangements, and therefore can outline certain pathways of the karyotype and
genome evolution in this enormous group of animals.[11, 23, 25, 26, 31, 43, 44, 53, 73, 76, 82, 86, 138, 139]
We expect that, either by itself or in combination with other methods, this tech-
nique will reveal many new details of the genome change at various scales of space
and time.
FISH—in Insect Chromosomes 329

FIGURE  27.1 Ancyra sp. (Hemiptera, Eurybrachidae): meiotic chromosomes at diakinesis

(2n = 12AA + X). Results of FISH with 18S rDNA and TTAGG telomere probes (green and
red signals, respectively) are shown.

ACKNOWLEDGEMENT
The authors are grateful to Boris A. Anokhin (Zoological Institute, Russian Academy
of Sciences) for his useful advice on practical aspects of performing FISH on insect
chromosomes.

REFERENCES
1. Grimaldi, D.; Engel, M.S. Evolution of the Insects. Cambridge University Press,
Cambridge; 2005, 755 p.
2. Stork, N.E. How many species of insects and other terrestrial arthropods are there on
Earth? Ann. Rev. Entomol. 2018, 63, 31–45.
3. Scudder, G.G.E. The importance of insects. In: Insect Biodiversity: Science and
Society; Foottit, R.G., Adler, P.H., Eds.; 2nd Edition, 1; Wiley Blackwell, Oxford, 2017,
pp. 9–43.
4. White, M.J.D. Animal Cytology and Evolution. 3rd Edition. Cambridge University
Press, Cambridge; 1973, 961 p.
5. Lukhtanov, V.A. Sex chromatin and sex chromosome systems in nonditrysian
Lepidoptera “Insecta”. J. Zool. Syst. Evol. Res. 2000, 38, 73–79.
6. Nokkala, S.; Kuznetsova, V.G.; Maryańska-Nadachowska, A.; Nokkala, C. Holocentric
chromosomes in meiosis. I. Restriction of the number of chiasmata in bivalents.
Chromosome Res. 2004, 12, 733–739.
330 Cytogenetics and Molecular Cytogenetics

7. Gokhman, V.E. Karyotypes of Parasitic Hymenoptera. Springer, Dordrecht; 2009, 183 p.

8. Blackmon, H.; Ross, L.; Bachtrog, D. Sex determination, sex chromosomes, and karyo-
type evolution in insects. J. Hered. 2017, 108, 78–93.
9. Kuznetsova, V.; Grozeva, S.; Gokhman, V. Telomere structure in insects: A review. J.
Zool. Syst. Evol. Res. 2020, 58(1), 127–158.
10. Kuznetsova, V.G.; Gavrilov-Zimin, I.A.; Grozeva, S.M.; Golub, N.V. Comparative anal-
ysis of chromosome numbers and sex chromosome systems in Paraneoptera (Insecta).
Comp. Cytogenet. 2021, 15, 279–327.
11. Kuznetsova, V.; Maryańska-Nadachowska, A.; Anokhin, B.; Shapoval, N.; Shapoval,
A. Chromosomal analysis of eight species of dragonfies (Anisoptera) and damselfies
(Zygoptera) using conventional cytogenetics and FISH: Insights into the karyotype evo-
lution of the ancient insect order Odonata. J. Zool. Syst. Evol. Res. 2021, 59, 387–399.
12. Sylvester, T.; Hjelmen, C.E.; Hanrahan, S.J.; Lenhart, P.A.; Johnston, J.S.; Blackmon,
H. Lineage-specifc patterns of chromosome evolution are the rule not the exception in
Polyneoptera insects. Proc. R. Soc. B. 2020, 287, 20201388.
13. Gokhman, V.E.; Kuznetsova, V.G. Comparative insect karyology: Current state and
applications. Entomol. Rev. 2006, 86, 352–368.
14. Martins, C.; Cabral-de-Mello, D.C.; Valente, G.T; Mazzuchelli, J.; de Oliveira, S.G.;
Pinhal, D. Animal Genomes under the Focus of Cytogenetics. Genetics—Research and
Issues. Nova Science Publishers, Inc., New York, NY; 2011, 154 p.
15. Lukhtanov, V.A.; Kuznetsova, V.G. What genes and chromosomes say about the origin
and evolution of insects and other arthropods. Russ. J. Genet. 2010, 46, 1115–1121.
16. Liehr, T. Classifcation of FISH probes. In: Fluorescence In Situ Hybridization (FISH):
Application Guide; Springer Protocols Handbooks; Liehr, T., Ed. 2nd Edition. Springer
Verlag, Berlin; 2017, pp. 43–48.
17. Alves-Silva, A.P.; Campos Barros, L.A.; Pompolo, S.G. General protocol of FISH for
insects. In: Fluorescence In Situ Hybridization (FISH): Application Guide; Springer
Protocols Handbooks; Liehr, T., Ed. 2nd Edition. Springer Verlag, Berlin; 2017,
pp. 459–466.
18. Liehr, T.; Weise, A. Background. In: Fluorescence In Situ Hybridization (FISH):
Application Guide; Springer Protocols Handbooks; Liehr, T., Ed. 2nd Edition. Springer
Verlag, Berlin; 2017, pp. 1–14.
19. Cabral-de-Mello, D.C.; Marec, F. Universal fuorescence in situ hybridization (FISH)
protocol for mapping repetitive DNAs in insects and other arthropods. Mol. Genet.
Genomics. 2021, 296, 513–526.
20. Masri, R.A.; Karagodin, D.A.; Sharma, A.; Sharakhova, M.V. A gene-based method for
cytogenetic mapping of repeat-rich mosquito genomes. Insects. 2021, 12, 138.
21. Schwarzacher, T.; Heslop-Harrison, J.S. Practical In Situ Hybridization. BIOS
Scientifc Publishers, Oxford; 2000, 203 p.
22. Speicher, M.R.; Carter, N.P. The new cytogenetics: Blurring the boundaries with
molecular biology. Nat. Rev. Genet. 2005, 6, 782–792.
23. Cabral-de-Mello, D.C. Beetles (Coleoptera). In: Protocols for Cytogenetic Mapping
of Arthropod Genomes; Sharakhov, I.V., Ed. CRC Press, Boca Raton, FL; 2015,
pp. 171–217.
24. Grozeva, S.; Kuznetsova, V.G.; Anokhin, B.A. Karyotypes, male meiosis and compara-
tive FISH mapping of 18S ribosomal DNA and telomeric (TTAGG)n repeat in eight
species of true bugs (Hemiptera, Heteroptera). Comp. Cytogenet. 2011, 5, 355–374.
25. Camacho, J.P.M.; Cabrero, J.; López-León, M.D.; Cabral-de-Mello, D.C.; Ruiz-Ruano,
F.J. Grasshoppers (Orthoptera). In: Protocols for Cytogenetic Mapping of Arthropod
Genomes; Sharakhov, I.V., Ed. CRC Press, Boca Raton, FL; 2015, pp. 381–438.
FISH—in Insect Chromosomes 331

26. Sharakhova, M.V.; George, P.; Timoshevskiy, V.; Sharma, A.; Peery, A.; Sharakhov, I.V.
Mosquitoes (Diptera). In: Protocols for Cytogenetic Mapping of Arthropod Genomes;
Sharakhov, I.V., Ed. CRC Press, Boca Raton, FL; 2015, pp. 93–170.
27. Angus, R.B.; Jeangirard, C.; Stoianova, D.; Grozeva, S.; Kuznetsova, V.G. A chromo-
somal analysis of Nepa cinerea Linnaeus, 1758 and Ranatra linearis (Linnaeus, 1758)
(Heteroptera, Nepidae). Comp. Cytogenet. 2017, 11, 641–657.
28. Karagyan, G.; Golub, N.; Sota, T. Cytogenetic characterization of periodical cicadas
(Hemiptera: Cicadidae: Magicicada). Eur. J. Entomol. 2020, 117, 474–480.
29. Yoshido, A.; Sahara, K.; Yasukochi, Y. Silk moths (Lepidoptera). In: Protocols for
Cytogenetic Mapping of Arthropod Genomes; Sharakhov, I.V., Ed. CRC Press, Boca
Raton, FL; 2015, pp. 219–256.
30. Sharakhova, M.V.; Timoshevskiy, V.A.; Yang, F.; Demin, S.I.; Severson, D.W.;
Sharakhov, I.V. Imaginal discs—a new source of chromosomes for genome mapping of
the yellow fever mosquito Aedes aegypti. PLoS Negl. Trop. Dis. 2011, 5, e1335.
31. Mandrioli, M.; Manicardi, G.C. Aphids (Hemiptera). In: Protocols for Cytogenetic
Mapping of Arthropod Genomes; Sharakhov, I.V., Ed. CRC Press, Boca Raton, FL;
2015, pp. 327–350.
32. Matsumoto, K.; Yamamoto, D.S.; Sumitani, M.; Lee, J.M.; Hatakeyama, M.; Oishi, K.
Detection of a single copy gene on a mitotic metaphase chromosome by fuorescence
in situ hybridization (FISH) in the sawfy, Athalia rosae (Hymenoptera). Arch. Ins.
Biochem. Physiol. 2002, 49, 34–40.
33. Grozeva, S.; Anokhin, B.A.; Kuznetsova, V.G. Bedbugs (Hemiptera). In: Protocols for
Cytogenetic Mapping of Arthropod Genomes; Sharakhov, I.V., Ed. CRC Press, Boca
Raton, FL; 2015, pp. 285–326.
34. König, C.; Paschke, S.; Pollmann, M.; Reinisch, R.; Gantert, C.; Weber, J.; Krogmann,
L.; Steidle, J.L.M.; Gokhman, V.E. Molecular and cytogenetic differentiation within
the Lariophagus distinguendus (Förster, 1841) species complex (Hymenoptera,
Pteromalidae). Comp. Cytogenet. 2019, 13, 133–145.
35. Palacios-Gimenez, O.M.; Carvalho, C.R.; Ferrari Soares, F.A.; Cabral-de-Mello, D.C.
Contrasting the chromosomal organization of repetitive DNAs in two Gryllidae crick-
ets with highly divergent karyotypes. PLoS One. 2015, 10, e0143540.
36. Heiskanen, M.; Kallioniemi, O.; Palotie, A. Fiber-FISH: Experiences and a refned pro-
tocol. Genet. Anal.: Biomol. Eng. 1996, 12, 179–184.
37. Raap, A.K.; Florijn, R.J.; Blonden, L.A.J.; Wiegant, J.; Vaandrager, J.; Vrolijk, H.;
Dunnen, J.; Tanke, H.J.; van Ommen, G.-J. Fiber FISH as a DNA mapping tool.
Methods. 1996, 9, 67–73.
38. Liehr, T. Homemade locus-specifc FISH probes: Bacterial artifcial chromosomes. In:
Fluorescence In Situ Hybridization (FISH): Application Guide; Springer Protocols
Handbooks; Liehr, T., Ed. 2nd Edition. Springer Verlag, Berlin; 2017, 101–106.
39. Yoshido, A.; Yasukochi, Y.; Marec, F.; Abe, H.; Sahara, K. FISH analysis of the W
chromosome in Bombyx mandarina and several other species of Lepidoptera by means
of B. mori W-BAC probes. J. Ins. Biotechnol. Sericol. 2007, 76, 1–7.
40. Nguyen, P.; Sýkorová, M.; Šíchová, J.; Kůta, V.; Dalíková, M.; Čapková Frydrychová,
R.; Neven, L.G.; Sahara, K.; Marec, F. Neo-sex chromosomes and adaptive potential in
tortricid pests. Proc. Natl. Acad. Sci. USA. 2013, 110, 6931–6936.
41. Carabajal Paladino, L.Z.; Nguyen, P.; Šíchová, J.; Marec, F. Mapping of single-copy
genes by TSA-FISH in the codling moth, Cydia pomonella. BMC Genet. 2014,
15, S15.
42. Liehr, T.; Kreskowski, K.; Ziegler, M.; Piaszinski, K.; Rittscher, K. The standard
FISH procedure. In: Fluorescence In Situ Hybridization (FISH): Application Guide;
332 Cytogenetics and Molecular Cytogenetics

