The general morphology and organization of human chromosomes, as well as their molecular and genomic composition. Chromosome analysis can be performed for clinical purposes by obtaining peripheral blood and stimulating T lymphocytes to prepare short- term cultures. After a few days, the dividing cells are arrested in metaphase with chemicals that inhibit the mitotic spindle, and chromosomes are fixed to glass slides and stained by one of several techniques, depending on the particular diagnostic procedure being performed. They are then ready for analysis.

Although ideal for rapid clinical analysis, cell cultures prepared from peripheral blood have the disadvantage of being short lived (3– 4 days). Long- term cultures suitable for permanent storage or further studies can be derived from a variety of other tissues. Skin biopsy, a minor surgical procedure, can provide samples of tissue that in culture produce fibroblasts, which can be used for a variety of bio chemical and molecular studies as well as for chromosome and genome analysis. White blood cells can also be trans formed in culture to form lymphoblastoid cell lines that are potentially immortal. Bone marrow has the advantage of containing a high proportion of dividing cells so that little if any culturing is required; however, it can be obtained only by the relatively invasive procedure of marrow biopsy. Its main use is in the diagnosis of suspected hematologic malignancies. Fetal cells derived from amniotic fluid (amniocytes) or obtained by chorionic villus biopsy can also be cultured successfully for cytogenetic, genomic, biochemical, or molecular analysis. Chorionic villus cells can also be analyzed directly after biopsy, without the need for culturing. Remarkably, small amounts of cell- free fetal DNA are found in the maternal plasma and can be tested by WGS.

Molecular analysis of the genome, including WGS, can be carried out on any appropriate clinical mate rial, provided that good- quality DNA can be obtained. Cells need not be dividing for this purpose, and thus it is possible to study DNA from tissue and tumor samples, for example, as well as from peripheral blood. Which approach is most appropriate for a particular diagnostic or research purpose is a rapidly evolving area as the resolution, sensitivity, and ease of chromosome and genome analysis increase (see Box1).

Box1.  CLINICAL INDICATIONS FOR CHROMOSOME AND GENOME ANALYSIS

Chromosome Identification

The different chromosomes in the genome can be identified cytologically by their characteristic banding pat terns after applying specific staining procedures. The most common of these, Giemsa banding (G- banding), was developed in the early 1970s and was the first widely used whole genome analytic tool for research and clinical diagnosis that may still apply. It has been the gold standard for the detection and characterization of structural and numerical genomic abnormalities in clinical diagnostic settings for both constitutional (postnatal or prenatal) and acquired (cancer) disorders.

G- banding and other staining procedures can be used to describe individual chromosomes and their variants or abnormalities, using an internationally accepted system of chromosome classification. Fig.1 is an ideogram of the banding pattern of a set of normal human chromosomes at metaphase, illustrating the alternating pattern of light and dark bands used for chromosome identification. The pattern of bands on each chromo some is numbered on each arm from the centromere to the telomere, as shown in detail in Fig. 2 for several chromosomes. The identity of any particular band (and thus the DNA sequences and genes within it) can be described precisely and unambiguously by use of this regionally based and hierarchic numbering system.

Fig1. Ideogram showing G- banding patterns for human chromosomes at metaphase, with ~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. Centromeres 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 Fig. 2. (Redrawn from Shaffer LG, McGowan- Jordan J, Schmid M, editors: ISCN 2013: an international system for human cytogenetic nomenclature, Basel, 2013, Karger.)

Fig2. Examples of G- banding patterns for chromosomes 5, 6, and 7 at the 550- band stage of condensation. Band numbers permit unambiguous identification of each G- dark or G- light band. The banding nomenclature indicates the chromosome number (1– 22,X,y), the short arm (p) or long arm (q), the region, band, and subband. For example, chromosome 5p15.2 is pronounced as “5- p- one- five- point- 2.” (Redrawn from Shaffer LG, McGowan- Jordan J, Schmid M, editors: ISCN 2013: an international system for human cytogenetic nomenclature, Basel, 2013, Karger.)

Human chromosomes are often classified into three types that can be easily distinguished at metaphase by the position of the centromere, the primary constriction visible at metaphase (see Fig. 1). Metacentric chromosomes have a central centromere and arms of approximately equal length, submetacentric chromosomes have an off- center centromere and arms of clearly different lengths, and acrocentric chromosomes have the centromere near one end. A potential fourth type of chromo some, telocentric, with the centromere at one end and only a single arm, does not occur in the normal human karyotype, but it is occasionally observed in chromo some rearrangements. The human acrocentric chromosomes (13, 14, 15, 21, and 22) have small, distinctive masses of chromatin known as satellites attached to their short arms by narrow stalks (called secondary con strictions). The stalks of these five chromosome pairs contain hundreds of copies of genes for ribosomal RNA (the major component of ribosomes) as well as a variety of repetitive sequences.

