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 (p. 31). Under the electron microscope chromosomes can be seen to have a rounded and rather irregular morphology (Figure 3.1). 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.

Figure 3.1 Electron micrograph of human chromosomes showing the centromeres and well-defined chromatids.

(Courtesy Dr. Christine Harrison. Reproduced from Harrison et al 1983 Cytogenet Cell Genet 35: 21–27; with permission of the publisher, S. Karger, Basel.)

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 (p. 39). 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 into short and long arms, designated p (= petite) and q (‘g’ = grande), respectively.

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 3.2). 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 3.2 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 (p. 33) 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 (p. 92). 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 3.3). In fact, slightly more male babies are born than females, although during childhood and adult life the sex ratio evens out at 1 : 1.

Figure 3.3 Punnett square showing sex chromosome combinations for male and female gametes.

The process of sex determination is considered in detail later (p. 101).

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 3.4).

Figure 3.4 Preparation of a karyotype.

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 3.5).

Figure 3.5 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 3.6). 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 3.7).

Figure 3.6 An idiogram showing the banding patterns of individual chromosomes as revealed by fluorescent and Giemsa staining.

Figure 3.7 A G-banded metaphase spread.

(Courtesy Mr. A. Wilkinson, Cytogenetics Unit, City Hospital, Nottingham, UK.)

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; see p. 35) 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.

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; see p. 274) 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 3.8). The group of disorders referred to as the microdeletion syndromes are described in Chapter 18. 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 3.8 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.

(Courtesy Catherine Delmege, Bristol Genetics Laboratory, Southmead Hospital, Bristol, UK.)

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 3.9), and for identifying the origin of additional chromosome material, such as small supernumerary markers or rings.

Figure 3.9 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 (p. 220). 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 3.10). 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 3.11). Similarly a deletion in the test sample was identified by a reduction in the green to red fluorescence ratio.

Figure 3.10 Comparative genomic hybridization (CGH) analysis showing areas of gene amplification and reduction (deletion) in tumor DNA. DAPI, diamidinophenylindole; FITC, fluorescein isothiocyanate.

(Courtesy Dr. Peter Lichter, German Cancer Research Center, Heidelberg, and Applied Imaging.)

Figure 3.11 Comparison of conventional and array comparative genomic hybridization (CGH). Both techniques involve the hybridization of differentially labeled normal and patient DNA, but the targets of the hybridization 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 data courtesy Dr. John Barber, National Genetics Reference Laboratory [Wessex], Salisbury, UK.)

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.

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 3.11). 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.

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 (p. 39). 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 3.13).

Figure 3.13 Stages of mitosis.

The Cell Cycle

The period between successive mitoses is known as the interphase of the cell cycle (Figure 3.14). 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 responsible for the variation in generation time between different cell populations. Cells that have stopped dividing, such as neurons, usually arrest in this phase and are said to have entered a noncyclic stage known as G₀.

Figure 3.14 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 (p. 14).

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 (p. 103) 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:

Figure 3.15 Stages of meiosis.

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 3.16).

Figure 3.16 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 (p. 44). The accumulating 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 (p. 17).

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 dominant mutations (p. 113) is consistent with the concept that many mutations arise as a consequence of DNA copy errors occurring during mitosis.

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 chromosome; hence, Down syndrome is often known as trisomy 21. Other autosomal trisomies compatible with survival to term are Patau syndrome (trisomy 13) (p. 275) and Edwards syndrome (trisomy 18) (p. 275). 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 chromosome (X or Y) has only mild phenotypic effects (p. 104).

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 3.17).

Figure 3.17 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.

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 3.4).

Table 3.4 Parental origin of meiotic error leading to aneuploidy

Chromosome Abnormality	Paternal (%)	Maternal (%)
Trisomy 13	15	85
Trisomy 18	10	90
Trisomy 21	5	95
45,X	80	20
47,XXX	5	95
47,XXY	45	55
47,XYY	100	0

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 (p. 50).

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 (p. 41). This is based on the well-documented association between advancing maternal age and increased incidence of Down syndrome in offspring (see Table 18.4; see p. 275). 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.

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 (p. 101). 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 3.18). 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 in Chapter 6.

Figure 3.18 Karyotype from products of conception of a spontaneous miscarriage showing triploidy.

