Chromosomes and Cell Division

Professor Varban Ganev, MD, PhD, DSc

Faculty of Medicine
Sofia University “St. Kliment Ohridski”
University Hospital “Lozenetz”
 Genetics and Genomics in Medicine
Strachan • Goodship • Chinnery
(2015) Garland Science

Essential Medical Genetics Emery's Elements of Medical

Tobias • Connor • Ferguson-Smith Genetics
(2011) Blackwell Published Ltd. Turnpenny • Ellard
(2011) Churchill Livingstone
 Let us not take it for granted that life exists more
fully in what is commonly thought big than in what
is commonly thought small.

VIRGINIA WOOLF
At the molecular or submicroscopic level, DNA can be regarded as the
basic template that provides a blueprint for the formation and
maintenance of an organism. DNA is packaged into chromosomes and at a
very simple level these can be considered as being made up of tightly coiled
long chains of genes. Unlike DNA, chromosomes can be visualized during
cell division using a light microscope, under which they appear as
threadlike structures or ‘colored bodies’. The word chromosome is derived
from the Greek chroma (= color) and soma (= body).
Chromosomes are the factors that distinguish one species from another
and that enable the transmission of genetic information from one
generation to the next. Their behavior at somatic cell division in mitosis
provides a means of ensuring that each daughter cell retains its own
complete genetic complement. Similarly, their behavior during gamete
formation in meiosis enables each mature ovum and sperm to contain a
unique single set of parental genes. Chromosomes are quite literally the
vehicles that facilitate reproduction and the maintenance of a species.
The study of chromosomes and cell division is referred to as cytogenetics.
Before the 1950s it was thought, incorrectly, that each human cell
contained 48 chromosomes and that human sex was determined by the
number of X chromosomes present at conception. Following the
development in 1956 of more reliable techniques for studying human
chromosomes, it was realized that the correct chromosome number in
humans is 46 and that maleness is determined by the presence of a Y
chromosome regardless of the number of X chromosomes present in each
cell. It was also realized that abnormalities of chromosome number and
structure could seriously disrupt normal growth and development. Table
4.1 highlights the methodological developments that have taken place
during the past 5 decades that underpin our current knowledge of human
cytogenetics.
Table 4.1 Development of methodologies for cytogenetics.
Human Chromosomes

Morphology
At the submicroscopic level, chromosomes consist of an extremely
elaborate complex, made up of supercoils of DNA, which has been likened
to the tightly coiled network of wiring seen in a solenoid. Under the
electron microscope chromosomes can be seen to have a rounded and
rather irregular morphology (Figure 4.1). However, most of our knowledge
of chromosome structure has been gained using light microscopy. Special
stains selectively taken up by DNA have enabled each individual
chromosome to be identified. These are best seen during cell division, when
the chromosomes are maximally contracted and the constituent genes can
no longer be transcribed.

At this time each chromosome can be seen to consist of two identical

strands known as chromatids, or sister chromatids, which are the result of
DNA replication having taken place during the S (synthesis) phase of the
cell cycle. These sister chromatids can be seen to be joined at a primary
constriction known as the centromere. Centromeres consist of several
hundred kilobases of repetitive DNA and are responsible for the movement
of chromosomes at cell division. Each centromere divides the chromosome
Figure 4.1 Electron micrograph of human chromosomes showing the
centromeres and well-defined chromatids.
The tip of each chromosome arm is known as the telomere. Telomeres play
a crucial role in sealing the ends of chromosomes and maintaining their
structural integrity. Telomeres have been highly conserved throughout
evolution and in humans they consist of many tandem repeats of a
TTAGGG sequence. During DNA replication, an enzyme known as
telomerase replaces the 5′ end of the long strand, which would otherwise
become progressively shorter until a critical length was reached when the
cell could no longer divide and thus became senescent. This is in fact part
of the normal cellular aging process, with most cells being unable to
undergo more than 50 to 60 divisions. However, in some tumors increased
telomerase activity has been implicated as a cause of abnormally
prolonged cell survival.

Morphologically chromosomes are classified according to the position of

the centromere. If this is located centrally, the chromosome is metacentric,
if terminal it is acrocentric, and if the centromere is in an intermediate
position the chromosome is submetacentric (Figure 4.2). Acrocentric
chromosomes sometimes have stalk-like appendages called satellites that
form the nucleolus of the resting interphase cell and contain multiple
repeat copies of the genes for ribosomal RNA.
Figure 4.2 Morphologically chromosomes are described as metacentric,
submetacentric, or acrocentric, depending on the position of the
centromere.
 Classification
Individual chromosomes differ not only in the position of the centromere,
but also in their overall length. Based on the three parameters of length,
position of the centromere, and the presence or absence of satellites, early
pioneers of cytogenetics were able to identify most individual
chromosomes, or at least subdivide them into groups labeled A to G on the
basis of overall morphology (A, 1–3; B, 4–5; C, 6–12 1 X; D, 13–15; E, 16–
18; F, 19–20; G, 21–22 1 Y). In humans the normal cell nucleus contains 46
chromosomes, made up of 22 pairs of autosomes and a single pair of sex
chromosomes—XX in the female and XY in the male. One member of each
of these pairs is derived from each parent. Somatic cells are said to have a
diploid complement of 46 chromosomes, whereas gametes (ova and sperm)
have a haploid complement of 23 chromosomes. Members of a pair of
chromosomes are known as homologs.
The development of chromosome banding enabled very precise recognition
of individual chromosomes and the detection of subtle chromosome
abnormalities. This technique also revealed that chromatin, the
combination of DNA and histone proteins that comprise chromosomes,
exists in two main forms. Euchromatin stains lightly and consists of genes
that are actively expressed. In contrast, heterochromatin stains darkly and
is made up largely of inactive, unexpressed, repetitive DNA.
 The Sex Chromosomes
The X and Y chromosomes are known as the sex chromosomes because of
their crucial role in sex determination. The X chromosome was originally
labeled as such because of uncertainty as to its function when it was
realized that in some insects this chromosome is present in some gametes
but not in others. In these insects the male has only one sex chromosome
(X), whereas the female has two (XX). In humans, and in most mammals,
both the male and the female have two sex chromosomes – XX in the
female and XY in the male. The Y chromosome is much smaller than the X
and carries only a few genes of functional importance, most notably the
testis-determining factor, known as SRY. Other genes on the Y
chromosome are known to be important in maintaining spermatogenesis.

