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.