Available Assays

Protein truncation assays (see chapter 2) of the APC gene have been commonly used as a commercial test for mutations in FAP families. This assay capitalizes on the frequency of truncating mutations as a cause of classic FAP. An advantage of this method is that a mutation resulting in a truncated protein is almost certainly deleterious. One drawback of this assay is that it is not reliable in detecting APC mutations in the extreme 5' or 3' ends of the gene. Missense variants and alterations outside the coding region are not detected with this technique. Protein truncation assays do not provide information regarding the specific mutation responsible for FAP in the family, so direct sequencing of the truncated region is necessary to identify the specific mutation.

Linkage analysis is used to identify FAP carriers. Linkage analysis requires the participation of affected and unaffected family members, and provides a clinical testing alternative for families that do not have APC mutations identifiable by protein truncation or by direct DNA sequencing. Linkage analysis is informative in 90% to 95% of families who are able to submit the appropriate samples for testing. However, not all families have a sufficient number of members who are willing or able to participate in genetic testing for linkage analysis to be informative. Additionally, individuals with de novo mutations may not be aided by linkage analysis, unless their children become affected. Linkage analysis also may prove problematic if the markers used are not informative (i.e., are homozygous) in the affected and unaffected family members.

Direct sequence analysis of the APC coding, flanking, and intronic regions is available. This method has the advantage of directly identifying the specific familial mutation. Mutations may be detected by automated methods utilizing gel or capillary electrophoresis. While DNA sequencing is the most comprehensive genetic test, it may miss mutations due to rearrangements or large insertions or deletions, or that result in altered expression. Sequencing also will reveal variants of uncertain clinical significance, which go undetected when indirect methods such as protein truncation and linkage analysis are used. Denaturing high-performance liquid chromatography (DHPLC), single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) are mutation screening methods that may be used in conjunction with sequencing for the detection of an APC mutation.

To detect large rearrangements in the APC gene, a quantitative polymerase chain reaction (PCR) assay may be used to amplify all exons of the APC gene. Genes that exhibit rearrangements will differ in the quantity of PCR product produced. Gene regions showing differences in the quantity of amplified PCR products are subsequently confirmed by additional methods such as long-range PCR with the use of intragenic single-nucleotide polymorphic (SNP) marker analysis, or Southern blot analysis.

Genetic testing for the I1307K variant is available by allele-specific oligonucleotide hybridization (ASOH) or by sequencing.

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