Hereditary nonpolyposis colorectal cancer: pitfalls in deletion screening in MSH2 and MLH1 genes

Abstract

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by a deficiency in DNA mismatch repair in consequence of germline mutations mainly in the genes MSH2 and MLH1. Around 10% of patients suspected of HNPCC are identified with large genomic deletions that cannot be detected by conventional methods of mutation screening. The recently developed multiplex ligation-dependent probe amplification (MLPA) proved to be an easy to perform method for deletion detection and is reliable when more than one exon is deleted. We show that, in some cases, apparent deletions of single exons may actually result from single base substitutions or small insertions/deletions in the hybridisation sequence of MLPA probes. We conclude that single exon deletions, detected by MLPA or multiplex PCR, should be validated with additional methods.

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

“…Moreover, the use of two independent semiquantitative methods substantially adds to the confidence in results obtained by a single method, especially in cases where a single exon is involved. In fact, the interpretation of a single semiquantitative method (e.g., MLPA, QMPSF, or NFMP-HPLC) may be hampered by artifacts such as those derived from nucleotide substitutions in the template that may interfere with the annealing of PCR primers generating a false indication of exon deletion [Wehner et al, 2005]. However, considering that undesired mismatches are unlikely to affect simultaneously different probes or primers, the results confirmed by two complementary methods, such as those obtained by MLPA and NFMP-HPLC in the present study, could be correctly interpreted even when a single exon is involved.…”

“…However, developers of MLPA do not recommend its use in a diagnostic procedure because artifacts might hamper the interpretation of the results, especially when the putative rearrangement involves a single exon. In fact, nucleotide substitutions located in the template may interfere with the annealing of MLPA probes and thus with semiquantitative analyses [Wehner et al, 2005]. Similar artifacts due to nucleotide substitutions in the template may also affect QMPSF.…”

“…In principle, the results obtained by applying a single semiquantitative method could be correctly interpreted when two or more contiguous exons are involved, since undesired mismatches are unlikely to simultaneously affect multiple oligonucleotides. Conversely, it is necessary that single exon deletions be verified by two independent methods [Taylor et al, 2003;Wehner et al, 2005;Sellner and Taylor, 2004]. Breakpoint characterization allows unequivocal confirmation of rearrangements and provides a PCR-based diagnostic tool to search for the mutation in at-risk family members.…”

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

“…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.…”

“…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.…”