Principles of Clinical Cytogenetics and Genome Analysis PDF

C H A P T E R 5 Principles of Clinical Cytogenetics and Genome Analysis Clinical cytogenetics is the study of chromosomes, their hundreds to thousands of genes found on individual structure, and their in...

C H A P T E R 5 Principles of Clinical Cytogenetics and Genome Analysis Clinical cytogenetics is the study of chromosomes, their hundreds to thousands of genes found on individual structure, and their inheritance, as applied to the prac- chromosomes or for smaller numbers of genes located tice of medicine. It has been apparent for over 50 years within a particular chromosome region. Application of that chromosome abnormalities—microscopically visible these principles to some of the most common and best- changes in the number or structure of chromosomes— known chromosomal and genomic disorders will then could account for a number of clinical conditions that be presented in Chapter 6. are thus referred to as chromosome disorders. With their focus on the complete set of genetic material, cytogeneticists were the first to bring a genome-wide INTRODUCTION TO CYTOGENETICS AND perspective to the practice of medicine. Today, chro­ mosome analysis—with increasing resolution and preci- GENOME ANALYSIS sion at both the cytological and genomic levels—is an The general morphology and organization of human important diagnostic procedure in numerous areas of chromosomes, as well as their molecular and genomic clinical medicine. Current genome analyses that use composition, were introduced in Chapters 2 and 3. To approaches to be explored in this chapter, including be examined by chromosome analysis for clinical pur- chromosomal microarrays and whole-genome sequenc- poses, cells must be capable of proliferation in culture. ing, represent impressive improvements in capacity The most accessible cells that meet this requirement are and resolution, but ones that are conceptually similar white blood cells, specifically T lymphocytes. To prepare to microscopic methods focusing on chromosomes a short-term culture that is suitable for cytogenetic anal- (Fig. 5-1). ysis of these cells, a sample of peripheral blood is Chromosome disorders form a major category of obtained, and the white blood cells are collected, placed genetic disease. They account for a large proportion of in tissue culture medium, and stimulated to divide. After all reproductive wastage, congenital malformations, a few days, the dividing cells are arrested in metaphase and intellectual disability and play an important role in with chemicals that inhibit the mitotic spindle. Cells are the pathogenesis of cancer. Specific cytogenetic disor- treated with a hypotonic solution to release the chro- ders are responsible for hundreds of distinct syndromes mosomes, which are then fixed, spread on slides, and that collectively are more common than all the single- stained by one of several techniques, depending on the gene diseases together. Cytogenetic abnormalities are particular diagnostic procedure being performed. They present in nearly 1% of live births, in approximately are then ready for analysis. 2% of pregnancies in women older than 35 years who Although ideal for rapid clinical analysis, cell cultures undergo prenatal diagnosis, and in fully half of all spon- prepared from peripheral blood have the disadvantage taneous, first-trimester abortions. of being short-lived (3 to 4 days). Long-term cultures The spectrum of analysis from microscopically visible suitable for permanent storage or further studies can be changes in chromosome number and structure to anom- derived from a variety of other tissues. Skin biopsy, a alies of genome structure and sequence detectable at the minor surgical procedure, can provide samples of tissue level of whole-genome sequencing encompasses literally that in culture produce fibroblasts, which can be used the entire field of medical genetics (see Fig. 5-1). In this for a variety of biochemical and molecular studies as chapter, we present the general principles of chromo- well as for chromosome and genome analysis. White some and genome analysis and focus on the chromo- blood cells can also be transformed in culture to form some mutations and regional mutations introduced in lymphoblastoid cell lines that are potentially immortal. the previous chapter. We restrict our discussion