Springer Protocols Handbooks; Liehr, T., Ed. 2nd Edition; Springer Verlag, Berlin;
2017, 109–118.
43. Gokhman, V.E.; Anokhin, B.A.; Kuznetsova, V.G. Distribution of 18S rDNA sites and
absence of the canonical TTAGG insect telomeric repeat in parasitoid Hymenoptera.
Genetica. 2014, 142, 317–322.
44. Kuznetsova, V.G.; Grozeva, S.M.; Hartung, V.; Anokhin, B.A. First evidence for
(TTAGG)n telomeric sequence and sex chromosome post-reduction in Coleorrhyncha
(Insecta, Hemiptera). Comp. Cytogenet. 2015, 9, 523–532.
45. Kuznetsova, V.G.; Khabiev, G.N.; Anokhin, B.A. Cytogenetic study on antlions
(Neuroptera, Myrmeleontidae): First data on telomere structure and rDNA location.
Comp. Cytogenet. 2016, 10, 647–656.
46. Kuznetsova, V.G.; Maryańska-Nadachowska, A.; Shapoval, N.A.; Anokhin, B.A.;
Shapoval, A.P.; Cytogenetic characterization of eight Odonata species originating from
the Curonian spit (the Baltic Sea, Russia) using C-banding and FISH with 18S rDNA
and telomeric (TTAGG)n probes. Cytogenet. Genome Res. 2018, 153, 147–157.
47. Lachowska-Cierlik, D.; Maryańska-Nadachowska, A.; Kuznetsova, V.; Picker, M. First
chromosomal study of Mantophasmatodea: Karyotype of Karoophasma biedouwense
(Austrophasmatidae). Eur. J. Entomol. 2015, 112, 599–605.
48. Vershinina, A.O.; Anokhin, B.A.; Lukhtanov, V.A. Ribosomal DNA clusters and telo-
meric (TTAGG)n repeats in blue butterfies (Lepidoptera, Lycaenidae) with low and
high chromosome numbers. Comp. Cytogenet. 2015, 9, 161–171.
49. Gokhman, V.E.; Kuznetsova, V.G. Presence of the canonical TTAGG insect telomeric
repeat in the Tenthredinidae (Symphyta) suggests its ancestral nature in the order
Hymenoptera. Genetica. 2018, 146, 341–344.
50. Maryańska-Nadachowska, A.; Kuznetsova, V.G.; Golub, N.V.; Anokhin, B.A. Detection
of telomeric sequences and ribosomal RNA genes in holokinetic chromosomes of
fve jumping plant-lice species: First data on the superfamily Psylloidea (Hemiptera:
Sternorrhyncha). Eur. J. Entomol. 2018, 115, 632–640.
51. Golub, N.; Anokhin, B.; Kuznetsova, V. Comparative FISH mapping of ribosomal
DNA clusters and TTAGG telomeric sequences to holokinetic chromosomes of eight
species of the insect order Psocoptera. Comp. Cytogenet. 2019, 13, 403–410.
52. Gapon, D.A.; Kuznetsova, V.G.; Maryańska-Nadachowska, A. A new species of the
genus Rhaphidosoma Amyot et Serville, 1843 (Heteroptera, Reduviidae), with data on
its chromosome complement. Comp. Cytogenet. 2021, 15, 467–505.
53. Palomeque, T.; Lorite, P. Satellite DNA in insects: A review. Heredity. 2008, 100,
564–573.
54. Wells, J.N.; Feschotte, C. A feld guide to eukaryotic transposable elements. Ann. Rev.
Genet. 2020, 54, 539–561.
55. Cabrero, J.; Camacho, J.P.M. Location and expression of ribosomal RNA genes in
grasshoppers: Abundance of silent and cryptic loci. Chromosome Res. 2008, 16,
595–607.
56. Warchałowska-Śliwa, E.; Grzywacz, B.; Maryańska-Nadachowska, A.; Karamysheva,
T.V.; Heller, K.-G.; Lehmann, A.W.; Lehmann, G.U.C.; Chobanov, D.P. Molecular and
classical chromosomal techniques reveal diversity in bushcricket genera of Barbitistini
(Orthoptera). Genome. 2013, 56, 667–676.
57. Sochorová, J.; Garcia, S.; Gálvez, F.; Symonová, R.; Kovařík, A. Evolutionary trends
in animal ribosomal DNA loci: Introduction to a new online database. Chromosoma.
2018, 127, 141–150.
58. Richard, G.F.; Kerrest, A.; Dujon, B. Comparative genomics and molecular dynamics
of DNA repeats in eukaryotes. Microbiol. Mol. Biol. Rev. 2008, 72, 686–727.
FISH—in Insect Chromosomes 333

59. Hillis, D.M.; Dixon, M.T. Ribosomal DNA: Molecular evolution and phylogenetic
inference. Quart. Rev. Biol. 1991, 66, 411–453.
60. Ferretti, A.B.S.M.; Ruiz-Ruano, F.J.; Milani, D.; Loreto, V.; Martí, D.A.; Ramos, E.;
Martins, C.; Cabral-de-Mello, D.C. How dynamic could be the 45S rDNA cistron? An
intriguing variability in a grasshopper species revealed by integration of chromosomal
and genomic data. Chromosoma. 2019, 128, 165–175.
61. Cabral-de-Mello, D.C.; Martins, C.; Souza, M.J.; Moura, R.C. Cytogenetic mapping of
5S and 18S rRNAs and H3 histone genes in 4 ancient Proscopiidae grasshopper spe-
cies: Contribution to understanding the evolutionary dynamics of multigene families.
Cytogenet. Genome Res. 2011, 132, 89–93.
62. Cabral-de-Mello, D.C.; Oliveira, S.G.; Moura, R.C.; Martins, C. Chromosomal orga-
nization of the 18S and 5S rRNAs and histone H3 genes in Scarabaeinae coleopterans:
Insights into the evolutionary dynamics of multigene families and heterochromatin.
BMC Genet. 2011, 12, 88.
63. Oliveira, N.L.; Cabral-de-Mello, D.C.; Rocha, M.F.; Loreto, V.; Martins, C.; Moura,
R.C. Chromosomal mapping of rDNAs and H3 histone sequences in the grasshopper
Rhammatocerus brasiliensis (Acrididae, Gomphocerinae): Extensive chromosomal
dispersion and co-localization of 5S rDNA/H3 histone clusters in the A complement
and B chromosome. Mol. Cytogenet. 2011, 4, 24.
64. Bueno, D.; Palacios-Gimenez, O.M.; Cabral-de-Mello, D.C. Chromosomal mapping of
repetitive DNAs in the grasshopper Abracris favolineata reveal possible ancestry of
the B chromosome and H3 histone spreading. PLoS One. 2013, 8, e66532
65. Palacios-Gimenez, O.M.; Castillo, E.R.; Martí, D.A.; Cabral-de-Mello, D. C. Tracking
the evolution of sex chromosome systems in Melanoplinae grasshoppers through chro-
mosomal mapping of repetitive DNA sequences. BMC Evol. Biol. 2013, 13, 167.
66. Palacios-Gimenez, O.M.; Cabral-de-Mello, D.C. Repetitive DNA chromosomal orga-
nization in the cricket Cycloptiloides americanus: A case of the unusual X1X20 sex
chromosome system in Orthoptera. Mol. Genet. Genomics. 2015, 290, 623–631.
67. Bardella, V.B.; Fernandes, J.A.M.; Cabral de Mello, D.C. Chromosomal evolutionary
dynamics of four multigene families in Coreidae and Pentatomidae (Heteroptera) true
bugs. Mol. Genet. Genomics. 2016, 291, 1919–1925.
68. Gusso Goll, L.; Matiello, R.R.; Artoni, R.F.; Vicari, M.R.; Nogaroto, V.; de Barros,
A.V.; Almeida, M.C. High-resolution physical chromosome mapping of multigene fam-
ilies in Lagria villosa (Tenebrionidae): Occurrence of interspersed ribosomal genes in
Coleoptera. Cytogenet. Genome Res. 2015, 146, 64–70.
69. Maryańska-Nadachowska, A.; Anokhin, B.A.; Gnezdilov, V.M.; Kuznetsova, V.G.
Karyotype stability in the family Issidae (Hemiptera, Auchenorrhyncha) revealed by
chromosome techniques and FISH with telomeric (TTAGG)n and 18S rDNA probes.
Comp. Cytogenet. 2016, 10, 347–369.
70. Maryańska-Nadachowska, A.; Kuznetsova, V.G.; Karamysheva, T.V. Chromosomal
location of rDNA clusters and TTAGG telomeric repeats in eight species of the spittle-
bug genus Philaenus (Hemiptera: Auchenorrhyncha: Aphrophoridae). Eur. J. Entomol.
2013, 110, 411–418.
71. Piccoli, M.C.A.; Bardella, V.B.; Cabral-de-Mello, D.C. Repetitive DNAs in Melipona
scutellaris (Hymenoptera: Apidae: Meliponidae): Chromosomal distribution and
test of multiple heterochromatin amplifcation in the genus. Apidologie. 2018, 49,
497–504.
72. Micolino, R.; Cristiano, M.P.; Travenzoli, N.P.; Lopes, D.M.; Cardoso, D.C.
Chromosomal dynamics in space and time: Evolutionary history of Mycetophylax ants
across past climatic changes in the Brazilian Atlantic coast. Sci. Rep. 2019, 9, 18800.
334 Cytogenetics and Molecular Cytogenetics

73. Menezes, R.S.T.; Cabral-de-Mello, D.C.; Milani, D.; Bardella, V.B.; Almeida, E.A.B.
The relevance of chromosome fssions for major ribosomal DNA dispersion in
hymenopteran insects. J. Evol. Biol. 2021, 34, 1466–1476.
74. Provazníková, I.; Hejníčková, M.; Visser, S.; Dalíková, M.; Carabajal Paladino, L.Z.;
Zrzavá, M.; Voleníková, A.; Marec, F.; Nguyen, P. Large-scale comparative analysis of
cytogenetic markers across Lepidoptera. Sci. Rep. 2021, 11, 12214.
75. Cabrero, J.; Bugrov, A.; Warchałowska-Śliwa, E.; López-León, M.D.; Perfectti, F.;
Camacho, J.P.M. Comparative FISH analysis in fve species of Eyprepocnemidine
grasshoppers. Heredity. 2003, 90, 377–381.
76. Nguyen, P.; Sahara, K.; Yoshido, A.; Marec, F. Evolutionary dynamics of rDNA clus-
ters on chromosomes of moths and butterfies (Lepidoptera). Genetica. 2010, 138,
343–354.
77. Travenzoli, N.M.; Lima, B.A.; Cardoso, D.C.; Dergam, J.A.; Fernandes-Salomão, T.M;
Lopes, D.M. Cytogenetic analysis and chromosomal mapping of repetitive DNA in
Melipona species (Hymenoptera, Meliponini). Cytogenet. Genome Res. 2019,158, 213–224.
78. Martí, E.; Milani, D.; Bardella, V.B.; Albuquerque, L.; Song, H.; Palacios-Gimenez,
O.M.; Cabral-de-Mello, D.C. Cytogenomic analysis unveils mixed molecular evolu-
tion and recurrent chromosomal rearrangements shaping the multigene families on
Schistocerca grasshopper genomes. Evolution. 2021, 75, 2027–2041.
79. Panzera, F.; Pita, S.; Lorite, P. Chromosome structure and evolution of Triatominae:
A review. In: Triatominae—The Biology of Chagas Disease Vectors; Guarneri, A.;
Lorenzo, M., Eds. Springer Nature Switzerland AG, Cham, 2021, pp. 65–99.
80. Pereira, J.A.; Travenzoli, N.M.; de Oliveira, M.P.; Werneck, H.A.; Fernandes Salomão,
T.M.; Lopes, D.M. Molecular cytogenetics in the study of repetitive sequences help-
ing to understand the evolution of heterochromatin in Melipona (Hymenoptera,
Meliponini). Genetica. 2021, 149, 55–62.
81. Teixeira, G.A.; de Aguiar, H.J.A.C.; Petitclerc, F.; Orivel, J.; Lopes, D.M.; Barros,
L.A.C. Evolutionary insights into the genomic organization of major ribosomal DNA
in ant chromosomes. Ins. Mol. Biol. 2021, 30, 340–354.
82. Sproul, J.S.; Barton, L.M.; Maddison, D.R. Repetitive DNA profles reveal evidence
of rapid genome evolution and refect species boundaries in ground beetles. Syst. Biol.
2020, 69, 1137–1148.
83. Maxson, R.; Cohn, R.; Kedes, L. Expression and organization of histone genes. Annu.
Rev. Genet. 1983, 17, 239–277.
84. Cabrero, J.; Lopez-Leon, M.D.; Teruel, M.; Camacho, J.P. Chromosome mapping of H3
and H4 histone gene clusters in 35 species of acridid grasshoppers. Chromosome Res.
2009, 17, 397–404.
85. Mandrioli, M.; Manicardi, G.C. Chromosomal mapping reveals a dynamic organiza-
tion of the histone genes in aphids (Hemiptera: Aphididae). Entomologia. 2013, 1, e2.
86. Toscani, M.A.; Pigozzi, M.I.; Papeschi, A.G.; Bressa, M.J. Histone H3 methylation and
autosomal vs. sex chromosome segregation during male meiosis in Heteroptera. Front.
Ecol. Evol. 2022, 10, 836786.
87. Cabral-de-Mello, D.C.; Moura, R.C.; Martins, C. Chromosomal mapping of repetitive
DNAs in the beetle Dichotomius geminatus provides the frst evidence for an asso-
ciation of 5S rRNA and histone H3 genes in insects, and repetitive DNA similarity
between the B chromosome and A complement. Heredity. 2010, 104, 393–400.
88. Cabral-de-Mello, D.C.; Moura, R.C.; Melo, A.S.; Martins, C. Evolutionary dynamics of
heterochromatin in the genome of Dichotomius beetles based on chromosomal analy-
sis. Genetica. 2011, 139, 315–325.
FISH—in Insect Chromosomes 335