In the female each ovum carries an X chromosome, whereas in the male

each sperm carries either an X or a Y chromosome. As there is a roughly
equal chance of either an X-bearing sperm or a Y-bearing sperm fertilizing
an ovum, the numbers of male and female conceptions are approximately
equal (Figure 4.3). In fact, slightly more male babies are born than
females, although during childhood and adult life the sex ratio evens out at
1:1.
The process of sex determination is considered in detail later.
Figure 4.3 Punnett square showing sex chromosome combinations for male
and female gametes.
Methods of Chromosome Analysis
It was generally believed that each cell contained 48 chromosomes until
1956, when Tjio and Levan correctly concluded on the basis of their
studies that the normal human somatic cell contains only 46 chromosomes.
The methods they used, with certain modifications, are now universally
employed in cytogenetic laboratories to analyze the chromosome
constitution of an individual, which is known as a karyotype. This term is
also used to describe a photomicrograph of an individual’s chromosomes,
arranged in a standard manner.

Chromosome Preparation
Any tissue with living nucleated cells that undergo division can be used for
studying human chromosomes. Most commonly circulating lymphocytes
from peripheral blood are used, although samples for chromosomal
analysis can be prepared relatively easily using skin, bone marrow,
chorionic villi, or cells from amniotic fluid (amniocytes).
In the case of peripheral (venous) blood, a sample is added to a small
volume of nutrient medium containing phytohemagglutinin, which
stimulates T lymphocytes to divide. The cells are cultured under sterile
conditions at 37°C for about 3 days, during which they divide, and
colchicine is then added to each culture. This drug has the extremely useful
property of preventing formation of the spindle, thereby arresting cell
division during metaphase, the time when the chromosomes are maximally
condensed and therefore most visible. Hypotonic saline is then added,
which causes the red blood cells to lyze and results in spreading of the
chromosomes, which are then fixed, mounted on a slide and stained ready
for analysis (Figure 4.4).

Chromosome Banding

Several different staining methods can be used to identify individual

chromosomes but G (Giemsa) banding is used most commonly. The
chromosomes are treated with trypsin, which denatures their protein
content, and then stained with a DNA-binding dye—also known as
‘Giemsa’—that gives each chromosome a characteristic and reproducible
pattern of light and dark bands (Figure 4.5).
Figure 4.4 Preparation of a karyotype.
Figure 4.5 A normal G-banded male karyotype.
G banding generally provides high-quality chromosome analysis with
approximately 400 to 500 bands per haploid set. Each of these bands
corresponds on average to approximately 6000 to 8000 kilobases (kb) (i.e.,
6 to 8 megabases [mb]) of DNA. High-resolution banding of the
chromosomes at an earlier stage of mitosis, such as prophase or
prometaphase, provides greater sensitivity with up to 800 bands per
haploid set, but is much more demanding technically. This involves first
inhibiting cell division with an agent such as methotrexate or thymidine.
Folic acid or deoxycytidine is added to the culture medium, releasing the
cells into mitosis. Colchicine is then added at a specific time interval, when
a higher proportion of cells will be in prometaphase and the chromosomes
will not be fully contracted, giving a more detailed banding pattern.

Karyotype Analysis

The next stage in chromosome analysis involves first counting the number
of chromosomes present in a specified number of cells, sometimes referred
to as metaphase spreads, followed by careful analysis of the banding
pattern of each individual chromosome in selected cells.
The banding pattern of each chromosome is specific and can be shown in
the form of a stylized ideal karyotype known as an idiogram (Figure 4.6).
The cytogeneticist analyzes each pair of homologous chromosomes, either
directly by looking down the microscope or using an image capture system
to photograph the chromosomes and arrange them in the form of a
karyogram (Figure 4.7).

Molecular Cytogenetics

Fluorescent In-Situ Hybridization

This diagnostic tool combines conventional cytogenetics with molecular
genetic technology. It is based on the unique ability of a portion of single-
stranded DNA (i.e., a probe) to anneal with its complementary target
sequence on a metaphase chromosome, interphase nucleus or extended
chromatin fiber. In fluorescent in-situ hybridization (FISH), the DNA
probe is labeled with a fluorochrome which, after hybridization with the
patient’s sample, allows the region where hybridization has occurred to be
visualized using a fluorescence microscope. FISH has been widely used for
clinical diagnostic purposes during the past 15 years and there are a
number of different types of probes that may be employed.
Figure 4.6 An idiogram showing the banding patterns of individual
chromosomes as revealed by fluorescent and Giemsa staining.
Figure 4.7 A G-banded metaphase spread.
 Different Types of FISH Probe

Centromeric probes
These consist of repetitive DNA sequences found in and around the
centromere of a specific chromosome. They were the original probes used
for rapid diagnosis of the common aneuploidy syndromes (trisomies 13, 18,
21) using non-dividing cells in interphase obtained from a prenatal
diagnostic sample of chorionic villi. In the present, quantitative fluorescent
polymerase chain reaction is more commonly used to detect these
trisomies.