to Bone marrow has the advantage of containing a high disorders due to genomic imbalance—either for the proportion of dividing cells, so that little if any culturing 57 58 THOMPSON & THOMPSON GENETICS IN MEDICINE Unit of resolution Approximate size Typical diagnostic approach Haploid genome s3,000,000,000 bp 109 Standard karyotyping 108 Whole chromosome 50-250,000,000 bp 107 Chromosome band (400-550-band stage) 5-15,000,000 bp Routine banding Increasing resolution Chromosome band (850-band stage) 1-3,000,000 bp High-resolution banding 106 Base pairs Comparative genome hybridization 105 Submicroscopic region 50-250,000 bp FISH analysis Chromosomal 104 microarrays 103 102 Whole-genome Nucleotide(s) 1-1,000 bp sequencing 10 1 Figure 5-1 Spectrum of resolution in chromosome and genome analysis. The typical resolution and range of effectiveness are given for various diagnostic approaches used routinely in chromo- some and genome analysis. See text for details and specific examples. FISH, Fluorescence in situ hybridization. is required; however, it can be obtained only by the Chromosome Identification relatively invasive procedure of marrow biopsy. Its The 24 types of chromosome found in the human main use is in the diagnosis of suspected hematological genome can be readily identified at the cytological level malignancies. Fetal cells derived from amniotic fluid by specific staining procedures. The most common of (amniocytes) or obtained by chorionic villus biopsy can these, Giemsa banding (G banding), was developed in also be cultured successfully for cytogenetic, genomic, the early 1970s and was the first widely used whole- biochemical, or molecular analysis. Chorionic villus genome analytical tool for research and clinical diagno- cells can also be analyzed directly after biopsy, without sis (see Figs. 2-1 and 2-10). It has been the gold standard the need for culturing. Remarkably, small amounts of for the detection and characterization of structural and cell-free fetal DNA are found in the maternal plasma numerical genomic abnormalities in clinical diagnostic and can be tested by whole-genome sequencing (see settings for both constitutional (postnatal or prenatal) Chapter 17 for further discussion). and acquired (cancer) disorders. Molecular analysis of the genome, including whole- G-banding and other staining procedures can be used genome sequencing, can be carried out on any appro­ to describe individual chromosomes and their variants priate clinical material, provided that good-quality or abnormalities, using an internationally accepted sys­ DNA can be obtained. Cells need not be dividing tem of chromosome classification. Figure 5-2 is an ideo- for this purpose, and thus it is possible to study DNA gram of the banding pattern of a set of normal human from tissue and tumor samples, for example, as well chromosomes at metaphase, illustrating the alternating as from peripheral blood. Which approach is most pattern of light and dark bands used for chromosome appropriate for a particular diagnostic or research identification. The pattern of bands on each chromo- purpose is a rapidly evolving area as the resolution, some is numbered on each arm from the centromere sensitivity, and ease of chromosome and genome analy- to the telomere, as shown in detail in Figure 5-3 for sis increase (see Box). several chromosomes. The identity of any particular  CHAPTER 5 — Principles of Clinical Cytogenetics and Genome Analysis 59 chro­mosomes, with an off-center centromere and arms CLINICAL INDICATIONS FOR CHROMOSOME AND GENOME ANALYSIS of clearly different lengths; and acrocentric chromo- somes, with the centromere near one end. A potential Chromosome analysis is indicated as a routine diagnostic fourth type of chromosome, telocentric, with the cen- procedure for a number of specific conditions encountered tromere at one end and only a single arm, does not in clinical medicine. Some general clinical situations indi- cate a need for cytogenetic and genome analysis: occur in the normal human karyotype, but it is occa- Problems of early growth and development. Failure to sionally observed in chromosome rearrangements. The thrive, developmental delay, dysmorphic facies, mul- human acrocentric chromosomes (chromosomes 13, 14, tiple malformations, short stature, ambiguous genita- 15, 21, and 22) have small, distinctive masses of