Cytogenetics

Methods of Chromosome Analysis

Carlos A. Bacino

Brendan Lee

Cytogenetic studies are usually performed on peripheral blood lymphocytes, although cultured fibroblasts obtained from a skin biopsy may also be used. Prenatal (fetal) chromosome studies are performed with cells obtained from the amniotic fluid (amniocytes), chorionic villus tissue, and fetal blood or, in the case of preimplantation diagnosis, by analysis of a blastomere (cleavage stage) biopsy, polar body biopsy, or blastocyst biopsy. Cytogenetic studies of bone marrow have an important role in tumor surveillance, particularly among patients with leukemia. These are useful to determine induction of remission and success of therapy or in some cases the occurrence of relapses.

Chromosome anomalies include abnormalities of number and structure and are the result of errors during cell division. There are 2 types of cell division: mitosis, which occurs in most somatic cells, and meiosis, which is limited to the germ cells. In mitosis, 2 genetically identical daughter cells are produced from a single parent cell. DNA duplication has already occurred during interphase in the S phase of the cell cycle (DNA synthesis). Therefore, at the beginning of mitosis the chromosomes consist of 2 double DNA strands joined together at the centromere, known as sister chromatids. Mitosis can be divided into 4 stages: prophase, metaphase, anaphase, and telophase. Prophase is characterized by condensation of the DNA. Also during prophase, the nuclear membrane and the nucleolus disappear and the mitotic spindle forms. In metaphase the chromosomes are maximally compacted and are clearly visible as distinct structures. The chromosomes align at the center of the cell, and spindle fibers connect to the centromere of each chromosome and extend to centrioles at the 2 poles of the mitotic figure. In anaphase the chromosomes divide along their longitudinal axes to form 2 daughter chromatids, which then migrate to opposite poles of the cell. Telophase is characterized by formation of 2 new nuclear membranes and nucleoli, duplication of the centrioles, and cytoplasmic cleavage to form the 2 daughter cells.

Meiosis begins in the female oocyte during fetal life and is completed years to decades later. In males it begins in a particular spermatogonial cell sometime between adolescence and adult life and is completed in a few days. Meiosis is preceded by DNA replication so that at the outset, each of the 46 chromosomes consists of 2 chromatids. In meiosis, a diploid cell (2n = 46 chromosomes) divides to form 4 haploid cells (n = 23 chromosomes). Meiosis consists of 2 major rounds of cell division. In meiosis I , each of the homologous chromosomes pair precisely so that genetic recombination , involving exchange between 2 DNA strands ( crossing over ), can occur. This results in reshuffling of the genetic information for the recombined chromosomes and allows further genetic diversity. Each daughter cell then receives 1 of each of the 23 homologous chromosomes. In oogenesis, one of the daughter cells receives most of the cytoplasm and becomes the egg, whereas the other smaller cell becomes the first polar body. Meiosis II is similar to a mitotic division but without a preceding round of DNA duplication (replication). Each of the 23 chromosomes divides longitudinally, and the homologous chromatids migrate to opposite poles of the cell. This produces 4 spermatogonia in males, or an egg cell and a 2nd polar body in females, each with a haploid (n = 23) set of chromosomes. Consequently, meiosis fulfills 2 crucial roles: It reduces the chromosome number from diploid (46) to haploid (23) so that on fertilization a diploid number is restored, and it allows genetic recombination.

Two common errors of cell division may occur during meiosis or mitosis, and either can result in an abnormal number of chromosomes. The 1st error is nondisjunction , in which 2 chromosomes fail to separate during meiosis and thus migrate together into one of the new cells, producing 1 cell with 2 copies of the chromosome and another with no copy. The 2nd error is anaphase lag , in which a chromatid or chromosome is lost during mitosis because it fails to move quickly enough during anaphase to become incorporated into 1 of the new daughter cells ( Fig. 98.1 ).

For chromosome analysis, cells are cultured (for varying periods depending on cell type), with or without stimulation, and then artificially arrested in mitosis during metaphase (or prometaphase), later subjected to a hypotonic solution to allow disruption of the nuclear cell membrane and proper dispersion of the chromosomes for analysis, fixed, banded, and finally stained. The most commonly used banding and staining method is the GTG banding (G-bands trypsin Giemsa), also known as G banding , which produces a unique combination of dark (G-positive) and light (G-negative) bands that permits recognition of all individual 23 chromosome pairs for analysis.

Metaphase chromosome spreads are first evaluated microscopically, and then their images are photographed or captured by a video camera and stored on a computer for later analysis. Humans have 46 chromosomes or 23 pairs, which are classified as autosomes for chromosomes 1-22, and the sex chromosomes , often referred as sex complement : XX for females and XY for males. The homologous chromosomes from a metaphase spread can then be paired and arranged systematically to assemble a karyotype according to well-defined standard conventions such as those established by International System for Human Cytogenetic Nomenclature (ISCN), with chromosome 1 being the largest and 22 the smallest. According to nomenclature, the description of the karyotype includes the total number of chromosomes followed by the sex chromosome constitution. A normal karyotype is 46,XX for females and 46,XY for males ( Fig. 98.2 ). Abnormalities are noted after the sex chromosome complement.