Chromosome-specific unique-sequence probes

These are specific for a particular single locus. Unique-sequence probes
are particularly useful for identifying tiny submicroscopic deletions and
duplications (Figure 4.8). The group of disorders referred to as the
microdeletion syndromes are described later. Another application is the
use of an interphase FISH probe to identify HER2 overexpression in breast
tumors to identify patients likely to benefit from Herceptin treatment.
Figure 4.8 Metaphase image of Williams (ELN) region
probe (Vysis), chromosome band 7q11.23, showing the
deletion associated with Williams syndrome. The normal
chromosome has signals for the control probe (green) and
the ELN gene probe (orange), but the deleted chromosome
shows only the control probe signal.
 Telomeric probes
A complete set of telomeric probes was been developed for all 24
chromosomes (i.e., autosomes 1 to 22 plus X and Y). Using these, a method
has been devised that enables the simultaneous analysis of the
subtelomeric region of every chromosome by means of only one
microscope slide per patient. This proved to be a useful technique for
identifying tiny ‘cryptic’ subtelomeric abnormalities, but has largely been
replaced with a quantitative polymerase chain reaction method, multiplex
ligation-dependent probe amplifications, that simultaneously measures
dosage for all the subtelomeric chromosome regions.

Whole-Chromosome paint probes

These consist of a cocktail of probes obtained from different parts of a
particular chromosome. When this mixture of probes is used together in a
single hybridization, the entire relevant chromosome fluoresces (i.e., is
‘painted’). Chromosome painting is extremely useful for characterizing
complex rearrangements, such as subtle translocations (Figure 4.9), and
for identifying the origin of additional chromosome material, such as small
supernumerary markers or rings.
Figure 4.9 Chromosome painting showing a reciprocal translocation
involving chromosomes 3 (red) and 20 (green).
 Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) was originally developed to

overcome the difficulty of obtaining good-quality metaphase preparations
from solid tumors. This technique enabled the detection of regions of allele
loss and gene amplification. Tumor or ‘test’ DNA was labeled with a green
paint, and control normal DNA with a red paint. The two samples were
mixed and hybridized competitively to normal metaphase chromosomes,
and an image captured (Figure 4.10). If the test sample contained more
DNA from a particular chromosome region than the control sample, that
region was identified by an increase in the green to red fluorescence ratio
(Figure 4.11). Similarly a deletion in the test sample was identified by a
reduction in the green to red fluorescence ratio.
Array CGH
Cytogenetic techniques are traditionally based on microscopic analysis.
However, the increasing application of microarray technology is also
having a major impact on cytogenetics. Although array CGH is a
molecular biology technique, it is introduced in this chapter because it has
evolved from metaphase CGH and is being used to investigate
chromosome structure.
Figure 4.10 Comparative genomic hybridization (CGH)
analysis showing areas of gene amplification and reduction
(deletion) in tumor DNA. DAPI, diamidinophenylindole;
FITC, fluorescein isothiocyanate.
Figure 4.11 Comparison of conventional and array comparative
genomic hybridization (CGH). Both techniques involve the
hybridization of diffe-rentially labeled normal and patient DNA,
but the targets of the hybridi-zation are metaphase chromosomes
and microarrays, respectively. The results show deletions of
chromosome 10q and deletion of three clones on a 1-Mb bacterial
artificial chromosome (BAC) array.
Array CGH also involves the hybridization of patient and reference DNA,
but metaphase chromosomes are replaced as the target by large numbers
of DNA sequences bound to glass slides (Figure 4.11). The DNA target
sequences have evolved from mapped clones (yeast artificial chromosome
[YAC], bacterial artificial chromosome [BAC], or P1-derived artificial
chromosome [PAC] or cosmid), to oligonucleotides. They are spotted on to
the microscope slides using robotics to create a microarray, in which each
DNA target has a unique location. Following hybridization and washing to
remove unbound DNA, the relative levels of fluorescence are measured
using computer software. Oligonucleotide arrays provide the highest
resolution and can include up to 1 million probes.

The application of microarray CGH has extended from cancer

cytogenetics to the detection of any type of gain or loss, including the
detection of subtelomeric deletions in patients with unexplained
intellectual impairment. Array CGH is faster and more sensitive than
conventional metaphase analysis for the identification of constitutional
rearrangements (with the exception of balanced translocations) and has
replaced conventional karyotyping as the first-line test in the investigation
of patients with severe developmental delay/learning difficulties and/or
congenital abnormalities.
Chromosome Nomenclature
By convention each chromosome arm is divided into regions and each
region is subdivided into bands, numbering always from the centromere
outwards (Figure 4.12). A given point on a chromosome is designated by
the chromosome number, the arm (p or q), the region, and the band (e.g.,
15q12). Sometimes the word region is omitted, so that 15q12 would be
referred to simply as band 12 on the long arm of chromosome 15.

A shorthand notation system exists for the description of chromosome

abnormalities (Table 4.2). Normal male and female karyotypes are
depicted as 46,XY and 46,XX, respectively. A male with Down syndrome as
a result of trisomy 21 would be represented as 47,XY,+21, whereas a
female with a deletion of the short arm of one number 5 chromosome (cri
du chat syndrome) would be represented as 46,XX,del(5p). A chromosome
report reading 46,XY,t(2;4)(p23;q25) would indicate a male with a
reciprocal translocation involving the short arm of chromosome 2 at
region 2 band 3 and the long arm of chromosome 4 at region 2 band 5.
Figure 4.12 X chromosome showing the short and long arms each
subdivided into regions and bands.
Table 4.2 Symbols used in describing a karyotype.
Cell Division
Mitosis

At conception the human zygote consists of a single cell. This undergoes

rapid division, leading ultimately to the mature human adult consisting of
approximately 1 × 1014 cells in total. In most organs and tissues, such as
bone marrow and skin, cells continue to divide throughout life. This
process of somatic cell division, during which the nucleus also divides, is
known as mitosis. During mitosis each chromosome divides into two
daughter chromosomes, one of which segregates into each daughter cell.
Consequently, the number of chromosomes per nucleus remains
unchanged.

Prior to a cell entering mitosis, each chromosome consists of two identical

sister chromatids as a result of DNA replication having taken place during
the S phase of the cell cycle. Mitosis is the process whereby each of these
pairs of chromatids separates and disperses into separate daughter cells.

Mitosis is a continuous process that usually lasts 1 to 2 hours, but for

descriptive purposes it is convenient to distinguish five distinct stages.
These are prophase, prometaphase, metaphase, anaphase, and telophase
Figure 4.13 Stages of mitosis.
 Prophase
During the initial stage of prophase, the chromosomes condense and the
mitotic spindle begins to form. Two centrioles form in each cell, from
which microtubules radiate as the centrioles move toward opposite poles of
the cell.