chro- lia, and intellectual disability are frequent findings in matin known as satellites attached to their short arms children with chromosome abnormalities. Unless there is a definite nonchromosomal diagnosis, chromosome by narrow stalks (called secondary constrictions). The and genome analysis should be performed for patients stalks of these five chromosome pairs contain hundreds presenting with any combination of such problems. of copies of genes for ribosomal RNA (the major com- Stillbirth and neonatal death. The incidence of ponent of ribosomes; see Chapter 3) as well as a variety chromosome abnormalities is much higher among of repetitive sequences. stillbirths (up to approximately 10%) than among live births (approximately 0.7%). It is also elevated In addition to changes in banding pattern, nonstain- among infants who die in the neonatal period ing gaps—called fragile sites—are occasionally observed (approximately 10%). Chromosome analysis should at particular sites on several chromosomes that are be performed for all stillbirths and neonatal deaths prone to regional genomic instability. Over 80 common that that do not have a clear basis to rule out a fragile sites are known, many of which are heritable chromosome abnormality. In such cases, karyotyping (or other comprehensive ways of scanning the variants. A small proportion of fragile sites are associ- genome) is essential for accurate genetic counseling. ated with specific clinical disorders; the fragile site These analyses may provide important information most clearly shown to be clinically significant is seen for prenatal diagnosis in future pregnancies. near the end of the long arm of the X chromosome Fertility problems. Chromosome studies are indicated in males with a specific and common form of X-linked for women presenting with amenorrhea and for couples with a history of infertility or recurrent mis- intellectual disability, fragile X syndrome      (Case 17), carriage. A chromosome abnormality is seen in one or as well as in some female carriers of the same genetic the other parent in 3% to 6% of cases in which there defect. is infertility or two or more miscarriages. Family history. A known or suspected chromosome or genome abnormality in a first-degree relative is an High-Resolution Chromosome Analysis indication for chromosome and genome analysis. Neoplasia. Virtually all cancers are associated with The standard G-banded karyotype at a 400- to 550- one or more chromosome abnormalities (see Chapter band stage of resolution, as seen in a typical metaphase 15). Chromosome and genome evaluation in the preparation, allows detection of deletions and duplica- tumor itself, or in bone marrow in the case of hema- tions of greater than approximately 5 to 10 Mb any- tological malignant neoplasms, can offer diagnostic or prognostic information. where in the genome (see Fig. 5-1). However, the Pregnancy. There is a higher risk for chromosome sensitivity of G-banding at this resolution may be lower abnormality in fetuses conceived by women of in regions of the genome in which the banding patterns increased age, typically defined as older than 35 years are less specific. (see Chapter 17). Fetal chromosome and genome anal- To increase the sensitivity of chromosome analysis, ysis should be offered as a routine part of prenatal care in such pregnancies. As a screening approach for the high-resolution banding (also called prometaphase most common chromosome disorders, noninvasive banding) can be achieved by staining chromosomes that prenatal testing using whole-genome sequencing is have been obtained at an early stage of mitosis (pro- now available to pregnant women of all ages. phase or prometaphase), when they are still in a rela- tively uncondensed state (see Chapter 2). High-resolution banding is especially useful when a subtle structural abnormality of a chromosome is suspected. Staining of band (and thus the DNA sequences and genes within it) prometaphase chromosomes can reveal up to 850 bands can be described precisely and unambiguously by use or even more in a haploid set, although this method is of this regionally based and hierarchical numbering frequently replaced now by microarray analysis (see system. later). A comparison of the banding patterns at three Human chromosomes are often classified into three different stages of resolution is shown for one