89. Nagoda, N.; Fukuda, A.; Nakashima, Y.; Mats, Y. Molecular characterization and evo-
lution of the repeating units of histone genes in Drosophila americana: Coexistence of
quartet and quintet units in a genome. Insect Mol. Biol. 2005, 14, 713–717.
90. Jonika, M.; Lo, J.; Blackmon, H. Mode and tempo of microsatellite evolution across
300 million years of insect evolution. Genes. 2020, 11, 945.
91. Ruiz-Ruano, F.J.; Cuadrado, A.; Montiel, E.E.; Camacho, J.P.; López-Leon, M.D. Next
generation sequencing and FISH reveal uneven and nonrandom microsatellite distribu-
tion in two grasshopper genomes. Chromosoma. 2015, 124, 221–234.
92. dos Santos, J.M.; Diniz, D.; Rodrigues, T.A.S.; Cioff, M.B.; Waldschmidt, A.M.
Heterochromatin distribution and chromosomal mapping of microsatellite repeats
in the genome of Frieseomelitta stingless bees (Hymenoptera: Apidae: Meliponini).
Florida Entomol. 2018, 101, 33–39.
93. Milani, D.; Cabral-de-Mello, D.C. Microsatellite organization in the grasshopper
Abracris favolineata (Orthoptera: Acrididae) revealed by FISH mapping: Remarkable
spreading in the A and B chromosomes. PLoS One. 2014, 9, e97956.
94. Elizeu, A.M.; Travenzoli, N.M.; Ferreira, R.P.; Lopes, D.M.; Tavares, M.G. Comparative
study on the physical mapping of ribosomal genes and repetitive sequences in Friesella
schrottkyi (Friese 1900) (Hymenoptera: Apidae, Meliponini). Zool. Anz. 2021, 292,
225–230.
95. Matera, A.G.; Terns, R.M.; Terns, M.P. Non-coding RNAs: Lessons from the small
nuclear and small nucleolar RNAs. Nat. Rev. Mol. Cell Biol. 2007, 8, 209–220.
96. Anjos, A.; Ruiz-Ruano, F.J.; Camacho, J.P.; Loreto, V.; Cabrero, J.; de Souza, M.J.;
Cabral-de-Mello, D.C. U1 snDNA clusters in grasshoppers: Chromosomal dynamics
and genomic organization. Heredity. 2015, 114, 207–219.
97. Milani, D.; Palacios-Gimenez, O.M.; Cabral-de-Mello, D.C. The U2 snDNA is a use-
ful marker for B chromosome detection and frequency estimation in the grasshopper
Abracris favolineata. Cytogenet. Genome Res. 2017, 151, 36–40.
98. Milani, D.; Ruiz-Ruano, F.J.; Camacho, J.P.M.; Cabral-de-Mello, D.C. Out of pat-
terns, the euchromatic B chromosome of the grasshopper Abracris favolineata is not
enriched in high-copy repeats. Heredity. 2021, 127, 475–483.
99. Plohl, M.; Luchetti, A.; Meštrović, N.; Mantovani, B. Satellite DNAs between selfsh-
ness and functionality: Structure, genomics and evolution of tandem repeats in centro-
meric (hetero)chromatin. Gene. 2008, 409, 72–82.
100. Huang, Y.C.; Lee, C.C.; Kao, C.Y.; Chang, N.C.; Lin, C.C.; Shoemaker, D.; Wang, J.
Evolution of long centromeres in fre ants. BMC Evol. Biol. 2016, 16, 189.
101. Mora, P.; Vela, J.; Ruiz-Mena, A.; Palomeque, T.; Lorite, P. Isolation of a pericen-
tromeric satellite DNA family in Chnootriba argus (Henosepilachna argus) with an
unusual short repeat unit (TTAAAA) for beetles. Insects. 2019, 10, 306.
102. Ruiz-Torres, L.; Mora, P.; Ruiz-Mena, A.; Vela, J.; Mancebo, F.J.; Montiel, E.E.;
Palomeque, T.; Lorite, P. Cytogenetic analysis, heterochromatin characterization and
location of the rDNA genes of Hycleus scutellatus (Coleoptera, Meloidae): A species
with an unexpected high number of rDNA clusters. Insects. 2021, 12, 385.
103. Fernandes, A.; Scudeler, P.A.S.; Diniz, D.; Foresti, F.; Campos, L.A.O.; Lopes, D.M.
Microdissection: A tool for bee chromosome studies. Apidologie. 2011, 42, 743–748.
104. Frydrychová, F.; Grossmann, P.; Trubač, P.; Vítková, M.; Marec, F. Phylogenetic distri-
bution of TTAGG telomeric repeats in insects. Genome. 2004, 47, 163–178.
105. Kuznetsova, V.; Grozeva, S.; Anokhin, B.A. The frst fnding of (TTAGG)n telomeric
repeat in chromosomes of true bugs (Heteroptera, Belostomatidae). Comp. Cytogenet.
2012, 6, 341–346.
336 Cytogenetics and Molecular Cytogenetics

106. Pita, S.; Panzera, F.; Mora, P.; Vela, J.; Palomeque, T.; Lorite, P. The presence of the
ancestral insect telomeric motif in kissing bugs (Triatominae) rules out the hypothesis
of its loss in evolutionarily advanced Heteroptera (Cimicomorpha). Comp. Cytogenet.
2016, 10, 427–437.
107. Chirino, M.G.; Dalíková, M.; Marec, F.R.; Bressa, M.J. Chromosomal distribution of
interstitial telomeric sequences as signs of evolution through chromosome fusion in six
species of the giant water bugs (Hemiptera, Belostoma). Ecol. Evol. 2017, 7, 5227–5235.
108. Mravinac, B.; Meštrović, N.; Čavrak, V.V.; Plohl, M. TCAGG, an alternative telomeric
sequence in insects. Chromosoma. 2011, 120, 367–376.
109. Menezes, R.S.T.; Bardella, V.B.; Cabral-de-Mello, D.C.; Lucena, D.A.A.; Almeida,
E.A.B. Are the TTAGG and TTAGGG telomeric repeats phylogenetically conserved in
aculeate Hymenoptera? Sci. Nat. 2017, 104, 85.
110. Lorite, P.; Maside, X.; Sanllorente, O.; Torres, M.I.; Periquet, G.; Palomeque, T. The
ant genomes have been invaded by several types of mariner transposable elements.
Naturwissenschaften. 2012, 99, 1007–1020.
111. Palacios-Gimenez, O.M.; Bueno, D.; Cabral-de-Mello, D.C. Chromosomal mapping
of two Mariner-like elements in the grasshopper Abracris favolineata {Orthoptera:
Acrididae} reveals enrichment in euchromatin. Eur. J. Entomol. 2014, 111,
329–334.
112. Li, Y.; Jing, X.A.; Aldrich, J.C.; Clifford, C.; Chen, J.; Akbari, O.A.; Ferree, P.M.
Unique sequence organization and small RNA expression of a “selfsh” B chromosome.
Chromosoma. 2017, 126, 753–768.
113. Amorim, I.C.; Costa, R.G.C.; Xavier, C.; Moura, R.C. Characterization and chro-
mosomal mapping of the DgmarMITE transposon in populations of Dichotomius
(Luederwaldtinia) sericeus species complex (Coleoptera: Scarabaeidae). Genet. Mol.
Biol. 2018, 41, 419–425.
114. Xavier, C.; Cabral-de-Mello, D.C.; Moura, R.C. Heterochromatin and molecular char-
acterization of DsmarMITE transposable element in the beetle Dichotomius schifferi
(Coleoptera: Scarabaeidae). Genetica. 2014, 142, 575–581.
115. Cunha, M.S.; Campos, L.A.O.; Lopes, D.M. Insights into the heterochromatin evolu-
tion in the genus Melipona (Apidae: Meliponini). Insectes Soc. 2020, 67, 391–398.
116. Fominaya, A.; Loarce, Y.; González, J.M.; Ferrer, E. Tyramide signal amplifcation:
Fluorescence in situ hybridization for identifying homoeologous chromosomes. In:
Plant Cytogenetics: Methods and Protocols; Kianian, S.F., Kianian, P.M.A., Eds.
Springer Science+Business Media, New York, NY; 2016, pp. 35–48.
117. Kirkpatrick, M. How and why chromosome inversions evolve. PLoS Biol. 2010, 8,
e1000501.
118. Thompson, M.J.; Jiggins, C.D. Supergenes and their role in evolution. Heredity. 2014,
113, 1–8.
119. Wang, J.; Wurm, Y.; Nipitwattanaphon, M.; Riba-Grognuz, O.; Huang, Y.-C.;
Shoemaker, D.; Keller, L. A Y-like social chromosome causes alternative colony orga-
nization in fre ants. Nature. 2013, 493, 664–668.
120. Fleming, J.G.W.; Summers, M.D. Polydnavirus DNA is integrated in the DNA of its
parasitoid wasp host. Proc. Natl. Acad. Sci. USA. 1991, 88, 9770–9774.
121. Belle, E.; Beckage, N.E.; Rousselet, J.; Poirié, M.; Lemeunier, F.; Drezen, J.-M.
Visualization of polydnavirus sequences in a parasitoid wasp chromosome. J. Virol.
2002, 76, 5793–5796.
122. Rütten, K.B.; Pietsch, C.; Olek, K.; Neusser, M.; Beukeboom, L.W.; Gadau, J.
Chromosomal anchoring of linkage groups and identifcation of wing size QTL using
FISH—in Insect Chromosomes 337

markers and FISH probes derived from microdissected chromosomes in Nasonia

(Pteromalidae: Hymenoptera). Cytogenet. Genome Res. 2004, 105, 126–133.
123. Bugrov, A.G.; Karamysheva, T.V.; Perepelov, E.A.; Elisaphenko, E.A.; Rubtsov, D.N.;
Warchałowska-Śliwa, E.; Tatsuta, H.; Rubtsov, N.B. DNA content of the B chromo-
somes in grasshopper Podisma kanoi Storozh. (Orthoptera, Acrididae). Chromosome
Res. 2007, 15, 315–325.
124. Teruel, M.; Cabrero, J.; Montiel, E.E.; Acosta, M.J.; Sánchez, A.; Camacho, J.P.M.
Microdissection and chromosome painting of X and B chromosomes in Locusta migra‑
toria. Chromosome Res. 2009, 17, 11–18.
125. Menezes-de-Carvalho, N.Z.; Palacios-Gimenez, O.M.; Milani, D.; Cabral-de-Mello,
D.C. High similarity of U2 snDNA sequence between A and B chromosomes in the
grasshopper Abracris favolineata. Mol. Genet. Genomics. 2015, 290, 1787–1792.
126. Gokhman, V.E.; Cioff, M.B.; König, C.; Pollmann, M.; Gantert, C.; Krogmann, L.;
Steidle, J.L.M.; Kosyakova, N.; Liehr, T.; Al-Rikabi, A. Microdissection and whole
chromosome painting confrm karyotype transformation in cryptic species of the
Lariophagus distinguendus (Förster, 1841) complex (Hymenoptera: Pteromalidae).
PLoS One. 2019, 14, e0225257.
127. Fuková, I.; Traut, W.; Vítková, M.; Nguyen, P.; Kubíčková, S.; Marec, F. Probing the
W chromosome of the codling moth, Cydia pomonella, with sequences from microdis-
sected sex chromatin. Chromosoma. 2007, 116, 135–145.
128. Bressa, M.J.; Papeschi, A.G.; Vítková, M.; Kubíčková, S.; Fuková, I.; Pigozzi, M.I.;
Marec, F. Sex chromosome evolution in cotton stainers of the genus Dysdercus
(Heteroptera: Pyrrhocoridae). Cytogenet. Genome Res. 2009, 125, 292–305.
129. Zrzavá, M.; Hladová, I.; Dalíková, M.; Šíchová, J.; Õunap, E.; Kubíčková, S.; Marec,
F. Sex chromosomes of the iconic moth Abraxas grossulariata (Lepidoptera,
Geometridae) and its congener A. sylvata. Genes. 2018, 9, 279.
130. Telenius, H.; Carter, N.P.; Bebb, C.E.; Nordenskjöld, M.; Ponder, B.A.J.; Tunnacliffe,
A. Degenerate oligonucleotide-primed PCR: General amplifcation of target DNA by a
single degenerated primer. Genomics. 1992, 8, 718–725.
131. Jetybayev, I.Y.; Bugrov, A.G.; Buleu, O.G.; Bogomolov, A.G.; Rubtsov, N.B. Origin and
evolution of the neo-sex chromosomes in Pamphagidae grasshoppers through chromo-
some fusion and following heteromorphization. Genes. 2017, 8, 323.
132. Beliveau, B.J.; Joyce, E.F.; Apostolopoulos, N.; Yilmaz, F.; Fonseka, C.Y.; McCole,
R.B.; Chang, Y.; Li, J.B.; Senaratne, T.N.; Williams, B.R.; Rouillard, J.-M.; Wu, C.
Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH
probes. Proc. Natl. Acad. Sci. USA. 2012, 109, 21301–21306.
133. Rosin, L.F.; Gil, J.Jr.; Drinnenberg, I.A.; Lei, E.P. Oligopaint DNA FISH reveals telo-
mere-based meiotic pairing dynamics in the silkworm, Bombyx mori. PLoS Genet.
2021, 17, e1009700.
134. Lopes, D.M.; Fernandes, A.; Diniz, D.; Scudeler, P.E.S.; Foresti, F.; Campos, L.A.O.
Similarity of heterochromatic regions in the stingless bees (Hymenoptera: Meliponini)
revealed by chromosome painting. Caryologia. 2014, 67, 222–226.
135. Jetybayev, I.Y.; Bugrov, A.G.; Dzuybenko, V.V.; Rubtsov, N.B. B chromosomes in grass-
hoppers: Different origins and pathways to the modern Bs. Genes. 2018, 9, 509.
136. Ferguson, K.B.; Visser, S.; Dalíková, M.; Provazníková, I.; Urbaneja, A.; Pérez-Hedo,
M.; Marec, F.; Werren, J.H.; Zwaan, B.J.; Pannebakker, B.A.; Verhulst, E.C. Jekyll
or Hyde? The genome (and more) of Nesidiocoris tenuis, a zoophytophagous preda-
tory bug that is both a biological control agent and a pest. Insect Mol. Biol. 2021, 30,
188–209.
338 Cytogenetics and Molecular Cytogenetics