Prometaphase
During prometaphase the nuclear membrane begins to disintegrate,
allowing the chromosomes to spread around the cell. Each chromosome
becomes attached at its centromere to a microtubule of the mitotic spindle.

Metaphase
In metaphase the chromosomes become aligned along the equatorial plane
or plate of the cell, where each chromosome is attached to the centriole by
a microtubule forming the mature spindle. At this point the chromosomes
are maximally contracted and, therefore, most easily visible. Each
chromosome resembles the letter X in shape, as the chromatids of each
chromosome have separated longitudinally but remain attached at the
centromere, which has not yet undergone division.
 Anaphase
In anaphase the centromere of each chromosome divides longitudinally
and the two daughter chromatids separate to opposite poles of the cell.

Telophase
By telophase the chromatids, which are now independent chromosomes
consisting of a single double helix, have separated completely and the two
groups of daughter chromosomes each become enveloped in a new nuclear
membrane. The cell cytoplasm also separates (cytokinesis), resulting in the
formation of two new daughter cells, each of which contains a complete
diploid chromosome complement.

The Cell Cycle

The period between successive mitoses is known as the interphase of the
cell cycle (Figure 4.14). In rapidly dividing cells this lasts for between 16
and 24 hours. Interphase commences with the G1 (G = gap) phase during
which the chromosomes become thin and extended. This phase of the cycle
is very variable in length and is re-sponsible for the variation in generation
time between different cell populations. Cells that have stopped divid-ing,
such as neurons, usually arrest in this phase and are said to have entered a
noncyclic stage known as G0.
Figure 4.14 Stages of the cell cycle. G1 and G2 are the first
and second ‘resting’ stages of interphase. S is the stage of
DNA replication. M, mitosis.
The G1 phase is followed by the S phase (S = synthesis), when DNA
replication occurs and the chromatin of each chromosome is replicated.
This results in the formation of two chromatids, giving each chromosome
its characteristic X-shaped configuration. The process of DNA replication
commences at multiple points on a chromosome.

Homologous pairs of chromosomes usually replicate in synchrony.

However, one of the X chromosomes is always late in replicating. This is
the inactive X chromosome that forms the sex chromatin or so-called Barr
body, which can be visualized during interphase in female somatic cells.
This used to be the basis of a rather unsatisfactory means of sex
determination based on analysis of cells obtained by scraping the buccal
mucosa—a ‘buccal smear’.

Interphase is completed by a relatively short G2 phase during which the

chromosomes begin to condense in preparation for the next mitotic
division.
 Meiosis

Meiosis is the process of nuclear division that occurs during the final stage
of gamete formation. Meiosis differs from mitosis in three fundamental
ways:

1. Mitosis results in each daughter cell having a diploid chromosome

complement (46). During meiosis the diploid count is halved so that each
mature gamete receives a haploid complement of 23 chromosomes.

2. Mitosis takes place in somatic cells and during the early cell divisions in
gamete formation. Meiosis occurs only at the final division of gamete
maturation.

3. Mitosis occurs as a one-step process. Meiosis can be considered as two

cell divisions known as meiosis I and meiosis II, each of which can be
considered as having prophase, metaphase, anaphase, and telophase
stages, as in mitosis (Figure 4.15).
Figure 4.15 Stages of meiosis.
 Meiosis I
This is sometimes referred to as the reduction division, because it is during
the first meiotic division that the chromosome number is halved.

Prophase I
Chromosomes enter this stage already split longitudinally into two
chromatids joined at the centromere. Homologous chromosomes pair and,
with the exception of the X and Y chromosomes in male meiosis, exchange
of homologous segments occurs between non-sister chromatids; that is,
chromatids from each of the pair of homologous chromosomes. This
exchange of homologous segments between chromatids occurs as a result
of a process known as crossing over or recombination. The importance of
crossing over in linkage analysis and risk calculation is considered later.

During prophase I in the male, pairing occurs between homologous

segments of the X and Y chromosomes at the tip of their short arms, with
this portion of each chromosome being known as the pseudoautosomal
region.
The prophase stage of meiosis I is relatively lengthy and can be subdivided
into five stages.

Leptotene
 Zygotene
Homologous chromosomes align directly opposite each other, a process
known as synapsis, and are held together at several points along their
length by filamentous structures known as synaptonemal complexes.

Pachytene
Each pair of homologous chromosomes, known as a bivalent, becomes
tightly coiled. Crossing over occurs, during which homologous regions of
DNA are exchanged between chromatids.

Diplotene
The homologous recombinant chromosomes now begin to separate but
remain attached at the points where crossing over has occurred. These are
known as chiasmata. On average, small, medium, and large chromosomes
have one, two, and three chiasmata, respectively, giving an overall total of
approximately 40 recombination events per meiosis per gamete.

Diakinesis
Separation of the homologous chromosome pairs proceeds as the
chromosomes become maximally condensed.
 Metaphase I
The nuclear membrane disappears and the chromosomes become aligned
on the equatorial plane of the cell where they have become attached to the
spindle, as in metaphase of mitosis.

Anaphase I
The chromosomes now separate to opposite poles of the cell as the spindle
contracts.

Telophase I
Each set of haploid chromosomes has now separated completely to
opposite ends of the cell, which cleaves into two new daughter gametes, so-
called secondary spermatocytes or oocytes.

Meiosis II
This is essentially the same as an ordinary mitotic division. Each
chromosome, which exists as a pair of chromatids, becomes aligned along
the equatorial plane and then splits longitudinally, leading to the
formation of two new daughter gametes, known as spermatids or ova.
 The Consequences of Meiosis

When considered in terms of reproduction and the maintenance of the

species, meiosis achieves two major objectives. First, it facilitates halving
of the diploid number of chromosomes so that each child receives half of
its chromosome complement from each parent. Second, it provides an
extraordinary potential for generating genetic diversity. This is achieved in
two ways:

1 When the bivalents separate during prophase of meiosis I, they do so

independently of one another. This is consistent with Mendel’s third law.
Consequently each gamete receives a selection of parental chromosomes.
The likelihood that any two gametes from an individual will contain
exactly the same chromosomes is 1 in 223, or approximately 1 in 8 million.