Although the internationally accepted system for human chromosome classification relies largely on the length and banding pattern of each chromosome, the position of the centromere relative to the ends of the chromosome also is a useful distinguishing feature ( Fig. 98.3 ). The centromere divides the chromosome in 2, with the short arm designated the p arm and the long arm designated the q arm . A plus or minus sign before the number of a chromosome indicates that there is an extra or missing chromosome, respectively. Table 98.2 lists some of the abbreviations used for the descriptions of chromosomes and their abnormalities. A metaphase chromosome spread usually shows 450-550 bands. Prophase and prometaphase chromosomes are longer, are less condensed, and often show 550-850 bands. High-resolution analysis may detect small chromosome abnormalities although has been mostly replaced by chromosome microarray studies (array CGH or aCGH).

Molecular techniques (e.g., FISH, CMA, aCGH) have filled a significant void for the diagnosing cryptic chromosomal abnormalities. These techniques identify subtle abnormalities that are often below the resolution of standard cytogenetic studies. Fluorescence in situ hybridization (FISH) is used to identify the presence, absence, or rearrangement of specific DNA segments and is performed with gene- or region-specific DNA probes. Several FISH probes are used in the clinical setting: unique sequence or single-copy probes, repetitive-sequence probes (alpha satellites in the pericentromeric regions), and multiple-copy probes (chromosome specific or painting) ( Fig. 98.4 A and B ). FISH involves using a unique, known DNA sequence or probe labeled with a fluorescent dye that is complementary to the studied region of disease interest. The labeled probe is exposed to the DNA on a microscope slide, typically metaphase or interphase chromosomal DNA. When the probe pairs with its complementary DNA sequence, it can then be visualized by fluorescence microscopy ( Fig. 98.5 ). In metaphase chromosome spreads, the exact chromosomal location of each probe copy can be documented, and often the number of copies (deletions, duplications) of the DNA sequence as well. When the interrogated segments (as in genomic duplications) are close together, only interphase cells can accurately determine the presence of 2 or more copies or signals since in metaphase cells, some duplications might falsely appear as a single signal.

Chromosome rearrangements <5 million bp (5 Mbp) cannot be detected by conventional cytogenetic techniques. FISH was initially used to detect deletions as small as 50-200 kb of DNA and facilitated the early clinical characterization of a number of microdeletion syndromes . Some FISH probes hybridize to repetitive sequences located in the pericentromeric regions. Pericentromeric probes are still widely used for the rapid identification of certain trisomies in interphase cells of blood smears, or even in the rapid analysis of prenatal samples from cells obtained through amniocentesis. Such probes are available for chromosomes 13, 18, and 21 and for the sex pair X and Y (see Fig. 98.4 C and D ). With regard to the detection of genomic disorders, FISH is no longer the first line of testing, and its role has also mostly changed to the confirmation of microarray findings. In summary, FISH is reserved for (1) confirmation studies of abnormalities detected by CMA, (2) rapid prenatal screening on interphase amniotic fluid cells, and (3) interphase blood smear for sex assignment of newborns who present with ambiguous genitalia.

Array comparative genomic hybridization (aCGH) and chromosomal microarray (CMA) are molecular-based techniques that involve differentially labeling the patient's DNA with a fluorescent dye (green fluorophore) and a normal reference DNA with a red fluorophore ( Fig. 98.6 ). Oligonucleotides (short DNA segments) encompassing the entire genome are spotted onto a slide or microarray grid. Equal amounts of the 2-label DNA samples are mixed, and the green:red fluorescence ratio is measured along each tested area. Regions of amplification of the patient's DNA display an excess of green fluorescence, and regions of loss show excess red fluorescence. If the patient's and the control DNA are equally represented, the green:red ratio is 1 : 1, and the tested regions appear yellow (see Chapter 96 , Fig. 96.5 ). The detection is currently possible at the single-exon resolution level, depending on the arrays used. In the near future, copy number detections may further evolve to be detected by next generation sequencing in the context of whole genome sequencing.

The many advantages of aCGH include its ability to test all critical disease-causing regions in the genome at once, detect duplications and deletions not currently recognized as recurrent disease-causing regions probed by FISH, and detect single-gene and contiguous gene deletion syndromes. Also, aCGH does not always require cell culture to generate sufficient DNA, which may be important in the context of prenatal testing because of timing. Limitations of aCGH are that it does not detect balanced translocations or inversions and may not detect low levels of chromosomal mosaicism. Among different types of aCGH, some are more targeted than others. Targeted aCGH can be an efficient way to detect clinically known cryptic chromosomal aberrations, which are typically associated with known disease phenotypes. Whole genome arrays target the entire genome and allow better and denser coverage in evenly spaced genomic regions. Its disadvantage is that interpretation of deletions or duplications may be difficult if it involves areas not previously known to be involved in disease.