137. Hejníčková, M.; Dalíková, M.; Potocký, P.; Tammaru, T.; Trehubenko, M.; Kubíčková,
S.; Marec, F.; Zrzavá, M. Degenerated, undifferentiated, rearranged, lost: High vari-
ability of sex chromosomes in Geometridae (Lepidoptera) identifed by sex chromatin.
Cells. 2021, 10, 2230.
138. Lukhtanov, V.A.; Shapoval, N.A.; Anokhin, B.A.; Saiftdinova, A.F.; Kuznetsova, V.G.
Homoploid hybrid speciation and genome evolution via chromosome sorting. Proc. R.
Soc. B. 2015, 282, 20150157.
139. Pita, S.; Panzera, F.; Sánchez, A.; Panzera, Y.; Palomeque, T.; Lorite, P. Distribution and
evolution of repeated sequences in genomes of Triatominae (Hemiptera-Reduviidae)
inferred from genomic in situ hybridization. PLoS One. 2014, 9, e114298.
 28 FISH—in Plant
Chromosomes
Susan Liedtke, Sarah Breitenbach
and Tony Heitkam

CONTENTS
Introduction............................................................................................................ 340
Samples/Tissues ..................................................................................................... 340
Description of Methods: Fixation .......................................................................... 341
Chemicals ..................................................................................................... 341
Fixation of Leaf Tissue ................................................................................. 341
Fixation of Primary Root Tissue................................................................... 341
Description of Methods: Preparation of Mitotic Plant Chromosomes................... 342
Chemicals ..................................................................................................... 343
Enzyme Treatment ........................................................................................ 343
Washing and Nuclei Purifcation .................................................................. 344
Preparation of the Dropping Preparation...................................................... 344
Description of Methods: Fluorescence In Situ Hybridization ............................... 344
Equipment..................................................................................................... 344
Chemicals ..................................................................................................... 345
FISH with Indirect Labeling......................................................................... 345
RNase Treatment .............................................................................. 345
Pepsin Treatment .............................................................................. 346
Fixation of Chromosomes................................................................. 346
Dehydration of the Preparations ....................................................... 346
Hybridization .................................................................................... 346
Washing (All Steps in Coplin Jars)................................................... 347
Antibody Reaction ............................................................................ 347
DAPI Staining................................................................................... 347
Fluorescence Microscopy ................................................................. 347
Typical Applications .............................................................................................. 348
Conclusions............................................................................................................ 350
Acknowledgements................................................................................................ 350
References.............................................................................................................. 350

DOI: 10.1201/9781003223658-28 339

340 Cytogenetics and Molecular Cytogenetics

INTRODUCTION
With the advent of fuorescence in situ hybridization (FISH) methods since the
1980s–1990s, we have rapidly gained insights into the structure, organization and evo-
lution of plant genomes.[1, 2] The potential of molecular cytogenetics was also recog-
nized in the plant breeding sector, often with a focus on hybridization, introgressions
and chromosomal additions. Now, with the advances in long-read sequencing and with
the new genome-editing techniques in place, the plant cytogenetics feld is reposition-
ing itself at the interface between microscopy and genomics. FISH methods are more
relevant than ever, for example to better resolve the inaccessible, repetitive genome
fraction,[3] the genomes’ three-dimensional organization,[4, 5] but also to identify
chromosomes,[6] assess haplotypes, predict recombination,[7] and determine genomic
variation among individuals.[8] On top, FISH may also assist the synthetic genomics
feld seeking to steer chromosome mutations and to build artifcial chromosomes.[9, 10]
Nevertheless, the application of FISH to plant chromosomes remains challenging.
First, plant genomes can contain over 80% of repetitive DNA,[11] and up to 149
gigabasepairs.[12] Although these numbers reside at the extreme ends of the corre-
sponding scales, they serve well to illustrate that plant genomes are often large and
repetitive. Therefore, they remain diffcult targets for sequence-based methods.
Second, only limited genome and haplotype information has been available for
many plants, leading to less fexibility in experimental design. This gap is now clos-
ing at high speed, with many new crop and wild plant genome builds being released
every year (see www.plabipd.de/timeline_view.ep). Similarly, many karyotyping
tools that were originally designed for human chromosomes are now fexible in
organism choice and can be used for plant chromosomes as well.
Finally, the preparation of plant chromosomes—a prerequisite for FISH—is not
straight-forward. Plants contain a high amount of fbers and their cells are enclosed
by a cell wall with a variable chemical composition, depending on species, tissue and
developmental stage. Finding the balance between cell wall degradation and removal
of cytoplasm on the one hand, and preserving chromosome integrity on the other
hand, is one of the key challenges of plant cytogenetics, a process which needs to be
adapted or even established for each new plant species and tissue coming to the lab.

SAMPLES/TISSUES
As most FISH experiments require spreads of mitotic chromosomes, typically,
dividing plant cells are harvested. In plants, mitotic cells occur in the meristems,
out of which the root apical meristems are best accessible. Hence, the wide majority
of plant cytogenetics labs uses root tip tissues to prepare mitotic chromosomes, with
primary roots being especially serviceable. However, for some plants this is not an
option: root crops for example accumulate carbohydrates in thickened roots, and
grown trees may have buried their root tips inaccessibly below the soil. For these
plants, very young leaves in leaf buds may provide a suitable alternative. Examples
for this are beet crops for which a barely visible young leaf can be picked from the
middle of the rosette (as performed for example by[13, 14]), and trees or shrubs such as
beeches or camellias, for which young shoot tips can be taken in spring season.[15, 16]
FISH—in Plant Chromosomes 341

To study meiosis, tissue of the developing fower has to be harvested; i.e., the anthers
for male meiosis and the ovules for female meiosis (please see for the collection of
protocols for the analysis of meiotic chromosomes by[17]).

DESCRIPTION OF METHODS: FIXATION

Due to the low fraction of mitotic cells in most tissues, a number of methods are sug-
gested to increase the amount of mitotic nuclei in the sample. Usually, prior to a fxa-
tion step, these methods involve antimitotic agents that arrest the chromosomes in
the metaphase and lead to stronger chromosome condensation. Among these, some
techniques use hydroxyquinoline and/or colchicine (e.g., for beets and crocus[8, 14]),
hydroxyurea (e.g., for conifers[18]) or nitrous oxide under pressure (e.g., for maize[19]).
In the following, hydroxyquinoline treatment prior to the fxation of leaves and roots
is laid out—one of the most commonly used methodologies.

CHEMICALS
• Fixative: methanol glacial acetic acid (3:1).
• Enrichment of metaphases: 8-hydroxyquinoline.

FIXATION OF LEAF TISSUE

1. For the preparation of mitotic metaphase chromosomes, meristems of
young leaves are fxed.
2. Remove approx. 1cm long leaves at the plant base with tweezers
(Figure 28.1A) after 4.5–5h of light-exposure (artifcial light in the climate
chamber or sunlight in the greenhouse).
3. Treat with 8-hydroxyquinoline for 2–3h to block formation of the mitotic
spindle and thus progression towards anaphase.
4. Fix leaf tissue in fxative at room temperature (RT).
5. Change the fxative regularly (every 30min to 2h) until green coloration of
leaves is no longer visible.
6. Incubate fxed leaves at −20°C for at least one night before attempting FISH.
7. Fixed leaves can be stored at −20°C for up to six months.

FIXATION OF PRIMARY ROOT TISSUE

1. Put seeds on wet paper and incubate for some nights. Water regularly and
observe germination.
2. After germination collect approx. 0.5–1.5 cm long root tips with tweezers.
3. Treat with 8-hydroxyquinoline for 2–3h to block spindle formation.
4. Fix roots in fxative for 30min at RT.
5. Replace solution with fresh fxative.
6. Incubate fxed leaves at −20°C for at least one night before attempting
FISH.
7. Fixed leaves can be stored at −20°C for up to six months.
342 Cytogenetics and Molecular Cytogenetics

FIGURE 28.1 Photographs of selected experimental details for chromosome fxation, prep-

aration and FISH.
A) The sugar beet plant shown harbors an approx. 1 cm long, young leaf at its base that pro-
vides suitable tissue for a chromosome preparation. After light-exposure for about 4.5–5
hours, this leaf can be taken with forceps for further experimentation.
B) With a Pasteur pipette, the nuclei pellet is suspended.
C) A mounted pipette with a height of 40–50 cm is used for the dropping method. (D) All
washing steps are performed in coplin jars on a shaking plate.
D) When working with small volumes, slides are typically incubated in a humid chamber.
Humidity is retained by wet tissues that are regularly changed to avoid moldiness. Slides
are slightly elevated to not come into contact with the wet tissue.
E) Plastic strips are used to carefully cover the slides before any incubation in the humid
chamber.

DESCRIPTION OF METHODS: PREPARATION OF MITOTIC

PLANT CHROMOSOMES
As with tissue fxation, there are many ways to prepare mitotic plant chromosomes,
including dropping,[20] squashing,[21] and tapping (followed by spreading using a nee-
dle).[22] For squashing and tapping, typically one slide results from a single root tip.
For dropping, several slides with comparable qualities can be received from a single
dropping suspension.
When establishing the chromosome preparation method for a new plant or tissue,
usually, those methods are compared amongst each other. Nevertheless, depending
FISH—in Plant Chromosomes 343

on the chromosomal confguration of the plant, some assumptions can be already

made: for nuclei with small to medium-sized chromosomes, such as beets, potato
or poplar, the dropping method usually works well; for species with large chromo-
somes, such as conifers or crocuses, the tapping method is usually preferred.
To obtain an insight into the standard methodology of our lab, we lay out the
dropping procedure next—usually the frst protocol we apply to any unknown plant.

CHEMICALS
• Enzyme solution: to remove the cell wall components, the plant tissue is
usually incubated in an enzyme solution. Please note that a wide range of
enzyme solutions with plenty variations exist. Depending on the species,
developmental stage and tissue, this solution needs to be adapted to the
tissue at hands. Here, we give one example which shows typical enzyme
components and concentrations, as applied to sugar beet,[13] camellia,[16] and
saffron crocus[8]:
4% (w/v) cellulase ‘Onozuka’ R-10 from Trichoderma viride (Serva 16419)
2% (w/v) cellulase from Aspergillus niger (Sigma C1184)
2% (w/v) cytohelicase from Helix pomatia (Sigma C8274)
OR
2% (w/v) hemicellulase from Aspergillus niger (Sigma H2125)
0.5% (w/v) pectolyase from Aspergillus japonicus (Sigma P3026)
5–20% (w/v or v/v) pectinase from Aspergillus niger (Sigma 17389 or Sigma
P4716)
• Enzyme buffer: 4mM citric acid, 6mM sodium citrate, prepared with dis-
tilled water, pH4.5 adjusted with HCl; if autoclaved, storable for up to
12 months at 4°C.
• Fixative: methanol glacial acetic acid (3:1).

