Chapter 063. Chromosome Disorders (Part 3)

Applications of FISHThe 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) Chapter 063. Chromosome Disorders (Part 3) Applications of FISH The majority of FISH applications involve hybridization of one or twoprobes of interest as an adjunctive procedure to conventional chromosomalbanding techniques. In this regard, FISH can be utilized to identify specificchromosomes, characterize de novo duplications or deletions, and clarify subtlechromosomal rearrangements. Its greatest utilization, however, is in the detectionof microdeletions (see below). Though conventional cytogenetic studies can detectsome microdeletions, initial detection and/or confirmation with FISH is essential.In fact, since appropriate FISH probes have become available, detection ofmicrodeletion 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 ininstances when metaphase chromosome preparations are not yet available (e.g.,amniotic fluid interphase analysis). Interphase analysis also increases the numberof cells available for examination, allows for investigation of nuclear organization,and provides results when cells do not progress to metaphase. One specializedtype of interphase analysis involves the application of FISH to paraffin-embeddedsections, thereby preserving the architecture of the tissue. The use of interphase FISH has increased recently, especially for analysesof amniocentesis samples. These studies are performed on uncultured amnioticfluid, typically using DNA probes specific for the chromosomes most commonlyidentified in trisomies (chromosomes 13, 18, 21, and the X and Y). These studiescan be performed rapidly (24–72 h) and will ascertain about 60% of theabnormalities detected prenatally. Another area in which interphase analysis isroutinely utilized is cancer cytogenetics (Chap. 79). Many site-specifictranslocations are associated with specific types of malignancies. For example,there are probes available for both the Abelson (Abl) oncogene and breakpointcluster region (bcr) involved in chronic myelogenous leukemia (CML); theseprobes are labeled in red and green, respectively; the fusion of these genes in CMLcombines the fluorescent colors and appears as a yellow hybridization signal. In addition to standard metaphase and interphase FISH analyses, a numberof enhanced techniques have been developed for specific types of analysis,including multicolor FISH techniques, reverse painting, comparative genomichybridization, and fiber FISH. Spectral karyotyping (SKY) and multicolor FISH(m-FISH) techniques use combinatorially labeled probes that create a unique colorfor individual chromosomes. This technology is useful in the identification ofunknown chromosome material (such as markers of duplications) but is mostcommonly used with the complex rearrangements seen in cancer specimens. Fiber FISH is a technique in which chromosomes are mechanicallystretched, using a variety of different methods. It provides a higher resolution ofanalysis than conventional FISH. Comparative genomic hybridization (CGH) is a method that can be usedonly when DNA is available from a specimen of interest. The entire DNAspecimen from the sample of interest is labeled in one color (e.g., green), and thenormal control DNA specimen is indicated by another color (e.g., red). These aremixed in equal amounts and hybridized to normal metaphase chromosomes. Thered-to-green ratio is analyzed by a computer program, which determines where theDNA of interest may have gains or losses of material. This technique is useful inthe analysis of tumors, particularly in those cases where cytogenetic analysis is notpossible. An extension of CGH promises to yield another major advance forexamining human chromosomes. Specifically, the development of CGH arraysuses protocols that are similar to standard CGH, except that test DNA ishybridized to DNAs that are spread on arrays, rather than hybridized to normalchromosomes. Depending on the type of array (most are constructed utilizingeither BACs or oligonucleotides), the resolution can be up to 150 kb, far greaterthan for standard chromosome analysis. This technology has been used to studycryptic chromosomal imbalances in patients with mental retardation and multiplecongenital anomalies, as well as in prenatal diagnosis. It has also been used todetect microdeletions and microduplications in cancer and in previouslyunidentified 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 thedevelopmental ...