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 are often at risk of producing children with an unbalanced chromosomal complement.

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 3.19).

Figure 3.19 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 3.9). 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 3.20). The key point to note is that each chromosome aligns with homologous material in the quadrivalent.

Figure 3.20 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 3.5). If alternate chromosomes segregate to each gamete, the gamete will carry a normal or balanced haploid complement (Figure 3.21) 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 3.20, 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.

Table 3.5 Patterns of segregation of a reciprocal translocation (see Figures 3.20 and 3.21)

Pattern of Segregation	Segregating Chromosomes	Chromosome Constitution in Gamete
2 : 2
Alternate	A + D	Normal
	B + C	Balanced translocation
Adjacent-1 (non-homologous centromeres segregate together)	A + C or B + D	Unbalanced, leading to a combination of partial monosomy and partial trisomy in the zygote
Adjacent-2 (homologous centromeres segregate together)	A + B or C + D
3 : 1
Three chromosomes	A + B + C A + B + D A + C + D B + C + D	Unbalanced, leading to trisomy in the zygote
One chromosome	A B C D	Unbalanced, leading to monosomy in the zygote

Figure 3.21 The different patterns of 2 : 2 segregation that can occur from the quadrivalent shown in Figure 3.20. (See Table 3.5.)

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 3.20 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 chromosome. This is sometimes referred to as tertiary trisomy. Experience has shown that, with this particular reciprocal translocation, tertiary trisomy for the derivative 22 chromosome is the only viable unbalanced product. All other patterns of malsegregation lead to early pregnancy loss. Unfortunately, tertiary trisomy for the derivative 22 chromosome is a serious condition in which affected 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 chromosomes (numbers 13, 14, 15, 21, and 22) at or close to their centromeres, with subsequent fusion of their long arms (see Figure 3.19). This is also referred to as centric fusion. The short arms of each chromosome are lost, this being of no clinical importance as they contain genes only for ribosomal RNA, for which there are multiple copies on the various other acrocentric chromosomes. The total chromosome number is reduced to 45. Because there is no loss or gain of important genetic material, this is a functionally balanced rearrangement. The overall incidence of Robertsonian translocations in the general population is approximately 1 in 1000, with by far the most common being fusion of the long arms of chromosomes 13 and 14 (13q14q).

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 3.22):

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.

Figure 3.22 Formation of a 14q21q Robertsonian translocation and the possible gamete chromosome patterns that can be produced at meiosis.

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.

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 3.23). 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 3.23 Chromosome painting showing a 14q21q Robertsonian translocation in a child with Down syndrome. Chromosome 21 is shown in blue and chromosome 14 in yellow.

(Courtesy Meg Heath, City Hospital, Nottingham, UK.)

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 (p. 113), and parents who are both homozygous for the same gene mutation causing an autosomal recessive disorder, such as sensorineural deafness.

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 3.24A). If it involves only one arm of the chromosome it is known as a paracentric inversion (Figure 3.24B).

Figure 3.24 A, Pericentric and, B, paracentric inversions.

(Courtesy Dr. J. Delhanty, Galton Laboratory, London.)

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 3.25A).

Figure 3.25 Mechanism of production of recombinant unbalanced chromosomes from, A, pericentric and, B, paracentric inversions by crossing over in an inversion loop.

(Courtesy Dr. J. Delhanty, Galton Laboratory, London.)

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 chromosomes that are either acentric or dicentric (Figure 3.25B). Acentric chromosomes, which strictly speaking should be known as chromosomal 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 compatible with survival of the embryo. Thus, overall, the likelihood that a balanced parental paracentric inversion will result in the birth of an abnormal baby is extremely low.

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 3.26). The two distal chromosomal fragments are lost so that, if the involved chromosome is an autosome, the effects are usually serious.

Figure 3.26 Partial karyotype showing a ring chromosome 9.

(Courtesy Meg Heath, City Hospital, Nottingham.)

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 arms of the X chromosome. This accounts for up to 15% of all cases of Turner syndrome (p. 277).

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 3.27), 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.

Figure 3.27 Generation of somatic mosaicism caused by mitotic non-disjunction.

Mosaicism can also exist at a molecular level if a new mutation arises in a somatic or early germline cell division (p. 120). 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 (p. 307) is an isolated case.