CYTOGENETICS

DEFINITION : Cytogenetic is the study of chromosomes and its

abnormalities: alteration in the number and structure.
MILESTONES IN CYTOGENETICS:
 Arnold: First observed chromosomes:1879
• Hansemann & Flemming Counted the chromosomes: 1891
• Winiwarter: Isolated X chromosome
• Painter: Isolated Y chromosome
• Tijio and Levan: 1956: Described correct chromosome number as 22
pair of autosomes and 2 sex chromosomes.
• Levan introduced the use of Colchicine to arrest mitosis at metaphase.
• Hsu,Makino & Nishimura and Hughes- Hypotonic technique: In 1952 in
karyotyping.
• Gall and Prudue described in situ hybridization techniques.
Chromosome : Chromosome Is a packaged and organized structure
containing the DNA of a living organism
Centromere
The centromere index is the ratio of short arm length to total arm length. Centromere
is the position in the chromosome where sister chromatids are separated into two
daughter cells which is obtained during mitosis.
Types of chromosomes
According to the location of centromere, the chromosomes are classified into 4
groups namely metacentric, submetacentric, acrocentric and telocentric.
Metacentric chromosome
A chromosome with the centromere at or near the middle is called metacentric. E.g.
1st, 3rd, 16th, 19th, and 20th are metacentric chromosomes in humans
submetacentric chromosome
A submetacentric chromosome has a centromere little away from the middle point.
e.g. 2nd, 4th to 12th, 17th, 18th, and X chromosomes.
Acrocentric chromosome
 Acrocentric chromosomes have centromeres very near the end. E.g. 13th, 14th, 15th,
21st, 22nd, and Y chromosomes are acrocentric in humans
Telocentric chromosome
Telocentric chromosomes are absent in human cells and the centromeres are at the
tip. It is not observed in humans.

INDICATIONS FOR CYTOGENETIC ANALYSIS

• Prenatal – pregnancies involving older (>35yrs) women.
• Confirmation or exclusion of diagnosis - known chromosomal
syndromes.
• Unexplained psychomotor retardation with or without dysmorphic
features.
• Abnormalities of sexual differentiation and development - ambiguous
genitalia.
• Recurrent miscarriage, stillbirth or spontaneous abortions.
• Females with proportionate short stature and primary amenorrhea.
• Parents and children of persons with chromosomal translocations,
deletions and duplications.
• Pregnancies at risk of aneuploidy from results of foetal ultrasound.
• Neoplastic conditions- soft tissues and haematological.
APPROACH TO THE DIAGNOSIS OF CYTOGENETIC
DISORDERS
1. Karyotyping
2. Insitu hybridization
3. Fluorescence insitu hybridization
4. Spectral karyotyping
5. Comparative genomic hybridization
KARYOTYPE
 • Standard display of stained and photographed chromosomes in
metaphase spread, arranged in pairs, in order of decreasing length.
• Human somatic cells - 22 pair of autosomes identical in male and
female
• sex chromosomes XX - female & XY - males.
Steps:

1. Sample collection and tissue culture

2. Arresting cells at metaphase
3. Swelling, separating and spreading chromosomes using hypotonic
solution
4. Separating chromosomes onto the slide
5. Staining or banding
6. Arranging the results- a karyotype

1.Sample collection and tissue culture

• Prenatal
- Amniotic fluid- 20ml
- Chorionic villi- 25mg of Vascularised and budding villi from chorion
frondosum
- PUBS (percutaneous umbilical blood sampling)
• Postnatal – Peripheral blood – 4 ml of heparinised –
Skin fibroblasts- 4mm diameter
– Bone marrow- 1 ml of heparinised bone marrow.
– Lymph node- 0.5 to 1 cm3
– Solid tumors- Part of specimen submitted for histopathological
examination.
Ideally 0.5-1 gm
Reagents
Tissue Culture media
RPMI1640(Rosewell Park Memorial Institute Medium)
Phytohemagglutinin : mitotic stimulant
Antibiotics: penicillin, streptomycin .
 • Short term culture: 1-3 days – blood, bonemarrow, chorionic villi
• Long term culture: 1-3 wks - other tissue types
Arrest of cell division: at metaphase, by Colchicine [Deacetyl methyl
colchicine] for 20 min.
