Testing Methods

High-Resolution Karyotype. The genetic test with the lowest resolution is the karyotype analysis. In this test, a sample of cells, for example blood leukocytes or amniotic fluid cells, is grown in tissue culture medium in the presence of mitotic inhibitors that arrest cells at metaphase. The cells are then "squashed" to spread the chromosomes on a microscopic slide. The chromosomes are then stained to reveal a pattern of bands along the length of the chromosome arms. This form of testing is able to assess whether the cells have the normal number of chromosomes and whether interchanges between the chromosomes (translocations) have occurred. Depending on their size, this analytical method can also detect some deletions or duplications of chromosomal regions. A typical high-resolution test can detect deletions and insertions of approximately 10 million base pairs or more. Smaller deletions or duplications (submicroscopic deletions) will not be detected, including the duplication found in most patients with Charcot-Marie-Tooth 1A (CMT1A) or Pelizaeus-Merzbacher disease (see Chaptei.3ยง,).

Fluorescent In Situ Hybridization. Fluorescent in situ hybridization analysis is a useful method for determining deletions or duplications of specific chromosomal regions. This technique involves staining chromosomes by hybridization with a nucleic acid probe. This probe is visualized by being covalently labeled with a fluorescent molecule that can be detected with a conventional fluorescence microscope. Typical probes range from a few thousand bases to tens of thousands of bases in length. In addition to staining, chromosomal spreads prepared as described above for conventional karyotype analysis can be used to stain chromosomes in interphase or nonmitotic cells. This method is advantageous because the DNA is not tightly wound into densely packed chromatin, so that relatively closely spaced fluorescent markers may be resolved microscopically. This technique may detect the 1.5 million base duplication in CMT1A, the approximately 1 million base duplications seen in most patients with Pelizaeus-Merzbacher disease, or the submicroscopic deletions responsible for the Smith-Magenis and the Prader-Willi/ Angelman syndromes (see Chapter.32 ).

Single Gene Mutation Screening. Mutation detection specificity and sensitivity concerns apply to molecular diagnostic testing as well as to conventional biochemical testing. It is important to understand the nature of mutations that are known to occur in a gene in question as well as the range of potential mutations that can occur for any gene. The types of mutations that can cause a genetic disease determine the comprehensiveness or sensitivity of a screening test. The trinucleotide repeat disorders represent a group of genetic diseases that can be screened easily and with great sensitivity, because the mutations are of a single type and occur in a single region of their respective genes. However, screening for disorders such as X-linked Charcot-Marie-Tooth disease (connexin 32 gene) or myotonia congenita (skeletal muscle chloride channel) is difficult because different affected families will often have unique, or private, mutations such as single base changes, or small insertions or deletions. Although caused by many private mutations, some genetic disorders can be screened for by relatively convenient tests due to the nature of their mutations. Whereas the responsible gene for Duchenne and Becker muscular dystrophies (dystrophin) is an extremely large gene with many exons, mutations are fairly sizable (tens or hundreds of bases) deletions in most cases (see Ch.aptei..3.6 ). Because particular gene regions are so-called hot spots for these deletions, polymerase chain reaction (PCR) simultaneous amplification (so-called multiplex PCR) of the dystrophin gene regions most often deleted is able to detect mutations in about 75 percent of cases. Neurofibromatosis 1, another genetic disorder affecting a large gene caused by many private mutations, may lead to the premature termination of protein synthesis of the neurofibromin protein (see Chapter.32 ). Because the neurofibromin gene is expressed in white blood cells, an assay, the premature protein truncation test, was developed to screen leukocyte RNA for the neurofibromin mRNA. The mRNA can be specifically amplified and then used as a template for in vitro protein synthesis. After electrophoresis

of the synthetically generated neurofibromin and detection by antibody on Western blot, mutations can be reliably inferred if the immunoreactive neurofibromin is smaller than the normal protein. The premature protein truncation assay is now commercially available and reported to detect mutations in the neurofibromatosis 1 gene in 70 percent of individuals who fulfill clinical criteria. When mutations are not found in a clinically affected individual, mutation detection is more problematic. Currently no commercial laboratory offers testing for mutations in the remaining 30 percent of neurofibromatosis 1 individuals.