ENZYME TREATMENT
1. Wash fxed leaves with distilled water on a platform shaker at 50 rpm for
5 min.
2. Transfer the leaf onto a petri dish with enzyme buffer and continue shaking
at 50 rpm for 5 min. Replace the enzyme buffer once and repeat the wash-
ing step once.
3. Incubate the leaf in a reaction tube with 100 µl enzyme solution for 3.5–4 h
at 37°C. In many cases, enzyme solution, timeframe and temperature have
to be modifed to meet the optimal balance between digesting the cell wall
and cytoplasm, but keeping the nuclei intact.
4. Macerate tissue with forceps and remove undigested components directly
with the forceps or a micropistil.
5. Pre-pipette macerated tissue up and down with 200 µl pipette. Be careful to
not create any air bubbles.
6. Incubate for another 15–20 min at 37°C and homogenize every 5 min with
a 200 µl pipette.
344 Cytogenetics and Molecular Cytogenetics

WASHING AND NUCLEI PURIFICATION

1. Fill up the reaction mix with enzyme buffer (add approx. 1 ml) and centri-
fuge at 2,500×g at 4°C for 5 min.
2. Locate the nuclei pellet without shaking the reaction tube. The pellet can be
at stuck to the side of the tube or can foat on the surface.
3. Carefully remove supernatant to approx. 500 µl with a Pasteur pipette
(Figure 28.1B) without disturbing the nuclei pellet, and replace it with fresh
enzyme buffer.
4. Resuspend the nuclei and centrifuge again.
5. Repeat washing steps with freshly prepared fxative three times.
6. Very carefully remove the supernatant down to 200 µl (depending on size
of nucleus pellet).
7. Rinse the walls of the reaction tube with 1–2 drops of fxative and resus-
pend the nuclei.

PREPARATION OF THE DROPPING PREPARATION

1. Use slides that have been cleaned with chromosulfuric acid (or use com-
mercially available adhesion microscope slides). Rinse the slides in ethanol
and air-dry.
2. Use a mounted pipette with a height between 40–50 cm (Figure  28.1C).
Drop 13–20 µl of the cell nucleus suspension onto a slide.
3. Blow briefy and strongly over the slide, and move it with a strong, deter-
mined action downwards once.
4. Rinse the slides with fxative.
5. Air-dry slides vertically in a slide holder at RT.
6. Examine the slides under a phase contrast microscope at 100–200× magni-
fcation and evaluate the quality and density of the nuclei preparation:
a. If the nuclei preparation is too dense, add some additional fxative to the
solution. In contrast, if only very few nuclei are on the slide, let some
fxative evaporate to concentrate the preparation.
b. If the frst slides are of low quality, usually the nuclei suspension will not
yield any useful slides. In this case, the chromosome preparation should
be attempted again. Instead, if the frst slides are of high quality, with
well-purifed nuclei and without too much cytoplasm, it is recommended
to prepare more slides from this nuclei preparation or to store the nuclei
suspension for further use.
7. Choose slides with well-spread mitotic chromosomes for FISH.

DESCRIPTION OF METHODS: FLUORESCENCE

IN SITU HYBRIDIZATION
EQUIPMENT
• Coplin jars: all washing steps are performed in Coplin jars (Figure 28.1D).
• Humid chamber (Figure 28.1E).
FISH—in Plant Chromosomes 345

• Two shaking water baths; one heated to 37°C and the other to 42°C.
• Thermoblock/thermoshaker.
• Pasteur pipettes: disposable, plastic.
• Incubator heated to 37°C.
• Touchdown in situ system (for the controlled heating and cooling of slides).
• Phase-contrast light microscope.
• Fluorescence microscope.

CHEMICALS
• Enzyme buffer: 4mM citric acid, 6mM sodium citrate, prepared with dis-
tilled water, pH4.5 adjusted with HCl; if autoclaved, storable for up to 12
months at 4°C.
• Fixative: methanol glacial acetic acid (3:1).
• 20×SSC: 3M NaCl, 0.3M sodium citrate, fll up to 1 l with distilled
water
• RNaseA: 0.1 µg/µl in 2×SSC (freshly prepared from a 10 µg/µl stock
solution)
• 0.01M HCl: dilute 0.25M HCl to 0.01M HCl with distilled water
• Pepsin solution: 10 µg/ml in 0.01M HCl (freshly prepare from a 500 µg/ml
stock solution).
• 4% paraformaldehyde (freshly prepared): heat 2.8g paraformaldehyde in
70 ml distilled water to 70°C in a water bath; add 50 µl 1M NaOH until the
solution becomes clear; cool to RT.
• 4×SSC/0.2% Tween 20: use stock solutions of 20×SSC and Tween 20
(100%); fll up with distilled water to 1 l.
• Formamide wash solution (freshly prepared): 20% formamide in
0.2×SSC.
• Blocking solution: use 10×Block from Roche, dilute to 1×Block in
4×SSC/0.2% Tween 20
• Antibody solution (prepare fresh):
• Dilute the anti-Dig solution 1:75.
• Dilute the Streptavidin solution 1:200 in 1× Blocking solution.
• 4′,6-diamidino-2-phenylindole (DAPI) solution: use a DAPI stock solution
(100 µg/ml in ultrapure water); dilute 1:50 in Citifuor (fnal concentration
2 µg/ml).

FISH WITH INDIRECT LABELING

RNase Treatment
1. Pipette 200µl of RNaseA onto each slide, cover with a plastic strip
(Figure 28.1F) and place slides into a humid chamber for 30 min at 37°C
(Figure 28.1E).
2. Carefully transfer the slides to a coplin jar, already flled with 2×SSC at
37°C. Only now, while the slides are submerged, remove plastic strips and
wash for 3×5min at 37°C on a shaking plate.
346 Cytogenetics and Molecular Cytogenetics

Pepsin Treatment
3. Pour off 2×SSC and replace with pre-warmed 0.01M HCl, followed by wash-
ing for 1min at 37°C. This is to equilibrate the preparations to an acidic pH.
4. Pipette 200 µl pepsin onto each slide, cover slide with plastic strips, and
place slides into a pre-warmed humid chamber for 15 min at 37°C. This
will serve to further digest the cytoplasm.
5. Carefully transfer the slides to a coplin jar, already flled with 2×SSC at
37°C. Only now, while the slides are submerged, remove plastic strips and
wash for 3×5min at 37°C on a shaking plate.

Fixation of Chromosomes
6. In a coplin jar, incubate the slides in 4% paraformaldehyde solution for 15
min at 37°C without shaking.
7. In a coplin jar, wash preparations in 2×SSC 3×10min at 37°C on a shaking plate.

Dehydration of the Preparations

8. In a coplin jar, dehydrate the slides in 70% ethanol for 3 min at RT on a
shaking plate.
9. In a coplin jar, dehydrate the slides in 96% ethanol for 3 min at RT on a
shaking plate.
10. Subsequent air-drying of vertically placed slides in a slide rack at RT.

Hybridization
The hybridization and washing stringencies are infuenced by a range of parameters,
e.g., the base composition of the probe, the formamide and salt concentration as well
as the temperature. Stringency guide values are laid out in Table 28.1. The following
protocol uses our standard procedure, which entails hybridization at approximately
76% and washing at 79% stringency (see Table 28.1).

11. Heat dextran sulfate to 70°C.

12. Hybridization solution per slide at 76% hybridization stringency:
Formamide (fnal concentration 50%) 15 µl
20×SSC (fnal concentration 2×SSC) 3 µl
Salmon sperm DNA (2µg/µl) 1 µl
10% SDS (fnal concentration 0.15%) 0.5 µl
Probe (biotin-labeled) and/or 0.5–3.5 µl
Probe (digoxigenin-labeled) 0.5–3.5 µl
Water x µl
→ Prepare solution up to here, then mix and centrifuge down
Dextran sulfate (50%) 6 µl
Total volume 30 µl
13. Prepare hybridization master mix:
• Mix formamide, 20×SSC, salmon sperm DNA and SDS, centrifuge and
distribute into the reaction tubes. The salmon sperm serves to block
unspecifc binding sites.
FISH—in Plant Chromosomes 347

• To pipette the viscous dextran sulfate, cut about 0.5 cm of a 200 µl

pipette tip. Add the dextran sulfate and mix by vortexing and spin-
ning down.
• Denature hybridization solution for 10 min at 70°C, cool in an ice bath.
• Pipette 30 µl hybridization solution onto the marked region and cover
bubble-free with plastic strip.
• Place slides in a touchdown in situ system and denature the chromosomes
and the probe, followed by cooling down to 37°C in a controlled manner
(70°C 8 min, 55°C 5 min, 50°C 2 min, 45°C 3 min, 37°C hold temperature).
• Transfer the preparations to a pre-warmed humid chamber overnight
at 37°C.

Washing (All Steps in Coplin Jars)

14. Heat 2×SSC and formamide washing solution to 42°C in a shaking water
bath.
15. Heat detection buffer (4×SSC with 0.2% Tween 20) to 37°C.
16. Immerse preparations in warm 2×SSC, carefully detach plastic strips, and
place into 2×SSC.
17. Pour off 2×SSC and replace with pre-warmed formamide washing
solution.
18. Wash in shaking water bath for 2×5 min at 42°C.
19. Wash with 2×SSC, in shaking water bath for 2×5 min at 42°C.
20. Wash with 2×SSC, in shaking water bath for 1×5 min at 37°C.

Antibody Reaction
21. Wash with 4×SSC/0.2% Tween 20 in a shaking water bath for 1×5 min at
37°C (to equilibrate the salt concentration).
22. Pipette 200 µl blocking solution onto each preparation, cover with plastic
strip to block non-specifc binding.
23. Place preparations in a humid chamber for 20 min at 37°C.
24. Carefully remove plastic strips, pipette 50 µl of antibody solution onto
each slide.
25. Cover again and place in a humid chamber for 1 h at 37°C.
26. Remove plastic strips, wash in 4×SSC/0.2% Tween 20 in a shaking
water bath for 3×10 min at 37°C. This serves to wash off the unbound
antibody.

DAPI Staining
27. Pipette 15 µl DAPI solution (in Citifuor) onto the slide and carefully cover
it with a large coverslip (50×24 mm). Try to avoid any air bubbles.
28. Squeeze out any excess liquid with flter paper; the cover slip should lose
the ability to move freely on the slide.

Fluorescence Microscopy
29. Observe the slides under a fuorescence microscope.
348 Cytogenetics and Molecular Cytogenetics

TABLE 28.1
Guide Values for Hybridization and Washing Stringency in Relation to
Temperature, Salt and Formamide Concentration
Formamide Concentration [%]

60 55 50 45 40 35 30 25 20 15 10 5 0
2×SSC 82 79 76 73
b) 70 67 64 61 57 54 51 48 45
Tm at 37 °Ca)

1×SSC 87 84 81 78 75 72 69 66 62 59 56 53 50
Stringency

0.5×SSC 92 89 86 83 80 77 74 71 67 64 61 58 55
0.2×SSC 98 95 92 89 86 83 80 77 74 71 68 65 62
0.1×SSC 103 100 97 94 91 86 85 82 79 74 73 70 67
2×SSC 87 84 81 78 75 72 69 66 62 59 56 53 50
Tm at 42 °C a)

1×SSC 92 89 86 83 80 77 74 71 67 64 61 58 55
Stringency

0.5×SSC 97 94 91 88 85 82 79 76 72 69 66 63 60
0.2×SSC 103 100 97 94 91 88 85 82 79 76 73 70 67
0.1×SSC 108 105 102 99 96 93 90 87 84 81 78 75 72
The exemplary hybridization stringency of Tm = 76% used in this protocol is marked.
a) based on the following equation: Tm = 81.5°C + 16.6logM + 0.41(%G+C)—0.61(% formamide)—

(600/n); where M = sodium concentration [mol/l], n = number of base pairs in smallest duplex
b) standard hybridization parameters in our laboratory

TYPICAL APPLICATIONS
FISH to plant chromosomes enables a range of different applications. Depending
on the question, the respective probes, plant tissues and cell division stages need to
be chosen. In the following, we showcase several examples for FISH applications in
plant chromosomes (Figure 28.2):

(1) One of the earliest FISH applications has been the unequivocal assign-
ment of chromosomes and karyotyping. For this, typically, mitotic chro-
mosomes are prepared and hybridized with a probe cocktail producing
chromosome-specifc signals. These probes can be repeat-derived,[8]
derived from long-insert clones,[14] or from bulked oligonucleotides.[6] As
example, we follow a research question that asks for the genetic origin
of a polyploid plant, namely the triploid saffron crocus. This crop’s trip-
loidization has occurred only once in history, likely in the time of the
Aegean Bronze Age, and has spread since then across the globe as a clonal
line.[23] Nevertheless, the parental species contribution of saffron has been
debated for nearly a century. In this case, repeat-based probes have been
used for comparative FISH between saffron and potential parental wild
crocuses (Figure  28.2A). This FISH-based strategy clearly revealed the
triploid saffron crocus emerged from cytotypes of wild Crocus cart‑
wrightianus[8]; a fnding that was also mirrored through genotyping-by-
sequencing strategies.[24] With increasing sequence information available,
FISH—in Plant Chromosomes 349