. Cells harvested – centrifugation
Incubated for 10min in hypotonic solution (dilute solution of KCl 0.075
mol) .
Cell fixation- 3:1 methanol/ glacial acetic acid mixture (carnoy’s) for
30min.
Staining : Trypsinization of the chromosomes prior to staining, weakens
the DNA-Protein interactions, add buffer (Na2HPO4 and NaH2PO4),
banding techniques done with the dyes.
Microscopic analysis and photography
Karyotype production (manual/automated)
Interpretation
Staining
Q-banding-
• The first banding method developed
• Uses quinacrine mustard or quinacrine dihydrochloride,
• creates a flourescent transfers band on exposure to UV light,
• Q- bands fade over time not routinely used .
G-banding
• Uses Giemsa dye to produce transverse bands light (G-C rich DNA) dark band
(A-T rich DNA)
• G-bands are identical to Q- bands
• G-banding is the most widely used banding technique for routine chromosome
analysis
• Around 400 bands per haploid genome seen.
Each band corresponds to 5-10megabases
R-banding
• Treating chromosomes with a hot alkaline solution before Giemsa staining
• Produces bands that are the reverse of G-bands, called R- bands.
• In R banding telomeres should appear as dark bands and their absence as the
result of deletion is more obvious.
C-banding
• selectively stains constitutive heterochromatin, and are located at all
centromeres and distal long arm of Y chromosome.
• Staining with giemsa followed by heat denaturation results in darkly staining
heterochromatic regions at centromere with light staining chromosome arms.
Nuclear organizing region(NOR) banding
• Specific chromosomal region that forms and maintain the nucleoli are called
NOR.
• NOR located on stalk of acrocentric chromosomes and contain gene for 18S
and 28S rRNA.
• Stained by Geimsa (N- banding) or silver impregnation ( Ag-NOR)
Successful cytogenetic analysis depends on –
- Cells must be in adequate numbers
- Analysis must be performed on viable cells in division
- Chromosomes must be separated from one another
- Chromosomes must be identified & characterized normal/abnormal
- Arranged according to the length in a decreasing order.
Karyotyping Chromosome from each metaphase spread are arranged in
prescribed order
– karyotype cells
– imaged, printed & karyotyped
ISCN International System for Human Cytogenetic Nomenclature.
• Centromere divide the chromosome into short arm and long am
• Chromosome arms divided into regions on the basis of landmarks.
The region adjacent to centromere of short arm and long arm are given number
1 as p1, q1 respectively, the next distal region is given 2 and so on
• The regions are subdivided into bands and the bands are subdivided into sub
bands as the resolution increases and the numbering done sequentially
ISCN of chromosome 11
IN SITU HYBRIDIZATION
• Hybridization refers to the binding or annealing of complementary
DNA or RNA sequences
• Main purpose – detection of specific nucleic acid sequences in
chromosomes.
• In early studies, radio isotopes were used as labels for nucleic acids, and
detection of hybridized sequence were done with autoradiography.
• As technology advanced, detection by enzymatic and fluorescent means
become available for quick and safe analysis.
• Uses- Detection of missing, additional chromosomes, chromosome
rearrangements and microdeletions.
FLUORESCENT IN SITU HYBRIDIZATION
The probe and metaphase target are denatured by a high temperature and
formamide. Probe is hybridized to the chromosomal target. Unbound
probe is removed by post hybridization washes. Bound probe is detected
by fluorescence microscopy
Types of probes
1] Centromere enumerating probe(CEP) - Bind to highly repitative
sequence alpha satellite sequences of centromere and produce strong
signals. Similar sequences in pericentric region results in cross
hybridization artifact.
2] Locus specific identifier(LSI) probe Target distinct chromosomal
region of interest and utilize single copy rather than repetitive DNA.
 3] Whole chromosomes probes Also known as chromosome painting
probes or chromosomes libraries, consists of thousands of overlapping
probes that recognize unique and moderately repetitive sequences along
the entire length of individual chromosomes
Advantages –
- Many more cells can be examined at a single time.
- Metaphases are not essential, so abnormalities can be detected in non dividing
cells.