At present, numerous methods are available to screen for genetic mutations. PCR amplification is a technique that is critical to many of these tests because it enables the generation of large amounts of DNA. Routine PCR is limited to the amplification of specific, small (up to several hundred base pairs in length) regions of a single gene bracketed by a pair of opposing DNA primers that define the origins and directions of DNA synthesis of a target gene. Because each strand of the double helix can be used as a template for DNA synthesis in each repetitive cycle of DNA denaturation, primer annealing, and DNA synthesis, a doubling in the number of amplified DNA regions occurs. Thus, a several billion-fold amplification of a gene region is readily achievable from a small amount of DNA from the visual 30- to 35-cycle amplification. As previously mentioned, PCR is the basis for detecting trinucleotide repeat expansion mutations and deletions of the dystrophin gene. It is also used to amplify segments of a gene suspected of having a mutation, and the PCR products are then used in a variety of assays to screen for the mutations.

Single-Strand Conformation Polymorphism Analysis. Single-strand conformation polymorphism analysis has the capability of detecting single base mutations and is a very valuable means of screening for small mutations. PCR products, generally a few hundred bases in length, when denatured and subjected to electrophoresis usually show measurable differences in electrophoretic migration, due to the differences in conformation of the dissociated single strands of DNA. Mutations in a gene may further alter the electrophoretic migration of dissociated DNA strands, which have been PCR amplified. This technique is the basis of single-strand conformation polymorphism analysis, which when properly designed, can detect at least 80 percent of point mutations as small as a single base difference within the region amplified. If precise definition of the mutation is required, direct sequencing of the PCR product is undertaken.

Allele Specific Oligonucleotide Hybridization. Allele specific oligonucleotide hybridization is primarily used to detect specific point mutations. With properly designed oligonucleotides, single-stranded DNA probes of about 20 bases in length can detect either the normal or mutant gene variant by hybridization of the probes to PCR-amplified DNA segments.

Direct Gene Sequencing. The definitive method for the detection of point mutations involves direct gene sequencing. Whereas for large genes (neurofibromatosis 1, dystrophin), such a direct screen is impractical with the current level of technology. It is the method of choice for certain small genes. For example, the entire prion protein gene-coding region conveniently lies within a single exon that can be amplified by PCR with a single pair of primers. Current methods can now readily scan the entire coding sequence of the gene in cases of suspected hereditary prion mutations. With relatively short and few exons, other genes, such as the proteolipid protein gene that is mutated in Pelizaeus-Merzbacher disease, are also amenable to direct sequencing for mutation screening.

Restriction Fragment Length Polymorphism Analysis. Mutations that alter the sequence of a gene may, by chance, create or abolish a sequence cleavable by bacterial proteins termed restriction endonucleases. These enzymes cut double-stranded DNA wherever a specific short, usually 4 to 10 bases, sequence occurs. The discovery of these enzymes was key to the advances made in recombinant DNA technology and modern molecular analysis. When a restriction site is created or abolished, the pattern of DNA fragments generated by the appropriate enzyme is also changed. The methods of gene hunting that rely on restriction fragment length polymorphism (RFLP) analysis take advantage of restriction site variation not only within the gene itself, but also at site variations that lie outside, yet nearby, the gene in question. For example, before the discovery of the Huntington disease gene, RFLP analysis of samples gathered from whole families was the mainstay of genetic diagnosis of presymptomatic individuals (see Chapter.34 ). Restriction site variations that lie outside but nearby the Huntington's disease gene were used to mark the abnormal chromosome with a high (over 95 percent) reliability, even though these restriction site polymorphisms were not the cause of the disease.

Southern Blot Hybridization. There are several neurogenetic syndromes that are caused by increased or decreased numbers of otherwise normal genes. Charcot-Marie-Tooth 1A and Pelizaeus-Merzbacher disease are most often caused by duplications of the peripheral myelin protein-22 (PMP22) and proteolipid protein genes, respectively (see Chapter.30 and Ch.a.2te.L3.6.). Hereditary neuropathy with predisposition to pressure palsies is due to a loss of one copy of the PMP22 gene. One method to detect these changes is to compare the signal intensity of the altered gene region with that of a gene region lying outside the duplicated or deleted portion. The original method used for this procedure relies on the standard Southern blot technique, in which genomic DNA (isolated from peripheral leukocytes) is digested with an appropriate restriction endonuclease. The DNA is then subjected to agarose gel electrophoresis and transferred to nylon membranes for hybridization to labeled gene probes. For dosage analysis, a gene probe spanning the junction of the normal and dose-altered genomic region is hybridized to the filter. If a restriction enzyme site exists that separates the normal from altered copy number regions, the probe hybridizes to a normal level to a DNA restriction fragment in the normal copy region. In contrast, if the copy number for the gene is increased or decreased, the probe hybridizes greater or lesser intensity, respectively.

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