The standard G- banded karyotype at a 400- to 550- band stage of resolution, as seen in a typical metaphase preparation, allows detection of deletions and duplications greater than ~5 to 10 Mb. However, the sensitivity of G- banding at this resolution may be lower in regions of the genome in which the banding patterns are less specific. High- resolution banding (also called prometaphase banding) can achieve 850 or more bands in a haploid set by staining chromosomes that have been obtained at an early stage of mitosis (prophase or prometaphase), when they are still in a relatively uncondensed state. Development of high- resolution chromosome analysis in the early 1980s allowed the discovery of a number of new microdeletion and microdyuplication syndrome, caused by smaller genomic rearrangements in the 2-to-3 Mb size range. However, the time- consuming and technically difficult nature of this cytogenetic method precludes its routine use for whole genome analysis.

In addition to changes in banding pattern, nonstaining gaps, called fragile sites, are heritable variants that can be observed at particular chromosome sites that are prone to regional genomic instability induced by stress on DNA replication. Over 100 common and rare (population frequency <5%) fragile sites are documented. Common fragile sites are postulated to drive genomic instability in cancer cells, and a small proportion of rare fragile sites are associated with specific clinical disorders. For example, the rare fragile site located at Xq27.3 is caused by an expansion of CGG repeats and is observed in patients with fragile X syndrome.

Fluorescence In Situ Hybridization (FISH)

Targeted high- resolution chromosome banding was largely replaced in the early 1990s by FISH, a method for detecting the presence or absence of a particular DNA sequence or for evaluating the number or structural organization of a chromosome or chromosomal region in situ (literally, “in place”) in the cell. This convergence of genomic and cytogenetic approaches—variously termed molecular cytogenetics or cytogenomics—dramatically expanded both the scope and precision of chromosome analysis in routine clinical practice.

FISH technology takes advantage of ordered col lections of recombinant large- insert DNA clones containing DNA from virtually any locus in the genome. Clones containing specific human DNA sequences can be labeled with a fluorescent dye and used as probes to detect the corresponding region of the genome in chromosome preparations or in interphase nuclei for a variety of research and diagnostic purposes (Fig. 3).

Fig3. Fluorescence in situ hybridization to human chromosomes at metaphase and interphase, with different types of DNA probe. (Top) Single- copy DNA probes specific for sequences within bands 4q12 (red fluorescence) and 4q31.1 (green fluorescence). (Bottom) Repetitive α- satellite DNA probes specific for the centromeres of chromosomes 18 (aqua), X (green), and y (red) used to count the number of each chromosome in this individual. (Images courtesy M. Katharine Rudd, Emory Genetics Laboratory, Atlanta, Georgia.)

Although FISH technology provides much higher resolution and specificity than G- banded chromosome analysis, it does not allow for efficient analysis of the entire genome. Its use is limited to targeting a specific genomic region based on a clinical diagnosis or suspicion, structural characterization of genomic imbalances and family follow-up studies.

Multiplex Ligation- Dependent Probe Amplification (MLPA)

MLPA is a targeted copy number assay used to detect exon- level deletions and duplications in a gene or targeted chromosome region. This method uses multiplex polymerase chain reaction (PCR) to amplify DNA sequences from multiple exons simultaneously in one PCR. The relative quantity of DNA sequence generated from each exon is then compared to amplification of control regions with normal copy number (two copies). Since the total quantity of amplified PCR product is directly proportional to the copy number of each targeted exon in the individual DNA sample, a heterozygous deletion (one copy) will produce approximately half as much PCR product when compared to regions with normal copy number (two copies), and a heterozygous duplication (three copies) will generate ~50% more PCR product when compared to regions with normal copy number. This method is limited by the number of targeted regions that can be included in one PCR assay and is not amenable to genome- wide copy number analysis. It is used to investigate a specific gene (e.g., DMD) or known recurrent microdeletion/ microduplication syndrome region (e.g., 22q11.2). MLPA can be combined with methylation analysis (MS- MLPA) to specifically amplify targeted chromo some regions that are methylated, to determine imprinting status. As described in Chapter 8, a subset of the genome is differentially imprinted depending on the parent of origin; and abnormal methylation of these regions can be identified by MS- MPLA to confirm a diagnosis of imprinting disorder (e.g., 15q11.2q13 in Prader- Willi and Angelman syndromes.