FIGURE 28.2 Application examples of FISH onto mitotic and meiotic chromosomes of plants. (A) The
DAPI-stained mitotic chromosomes of triploid saffron are shown in grey. In total, 72 chromosomal
landmarks are produced by hybridization of the four satellite DNAs, CroSat1 (blue), CroSat2 (green),
CroSat3 (light blue), CroSat4 (red), the 18S rRNA gene (yellow) and the 5S rRNA gene (white). This
six-color hybridization of repetitive DNA probes allows the unequivocal identifcation of saffron’s
chromosomes.[8] The scale bar is 10 µm. (B) The DAPI-stained mitotic chromosomes of banana (Musa
acuminata ssp. malaccensis) are shown in blue. This example of chromosome painting targets the chro-
mosomes 6 (red) and 7 (green). For this, bulked oligonucleotide probes were derived from the genome
assembly and hybridized to allow the clear identifcation of chromosome 6– and chromosome 7–derived
regions.[25] The scale bar is 5 µm. (C) The DAPI-stained mitotic chromosomes of poplar (Populus tricho‑
carpa) are shown in blue. To assess the suitability of repeat-derived multilocus markers, it is important
to verify their spread across all chromosomes and chromosome regions. In this example, a probe derived
from the abundant transposable element SaliS-I (red) shows hybridization signals along all poplar chro-
mosomes.[26] The scale bar is 5 µm. (D–H) In this example, the closure of the sugar beet genome assem-
bly has been evaluated by hybridizing a terminal marker (red), the telomere (blue), and a subtelomeric
satellite DNA (yellow) to meiotic chromosome spreads of sugar beet. Based on the distance between
the terminal marker and the distal repeats, the closeness of the terminal marker to the telomere can be
evaluated. (D) Schematic representation of the hybridized probes (red, yellow, blue) onto the ends of a
pachytene chromosome (grey). (E, F) Terminal markers of the chromosome arms 1N (E, arrowhead)
and 9N (G, arrowhead) have been hybridized to pachytene spreads.[14] The close-up of 1N (F) shows the
overlapping of signals, indicating the closeness of the 1N terminal marker to the end of the chromosome.
In contrast, the close-up of 9N shows a large distance between the marker and the telomere, indicating
missing sequence information in the assembly of chromosome 9.

Source: (A) Saffron crocus FISH image reused from [8], Figure 2Q with permission from John Wiley
and Sons; (B) Banana FISH image reused from[25] under CC-BY; (C) Poplar FISH image reused from[26]
under CC-BY; (E–H) Figure 5 reused from[14] with permission from John Wiley and Sons.

it is also possible to generate bulked oligonucleotide probes, which are

directly derived from a genome assembly. As an example, the hybridiza-
tion of oligonucleotide-derived painting probes across two banana chro-
mosomes is reproduced (Figure 28.2B).[25]
350 Cytogenetics and Molecular Cytogenetics

(2) Another typical FISH application in plants is the assessment of repetitive

molecular markers and their distribution across all chromosomes. In the
example, a specifc repetitive element is hybridized onto the chromosomes
of poplar (Figure 28.2C).[26] This repeat’s dispersed occurrence along all
chromosomes shows this repeat’s suitability to act as molecular marker in
a multilocus marker system.[27]
(3) Lastly, hybridization of long-insert clones onto meiotic chromosomes
can help to assess the physical closure of sequence assemblies. For this,
the distance between the terminal marker and the telomeric region is
assessed, as was for example done for sugar beet (Figure  28.2D).[14]
Whereas the assembly of chromosome arm 1N seems terminally closed
(Figures 28.2E, 28.2F), the hybridization onto chromosome arm 9N sug-
gests long genomic regions that are not covered by the assembly (Figures
28.2G, 28.2H).

CONCLUSIONS
Taken together, for the analysis of plant genome structure and evolution as well as for
plant breeding, FISH is still a powerful technique.

ACKNOWLEDGEMENTS
The protocols laid out in the chapter have been established in the Dresden cytogenet-
ics lab, initially by the late Thomas Schmidt and the late Ines Walter. We remember
their efforts in setting up plant cytogenetics in Dresden and still admire their eye to
detail.

REFERENCES
1. Jiang, J. Fluorescence in situ hybridization in plants: Recent developments and future
applications. Chromosome Res. 2019, 27, 153–165.
2. Heslop-Harrison, J.S.; Schwarzacher, T. Organization of the plant genome in chromo-
somes. Plant J. 2011, 66, 18–33.
3. Naish, M.; Alonge, M.; Wlodzimierz, P.; Tock, A.J.; Abramson, B.W.; Schmücker, A.;
Mandáková, T.; Jamge, B.; Lambing, C.; Kuo, P.; Yelina, N.; Hartwick, N.; Colt, K.;
Smith, L.M.; Ton, J.; Kakutani, T.; Martienssen, R.A.; Schneeberger, K.; Lysak, M.A.;
Berger, F.; Bousios, A.; Michael, T.P.; Schatz, M.C.; Henderson, I.R. The genetic and
epigenetic landscape of the Arabidopsis centromeres. Science. 2021, 374, eabi7489.
4. Perničková, K.; Koláčková, V.; Lukaszewski, A.J.; Fan, C.; Vrána, J.; Duchoslav, M.;
Jenkins, G.; Phillips, D.; Šamajová, O.; Sedlářová, M.; Šamaj, J.; Doležel, J.; Kopecký,
D. Instability of alien chromosome introgressions in wheat associated with improper
positioning in the nucleus. Int. J. Mol. Sci. 2019, 20, 1448.
5. Lysak, M.A. Celebrating Mendel, McClintock, and Darlington: On end-to-end chromo-
some fusions and nested chromosome fusions. Plant Cell. 2022, 34, 2475–2491.
6. Braz, G.T.; He, L.; Zhao, H.; Zhang, T.; Semrau, K.; Rouillard, J.-M.; Torres, G.A.; Jiang,
J. Comparative oligo-FISH mapping: An effcient and powerful methodology to reveal
karyotypic and chromosomal evolution. Genetics. 2018, 208, 513.
FISH—in Plant Chromosomes 351

7. do Vale Martins, L.; Yu, F.; Zhao, H.; Dennison, T.; Lauter, N.; Wang, H.; Deng, Z.;
Thompson, A.; Semrau, K.; Rouillard, J.M.; Birchler, J.A.; Jiang, J. Meiotic crossovers
characterized by haplotype-specifc chromosome painting in maize. Nat. Commun.
2019, 10, 4604.
8. Schmidt, T.; Heitkam, T.; Liedtke, S.; Schubert, V.; Menzel, G. Adding color to a cen-
tury-old enigma: Multi-color chromosome identifcation unravels the autotriploid nature
of saffron (Crocus sativus) as a hybrid of wild Crocus cartwrightianus cytotypes. New
Phytol. 2019, 222, 1965–1980.
9. Beying, N.; Schmidt, C.; Pacher, M.; Houben, A.; Puchta, H. CRISPR-Cas9-mediated
induction of heritable chromosomal translocations in Arabidopsis. Nat. Plants. 2020, 6,
638–645.
10. Yu, W.; Yau, Y.-Y.; Birchler, J.A. Plant artifcial chromosome technology and its poten-
tial application in genetic engineering. Plant Biotechnol. J. 2016, 14, 1175–1182.
11. Schnable, P.S.; Ware, D.; Fulton, R.S.; Stein, J.C.; Wei, F.; Pasternak, S.; Liang, C.;
Zhang, J.; Fulton, L.; Graves, T.A.; Minx, P.; Reily, A.D.; Courtney, L.; Kruchowski,
S.S.; Tomlinson, C.; Strong, C.; Delehaunty, K.; Fronick, C.; Courtney, B.; Rock, S.M.;
Belter, E.; Du, F.; Kim, K.; Abbott, R.M.; Cotton, M.; Levy, A.; Marchetto, P.; Ochoa, K.;
Jackson, S.M.; Gillam, B.; Chen, W.; Yan, L.; Higginbotham, J.; Cardenas, M.; Waligorski,
J.; Applebaum, E.; Phelps, L.; Falcone, J.; Kanchi, K.; Thane, T.; Scimone, A.; Thane, N.;
Henke, J.; Wang, T.; Ruppert, J.; Shah, N.; Rotter, K.; Hodges, J.; Ingenthron, E.; Cordes,
M.; Kohlberg, S.; Sgro, J.; Delgado, B.; Mead, K.; Chinwalla, A.; Leonard, S.; Crouse,
K.; Collura, K.; Kudrna, D.; Currie, J.; He, R.; Angelova, A.; Rajasekar, S.; Mueller,
T.; Lomeli, R.; Scara, G.; Ko, A.; Delaney, K.; Wissotski, M.; Lopez, G.; Campos, D.;
Braidotti, M.; Ashley, E.; Golser, W.; Kim, H.; Lee, S.; Lin, J.; Dujmic, Z.; Kim, W.;
Talag, J.; Zuccolo, A.; Fan, C.; Sebastian, A.; Kramer, M.; Spiegel, L.; Nascimento, L.;
Zutavern, T.; Miller, B.; Ambroise, C.; Muller, S.; Spooner, W.; Narechania, A.; Ren, L.;
Wei, S.; Kumari, S.; Faga, B.; Levy, M.J.; McMahan, L.; Van Buren, P.; Vaughn, M.W.;
Ying, K.; Yeh, C.T.; Emrich, S.J.; Jia, Y.; Kalyanaraman, A.; Hsia, A.P.; Barbazuk, W.B.;
Baucom, R.S.; Brutnell, T.P.; Carpita, N.C.; Chaparro, C.; Chia, J.M.; Deragon, J.M.;
Estill, J.C.; Fu, Y.; Jeddeloh, J.A.; Han, Y.; Lee, H.; Li, P.; Lisch, D.R.; Liu, S.; Liu, Z.;
Nagel, D.H.; McCann, M.C.; SanMiguel, P.; Myers, A.M.; Nettleton, D.; Nguyen, J.;
Penning, B.W.; Ponnala, L.; Schneider, K.L.; Schwartz, D.C.; Sharma, A.; Soderlund,
C.; Springer, N.M.; Sun, Q.; Wang, H.; Waterman, M.; Westerman, R.; Wolfgruber, T.K.;
Yang, L.; Yu, Y.; Zhang, L.; Zhou, S.; Zhu, Q.; Bennetzen, J.L.; Dawe, R.K.; Jiang, J.;
Jiang, N.; Presting, G.G.; Wessler, S.R.; Aluru, S.; Martienssen, R.A.; Clifton, S.W.;
McCombie, W.R.; Wing, R.A.; Wilson, R.K. The B73 maize genome: Complexity, diver-
sity, and dynamics. Science. 2009, 326, 1112–1115.
12. Pellicer, J.; Fay, M.F.; Leitch, I.J. The largest eukaryotic genome of them all? Bot. J.
Linn. Soc. 2010, 164, 10–15.
13. Schmidt, N.; Seibt, K.M.; Weber, B.; Schwarzacher, T.; Schmidt, T.; Heitkam, T. Broken,
silent, and in hiding: Tamed endogenous pararetroviruses escape elimination from the
genome of sugar beet (Beta vulgaris). Ann. Bot. 2021, 128, 281–299.
14. Paesold, S.; Borchardt, D.; Schmidt, T.; Dechyeva, D. A sugar beet (Beta vulgaris L.)
reference FISH karyotype for chromosome and chromosome-arm identifcation, integra-
tion of genetic linkage groups and analysis of major repeat family distribution. Plant J.
2012, 72, 600–611.
15. Anamthawat-Jónsson, K. Preparation of chromosomes from plant leaf meristems for
karyotype analysis and in situ hybridization. Methods Cell Sci. 2004, 25, 91–95.
16. Heitkam, T.; Petrasch, S.; Zakrzewski, F.; Kögler, A.; Wenke, T.; Wanke, S.; Schmidt, T.
Next-generation sequencing reveals differentially amplifed tandem repeats as a major
352 Cytogenetics and Molecular Cytogenetics

genome component of Northern Europe’s oldest Camellia japonica. Chromosome Res.

