Analysis of hMLH1 and hMSH2 Gene Dosage Alterations in Hereditary Nonpolyposis Colorectal Cancer Patients by Novel Methods

Abstract

Hereditary nonpolyposis colorectal cancer is an autosomal dominant disorder characterized by the early onset of tumors in the setting of few polyps. The average age of colon cancer diagnosis in individuals with hereditary nonpolyposis colorectal cancer is in the early to middle 40s, although many tumors may occur in the 20s or even in teenage years. In addition to colorectal cancer, several other tumor types, including endometrial, gastric, and ovarian are observed at an increased frequency in families with this disease.

Frequency of Deletions of EPCAM (TACSTD1) in MSH2-Associated Lynch Syndrome Cases

“…23,24 This kit contains oligonucleotide probes targeting EPCAM/TACSTD1 exons 3, 8, 9, and two probes in the intervening region between EPCAM/TACSTD1 and MSH2: one 3 kb downstream and one 2.5 kb upstream from the MSH2 gene. Further delineation of the deletion breakpoints was not performed.…”

“…22 In this study, we determined the frequency of this novel mechanism for MSH2 inactivation and correlated results of multiplex ligation-dependent probe amplification (MLPA) 23,24 testing with results of the MSH2 promoter hypermethylation test. Although specific endpoints of the deletions were not determined, the loss of material in the 3= untranslated region is similar to the different size deletions previously reported.…”

Confirmation of Single Exon Deletions in MLH1 and MSH2 Using Quantitative Polymerase Chain Reaction

“…6,10 Due to the complex nature of the MLPA assay, however, this technique can be sensitive to DNA quality, concentration, and extraction method. 10 Given the implications of an incorrect disease diagnosis for individuals and potentially affected family members, some form of confirmation of a deletion is desirable. In the case of multiexon deletions, this confirmation is provided by the results from neighboring exons, since the MLPA probe sets for each exon bind independently of each other.…”

“…5,7,8 Genomic rearrangements are particularly prevalent in the MSH2 gene, where they comprise up to 45% of mutations. 9 Since large deletions cannot be detected by standard exonic sequencing, other methods such as Southern blotting, 5,10 long-range polymerase chain reaction (PCR), 11 quantitative multiplex PCR, 8 and multiplex ligation-dependent probe amplification (MLPA) [12][13][14][15] have been used to detect deletions spanning one or more exons.…”

“…12 However, while MLPA detection of deletions of multiple sequential exons is widely accepted, there is concern over the reliability of MLPA results showing single exon deletions. 10,13 Given the clinical implications of diagnoses of diseases such as Lynch syndrome, it is important that any detection method have built-in redundancy to guard against falsepositive results. For samples with multiple contiguous deleted exons, dosage ratios of adjacent deleted exons provide internal confirmation of results.…”