- Can be performed in a shorter period of time.
- Abnormalities that cannot be detected by conventional cytogenetic analysis
may be detected.
Main disadvantage –
- Only those abnormalities that are specifically sought will be found whereas
conventional analysis permits all chromosomes to be evaluated.
SPECTRAL KARYOTYPING
(multicolor fluorescence in-situ hybridization)
• 24-colour, multi-chromosomal painting assay that allows visualization of all
human chromosomes in one experiment.
• Uses –
1. Ability to detect complex chromosomal rearrangements.
2. Identifies marker chromosomes– makes this highly sensitive and valuable
tool for identifying recurrent chromosomal abnormalities.
SPK
Spectral karyotyping chromosomes of a single cell Pepsin treatment( at 37
degree C for 3-5 min) labeled with a different combination of fluorescent dyes
and allowed to hybridize, specific for each chromosome Imaged immediately
and Spectral karyotype done using SPK View software
Comparative genomic hybridization 2 genomes Test DNA Normal DNA
Labeled with 2 different fluorescence(green and red) dyes Allowed to hybridize
2 samples are equal focal deletion or Produce yellow fluorescence duplication
fluorescence skewed towards green or red.
• Uses
- Has higher sensitivity
- Can be performed using DNA extracted from fixed as well as tumor sample
- Technique makes it possible to perform a genome wide scan for structural
alteration even on those cases for which other cytological analysis is not
feasible or successful
CHROMOSOMALABNORMALITIES NUMERICAL STRUCTURAL
Karyotype with abnormal no. of chromosomes Include chromosome losses &
gains Alterations in structure Include loss, rearrangements or gain of
chromosome segments
1.Numerical abnormalities
• Haploid- gametes 23 or N
• Diploid- 46 or 2N
• Euploid- exact multiple of N eg: 3N (triploid), 4N (tetraploid)
• Aneuploid- indicates noneuploid, loss or gain of single chromosomes eg:
monosomy, trisomy
• Most common mechanism of aneuploidy- nondisjunction of chromosomes
• Nondisjunction in Meiosis I, results in 2 gametes with parental
chromosomes that fail to separate and 2 nullisomic gametes
• Nondisjunction in meiosis II results in 1 gamete with two identical
chromosomes, 1 nullisomic and 2 normal gametes.
• Monosome: fertilization of nullisomic with normal gamete
• Trisome: fertilization of gamete retaining both paternal and maternal or
both copies of either maternal or paternal with normal gamete
• Mosaicism: Nondisjunction when occurs in mitosis, a condition where
individual has two or more cell lines of different chromosomal
constitution derived from same zygote.
2.Structural abnormalities
• Translocation
- Reciprocal translocation - Robertsonian translocation
• Inversion
- Paracentric inversion
- Pericentric inversion
• Deletion
- Terminal deletion
- Inserstitial deletion
• Microdeletions :
- Subtype of chromosome deletion that can be observed only in banded
chromosomes or in some cases using molecular genetic approaches.
• Duplications:
- Intra chromosomal gain of chromatin lying in the same linear
orientation (direct) reverse orientation (inverted ) with respect to
centromere.
• Insertion
- Interchromosome insertion - Intrachromosome insertion
- Direct insertion
- Inverted insertion
• Isochromosomes
– either two identical short arms or two identical long arms. This occurs
as a result of transfers split instead of longitudinal split during meiosis
and mitosis.
• Ring chromosome
- these are formed when a break occurs on each arm of chromosomes
followed by fusion of the exposed ends to create a circular structure. The
distal fragments are lost because they lack the centromere.
• Uniparental disomy:
- A condition in which one parent has contributed 2 copies of
chromosome and other parent has contributed no copies. - Ex: Prader-
Willi syndrome Angelman syndrome
CYTOGENETIC ABNORMALITIES
1. Chromosomal disorders
- Autosomes - Sex chromosomes
2. Cancer cytogenetics
- Soft tissue tumors
- Hematological disorders
DOWNS SYNDROME
• John Langdon Down in1866
• Trisomy of chromosome 21
• 1 in 700 live births
• Major cause of mental retardation
• Maternal age has a strong influence – as the age increases the risk of
down syndrome increases
Trisomy 21 Karyotype and FISH
General Hypotonia with tendency to keep mouth open. Protruding
tongue Craniofacial Brachycephaly with flat occiput. Mild microcephaly.