Genome Analysis Using Microarrays Chromosome microarray analysis (CMA) has replaced G- banded karyotype as the

frontline diagnostic test to detect genome- wide copy number imbalances for most clinical applications. CMA simultaneously queries the whole genome on a glass slide containing regularly spaced DNA probes that represent loci across the entire genome. This technology detects relative copy number gains and losses in a genome- wide manner by hybridizing equal amounts of control and subject DNA to the DNA probes and calculating the ratio of each DNA sample hybridized to each probe. Microarray probes showing equal ratio of subject and control DNA indicate normal copy number at the respective genomic loci. An excess of subject DNA indicates copy number gain, whereas underrepresentation of subject DNA indicates copy number loss at the genomic loci represented by the microarray probes (Fig. 4). Microarray platforms may contain copy number probes (see earlier). Alternatively, they may comprise single nucleotide polymorphism (SNP) probes that contain versions of sequences corresponding to the variant alleles (as introduced in Chapter 4). The data from SNP probes can be plotted on an allele difference plot, which indicates whether a specific SNP locus is homozygous for the A allele (AA), homozygous for the B allele (BB), or heterozygous (AB). Normal copy number across a chromosome typically shows the three allele combinations of AA, AB, and BB along its length (see Fig. 4). A genomic region of homozygosity (ROH), with AA and BB but no AB track, can be observed when the chromosome region is identical by decent (due to parental consanguinity) or when there is uniparental disomy (UPD) with both copies of the chromosome inherited from one parent. The identification of several ROHs across the genome of a patient and involving multiple chromosomes suggests parental consanguinity and raises the possibility of a recessive disorder. While an ROH affecting only one chromo some raises the possibility of UPD, parental genotype analysis is required to confirm that the ROH is in fact due to UPD.

Fig4. Chromosome microarray analysis to detect copy number variants and regions of homology. (A) Chromosome 17: G-banding ideogram, followed by an example of copy number and single nucleotide polymorphism (SNP) microarray output, showing the Log2 ratio of fluorescence intensity and allele difference plots. DNA probes (blue dots) with a Log2 ratio of 0 indicate diploid copy number. In chromosome region 17p11.2, consecutive probes with a Log2 ratio of − 1 indicate a heterozygous deletion of ~3.7 Mb, associated with Smith- Magenis syndrome. (B) Chromosome 18: SNP microarray output plot showing a region of homozygosity of ~6.052 Mb. The total copy number is unaffected, but the allele difference plot shows a stretch of only homozygous genotypes (AA or BB) with no heterozygous genotypes (AB). (Microarray images courtesy of Genome Diagnostics, The Hospital for Sick Children.)

For routine clinical testing of suspected chromo some disorders, probe spacing on the array provides a resolution as high as 100 kb over the entire unique portion of the human genome. A higher density of probes can be used to achieve even higher resolution (20 kb) over regions of particular clinical interest, such as those associated with known developmental disorders or congenital anomalies. This approach is being used in clinical laboratories to provide high- resolution analysis of targeted clinically significant genes and lower resolution backbone coverage across the rest of the genome. Microarrays have been used successfully to identify chromosome and genome abnormalities in children with unexplained developmental delay, intellectual disability, or birth defects, revealing a number of pathogenic genomic alterations that were not detectable by conventional G- banding. Based on this significantly increased yield (1– 3% from karyotype versus 15– 20% from microarray), genome- wide arrays have replaced the G- banded karyotype as the routine frontline test for these patient populations.

Two important limitations of this technology bear mentioning, however. First, array- based methods measure only the relative copy number of DNA sequences but not whether they have been translocated or rearranged from their normal position(s) in the genome. Thus further characterization of copy number variants (CNVs) by karyotyping or FISH is important to determine the nature of an abnormality and thus its risk for recurrence for other family members. Second, high- resolution genome analysis can reveal variants in particular, small differences in copy number, that are of uncertain clinical significance. An increasing number of such variants are being documented and catalogued even from the general population. As we saw in Chapter 4, many are likely to be benign CNVs. Their existence underscores the unique nature of an individual’s genome and emphasizes the diagnostic challenge of assessing what is considered normal and what is likely to be pathogenic.

Genome Analysis by Whole Genome Sequencing

 On the same spectrum as cytogenetic and microarray analysis, the ultimate resolution for clinical tests to detect chromosomal and genomic disorders would be to sequence genomes in their entirety. Indeed, as the efficiency of WGS has increased and its costs have fallen, it is becoming increasingly practical to sequence samples in a clinical setting.