2 As a result of crossing over, each chromatid usually contains portions of

DNA derived from both parental homologous chromosomes. A large
chromosome typically consists of three or more segments of alternating
parental origin. The ensuing probability that any two gametes will have an
identical genome is therefore infinitesimally small. This dispersion of DNA
into different gametes is sometimes referred to as gene shuffling.
Gametogenesis

The process of gametogenesis shows fundamental differences in males and

females (Table 4.3). These have quite distinct clinical consequences if
errors occur.

Table 4.3 Differences in gametogenesis in males and females.

 Oogenesis

Mature ova develop from oogonia by a complex series of intermediate

steps. Oogonia themselves originate from primordial germ cells by a
process involving 20 to 30 mitotic divisions that occur during the first few
months of embryonic life. By the completion of embryogenesis at 3 months
of intrauterine life, the oogonia have begun to mature into primary oocytes
that start to undergo meiosis. At birth all of the primary oocytes have
entered a phase of maturation arrest, known as dictyotene, in which they
remain suspended until meiosis I is completed at the time of ovulation,
when a single secondary oocyte is formed. This receives most of the
cytoplasm. The other daughter cell from the first meiotic division consists
largely of a nucleus and is known as a polar body. Meiosis II then
commences, during which fertilization can occur. This second meiotic
division results in the formation of a further polar body (Figure 4.16).
Figure 4.16 Stages of oogenesis and spermatogenesis. n, haploid
number.
It is probable that the very lengthy interval between the onset of meiosis
and its eventual completion, up to 50 years later, accounts for the well
documented increased incidence of chromosome abnormalities in the
offspring of older mothers. The accumulated effects of “wear and tear” on
the primary oocyte during the dictyotene phase probably damage the cell’s
spindle formation and repair mechanisms, thereby predisposing to non-
disjunction.

Spermatogenesis
In contrast, spermatogenesis is a relatively rapid process with an average
duration of 60 to 65 days. At puberty spermatogonia, which will already
have undergone approximately 30 mitotic divisions, begin to mature into
primary spermatocytes which enter meiosis I and emerge as haploid
secondary spermatocytes. These then undergo the second meiotic division
to form spermatids, which in turn develop without any subsequent cell
division into mature spermatozoa, of which 100 to 200 million are present
in each ejaculate.

Spermatogenesis is a continuous process involving many mitotic divisions,

possibly as many as 20 to 25 per annum, so that mature spermatozoa
produced by a man of 50 years or older could well have undergone several
hundred mitotic divisions. The observed paternal age effect for new
Chromosome abnormalities

Specific disorders caused by chromosome abnormalities are considered

later. In this section, discussion is restricted to a review of the different
types of abnormality that may occur. These can be divided into numerical
and structural, with a third category consisting of different chromosome
constitutions in two or more cell lines (Box 4.1).
Numerical Abnormalities
Numerical abnormalities involve the loss or gain of one or more
chromosomes, referred to as aneuploidy, or the addition of one or more
complete haploid complements, known as polyploidy. Loss of a single
chromosome results in monosomy. Gain of one or two homologous
chromosomes is referred to as trisomy or tetrasomy, respectively.
Trisomy
The presence of an extra chromosome is referred to as trisomy. Most cases
of Down syndrome are due to the presence of an additional number 21
chromo-some; hence, Down syndrome is often known as trisomy 21. Other
autosomal trisomies compatible with survival to term are Patau syndrome
(trisomy 13) and Edwards syndrome (trisomy 18). Most other autosomal
trisomies result in early pregnancy loss, with trisomy 16 being a
particularly common finding in first-trimester spontaneous miscarriages.
The presence of an additional sex chromo-some (X or Y) has only mild
Trisomy 21 is usually caused by failure of separation of one of the pairs of
homologous chromosomes during anaphase of maternal meiosis I. This
failure of the bivalent to separate is called non-disjunction. Less often,
trisomy can be caused by non-disjunction occurring during meiosis II
when a pair of sister chromatids fails to separate. Either way the gamete
receives two homologous chromosomes (disomy); if subsequent
fertilization occurs, a trisomic conceptus results (Figure 4.17).

The origin of non-disjunction

The consequences of non-disjunction in meiosis I and meiosis II differ in
the chromosomes found in the gamete. An error in meiosis I leads to the
gamete containing both homologs of one chromosome pair. In contrast,
non-disjunction in meiosis II results in the gamete receiving two copies of
one of the homologs of the chromosome pair. Studies using DNA markers
have shown that most children with an autosomal trisomy have inherited
their additional chromosome as a result of non-disjunction occurring
during one of the maternal meiotic divisions (Table 4.4).

Non-disjunction can also occur during an early mitotic division in the

developing zygote. This results in the presence of two or more different cell
lines, a phenomenon known as mosaicism.
Figure 4.17 Segregation at meiosis of a single pair of chromosomes in, A,
normal meiosis, B, non-disjunction in meiosis I, and, C, non-disjunction in
meiosis II.
Table 4.4 Parental origin of meiotic error leading to aneuploidy.
 The cause of non-disjunction
The cause of non-disjunction is uncertain. The most favored explanation is
that of an aging effect on the primary oocyte, which can remain in a state
of suspended inactivity for up to 50 years. This is based on the well-
documented association between advancing maternal age and increased
incidence of Down syndrome in offspring. A maternal age effect has also
been noted for trisomies 13 and 18.