A frequently used array in the clinical setting is the single nucleotide polymorphism (SNP) . SNPs are polymorphic variations between 2 nucleotides, and when analyzed in massive parallel fashion, they can provide valuable clinical information. Several million SNPs normally occur in the human genome. SNP arrays can help with the detection of uniparental disomies (i.e., genetic information derived from only 1 parent), as well as consanguinity in the family. Many arrays currently used in clinical practice combine the use of oligonucleotides for the detection of copy number variations in conjunction with SNPs. There are many copy number variations causing deletion or duplication in the human genome. Thus, most detected genetic abnormalities, unless associated with well-known clinical phenotypes, require parental investigations because a detected copy number variation that is inherited could be benign or an incidental polymorphic variant. A de novo abnormality (i.e., one found only in the child and not the parents) is often more significant if it is associated with an abnormal phenotype found only in the child, and if it involves genes with important functions.

aCGH is a very valuable technology alone or when combined with FISH and conventional chromosome studies ( Fig. 98.7 ).

Down Syndrome and Other Abnormalities of Chromosome Number

Brendan Lee

Down Syndrome

Trisomy 21 is the most common genetic etiology of moderate intellectual disability. The incidence of Down syndrome in live births is approximately 1 in 733; the incidence at conception is more than twice that rate; the difference is accounted for by early pregnancy losses. In addition to cognitive impairment, Down syndrome is associated with congenital anomalies and characteristic dysmorphic features ( Figs. 98.8 and 98.9 and Table 98.4 ). Although there is variability in the clinical features, the constellation of phenotypic features is fairly consistent and permits clinical recognition of trisomy 21. Affected individuals are more prone to congenital heart defects (50%) such as atrioventricular septal defects, ventricular septal defects, isolated secundum atrial septal defects, patent ductus arteriosus, and tetralogy of Fallot. Pulmonary complications include recurrent respiratory infections, sleep-disordered breathing, laryngo- and tracheobronchochomalacia, tracheal bronchus, pulmonary hypertension, and asthma. Congenital and acquired gastrointestinal anomalies (celiac disease) and hypothyroidism are common ( Table 98.5 ). Other abnormalities include megakaryoblastic leukemia, immune dysfunction, diabetes mellitus, seizures, alopecia, juvenile idiopathic arthritis, and problems with hearing and vision. Alzheimer disease–like dementia is a known complication that occurs as early as the 4th decade and has an incidence 2-3 times higher than sporadic Alzheimer disease. Most males with Down syndrome are sterile, but some females have been able to reproduce, with a 50% chance of having trisomy 21 pregnancies. Two genes ( DYRK1A, DSCR1 ) in the putative critical region of chromosome 21 may be targets for therapy.

Table 98.4

Clinical Features of Down Syndrome in the Neonatal Period

Central Nervous System

• Hypotonia *

  * Hall's criteria to aid in diagnosis.

• Developmental delay

• Poor Moro reflex *

Craniofacial

• Brachycephaly with flat occiput

• Flat face *

• Upward slanted palpebral fissures *

• Epicanthal folds

• Speckled irises (Brushfield spots)

• Three fontanels

• Delayed fontanel closure

• Frontal sinus and midfacial hypoplasia

• Mild microcephaly

• Short, hard palate

• Small nose, flat nasal bridge

• Protruding tongue, open mouth

• Small dysplastic ears *

Cardiovascular

• Endocardial Cushing defects

• Ventricular septal defect

• Atrial septal defect

• Patent ductus arteriosus

• Aberrant subclavian artery

• Pulmonary hypertension

Musculoskeletal

• Joint hyperflexibility *

• Short neck, redundant skin *

• Short metacarpals and phalanges

• Short 5th digit with clinodactyly *

• Single transverse palmar creases *

• Wide gap between 1st and 2nd toes

• Pelvic dysplasia *

• Short sternum

• Two sternal manubrium ossification centers

Gastrointestinal

• Duodenal atresia

• Annular pancreas

• Tracheoesophageal fistula

• Hirschsprung disease

• Imperforate anus

• Neonatal cholestasis

Cutaneous

• Cutis marmorata

Table 98.5

Additional Features of Down Syndrome That Can Develop or Become Symptomatic With Time

Neuropsychiatric

• Developmental delay

• Seizures

• Autism spectrum disorders

• Behavioral disorders (disruptive)

• Depression

• Alzheimer disease

Sensory

• Congenital or acquired hearing loss

• Serous otitis media

• Refractive errors (myopia)

• Congenital or acquired cataracts

• Nystagmus

• Strabismus

• Glaucoma

• Blocked tear ducts

Cardiopulmonary

• Acquired mitral, tricuspid, or aortic valve regurgitation

• Endocarditis

• Obstructive sleep apnea

Musculoskeletal

• Atlantoaxial instability

• Hip dysplasia

• Slipped capital femoral epiphyses

• Avascular hip necrosis

• Recurrent joint dislocations (shoulder, knee, elbow, thumb)

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