The most widely used WGS approach generates mil lions of short- sequence reads that range between 100 and 500 bp in length, depending on the sequencing platform.

An individual’s genome is represented by overlapping sequence reads, with typically 30 to 40 reads corresponding to any particular segment of the genome. A genomic region or chromosome with an abnormally low or high representation of those sequence reads is likely to have a numeric or structural abnormality of that genomic region. To detect numeric abnormalities of an entire chromo some it is generally not necessary to sequence a genome to completion; even a limited number of sequences that align to a particular chromosome of interest should reveal whether those sequences are found in the expected number (e.g., equivalent to two copies per diploid genome for an autosome) or whether they are significantly overrepresented or underrepresented (Fig. 5). This concept is now being applied to the prenatal diagnosis of fetal chromo some imbalance.

Fig5. Strategies for detection of numeric and structural chromosome abnormalities by whole genome sequence analysis. Although only a small number of reads are illustrated schematically here, in practice, many millions of sequence reads are analyzed and aligned to the reference genome to obtain statistically significant support for a diagnosis of aneuploidy or a structural chromosome abnormality. (A) Alignment of sequence reads from a patient’s genome to the reference sequence of three individual chromosomes. Overrepresentation of sequences from the red chromosome indicates that the patient is aneuploid for this chromosome. (B) Alignment of sequence reads from a patient’s genome to the reference sequence of two chromosomes reveals a number of reads that contain contiguous sequences from both chromosomes. This indicates a translocation in the patient’s genome involving the blue and orange chromosomes at the positions designated by the dotted lines.

To detect balanced rearrangements of the genome, however, in which DNA in the genome is neither gained nor lost, a more complete genome sequence is required. Here, instead of sequence reads that align perfectly to the reference human genome sequence, one finds rare sequence reads that align to two different and normally noncontiguous regions in the reference sequence (whether on the same chromosome or on different chromosomes) (see Fig. 5). This approach has been used to identify the specific genes involved in some cancers, and in children with various congenital defects due to trans locations, involving the juxtaposition of sequences that are normally located on different chromosomes. More recently, bioinformatics algorithms have been developed to estimate the size of trinucleotide repeat expansions and provide the opportunity to assay known clinically significant loci (such as those involved in fragile X syn drome or Huntington disease) as part of the WGS diagnostic test.

Clinical laboratories are beginning to implement WGS to accurately detect sequence- level variants (single nucleotide variants); insertions/ deletions (indels) up to 50 bp and CNVs for genetically heterogeneous disorders; however, CMA and whole exome sequencing have so far been the predominant tests used for this purpose due to their lower cost. Exome sequencing (ES) generates sequence reads from protein- coding exons, which represent ~1.5% of the genome. This provides accurate detection of exonic sequence– level variants; however, detection of CNVs is less accurate by ES than by WGS because the number of sequence reads generated for each exon can be less consistent, and there is consider able uncertainty of CNV breakpoints due to the large unsequenced chromosome regions between exons. In addition, ES cannot detect balanced rearrangements and noncoding variants. As the cost of WGS continues to decrease, it will replace ES and CMA in genomic diagnostics, providing a much more complete representation of all the variants within an individual’s genome.

Although WGS short- read technologies provide a considerable improvement over microarray and ES, the short- read lengths limit the ability to resolve complex structural variation, repetitive regions, and genes with homologous sequence in other regions of the genome (e.g., pseudogenes). The emergence of long- read sequencing technologies that generate sequence reads greater than 10 kb has made it possible to begin to address many of these challenges that can potentially involve clinically relevant genes. Notably, this technology is able to (1) sequence genes without interference of homologous sequence from pseudogenes, (2) provide haplotypes and phase variants across large stretches of DNA (>10 kb), (3) sequence large repeat expansions and identify intervening sequence that may affect phenotype and repeat stability (e.g. involved in DMPK, the genes for myotonic dystrophy), and (4) identify balanced and unbalanced translocations, insertions, deletions, duplications, and inversions.

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مواضيع ذات صلة

Methods to Detect Fetal Chromosomal Abnormalities

The Origin and Frequency of Different Types of Mutation

Noninvasive Prenatal Screening by Analysis of Cell- Free Fetal DNA

Screening for Down Syndrome and Other Aneuploidies

Screening for Neural Tube Defects

Genetic Alterations Associated with Acromegaly

The Profession of Genetic Counseling

Applying Genomics to Individualize Cancer Therapy

The Role of Epigenetics in Cancer

Screening for Genetic Susceptibility to Disease

Pharmacogenomics

Monogenic Diseases Show One of Three Patterns of Inheritance