chromo- types that can be easily distinguished at metaphase by some in Figure 5-4, demonstrating the increase in diag- the position of the centromere, the primary constriction nostic precision that one obtains with these longer visible at metaphase (see Fig. 5-2): metacentric chro- chromosomes. Development of high-resolution chromo- mosomes, with a more or less central centromere and some analysis in the early 1980s allowed the discovery arms of approximately equal length; submetacentric of a number of new so-called microdeletion syndromes 60 THOMPSON & THOMPSON GENETICS IN MEDICINE 1 36.2 35 2 3 34.2 26 33 24 24 6 7 11 31 22 22 p 4 5 24 8 9 10 16 22 21 12 15.2 22 23 14 14 21 14 14 15.3 15.1 14 21.2 14 21 12 12 12 12 12 12 13 12 12 12 12 12 12 12 12 12 12 13.1 13 12 12 12 14 21 14.1 13.3 14 14.3 16 21 21.1 21 22 22 14 14 22 24 21.3 22 21 24 24 21 23 22 q 26 22 31 31 24 23 23 23 31 26.1 28 33 25 24 33 24 26.3 31.2 24.2 24.2 32 28 26 35 32 32 41 34 34 34 43 36 X 16 22.2 17 19 20 13 14 15 13.2 18 21 22 21 p 12 11.3 13.2 12 11.3 Y 12 11.3 12 12 11.2 12 12 11.22 12 14 12 12 12 21 13 12 13.2 21 21.2 13.2 12 21 21 22 13.4 21 23 22 21 24 q 23 23 23 31 25 31 26.2 25 33 27 Figure 5-2 Ideogram showing G-banding patterns for human chromosomes at metaphase, with approximately 400 bands per haploid karyotype. As drawn, chromosomes are typically represented with the sister chromatids so closely aligned that they are not recognized as distinct entities. Cen- tromeres are indicated by the primary constriction and narrow dark gray regions separating the p and q arms. For convenience and clarity, only the G-dark bands are numbered. For examples of full numbering scheme, see Figure 5-3. See Sources & Acknowledgments. caused by smaller genomic deletions or duplications in expanded both the scope and precision of chromosome the 2- to 3-Mb size range (see Fig. 5-1). However, the analysis in routine clinical practice. time-consuming and technically difficult nature of this FISH technology takes advantage of the availability method precludes its routine use for whole-genome of ordered collections of recombinant DNA clones con- analysis. taining DNA from around the entire genome, generated originally as part of the Human Genome Project. Clones containing specific human DNA sequences can be used Fluorescence In Situ Hybridization as probes to detect the corresponding region of the Targeted high-resolution chromosome banding was genome in chromosome preparations or in interphase largely replaced in the early 1990s by fluorescence in nuclei for a variety of research and diagnostic purposes, situ hybridization (FISH), a method for detecting the as illustrated in Figure 5-5: presence or absence of a particular DNA sequence or DNA probes specific for individual chromosomes, for evaluating the number or organization of a chromo- chromosomal regions, or genes can be labeled with some or chromosomal region in situ (literally, “in place”) different fluorochromes and used to identify particu- in the cell. This convergence of genomic and cytoge- lar chromosomal rearrangements or to rapidly diag- netic approaches—variously termed molec­ular cytoge- nose the existence of an abnormal chromosome netics, cytogenomics, or chromonomics—dramatically number in clinical material.  CHAPTER 5 — Principles of Clinical Cytogenetics and Genome Analysis 61 Metaphase Interphase 15.3 15.2 25 15.1 24 p 14 23 22 22.3 13.3 22.2 Locus-specific 13.2 22.1 21 13.1 probes 12 21.3 11 15.2 15.3 11.1 21.2 15.1 11.2 21.1 14 12 12 13 11.2 13.1 11.1 11.2 12 13.2 11 11.1 13.3 12 11.1 13 11.21 14 11.22 14 11.23 15 Satellite DNA 15 probes 21 16.1 21.1 16.2 q 22 16.3 21.2 21.3 23.1 21 23.2 22 23.3 22.1 22.2 31.1 31.1 22.3 31.2 Figure 5-5 Fluorescence in situ hybridization to human chro­ 31.2 23.1 23.2 23.3 31.3 mosomes at metaphase and interphase, with different types of 31.3 DNA probe. Top, Single-copy DNA probes specific for sequences 32 24 32 33.1 within bands 4q12 (red fluorescence) and 4q31.1 (green fluores- 33.2 33.3 33 25.2 25.1 34 cence). Bottom, Repetitive α-satellite DNA probes specific for the 25.3 34 26 35 centromeres of chromosomes 18 (aqua), X (green), and Y (red). 35.1 36 See Sources & Acknowledgments. 