Upslanting palpebral fissures Late closure of fontanelles. Aplasia of
frontal sinus. Low nasal bridge Inner epicanthal folds.
Eyes Speckling of iris (Brushfield spots)
Fine lens opacity, refractive error Nystagmus, strabismus, blocked tear
duct Ears Small, overfolding of upper helix Small or absent ear lobes
Hearing loss Skin loose folds in posterior neck (infancy) Cutis marmorata
– extremities
Hands Short metacarpal and phalanges Hypoplasia of mid phalanx of 5th
finger Single palmar deep flexion crease-simian crease
Feet Wide gap between 1st and 2nd toes rocker-bottom feet Cardiac
Endocardial cushion defects-40% VSD,PDA,ASD,MVP AR by 20yrs of
age
EDWARD SYNDROME
• TRISOMY 18 SYNDROME
• 1 per 6,000 newborn babies,<5% survive to term
• 47,XY/XX+18 • Second most common autosomal trisomy
Trisomy 18 syndrome
Karyotype and FISH
General Prenatal growth deficiency Craniofacial Characteristic facial
features Small ear, small mouth, Retrognathia
Hands and feet clenched hand, overlapping of fingers Nail hypoplasia,
short big toes rocker bottom feet Thorax short sternum, small nipples
Abdominal wall unbilical hernia, small pelvis Omphalocele-protrusion of
bowel into umbilical cord others VSD,cryptorchidism, hirsutism.
PATAU SYNDROME TRISOMY 13 SYNDROME
1 in every 5,000 births 47,XY,+13 Craniofacial Mental retardation
Microcephaly, microphthalmia,coloboma of iris Cleft lip, palate/both
Abnormal helices, low set ears Skin Capillary hemangioma, loose skin
Hands & feet Distal palmar triradii,flexion of fingers Polydactyly Cardiac
VSD,PDA. Others Cryptorchidism,bicornuate uterus
Trisomy 13 syndrome
Karyotype and FISH
TURNERS SYNDROME •
45X SYNDROME - X0,
• Complete or partial monosomy of X chromosome characterized by
hypogonadism in phenotypic females.
• Henry Turner – 1938 • 1 in 2000 live born females.
Karyotyping in Turner
Classic facial features Epicanthal folds Down-slanting palpebral fissures
Flat nasal bridge Receding chin Low set ears Excessive nuchal skin folds.
Webbed neck
KLINEFELTERS (XXY) SYNDROME
• Male hypogonadism
• 2 or more X chromosomes and one or more Y chromosomes.
• Harry Klinefelter – 1942
• Most common cause of hypogonadism and infertility
• 1 in 500 males affected
• Classic pattern – 47XXY karyotype in 82% of cases.
• Other mosaic patterns – 46XY/47XXY, 47XXY/48XXXY,
48XXXY/49XXXXY.
Karyotype 47,XXY
Performance Normal to low IQ Delayed speech, Poor memory Behavioral
problems Problems with psychosocial adjustment Growth Long limbs,
Low upper to lower segment ratio Tall and slim stature Gonads
Hypogonadism,Hypogenitalism Others Elbow dysplasia, FSH and
Estradiol Testosterone,Gynecomastia
XXXXX SYNDROME PENTA X SYNDROME
• First described by Dr.Nirmala kesaree and Wooly in 1963.
• Found the abnormality in prisoners in America.
Karyotype of XXXXX
 Mental retardation
 • Prenatal growth retardation
 • Short stature
 • Microcephaly
 • Hypertelorism
 • Low set ears
  • Mongoloid slant
 • Abnormal behavior
 • Clinodactyly of fingers
CRI-DU-CHAT SYNDROME(5p-)
• Deletion of short arm of chromosome 5 (5p-)
• Critical region : 5p15
• 1 in 15,000 to 1 in 50,000
Microcephaly Round face Hypertelorism Epicanthal folds Downward
slanting of palpebral fissures Strabismus – often divergent Low set/poorly
formed ears Facial asymmetry Cat like cry- mewing of a cat, due to
abnormal laryngeal development, become less pronounced with increasing
age
. PRADER-WILLI SYNDROME
• 1 in 15,000.
