76.1 Methods of Chromosome Analysis

Cytogenetic studies are usually performed on peripheral blood lymphocytes, although cultured fibroblasts may also be used. Prenatal (fetal) chromosome studies are performed with cells obtained from the amniotic fluid, chorionic villus tissue, and fetal blood or, in the case of preimplantation diagnosis, by analysis of a blastomere. 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 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 a reshuffling of the genetic information on the recombined chromosomes and allows further genetic diversity. Each daughter cell then receives 1 of each of the 23 homologous chromosomes. In oogenesis, 1 of the daughter cells receives most of the cytoplasm and becomes the egg, whereas the other smaller cell becomes the 1st 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 upon fertilization a diploid number is restored, and it allows genetic recombination.

Two errors of cell division commonly occur during meiosis or mitosis, and either can result in an abnormal number of chromosomes. The 1st is nondisjunction, in which 2 chromosomes fail to separate during meiosis and thus migrate together into 1 of the new cells, producing 1 cell with 2 copies of the chromosome and another with no copy. The 2nd 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. 76-1).

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 on subjected to a hypotonic solution to allow 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.

Other banding techniques such as Q-banding using quinacrine, reverse (R-banding) using acridine orange, and C-banding (constitutive heterchromatin) using barium hydroxide are available for use in certain circumstances but are losing ground to molecular technologies. Metaphase chromosome spreads are 1st evaluated microscopically, and then their images are photographed or captured by a video camera and stored on a computer to be later analyzed. Humans have 46 chromosomes or 23 pairs, which are classified as autosomes for chromosomes 1 to 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 like 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. 76-2). 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. 76-3). The centromere divides the chromosome in 2, with the short arm designated as the p arm and the long arm designated as the q arm. A plus or minus sign before the number of a chromosome indicate that there is an extra or missing chromosome, respectively. Table 76-1 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 is useful for detecting subtle chromosome abnormalities that might otherwise go unrecognized.

Molecular techniques such as fluorescence in situ hybridization (FISH) and comparative array genomic hybridization studies (conventional CGH and array CGH [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. 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). 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, that has been previously treated (denatured) to allow the DNA to become single stranded and to permit hybridization. When the probe pairs with its complementary DNA sequence, it can be then visualized by fluorescence microscopy (Fig. 76-4). 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, if they are not too close to each other; whereas in interphase cells, only the number of copies of a particular DNA segment can be determined. 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. In metaphase cells, some duplications might falsely appear as a single signal.

Metaphase and interphase FISH are particularly useful for detecting very small deletions that might escape notice with G-band analysis. In most cases the probe used for identification is used in conjunction with a control probe with a known location nearby the region studied. This allows correct identification of the hybridized signal to the right chromosome, and in some cases identification to the rearranged chromosome. With high-resolution chromosome analysis it is very difficult to recognize deletions of <5 million bp (5 Mb); FISH can reliably detect deletions as small as 50 to 200 kb of DNA. This has allowed the clinical characterization of a number of microdeletion syndromes. In addition to gene- or locus-specific probes, complex mixtures of DNA from a chromosome arm or an entire chromosome are available for fluorescence staining of large chromosome sections or entire chromosomes. The probe mixtures are referred to as chromosome paints (Fig. 76-5A and B). Other probes hybridize to repetitive sequences located to the pericentromeric regions. These probes are useful 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 (Fig. 76-5C and D).

Spectral karyotyping (SKY) and multicolor FISH (M-FISH) are similar molecular cytogenetic techniques that use 24 different chromosome painting probes and 5 fluorochromes to simultaneously visualize every chromosome in a metaphase spread. Each of the 24 different chromosome paints is labeled with a different combination of the 5 fluorescent dyes, which emit at different wavelengths. Each of the 22 autosomes and the X and Y chromosomes has its own unique spectrum of wavelengths of fluorescence. Special filters, cameras, and image-processing software are required to identify each chromosome. SKY and M-FISH are especially useful for identifying the complex chromosome rearrangements found in many tumors. This technique requires very special and costly equipment and is being displaced by comparative aCGH.

Comparative genomic hybridization (CGH) is a molecular-based technique that involves differentially labeling the patient’s DNA with a fluorescent dye (green) and a normal reference DNA with another fluorescent dye (red) (Fig. 76-6). Equal amounts of the two-label DNA samples are mixed and then used as a painting probe for FISH with normal metaphase chromosomes. The ratio of green : red fluorescence is measured along each chromosome. 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 chromosomes appear yellow.

A modified version of this technology, aCGH, uses DNA spotted onto a slide or microarray grid. In this case, instead of metaphase chromosomes, segments of DNA are represented by BACs (bacterial artificial chromosomes) and PACs (P1 artificial chromosomes) or oligonucleotides (short DNA segments of varied size) distributed in a microarray that resembles the chromosomes in a metaphase. The detection could reach the size of the BACs, PACs, and oligonucleotides and ranges in resolution from approximately 50 to 200 kb (even smaller if using cosmids or shorter polymerase chain reaction (PCR) products used as probes).

There are many advantages of aCGH. They can test all critical disease causing regions in the genome at once; FISH requires the clinical knowledge and tests only 1 area at a time. aCGH can detect duplications and deletions not currently recognized as recurrent disease-causing regions probed by FISH. aCGH can detect single and contiguous gene deletion syndromes. aCGH does not always require cell culture to generate sufficient DNA, something that may be important in the context of prenatal testing because of timing. There are disadvantages to aCGH: It does not detect balanced translocations, inversions, or very low-levels of mosaicism.

There are 2 different types of aCGH, targeted and whole-genome arrays. aCGH is the equivalent of doing thousands of FISH experiments in a single sample at once. Targeted aCGH is an effective and efficient technique for detecting clinically known cryptic chromosomal aberrations, which are typically associated with known disease phenotypes; many of these arrays have expanded detection to areas potentially susceptible to recurring deletion or duplication. Whole-genome array targets the entire genome. The advantage of this latter technique is that it allows better and denser coverage of the entire genome in evenly spaced portions; its disadvantage is that interpretation of deletions or duplications may be difficult if it involves areas not previously known to be involved in disease.

There are many copy number variations (CNVs) causing deletion or duplication in the human genome. Thus, most detected genetic abnormalities, unless associated with very well known clinical phenotypes, require parental investigations because a detected CNV that is inherited might turn out to be an incidental polymorphic variant. A de novo abnormality (i.e., 1 found only in the child and not the parents) is often more significant if it is associated with an abnormal phenotype 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. 76-7).

76.2 Down Syndrome and Other Abnormalities of Chromosome Number

Trisomy 8, mosaicism 1/20,000 births Long face, high prominent forehead, wide upturned nose, thick everted lower lip, microretrognathia, low-set ears, high arched, sometimes cleft palate; osteoarticular anomalies common (camptodactyly of 2nd to 5th digits, small patella); deep plantar and palmar creases; moderate mental retardation