35.2 27 35.3 5 6 7 target a specific genomic region based on a clinical Figure 5-3 Examples of G-banding patterns for chromosomes 5, 6, and 7 at the 550-band stage of condensation. Band numbers diagnosis or suspicion. permit unambiguous identification of each G-dark or G-light band, for example, chromosome 5p15.2 or chromosome 7q21.2. See Sources & Acknowledgments. Genome Analysis Using Microarrays Although the G-banded karyotype remains the front- line diagnostic test for most clinical applications, it has been complemented or even replaced by genome-wide p approaches for detecting copy number imbalances at higher resolution (see Fig. 5-1), extending the concept of targeted FISH analysis to test the entire genome. Instead of examining cells and chromosomes in situ one probe at a time, chromosomal microarray techniques q simultaneously query the whole genome represented as an ordered array of genomic segments on a microscope slide containing overlapping or regularly spaced DNA X segments that represent the entire genome. In one Figure 5-4 The X chromosome: ideograms and photomicro- approach based on comparative genome hybridization graphs at metaphase, prometaphase, and prophase (left to right). (CGH), one detects relative copy number gains and See Sources & Acknowledgments. losses in a genome-wide manner by hybridizing two samples—one a control genome and one from a patient—to such microarrays. An excess of sequences Repetitive DNA probes allow detection of satellite from one or the other genome indicates an overrepre- DNA or other repeated DNA elements localized to sentation or underrepresentation of those sequences in specific chromosomal regions. Satellite DNA probes, the patient genome relative to the control (Fig. 5-6). An especially those belonging to the α-satellite family of alternative approach uses “single nucleotide polymor- centromere repeats (see Chapter 2), are widely used phism (SNP) arrays” that contain versions of sequences for determining the number of copies of a particular corresponding to the two alleles of various SNPs around chromosome. the genome (as introduced in Chapter 4). In this case, Although FISH technology provides much higher the relative representation and intensity of alleles in dif- resolution and specificity than G-banded chromosome ferent regions of the genome indicate if a chromosome analysis, it does not allow for efficient analysis of the or chromosomal region is present at the appropriate entire genome, and thus its use is limited by the need to dosage (see Fig. 5-6). 62 THOMPSON & THOMPSON GENETICS IN MEDICINE Microarray with 1.2 DNA segments 0.6 Log2 ratio Gain 0 -0.6 No change -1.2 B Position along the genome Loss Test Reference DNA DNA A +4 +1 +2 Mean ratio (logR) -1 0 -2 -4 p22.32 p22.2 p22.12 p21.3 p21.1 p11.3 p11.22 q12 q13.2 q21.1 q21.31 q21.33 q22.2 q23 q26 q26.2 q27.1 q27.3 C Figure 5-6 Chromosomal microarray to detect chromosome and genomic dosage. A, Schematic of an array assay based on comparative genome hybridization (CGH), where a patient’s genome (denoted in green) is cohybridized to the array with a control reference genome (denoted in red). The probes are mixed and allowed to hybridize to their complementary sequences on the array. Relative intensities of hybridization of the two probes are measured, indicating equivalent dosage between the two genomes (yellow) or a relative gain (green) or loss (red) in the patient sample. B, A typical output plots the logarithm of the fluorescence ratios as a function of the position along the genome. C, Array CGH result for a patient with Rett syndrome      (Case 40), indicating a duplication of approximately 800 kb in band Xq28 containing the MECP2 gene. LogR of fluo- rescence ratios are plotted along the length of the X chromosome. Each dot represents the ratio for an individual sequence on the array. Sequences corresponding to the MECP2 gene and its sur- rounding region are duplicated in the patient’s genome, leading to an increased ratio, indicated by the green arrow and shaded box in that region of the chromosome. See Sources & Acknowledgments. For routine clinical testing of suspected chromosome conventional G banding. Based on this significantly disorders, probe spacing on the array provides a resolu- increased yield, genome-wide arrays are replacing the tion as high as 250 kb over the entire unique portion of G-banded karyotype as the routine frontline test for the human genome. A higher density of probes can be certain patient populations. used to achieve even higher resolution (