2015, 23, 791–806.
17. Pradillo, M.; Heckmann, S. Plant Meiosis: Methods and Protocols. Humana, New York,
NY; 2020.
18. Nkongolo, K.K.; Klimaszewska, K. Karyotype analysis and optimization of mitotic
index in Picea mariana (black spruce) preparations from seedling root tips and embryo-
genic cultures. Heredity. 1994, 73, 11–17.
19. Albert, P.S.; Zhang, T.; Semrau, K.; Rouillard, J.-M.; Kao, Y.-H.; Wang, C.-J.R.; Danilova,
T.V.; Jiang, J.; Birchler, J.A. Whole-chromosome paints in maize reveal rearrangements,
nuclear domains, and chromosomal relationships. Proc. Natl. Acad. Sci. USA. 2019, 116,
1679–1685.
20. Schwarzacher, T.; Heslop-Harrison, P. Practical In Situ Hybridization. BIOS Scientifc
Publishers, Oxford; 2000.
21. Schwarzacher, T.; Leitch, A.R. Enzymatic treatment of plant material to spread chromo-
somes for in situ hybridization. In Protocols for Nucleic Acid Analysis By Nonradioactive
Probes; Isaac, P.G., Ed. Humana Press, Totowa, NJ, 1994, pp. 153–160.
22. Fukui, K.; Iijima, K. Somatic chromosome map of rice by imaging methods. Theor.
Appl. Genet. 1991, 81, 589–596.
23. Kazemi-Shahandashti, S.-S.; Mann, L.; El-Nagish, A.; Harpke, D.; Nemati, Z.; Usadel,
B.; Heitkam, T. Ancient artworks and crocus genetics both support saffron’s origin in
early Greece. Front. Plant Sci. 2022, 13, 834416.
24. Nemati, Z.; Harpke, D.; Kerndorff, H.; Gemicioglu, A.; Blattner, F.R. Saffron (Crocus
sativus) is an autotriploid that evolved in Attica (Greece) from wild Crocus cartwrightia‑
nus. Mol. Phylogenet. Evol. 2019, 136, 14–20.
25. Šimoníková, D.; Němečková, A.; Karafátová, M.; Uwimana, B.; Swennen, R.; Doležel,
J.; Hřibová, E. Chromosome painting facilitates anchoring reference genome sequence
to chromosomes in situ and integrated karyotyping in banana (Musa spp.). Front. Plant
Sci. 2019, 10, 1503.
26. Kögler, A.; Seibt, K.M.; Heitkam, T.; Morgenstern, K.; Reiche, B.; Brückner, M.; Wolf,
H.; Krabel, D.; Schmidt, T. Divergence of 3′ ends as driver of Short Interspersed Nuclear
Element (SINE) evolution in the Salicaceae. Plant J. 2020, 103, 443–458.
27. Reiche, B.; Kögler, A.; Morgenstern, K.; Brückner, M.; Weber, B.; Heitkam, T.; Seibt,
K.M.; Tröber, U.; Meyer, M.; Wolf, H.; Schmidt, T.; Krabel, D. Application of retrotrans-
poson-based Inter-SINE Amplifed Polymorphism (ISAP) markers for the differentia-
tion of common poplar genotypes. Can. J. For. Res. 2021, 51, 1650–1663.
29 FISH—and
CRISPR/CAS9
Thomas Liehr

CONTENTS
Introduction ............................................................................................................ 353
Samples/Tissues ..................................................................................................... 354
Description of Method ........................................................................................... 354
Yields ..................................................................................................................... 355
Conclusions ............................................................................................................ 355
References .............................................................................................................. 355

INTRODUCTION
Fluorescence in situ hybridization (FISH) is an approach that enables the mapping of
DNA-probes in nuclei and/or on chromosomes. This is an option being not available
until ~50 years ago; still research and diagnostic possibilities of FISH-technique are
still far from being fully utilized.[1] A major shortcut of FISH is that it normally can
only be done on fxed, dead and in most cases heat denatured tissues. In other words,
native chromatin structure is impaired and insights into in vivo situation of a living
and metabolic active cell, is not possible.[2]
However, results similar to those obtained by normal FISH approach can be
achieved on fxed and on living cells by taking advantage of the so-called “gene scis-
sor system” based on CRISPR/Cas9, being originally a defense system of bacterial
cells against foreign viral DNA. CRISPR stands for “Clustered Regularly Interspaced
Short Palindromic Repeats” and Cas9 is a CRISPR-associated gene #9.[3]
The CRISPR/Cas9 system is used for targeted genomic editing, and high suc-
cess rates are reported for such experiments.[3–4] However, far from all high expecta-
tions it was clear from the beginning that even rates of ~99.9% correct targeting of
a gene defect in a patient is not specifc enough to justify a responsible use in gene
therapy.[5] Furthermore, recent research showed that CRISPR/Cas9 is able to induce
chromothripsis, an effect which is defnitely unwanted, deleterious and/or a starting
point of malignifcation of cells.[6]
Still, for the topic of this book, FISH and the new possibilities in connection with
the “gene scissor system” have to be mentioned. First there is (a) FISH-like labeling
of living cells,[7–8] requiring a sophisticated cell-biological equipment and experience,
being not related to what a “standard” molecular cytogenetic laboratory normally
can do. Besides there are yet three other CRISPR/Cas9 system based approaches on

DOI: 10.1201/9781003223658-29 353

354 Cytogenetics and Molecular Cytogenetics

fxed cells. The so-called (b) “Genome Oligopaint via Local Denaturation” (GOLD)
FISH assay can detect single copy sequences using Cas9-RNA and local (relatively
low temperature) denaturation.[9] However, CAS-FISH-,[10] and RNA-guided endo-
nuclease-in situ labeling (RGEN-ISL) (synonym CRISPR-FISH) protocols,[11] allow
detection of repetitive DNA in fxed cells, without denaturation.
Here exclusively the protocol for CRISPR-FISH is treated.[11]

SAMPLES/TISSUES
CRISPR-FISH is done in fxed cells.[11] Here a ribonucleoprotein (RNP) consisting
of a target-specifc CRISPR RNA (crRNA), a fuorescent-labelled transactivating
crRNA (tracrRNA), and Cas9 endonuclease are used. Several fuorochromes can be
applied also in parallel in this approach.[11]

DESCRIPTION OF METHOD
The method of CRISPR-FISH is described acc. to[11].

1. To use the Alt-R CRISPR-Cas9 system, functional guide RNA (gRNA)

for the target region can be constructed and commercially prepared
(e.g. at Integrated DNA Technologies, https://eu.idtdna.com). Target-
DNA-sequence (~20-mer) can be selected by support of web base tool
Crisprdirect (https://crispr.dbcls.jp/). crRNA and fuorochrome-labelled
trans-activating crRNA (tracrRNA) form the two-part guide RNA.
Therefore . . .
2. dissolve lyophilized crRNA and tracrRNA-fuorochrome to 100 μM in
nuclease-free Buffer 1(30 mM Hepes, pH 7.5; 100 mM CH3COOK), each,
and store separately (−20°C).
3. Mix 1 μl 100 μM crRNA and 1 μl 100 μM tracrRNA-fuorochrome plus 8
μl Buffer 1 to get 10µM gRNA.
4. Denature gRNA mix at 95°C (5 min) and hybridize for 10 min at 37°C
(can be stored at −20°C).
5. Mix 1 µl gRNA from step 4 with
– Buffer 2 =
– 6.25 μM dCas9 protein (D10A and H840A; Novateinbio, www.nova-
teinbio.com, # PR-137213)
– 10 μl 10× buffer (200 mM Hepes (pH 7.5), 1 M KCl, 50 mM MgCl2,
50% (v/v) glycerol, 10% bovine serum albumin, 1% Tween 20)
– 10 μl 10 mM dithiothreitol
– 80 μl double distilled water.
6. Incubate the mix from step 5 for 10 min at 26°C; store at 4°C.
7. Prepare cells acc. to standard procedures and spin them after correspond-
ing fxation on slides.
8. Transfer slides in 1×PBS, 4°C.
9. Add to a slide each 100 µl of Buffer 2 at room temperature for 2 min.
FISH—and CRISPR/CAS9 355

10. Tip over the slide(s) to remove Buffer 2, add ~30 µl of fuid from step 6,
and cover with coverslip.
11. Incubate at 26°C, acc. to species and DNA-target-sequence, for 2–4 hours
in a humid chamber.
12. Wash slide(s) in 5 min in 1×PBS on ice.
13. Fix slide(s) 5 min in 4% formaldehyde/1×PBS on ice.
14. Wash with 1×PBS (5 min on ice)
15. Dehydrated in an ethanol series (70%, 90%, 96%; 2 min each) at room
temperature.
16. Counterstain with 4′,6-diamino-2-phenylindole (DAPI) in mounting
medium (Vectashield, Vector Laboratories, Burlingame, CA, USA).
17. Evaluate in a suited fuorescence microscope—laser scanning microscopy
is recommended.

YIELDS
Similar results as by standard FISH using repetitive probes (as telomeric or centro-
meric sequences) are possible using CRISPR-FISH. The advantage of CRISPR-FISH
is that tissue is not denatured. Thus, e.g. an in parallel immunostaining can be done
targeting non-degraded proteins.[12]

CONCLUSIONS
Results obtainable by taking advantage of CRISPR/Cas9 “gene scissor system” sys-
tem as discussed in this chapter remember pictures known from standard FISH-
approaches. Still the way how they are obtained is a completely different one. Thus,
the use of the designation CRISPR-FISH is traceable, but may not be the best choice
in terms of potentially misleading molecular cytogenetic labs to make them think
CRISPR-FISH is an easily adaptable alternative for them. The latter seems yet not to
be the case, and CRISPR-FISH is only to be introduced in conditions well suited for
standard FISH with a medium to high investment of time and resources.

REFERENCES
1. Liehr, T. Molecular cytogenetics in the era of chromosomics and cytogenomic
approaches. Front. Genet. 2021, 12, 720507.
2. Potlapalli, B.P.; Schubert, V.; Metje-Sprink, J.; Liehr, T.; Houben, A. Application of Tris-
HCl allows the specifc labeling of regularly prepared chromosomes by CRISPR-FISH.
Cytogenet. Genome Res. 2020, 160, 156–165.
3. Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 2018, 9,
911.
4. Li, H.; Yang, Y.; Hong, W.; Huang, M.; Wu, M.; Zhao, X. Applications of genome edit-
ing technology in the targeted therapy of human diseases: Mechanisms, advances and
prospects. Signal Transduct. Target. Ther. 2020, 5, 1.
5. Uddin, F.; Rudin, C.M.; Sen, T. CRISPR gene therapy: Applications, limitations, and
implications for the future. Front. Oncol. 2020, 10, 1387.
356 Cytogenetics and Molecular Cytogenetics

6. Leibowitz, M.L.; Papathanasiou, S.; Doerfer, P.A.; Blaine, L.J.; Sun, L.; Yao. Y.; Zhang,
C.Z.; Weiss, M.J.; Pellman, D. Chromothripsis as an on-target consequence of CRISPR-
Cas9 genome editing. Nat. Genet. 2021, 53, 895–905.
7. Wang, H.; Xu, X.; Nguyen, C.M.; Liu, Y.; Gao, Y.; Lin, X.; Daley, T.; Kipniss, N.H.;
La Russa, M.; Qi, L.S. CRISPR-mediated programmable 3D genome positioning and
nuclear organization. Cell. 2018, 175, 1405–1417.
8. Wang, H.; Nakamura, M.; Abbott, T.R.; Zhao, D.; Luo, K.; Yu, C.; Nguyen, C.M.; Lo, A.;
Daley, T.P.; La Russa, M.; Liu, Y.; Qi, L.S. CRISPR-mediated live imaging of genome
editing and transcription. Science. 2019, 365, 1301–1305.
9. Wang, Y.; Cottle, W.T.; Wang, H.; Feng, X.A.; Mallon, J.; Gavrilov, M.; Bailey, S.; Ha, T.
Genome oligopaint via local denaturation fuorescence in situ hybridization. Mol. Cell.
2021, 81, 1566–1577.
10. Deng, W.; Shi, X.; Tjian, R.; Lionnet, T.; Singer, R.H. CASFISH: CRISPR/Cas9-
mediated in situ labeling of genomic loci in fxed cells. Proc. Natl. Acad. Sci. U. S. A.
2015, 112, 11870–11875.
11. Ishii, T.; Schubert, V.; Khosravi, S.; Dreissig, S.; Metje-Sprink, J.; Sprink, T.; Fuchs, J.;
Meister, A.; Houben, A. RNA-guided endonuclease—in situ labelling (RGEN-ISL): A
fast CRISPR/Cas9-based method to label genomic sequences in various species. New
Phytol. 2019, 222, 1652–1661.
12. Ishii, T.; Kiyotaka, N.; Houben, A. Application of CRISPR/Cas9 to visualize defned
genomic sequences in fxed chromosomes and nuclei. In: Cytogenomics; Liehr, T., Eds.
Academic Press, New York, 2021, pp. 147–153.
Index
Page locators in bold indicate a table. Page locators in italics indicate a fgure.