It is not known how or why advancing maternal age predisposes to non-

disjunction, although research has shown that absence of recombination in
prophase of meiosis I predisposes to subsequent non-disjunction. This is
not surprising, as the chiasmata that are formed after recombination are
responsible for holding each pair of homologous chromosomes together
until subsequent separation occurs in diakinesis. Thus failure of chiasmata
formation could allow each pair of homologs to separate prematurely and
then segregate randomly to daughter cells. In the female, however,
recombination occurs before birth whereas the non-disjunctional event
occurs any time between 15 and 50 years later. This suggests that at least
two factors can be involved in causing non-disjunction: an absence of
recombination between homologous chromosomes in the fetal ovary, and
an abnormality in spindle formation many years later.
 Monosomy
The absence of a single chromosome is referred to as monosomy.
Monosomy for an autosome is almost always incompatible with survival to
term. Lack of contribution of an X or a Y chromosome results in a 45,X
karyotype, which causes the condition known as Turner syndrome.

As with trisomy, monosomy can result from non-disjunction in meiosis. If

one gamete receives two copies of a homologous chromosome (disomy), the
other corresponding daughter gamete will have no copy of the same
chromosome (nullisomy). Monosomy can also be caused by loss of a
chromosome as it moves to the pole of the cell during anaphase, an event
known as anaphase lag.

Polyploidy
Polyploid cells contain multiples of the haploid number of chromosomes
such as 69, triploidy, or 92, tetraploidy. In humans, triploidy is found
relatively often in material grown from spontaneous miscarriages, but
survival beyond mid-pregnancy is rare. Only a few triploid live births have
been described and all died soon after birth.
Triploidy can be caused by failure of a maturation meiotic division in an
ovum or sperm, leading, for example, to retention of a polar body or to the
formation of a diploid sperm. Alternatively it can be caused by fertilization
of an ovum by two sperm: this is known as dispermy. When triploidy
results from the presence of an additional set of paternal chromosomes, the
placenta is usually swollen with what are known as hydatidiform changes.
In contrast, when triploidy results from an additional set of maternal
chromosomes, the placenta is usually small.

Triploidy usually results in early spontaneous miscarriage (Figure 4.18).

The differences between triploidy due to an additional set of paternal
chromosomes or maternal chromosomes provide evidence for important
‘epigenetic’ and ‘parent of origin’ effects with respect to the human
genome. These are discussed in more detail later.

Structural Abnormalities
Structural chromosome rearrangements result from chromosome
breakage with subsequent reunion in a different configuration. They can
be balanced or unbalanced. In balanced rearrangements the chromosome
complement is complete, with no loss or gain of genetic material.
Consequently, balanced rearrangements are generally harmless with the
exception of rare cases in which one of the breakpoints damages an
important functional gene. However, carriers of balanced rearrangements
Figure 4.18 Karyotype from products of
conception of a spontaneous miscarriage showing
triploidy.
When a chromosome rearrangement is unbalanced the chromosomal
complement contains an incorrect amount of chromosome material and
the clinical effects are usually serious.

Translocations
A translocation refers to the transfer of genetic material from one
chromosome to another. A reciprocal translocation is formed when a break
occurs in each of two chromosomes with the segments being exchanged to
form two new derivative chromosomes. A Robertsonian translocation is a
particular type of reciprocal translocation in which the breakpoints are
located at, or close to, the centromeres of two acrocentric chromosomes
(Figure 4.19).

Figure 4.19 Types of translocation.

 Reciprocal translocations
A reciprocal translocation involves breakage of at least two chromosomes
with exchange of the fragments. Usually the chromosome number remains
at 46 and, if the exchanged fragments are of roughly equal size, a
reciprocal translocation can be identified only by detailed chromosomal
banding studies or FISH (see Figure 4.9). In general, reciprocal
translocations are unique to a particular family, although, for reasons that
are unknown, a particular balanced reciprocal translocation involving the
long arms of chromosomes 11 and 22 is relatively common. The overall
incidence of reciprocal translocations in the general population is
approximately 1 in 500.

Segregation at meiosis
The importance of balanced reciprocal translocations lies in their behavior
at meiosis, when they can segregate to generate significant chromosome
imbalance. This can lead to early pregnancy loss or to the birth of an
infant with multiple abnormalities. Problems arise at meiosis because the
chromosomes involved in the translocation cannot pair normally to form
bivalents. Instead they form a cluster known as a pachytene quadrivalent
(Figure 4.20). The key point to note is that each chromosome aligns with
homologous material in the quadrivalent.
Figure 4.20 How a balanced reciprocal translocation involving
chromosomes 11 and 22 leads to the formation of a quadrivalent at
pachytene in meiosis I. The quadrivalent is formed to maintain
homologous pairing.
 2 : 2 Segregation

When the constituent chromosomes in the quadrivalent separate during

the later stages of meiosis I, they can do so in several different ways (Table
4.5). If alternate chromosomes segregate to each gamete, the gamete will
carry a normal or balanced haploid complement (Figure 4.21) and with
fertilization the embryo will either have normal chromosomes or carry the
balanced rearrangement. If, however, adjacent chromosomes segregate
together, this will invariably result in the gamete acquiring an unbalanced
chromosome complement. For example, in Figure 4.20, if the gamete
inherits the normal number 11 chromosome (A) and the derivative number
22 chromosome (C), then fertilization will result in an embryo with
monosomy for the distal long arm of chromosome 22 and trisomy for the
distal long arm of chromosome 11.

3 : 1 Segregation

Another possibility is that three chromosomes segregate to one gamete

with only one chromosome in the other gamete. If, for example, in Figure
4.20 chromosomes 11 (A), 22 (D) and the derivative 22 (C) segregate
together to a gamete that is subsequently fertilized, this will result in the
embryo being trisomic for the material present in the derivative 22
Table 4.5 Patterns of segregation of a reciprocal
translocation (see Figures 4.20 and 4.21).
Figure 4.21 The different patterns of 2 : 2 segregation
that can occur from the quadrivalent shown in Figure
Experience has shown that, with this particular reciprocal translocation,
tertiary tri-somy for the derivative 22 chromosome is the only viable
unbalanced product. All other patterns of malsegregation lead to early
pregnancy loss. Unfortunately, terti-ary trisomy for the derivative 22
chromosome is a serious condition in which affect-ed children have multiple
congenital abnormalities and severe learning difficulties.