• Mechanism: Deletion of 15q at q11-q13(paternally derived)-75% Maternal
UPD – 2 maternal, no paternal copies of 15q – 20% Chromosomal
translocation involving proximal 15q – 5%
Craniofacial Almond shaped eyes.
Upslanting palpebral fissures Strabismus
Thin upper lip Performance
Mental retardation mild 63%,moderate 31% Excess appetite ,obsession with
eating, obesity Hands and feet Small Narrow hands, straight ulnar border,
Genitalia- Small penis, cryptorchidism, Hypoplastic labia minora & clitoris,
Hypogonadism
• HAPPY PUPPET SYNDROME – abnormal puppet like gait, characteristic
facies, paroxysms of laughter, due to brain stem defect – not apparently
associated with happiness
• Mechanism Deletion of 15q11 ( maternal origin )
-75% Paternal uniparental disomy in 2% Imprinting mutation 2%
Happy disposition, an open mouth expression, widely spaced teeth, and a
pronounced mandible
CLINICAL APPLICATIONS OF CYTOGENETICS IN
HEMATOLOGICAL DISORDERS
• Confirmation of the diagnosis of CML
• Confirmation of blast crisis of CML
• Diagnosis of Acute leukemias
• Diagnosis of lymphoproliferative disorders
• Diagnosis of non hodgkins lymphomas
Chronic Myeloid Leukaemia-(Ph+)
• t(9:22)(q34;q11)/BCR-ABL abnormality- Philadelphia chromosome ,
identified in approximately 92 % of CML patients Other abnormalities
• Del(9q) • +8 • i(17q)
Polycythemia vera (PV)
• The most common anomalies are - +8, - –7, or a del(7q) - del(11q) -
del(13q) - del(20q).
Acute Myeloid Leukaemia
• Broadly classified as being favorable, intermediate or poor prognostic
types Eg –extensive numerical/structural karyotype abnormality
aggressive myelodysplastic background t(15:17) t(8:21) favorable inv (16)
or related t(16:16)
Acute Lymphoblastic Leukaemia
• most common - t(9:22)
• Tumor genetics →used for risk evaluation - hyperdiploidy(>52 chr)
→favorabble - t(12:21) → favorable - t(1:19) → unfavorable - t(9:22)→
unfavorable - 11q23 translocation like t(4;11), t(11;19) → unfavorable -
hypodiploidy(<40 chr) →unfavorable
Lymphoproliferative disorders
• CLL - Only 50 % of CLL patients have detectabl chromosomal
abnormalities - Trisomy 12 – more common – worse prognosis - Less
often structural abnormalities seen – del(13q), del(14q), del(17p)/- 17,
del(6q)
• Multiple myeloma structural abnormalities of chromosome- t(14;16),
t(4;14)
Non hodgkin’s lymphoma(NHL)
• t(14;18) in follicular lymphoma
• t(8;14), t(2;8) and t(8;22) in Burkitt lymphoma • t(11;14) in mantle cell
lymphoma
• t(3;22)or t(3;14) in diffuse large B-cell lymphomas (DLBCL)
• t(2;5) or t(1;2) in Anaplastic large cell lymphoma (ALCL)
NEED OF CYTOGENETICS IN SOFT TISSUE TUMORS
• Understanding of soft tissue tumor biology
• A substantial set of soft tissue tumors contain specific karyotypic
abnormalities and thus helps in diagnosis
• Provide insight into pathogenesis, classification, prognostic factors.
A Karyotype from a lipoma shows the most common rearrangement
t(3;12)(q27;q15)
Karyotype of a Ewing’s tumor showing translocation of chromosome 11
and 22, chromosome 3 on right side is shorter than its partner because of a
deletion.
Karyotype of a benign schwannoma with monosomy of chromosome 22
Complex Karyotype of a malignant peripheral nerve sheeth tumor
showing aneuploid with numerous chromosomal gain, losses and
rearrangement.
Karyotype of dermatofibrosarcoma protuberans showing supernumerary
ring chromosome.