No abstract

In order to assess whether variations affecting DNA mismatch repair (MMR) genes are pathogenic and hence predisposing to Lynch syndrome (LS), a three-step assessment model has been proposed. Where LS is suspected based on family history, STEP1 is dedicated to the identification of the causative MMR gene and the variation within it. Thereafter, in STEP2 of the assessment model, the effect of the variation on the function of the protein is assessed in an in vitro MMR and in silico assays. Where LS cannot be confirmed or ruled out in STEP2, the more specific biochemical laboratory assays such as analyzing the effect of the variation on expression, localization, and interaction of the protein are required in STEP3. Here, we verified the proposed three-step assessment model and its ability to distinguish pathogenic MMR variations from variants of uncertain significance (VUS) by utilizing the clinical as well as the laboratory and in silico data of 37 MLH1, 26 MSH2, and 11 MSH6 variations. The proposed model was shown to be appropriate and proceed logically in assessing the pathogenicity of MMR variations. In fact, for MMR deficient MSH2 and MLH1 variations the first two steps seem to be sufficient as STEP3 provides no imperative information concerning the variant pathogenicity. However, the importance of STEP3 is seen in the assessment of MMR proficient variations showing discrepant in silico results as their pathogenicity cannot be confirmed or ruled out after STEP2. MSH6 variations may be applicable to the model if appropriate selection in terms of ruling out MLH1 and MSH2 variations and MLH1 promoter hypermethylation is ensured prior to the completion of STEP2. In conclusion, taking into consideration the susceptibility gene the three-step model can be utilized in an appropriate and efficient manner to determine the pathogenicity of MMR gene variations. Hum Mutat 32:107–115, 2011. © 2010 Wiley-Liss, Inc.

BACKROUND: The target substrates of DNA mismatch recognising factors MutSa (MSH2 þ MSH6) and MutSb (MSH2 þ MSH3) have already been widely researched. However, the extent of their functional redundancy and clinical substance remains unclear. Mismatch repair (MMR)-deficient tumours are strongly associated with microsatellite instability (MSI) and the degree and type of MSI seem to be dependent on the MMR gene affected, and is linked to its substrate specificities. Deficiency in MSH2 and MSH6 is associated with both mononucleotide and dinucleotide repeat instability. Although no pathogenic MSH3 mutations have been reported, its deficiency is also suggested to cause low dinucleotide repeat instability. METHODS: To assess the substrate specificities and functionality of MutSa and MutSb we performed an in vitro MMR assay using three substrate constructs, GT mismatch, 1 and 2 nucleotide insertion/deletion loops (IDLs) in three different cell lines. RESULTS: Our results show that though MutSa alone seems to be responsible for GT and IDL1 repair, MutSa and MutSb indeed have functional redundancy in IDL2 repair and in contrast with earlier studies, MutSb seems to exceed MutSa. CONCLUSION: The finding is clinically relevant because the strong role of MutSb in IDL2 repair indicates MSH3 deficiency in tumours with low dinucleotide and no mononucleotide repeat instability.

Lynch syndrome (LS), the most common familial colon cancer, is associated with mismatch repair (MMR) malfunction. As mutation carriers inherit one normal and one defected MMR gene allele, cancer risk can be considered as limited amount of normal MMR gene product. How reductions in different MMR gene expressions affect MMR capability is, however, not known. The in vitro MMR assay is a method for the pathogenicity assessment of MMR gene variants causing functional or expressional defects and thus also suitable to evaluate the effects of reduced expression of normal mRNA. Here, the assay was applied to quantify repair efficiencies of human cells retaining varying expression levels (25%/50%/75%) of the main LS susceptibility genes MLH1, MSH2, or MSH6. Compared with the shRNA knockdown control, already a 50% reduction in mRNA levels could be detected as decreased MMR function although without statistical significance in MLH1. In MSH2 and MLH1, total loss of MMR was achieved with 25% expression, whereas in MSH6 and MSH2, the repair capability decreased significantly already with 75% expression. Our results provide a preliminary indication of relative expressions required for wild-type function and suggest that the in vitro MMR assay could be used to recognize expression levels indicative of LS.

PMS2 is one of the four susceptibility genes in Lynch syndrome (LS), the most common cancer syndrome in the world. Inherited mutations in DNA mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, account for approximately 90% of LS, while a relatively small number of LS families segregate a PMS2 mutation. This and the low cancer penetrance in PMS2 families suggest that PMS2 is only a moderate or low‐risk susceptibility gene. We have previously shown that even a partial expression decrease in MLH1, MSH2, or MSH6 suggests that heterozygous LS mutation carriers have MMR malfunction in constitutive tissues. Whether and how PMS2 expression decrease affects the repair capability is not known. Here, we show that PMS2 knockdown cells retaining 19%, 33%, or 53% of PMS2 expression all have significantly reduced MMR efficiency. Surprisingly, the cells retaining expression levels comparable to PMS2 mutation carriers indicate the lowest repair efficiency.