A bacterial artifcial chromosome (BAC), 36,

49–50, 132, 173, 226, 308
aberration types, 78, 83, 164, 166 banding
aCGH (array-comparative genomic hybridization) cytogenetics, clinical application, 16
advantages/limitations, 232 methods, FISH based, 152, 154, 166–167
applications, 232–234 molecular basis of, 10
clinical case studies, 234, 236–238, 240, multicolor, 46, 166, 184
242–245 protein-based, 3
copy number variant, detection, 138, 225 techniques, 8–9, 11
results, 228 traditional, of chromosomes, 10, 73, 87, 89,
techniques, 226–227, 229–230, 246 125, 128, 134, 264
acrocentric chromosomes, 9, 11, 15, 132, 134, Barr-body, 8, 201
137, 159, 164 birds, 263–264
acute leukemia (AL), 72, 76, 82 bone marrow (BM)
acute lymphoblastic leukemia (ALL), 72, 79, 91 excess proliferation, 72
acute myeloid leukemia (AML), 18, 20, 58, 61, plasma cells, 59–60
72, 76, 91 sampling, 152, 166, 253, 274
acute promyelocytic leukemia (APL), 20, 58, 77 BPDE (benzo[a]pyrene-diolepoxide),
alveolar rhabdomyosarcoma, 64 213–214
alveolar soft-part sarcoma, 64 breakpoint cluster region protein (BCR)
amniocytes, uncultured, 53, 143 fusion protein, common, 78, 81, 90
amplifcation genes, 51, 57–58, 73, 75, 95
genome, whole (see whole genome probe, 94
amplifcation) breast cancer, 64, 173, 213, 252, 254
intrachromosomal, 18, 58, 81 Burkitt lymphoma (BL), 60, 80, 91
rolling circle, 214
aneuploidies
abortion causing, 118, 123 C
common, 50, 53, 54, 64 C-banding, 2, 3, 10, 11, 15, 264
most frequent, 16–17 cardiovascular disease, 172
aneuploidy Cas9 (CRISPR associate gene), 353–354
common, rapid prenatal prescreening, 53, 54, central nervous system (CNS), 72, 80, 234
111, 128 charge coupled device (CCD) camera, 45, 164,
human sperm, 112 314
partial, miscarriage causing, 123, 125 chorionic villus sampling, 20, 53, 123
screening procedures, 140, 182, 233 chromosomal alterations, 76, 124, 135
AneuVysion, 53 chromosomal microarray analysis (CMA), 2, 4,
Angelman syndrome, 53, 131 37, 52, 56, 154, 159–160
angiomatoid fbrous histiocytoma, 64 chromosome
assisted reproduction techniques (ARTs), 111 acrocentric chromosomes, 9, 11, 15, 132, 134,
ataxia telangiectasia (AT), 163, 165 137, 156, 164
Atlas of Genetics and Cytogenetics in Oncology insect, 319
and Hematology, 19–20, 21, 60 lampbrush, 307
autism, 172, 237 Philadelphia, 8, 18, 57, 80
plant, 339
B ring chromosomes, 129
segregation, 17, 122–123, 127
B-cell precursor acute lymphoblastic leukemia sex (see sex chromosomes)
(B-ALL), 18, 58, 62 structure, 7–8, 65, 319–320

357
358 Index

chromosome abnormalities double-strand breaks (DSB), 172, 212, 252,

detection of, 16–18 297
numerical and structural, 50, 53, 64, 182 genomic, 9, 32, 125, 213, 226, 269–270, 273,
recurrent/common, 73, 124 328
chromosome heteromorphism, 14 human genomic, 49
chromosome instability (CIN), 181–182, 183, 184 isolation, 27
chronic lymphocytic leukemia (CLL), 57, 62, 75 probe, 45, 75, 182, 273, 308, 314, 321
chronic myelogenous leukemia (CML), 18, 57, sequences, repetitive, 14, 130, 282, 300,
61, 75–76 325–326
chronic myelomonocytic leukemia (CMML), 76 sequences, specifc, 212, 264
CO-FISH (chromosome orientation), 191, 194, short tandem repeat (STR), 117
196 Down syndrome, 8, 17, 53, 54, 82, 112, 126
colorectal cancer, 252
combined ratio labeling FISH (COBRA-FISH), E
151
Comet Assay (CA), 212, 217 Edwards syndrome, 53, 54, 112, 126, 127
Comet-FISH, 212–214, 216–217 Emanuel syndrome, 55
comparative genomic hybridization (CGH), 2, endothelial cells, 253
4, 37 epidermal growth factor receptor (EGFR), 64,
array-based, 225 213
complex karyotypes (CC), 20, 83, 153 epilepsy, 172
contamination, 31, 53, 252 epithelial cells, 213, 215, 253, 254
copy number variations (CNVs), 171, 230, 253 Ewing sarcoma, 64
COVID-19, 252–253 extraskeletal myxoid chondrosarcoma, 64
Cri-du-chat syndrome, 54, 129, 130
CRISPR (clustered regularly interspaced short F
palindromic repeats), 353
cytogenetic fuorescence in situ hybridization (FISH), 2, 3,
alterations, 76, 79, 171 27, 35, 49
banding, 3, 7, 151 3D-FISH, 201–202
prenatal, 17, 53 applications, 38, 138, 181–182, 348
assays, 50–52, 55–60
D banding, 152, 154, 166–167
favorable prognosis Leukemia, 91–94
Database of Genomic Variants, 227 fsh-based assays, 43, 182
de novo (frst occurrence), 124, 126, 132, 227, multicolor, 28, 118, 151, 158, 288, 327
242 principle of, 35
clinical effect sSMC, 55, 158 probe sets, 37, 118, 157, 160 see also probes
incidence of in CNV, 138–140, 171, 178 protocol, 38, 87, 289, 292
DECIPHER (database), 227 sequential, 293
degenerate universal primer (DOP-PCR), 27, 31, in tissues, 105
272 unfavorable prognosis leukemia, 94–95
deletion validation/verifcation of clinical testing,
interstitial, 19, 129 50–52
occurrences of, 12, 51, 56, 59, 75, 82 fuorescence microscopy, 44, 347
terminal, 18, 56, 129–130, 139–140 follicular lymphoma (FL), 59, 63
desmoplastic round cell tumor, 64 formalin-fxed/paraffn-embedded (FFPE) tissue,
diandry, 124 105, 107
diffuse large B-cell lymphoma (DLBCL), 59–60
DiGeorge syndrome (DGS), 53–54, 54, 131, 132 G
digyny, 124
disability, intellectual, 8, 17, 125–126, 172, 232, G-banding
237, 240, 242–243 karyotyping, 73, 94
disease, 57–58, 61–62 see also specifc disease normal population, 12
DNA protocol, 86
AT-rich, 10, 11 gene fusion, 83, 90–91
cloning, 2 gene mapping, 39, 74
Index 359

gene-targeted probes, 49–50 Langer-Giedion syndrome, 53, 54

Genome Oligopaint via Local Denaturation leukemia
(GOLD), 354 diagnostics, 90–91
genomic disorders, 54, 130, 233, 246 karyotypic alterations, 77
genotype-phenotype correlation, 18, 227, 229, lissencephaly, 53, 253
232, 237 locus-specifc probes (LSPs), 36–37, 105
germline-restricted chromosome (GRC), 300 low copy repeats (LCRs), 54, 130, 172
GISH (genomic in situ hybridization), 282, 328 low-grade fbromyxoid sarcoma, 64
GTG-banding, 3, 29–30, 117, 126–127, 130, 132, lung cancer, 252
144, 145, 233 lymph nodes, 60, 72, 75

H M
hematological neoplasms, 19, 95–96 malignant cells, 73, 77, 213
hematology, 60, 73 maternal
hematopoietic stem cells (HSCs), 75–76 chromosome separation, 122
heparin (anticoagulant), 83, 216 testing, cytogenetic, 17, 53
homologous recombination (HR), 54, 122, 130, mesenchymal stem cells (MSCs), 253
172, 190, 203 MGMT (O6-methylguanine-DNA
Human Genome Project, 49, 74, 157 methyltransferase), 212–213
human papilloma virus (HPV), 243 microdeletion, detection of, 53, 54, 64, 131
microduplication syndromes, 53
I Miller-Dieker lissencephaly syndrome (MDLS),
53
imaging/image analysis, 45–46, 212, 313 mitochondria, 251–254
immuno-FISH, 184, 298–300, 301, 303 molecular combing, 2, 4, 37, 207, 208, 209
immunocytochemistry (ICC), 297–299, 302 momiome (mobile functions of mitochondria
infertility, human sperm and, 14, 55, 111–112, and the mitochondrial genome), 253
124, 128, 135, 148 monoclonal gammopathy of undetermined
insect signifcance (MGUS), 59–60
chromosome painting, 327 mosaicism
chromosome preparation, 323–324 chromosomal, 45
chromosome, FISH protocol, 321 gonadal, 18
sequence mapping, 325–326 somatic, 16, 55
interphase cells, 35, 56, 105, 125, 152, 202 multicolor banding (MCB), 153–154
interphase FISH, 60, 105, 112–114, 181–182 interphase chromosome-specifc, 184
inversion myelodysplastic syndrome (MDS), 18, 20, 58, 61
paracentric, 135, 136, 240, 241 myxoid round cell liposarcoma, 64
pericentric, 14, 136–137, 238, 240
isochromosomes, 124, 128–129, 133–134 N

K Nick translation, 36, 174, 272, 283, 300, 310

Nijmegen breakage syndrome (NBS), 20,
karyotype 163–164, 165
complex, 20, 76, 83 nonallelic homologous recombination (NAHR), 54
haploid, 152–153 NOR silver staining, 2, 3
high resolution, 138 nuclear DNA (nDNA), 252–253, 255
normal, 76, 94, 142, 145, 147, 246 nucleic acid, 74, 125, 185, 191, 230, 310–312
routine, 58 nucleolus organizing regions (NOR), 36, 158
kidneys, 72 numerical alterations, 77, 123, 125
Klinefelter syndrome, 8, 17, 112, 127, 128 NUMTs (nuclear mtDNA), 252, 254

L O
lampbrush chromosomes (LBC), 314 Okazaki fragments, 190
lateral loops, 308, 313, 314, 315 Online Mendelian Inheritance in Man (OMIM),
methods for obtaining, 309 163
360 Index

oxidative small lymphocytic lymphoma (SLL), 59

damage, 198 small nuclear RNAs (snRNAs), 287, 326
lesions, 190 small supernumerary marker chromosomes
stress, 190, 213 (sSMC), 55, 132, 134, 158, 159, 160
oxidative phosphorylation (OXPHOS), 253 spectral karyotyping (SKY), 151, 154, 158, 166
spleen, 72, 75, 285
P structural rearrangements
balanced, 124, 135, 142, 144, 238, 240
P1-derived artifcial chromosomes (PACs), 125 unbalanced, 129, 143, 144, 145
Pallister-Hall syndrome, 253 supermale syndrome, 127
Pallister-Killian syndrome (PKS), 55 suspension-FISH (S-FISH), 201–203
partial chromosome paints, 37, 105 synaptonemal complex (SC), 297–300
Pätau syndrome, 54, 126, 127
peptide nucleic acid (PNA), 191 T
Phelan-McDermid syndrome, 54, 140
Philadelphia chromosome, 8, 18, 57, 80 T-banding, 10, 11
plasma cell myeloma (PCM)., 59–60 T-cell acute lymphoblastic leukemia (T-ALL),
pod-FISH (parental-origin-determination- FISH), 59, 80–82
173 tetrasomies, 76
pre-implantation genetic testing (PGT), 111 topologically associating domains (TADs), 201
prenatal screening, rapid, 17, 53, 64 translocation t
primed in situ hybridization (PRINS), 4, 35 balanced, 18, 19, 55–56
reciprocal, 18, 56–58, 73, 77–78
Q translocations
balanced, 17, 56, 82
Q-banding, 9–10, 11 variant, 57, 94
QFISH (quantitative fuorescence in situ transposable elements (TEs), 287
hybridization), 44–46, 183, 191, 194 Triple X syndrome, 127
trisomies, 18, 77, 80, 83, 112, 119, 125, 137, 147
R tumors, solid, 60, 64, 105, 226, 243
Turner syndrome, 8, 17, 53, 112, 127, 128, 130,
R-banding, 10, 11 134
radio sensitivity, individual, 163–165, 167
radioactive nucleic acid, 9, 49, 74, 308 U
radiotherapy, 163, 166
Robertsonian translocation, 14, 17, 126–127, 129, uniparental disomy (UPD), 55, 142, 147, 158,
136, 137 160, 230
Usher syndrome, 253
S
V
Sanger sequencing, 2
sarcoma, 64, 108 vascular smooth muscle cells (VSMCs), 253
schizophrenia, 172 verifcation and validation, 50–52
sequencing
genome, 3, 11–12, 157, 252, 327, 348 W
long read, 201, 340
next generation (NGS), 246, 286 WAGR syndrome, 53
second/third generation, 2 Wilms tumor aniridia gonad blastoma retardation
sex chromosomes (WAGR), 53
abnormalities, 18, 19, 60, 112, 119, 127, 226
aneuploidies, 17, 54, 123 Y
systems, 282, 287
shelterin protein, 189 yeast artifcial chromosomes (YACs), 125