Chapter 063. Chromosome Disorders (Part 3)

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Applications of FISH The majority of FISH applications involve hybridization of one or two probes of interest as an adjunctive procedure to conventional chromosomal banding techniques. In this regard, FISH can be utilized to identify specific chromosomes, characterize de novo duplications or deletions, and clarify subtle chromosomal rearrangements. Its greatest utilization, however, is in the detection of microdeletions (see below). Though conventional cytogenetic studies can detect some microdeletions, initial detection and/or confirmation with FISH is essential. In fact, since appropriate FISH probes have become available, detection of microdeletion syndromes has increased significantly. In addition to metaphase FISH, cells can be analyzed...

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Chapter 063. Chromosome Disorders (Part 3) Applications of FISH The majority of FISH applications involve hybridization of one or two probes of interest as an adjunctive procedure to conventional chromosomal banding techniques. In this regard, FISH can be utilized to identify specific chromosomes, characterize de novo duplications or deletions, and clarify subtle chromosomal rearrangements. Its greatest utilization, however, is in the detection of microdeletions (see below). Though conventional cytogenetic studies can detect some microdeletions, initial detection and/or confirmation with FISH is essential. In fact, since appropriate FISH probes have become available, detection of microdeletion syndromes has increased significantly. In addition to metaphase FISH, cells can be analyzed at a variety of stages. Interphase analysis, for example, can be used to make a rapid diagnosis in instances when metaphase chromosome preparations are not yet available (e.g.,
amniotic fluid interphase analysis). Interphase analysis also increases the number of cells available for examination, allows for investigation of nuclear organization, and provides results when cells do not progress to metaphase. One specialized type of interphase analysis involves the application of FISH to paraffin-embedded sections, thereby preserving the architecture of the tissue. The use of interphase FISH has increased recently, especially for analyses of amniocentesis samples. These studies are performed on uncultured amniotic fluid, typically using DNA probes specific for the chromosomes most commonly identified in trisomies (chromosomes 13, 18, 21, and the X and Y). These studies can be performed rapidly (24–72 h) and will ascertain about 60% of the abnormalities detected prenatally. Another area in which interphase analysis is routinely utilized is cancer cytogenetics (Chap. 79). Many site-specific translocations are associated with specific types of malignancies. For example, there are probes available for both the Abelson (Abl) oncogene and breakpoint cluster region (bcr) involved in chronic myelogenous leukemia (CML); these probes are labeled in red and green, respectively; the fusion of these genes in CML combines the fluorescent colors and appears as a yellow hybridization signal. In addition to standard metaphase and interphase FISH analyses, a number of enhanced techniques have been developed for specific types of analysis, including multicolor FISH techniques, reverse painting, comparative genomic hybridization, and fiber FISH. Spectral karyotyping (SKY) and multicolor FISH
(m-FISH) techniques use combinatorially labeled probes that create a unique color for individual chromosomes. This technology is useful in the identification of unknown chromosome material (such as markers of duplications) but is most commonly used with the complex rearrangements seen in cancer specimens. Fiber FISH is a technique in which chromosomes are mechanically stretched, using a variety of different methods. It provides a higher resolution of analysis than conventional FISH. Comparative genomic hybridization (CGH) is a method that can be used only when DNA is available from a specimen of interest. The entire DNA specimen from the sample of interest is labeled in one color (e.g., green), and the normal control DNA specimen is indicated by another color (e.g., red). These are mixed in equal amounts and hybridized to normal metaphase chromosomes. The red-to-green ratio is analyzed by a computer program, which determines where the DNA of interest may have gains or losses of material. This technique is useful in the analysis of tumors, particularly in those cases where cytogenetic analysis is not possible. An extension of CGH promises to yield another major advance for examining human chromosomes. Specifically, the development of CGH "arrays" uses protocols that are similar to standard CGH, except that test DNA is hybridized to DNAs that are spread on arrays, rather than hybridized to normal
chromosomes. Depending on the type of array (most are constructed utilizing either BACs or oligonucleotides), the resolution can be up to 150 kb, far greater than for standard chromosome analysis. This technology has been used to study cryptic chromosomal imbalances in patients with mental retardation and multiple congenital anomalies, as well as in prenatal diagnosis. It has also been used to detect microdeletions and microduplications in cancer and in previously unidentified genomic disorders. Although this technology is still in development, its use is anticipated to increase in the near term. Indications for Cytogenetic Analysis Primary indications for karyotypic analysis vary according to the developmental stage/age of the conceptus/individual under investigation. One especially important application is in prenatal diagnosis (particularly for pregnancies involving older women), assaying for chromosomal abnormalities in either chorionic villi of first-trimester fetuses or amniotic fluid of second-trimester fetuses. Tissue specimens from spontaneously aborted fetuses or stillbirths can also be examined for chromosome abnormalities. Interphase cytogenetics (using FISH) is increasingly being used to study individual blastomeres of preimplantation embryos (with in vitro fertilization–derived pregnancies). This makes it possible to detect aneuploid or structurally unbalanced embryos or, in the case of sex-linked disorders, to identify male conceptuses; such embryos would not be used to initiate pregnancies.
Among infants and children, peripheral blood is examined, most often in individuals with specific phenotypic abnormalities. For example, karyotypic analysis can be used for the confirmation or exclusion of a specific chromosomal syndrome (e.g., trisomy 21); in patients with unexplained psychomotor retardation with or without dysmorphic features; in cases of monogenic disorders associated with mental retardation and/or dysmorphic features; and with abnormalities of sexual differentiation and development. In adults, peripheral blood can be examined in patients with infertility or recurrent miscarriages, since chromosome abnormalities can lead to meiotic arrest or to genetically unbalanced gametes. An important branch of cytogenetics is concerned with analyses of bone marrow, unstimulated peripheral blood, and lymph nodes of tumors, as chromosomal abnormalities are a common correlate of leukemia, lymphoma, and solid tumors (Chap. 79).