Risks in reciprocal translocations

When counseling a carrier of a balanced translocation it is necessary to
consider the particular rearrangement to determine whether it could result in
the birth of an abnormal baby. This risk is usually somewhere between 1%
and 10%. For carriers of the 11;22 translocation discussed, the risk has been
shown to be 5%.

Robertsonian translocations
A Robertsonian translocation results from the breakage of two acrocentric
chromo-somes (numbers 13, 14, 15, 21, and 22) at or close to their
centromeres, with subse-quent fusion of their long arms (see Figure 4.19).
This is also referred to as centric fusion. The short arms of each chromosome
are lost, this being of no clinical impor-tance as they contain genes only for
ribosomal RNA, for which there are multiple copies on the various other
acrocentric chromosomes. The total chromosome num-ber is reduced to 45.
Because there is no loss or gain of important genetic material, this is a
 Segregation at meiosis

As with reciprocal translocations, the importance of Robertsonian

translocations lies in their behavior at meiosis. For example, a carrier of a
14q21q translocation can produce gametes with (Figure 4.22):

1. A normal chromosome complement (i.e., a normal 14 and a normal 21).

2. A balanced chromosome complement (i.e., a 14q21q translocation
chromosome).
3. An unbalanced chromosome complement possessing both the translocation
chromosome and a normal 21. This will result in the fertilized embryo having
Down syndrome.
4. An unbalanced chromosome complement with a normal 14 and a missing
21.
5. An unbalanced chromosome complement with a normal 21 and a missing
14.
6. An unbalanced chromosome complement with the translocation
chromosome and a normal 14 chromosome.

The last three combinations will result in zygotes with monosomy 21,
monosomy 14, and trisomy 14, respectively. All of these combinations are
incompatible with survival beyond early pregnancy.
Figure 4.22 Formation of a 14q21q Robertsonian translocation and
the possible gamete chromosome patterns that can be produced at
meiosis.
Translocation Down syndrome

The major practical importance of Robertsonian translocations is that they

can predispose to the birth of babies with Down syndrome as a result of the
embryo inheriting two normal number 21 chromosomes (one from each
parent) plus a translocation chromosome involving a number 21 chromosome
(Figure 4.23). The clinical consequences are exactly the same as those seen in
pure trisomy 21. However, unlike trisomy 21, the parents of a child with
translocation Down syndrome have a relatively high risk of having further
affected children if one of them carries the rearrangement in a balanced
form.

Figure 4.23 Chromosome painting showing a 14q21q Robertsonian

translocation in a child with Down syndrome. Chromosome 21 is shown in
blue and chromosome 14 in yellow.
Consequently, the importance of performing a chromosome analysis in a
child with Down syndrome lies not only in confirmation of the diagnosis, but
also in identification of those children with a translocation. In roughly two-
thirds of these latter children with Down syndrome, the translocation will
have occurred as a new (de novo) event in the child, but in the remaining one-
third one of the parents will be a carrier. Other relatives might also be
carriers. Therefore it is regarded as essential that efforts are made to identify
all adult translocation carriers in a family so that they can be alerted to
possible risks to future offspring. This is sometimes referred to as
translocation tracing, or ‘chasing’.

Risks in Robertsonian translocations

Studies have shown that the female carrier of either a 13q21q or a 14q21q
Robertsonian translocation runs a risk of approximately 10% for having a
baby with Down syndrome, whereas for male carriers the risk is 1% to 3%.
It is worth sparing a thought for the unfortunate carrier of a 21q21q
Robertsonian translocation. All gametes will be either nullisomic or disomic
for chromosome 21. Consequently, all pregnancies will end either in
spontaneous miscarriage or in the birth of a child with Down syndrome. This
is one of the very rare situations in which offspring are at a risk of greater
than 50% for having an abnormality. Other examples are parents who are
both heterozygous for the same autosomal dominant disorder, and parents
 Deletions
A deletion involves loss of part of a chromosome and results in monosomy for
that segment of the chromosome. A very large deletion is usually
incompatible with survival to term, and as a general rule any deletion
resulting in loss of more than 2% of the total haploid genome will have a
lethal outcome.

Deletions are now recognized as existing at two levels. A ‘large’ chromosomal

deletion can be visualized under the light microscope. Such deletion
syndromes include Wolf-Hirschhorn and cri du chat, which involve loss of
material from the short arms of chromosomes 4 and 5, respectively.
Submicroscopic microdeletions were identified with the help of high-
resolution prometaphase cytogenetics augmented by FISH studies and
include Prader-Willi and Angelman syndromes.

Insertions
An insertion occurs when a segment of one chromosome becomes inserted
into another chromosome. If the inserted material has moved from elsewhere
in another chromosome then the karyotype is balanced. Otherwise an
insertion causes an unbalanced chromosome complement. Carriers of a
balanced deletion–insertion rearrangement are at a 50% risk of producing
unbalanced gametes, as random chromosome segregation at meiosis will
Inversions

An inversion is a two-break rearrangement involving a single chromosome in

which a segment is reversed in position (i.e., inverted). If the inversion
segment involves the centromere it is termed a pericentric inversion (Figure
4.24A). If it involves only one arm of the chromosome it is known as a
paracentric inversion (Figure 4.24B).

Figure 4.24 A, Pericentric and, B, paracentric

inversions.
Inversions are balanced rearrangements that rarely cause problems in
carriers unless one of the breakpoints has disrupted an important gene. A
pericentric inversion involving chromosome number 9 occurs as a common
structural variant or polymorphism, also known as a heteromorphism, and is
not thought to be of any functional importance. However, other inversions,
although not causing any clinical problems in balanced carriers, can lead to
significant chromosome imbalance in offspring, with important clinical
consequences.

Segregation at meiosis
Pericentric inversions
An individual who carries a pericentric inversion can produce unbalanced
gametes if a crossover occurs within the inversion segment during meiosis I,
when an inversion loop forms as the chromosomes attempt to maintain
homologous pairing at synapsis. For a pericentric inversion, a crossover
within the loop will result in two complementary recombinant chromosomes,
one with duplication of the distal non-inverted segment and deletion of the
other end of the chromosome, and the other having the opposite arrangement
(Figure 4.25A).
If a pericentric inversion involves only a small proportion of the total length
of a chromosome then, in the event of crossing over within the loop, the
duplicated and deleted segments will be relatively large. The larger these are,
the more likely it is that their effects on the embryo will be so severe that
miscarriage ensues. For a large pericentric inversion, the duplicated and
deleted segments will be relatively small so that survival to term and beyond
becomes more likely. Thus, in general, the larger the size of a pericentric
inversion the more likely it becomes that it will result in the birth of an
abnormal infant.
The pooled results of several studies have shown that a carrier of a balanced
pericentric inversion runs a risk of approximately 5% to 10% for having a
child with viable imbalance if that inversion has already resulted in the birth
of an abnormal baby. The risk is nearer 1% if the inversion has been
ascertained because of a history of recurrent miscarriage.
Paracentric inversions
If a crossover occurs in the inverted segment of a paracentric inversion, this
will result in recombinant chromo-somes that are either acentric or dicentric
(Figure 4.25B). Acentric chromosomes, which strictly speaking should be
known as chro-mosomal fragments, cannot undergo mitotic division, so that
survival of an embryo with such a rearrangement is extremely uncommon.
Dicentric chromosomes are inherently unstable during cell division and are,
therefore, also unlikely to be com-patible with survival of the embryo. Thus,
Figure 4.25 Mechanism of production of recombinant unbalanced
chromosomes from, A, pericentric and, B, paracentric inversions by
crossing over in an inversion loop.
Ring Chromosomes
A ring chromosome is formed when a break occurs on each arm of a
chromosome leaving two ‘sticky’ ends on the central portion that reunite as a
ring (Figure 4.26). The two distal chromosomal fragments are lost so that, if
the involved chromosome is an autosome, the effects are usually serious.

Figure 4.26 Partial karyotype showing a ring

chromosome 9.
Ring chromosomes are often unstable in mitosis so that it is common to find a
ring chromosome in only a proportion of cells. The other cells in the
individual are usually monosomic because of the absence of the ring
chromosome.

Isochromosomes
An isochromosome shows loss of one arm with duplication of the other. The
most probable explanation for the formation of an isochromosome is that the
centromere has divided transversely rather than longitudinally. The most
commonly encountered isochromosome is that which consists of two long
 Mosaicism and Chimerism (Mixoploidy)
Mosaicism

Mosaicism can be defined as the presence in an individual, or in a tissue, of

two or more cell lines that differ in their genetic constitution but are derived
from a single zygote, that is, they have the same genetic origin. Chromosome
mosaicism usually results from non-disjunction in an early embryonic mitotic
division with the persistence of more than one cell line. If, for example, the
two chromatids of a number 21 chromosome failed to separate at the second
mitotic division in a human zygote (Figure 4.27), this would result in the four-
cell zygote having two cells with 46 chromosomes, one cell with 47
chromosomes (trisomy 21), and one cell with 45 chromosomes (monosomy
21). The ensuing cell line with 45 chromosomes would probably not survive,
so that the resulting embryo would be expected to show approximately 33%
mosaicism for trisomy 21. Mosaicism accounts for 1% to 2 % of all clinically
recognized cases of Down syndrome.

Mosaicism can also exist at a molecular level if a new mutation arises in a

somatic or early germline cell division. The possibility of germline or gonadal
mosaicism is a particular concern when counseling the parents of a child in
whom a condition such as Duchenne muscular dystrophy is an isolated case.
Figure 4.27 Generation of somatic mosaicism caused by mitotic non-
disjunction.
 Chimerism
Chimerism can be defined as the presence in an individual of two or more
genetically distinct cell lines derived from more than one zygote; that is, they
have different genetic origin. The word chimera is derived from the
mythological Greek monster that had the head of a lion, the body of a goat
and the tail of a dragon. Human chimeras are of two kinds: dispermic
chimeras and blood chimeras.

Dispermic chimeras
These are the result of double fertilization whereby two genetically different
sperm fertilize two ova and the resulting two zygotes fuse to form one
embryo. If the two zygotes are of different sex, the chimeric embryo can
develop into an individual with true hermaphroditism and a XX/XY
karyotype. Mouse chimeras of this type can now be produced experimentally
in the laboratory to facilitate the study of gene transfer.

Blood chimeras
Blood chimeras result from an exchange of cells, via the placenta, between
non-identical twins in utero. For example, 90% of one twin’s cells can have
an XY karyotype with red blood cells showing predominantly blood group B,
whereas 90% of the cells of the other twin can have an XX karyotype with
red blood cells showing predominantly blood group A. It has long been
recognized that, when twin calves of opposite sex are born, the female can
FURTHER READING
Barch MJ Knutsen T Spurbeck JL The AGT cytogenetics laboratory manual.
3rd ed 1997 Lippincott-Raven Philadelphia
Gersen SL Keagle MB The principles of clinical cytogenetics. 3rd ed 2011
Humana Press Totowa, NJ
Shaffer LG Slovak ML Campbell LJ An international system for human
cytogenetic nomenclature. 2009 Karger Basel
Rooney DE, Czepulkowski BH: Human chromosome preparation. Essential
techniques. 1997 John Wiley Chichester, UK
Speicher MR, Carter NP: The new cytogenetics: blurring the boundaries
with molecular biology. Nat Rev Gen. 6: 782-792 2006
Therman E, Susman M: Human chromosomes. Structure, behavior and
effects. 3rd ed 1993 Springer New York
Tjio JH, Levan A: The chromosome number of man. Hereditas. 42: 1-6 1956

WEBSITE
National Center for Biotechnology Information. Microarrays: chipping away
at the mysteries of science and medicine. Online.
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html