A putative Lynch syndrome family carrying MSH2 and MSH6 variants of uncertain significance-functional analysis reveals the pathogenic one.

Inherited pathogenic mutations in the mismatch repair (MMR) genes, MSH2, MLH1, MSH6, and PMS2 predispose to Lynch syndrome (LS). However, the finding of a variant or variants of uncertain significance (VUS) in affected family members complicates the risk assessment. Here, we describe a putative LS family carrying VUS in both MSH2 (c.2768T>A, p.Val923Glu) and MSH6 (c.3563G>A, p.Ser1188Asn). Two colorectal cancer (CRC) patients were studied for mutations and identified as carriers of both variants. In spite of a relatively high mean age of cancer onset (59.5 years) in the family, many CRC patients and the tumor pathological data suggested that the missense variation in MSH2, the more common susceptibility gene in LS, would be the predisposing alteration. However, MSH2 VUS was surprisingly found to be MMR proficient in an in vitro MMR assay and a tolerant alteration in silico. By supplying evidence that instead of MSH2 p.Val923Glu the MSH6 p.Ser1188Asn variant is completely MMR-deficient, the present study confirms the previous findings, and suggests that MSH6 (c.3563G>A, p.Ser1188Asn) is the pathogenic mutation in the family. Moreover, our results strongly support the strategy to functionally assess all identified VUS before predictive gene testing and genetic counseling are offered to a family.

Functional characterization of rare missense mutations in MLH1 and MSH2 identified in Danish colorectal cancer patients.

Recently, we have performed a population based study to analyse the frequency of colorectal cancer related MLH1 and MSH2 missense mutations in the Danish population. Half of the analyzed mutations were rare and most likely only present in the families where they were identified originally. Some of the missense mutations were located in conserved regions in the MLH1 and MSH2 proteins indicating a relation to disease development. In the present study, we functionally characterized 10 rare missense mutations in MLH1 and MSH2 identified in 13 Danish CRC families. To elucidate the pathogenicity of the missense mutations, we carried out in vitro functional analyses. The missense mutations were analyzed for their effect on protein expression and repair efficiency. The results of the functional analysis were correlated with clinical data on the families carrying these mutations. Eight missense mutations resulted in proteins with expression and repair efficiency similar to the wild type. One missense mutation (MSH2 p.Met688Val) caused reduced protein expression and one (MSH2 p.Leu187Arg) caused both reduced protein expression and repair deficiency. The MSH2 p.Leu187Arg mutation was found in an Amsterdam II family presenting with high microsatellite instability and loss of MSH2 and MSH6 proteins in tumours. In conclusion, only 1/10 missense mutations displayed repair deficiency and could be classified as pathogenic. No final conclusion can be drawn on the MSH2 p.Met688Val mutation, which caused reduced protein expression. Although, no deficiencies have been identified in the proteins harbouring the other missense mutations, pathogenicity of these variants cannot be unambiguously excluded.

The first functional study of MLH3 mutations found in cancer patients.

The MLH3 gene is one of the five mismatch repair (MMR) genes associated with hereditary nonpolyposis colorectal cancer (HNPCC). Eighteen different inherited MLH3 mutations have been reported as pathogenic in an international mutation database. In several cases, a mutation was found in a patient without a family history suggestive of inherited cancer susceptibility. In some cases, a similar mutation was also found in sporadic patients and/or healthy controls. Four patients carried an MLH3 mutation together with another inherited MMR gene variation. No functional analyses have been performed to assess the pathogenicity of these 18 mutations. MLH3 has been assumed to be less important in MMR than the other HNPCC susceptibility genes MSH2, MSH6, MLH1, and PMS2, and accordingly a low-risk gene for colorectal cancer (CRC). To assess the significance of the inherited sequence variations in MLH3, we functionally characterized seven missense mutations (Q24E, R647C, S817G, G933C, W1276R, A1394T, E1451K) scattered throughout the MLH3 polypeptide. The mutations were found in CRC or endometrial cancer patients and reported as pathogenic. Our study showed that the seven mutated MLH3 proteins, in complex with their counterpart MLH1 (MutLgamma), repaired mismatches as the wild type MutLgamma but worse than a heterodimer of MLH1 and PMS2 (MutLalpha). The results confirm that MutLgamma is a less efficient MMR complex than MutLalpha and show that the MLH3 mutations alone do not interfere with MMR. Further studies are needed to evaluate the pathogenicity of MLH3 mutations in compound with other MMR mutations.

Conditional nuclear localization of hMLH3 suggests a minor activity in mismatch repair and supports its role as a low-risk gene in HNPCC.

DNA mismatch repair (MMR) mechanism contributes to the maintenance of genomic stability. Loss of MMR function predisposes to a mutator cell phenotype, microsatellite instability (MSI) and cancer, especially hereditary non-polyposis colorectal cancer (HNPCC). To date, five MMR genes, hMSH2, hMSH6, hMLH1, hPMS2, and hMLH3 are associated with HNPCC. Although, hMLH3 is suggested to be causative in HNPCC, its relevance to MMR needs to be confirmed to reliably assess significance of the inherited sequence variations in it. Recently, a human heterodimer hMLH1/hMLH3 (hMutLgamma) was shown to be able to assist hMLH1/hPMS2 (hMutLalpha) in the repair of mismatches in vitro. To repair mismatches in vivo, hMLH3 ought to localize in the nucleus. Our immunofluorescence analyses indicated that when all the three MutL homologues are natively expressed in human cells, endogenous hMLH1 and hPMS2 localize in the nucleus, whereas hMLH3 stays in the cytoplasm. Absence of hPMS2 and co-expression of hMLH3 with hMLH1 results in its partial nuclear localization. Our results are clinically relevant since they show that in the nuclear localization hMLH3 is dependent on hMLH1 and competitive with hPMS2. The continuous nuclear localization of hMLH1 and hPMS2 suggests that in vivo, hPMS2 (hMutLalpha) has a major activity in MMR. In absence of hPMS2, hMLH3 (hMutLgamma) is located in the nucleus, suggesting a conditional activity in MMR and supporting its role as a low-risk gene in HNPCC.

Functional significance and clinical phenotype of nontruncating mismatch repair variants of MLH1.

BACKGROUND & AIMS: Germline mutations in mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer. A significant proportion of mutations are nontruncating and associated with a variability of clinical phenotype and microsatellite instability and with occasional presence of residual protein in tumor tissue that suggests impaired functional activity but not total lack of mismatch repair. To address pathogenic significance and mechanism of pathogenicity, we studied the functionality of 31 nontruncating MLH1 mutations found in clinically characterized colorectal cancer families and 3 other variations listed in a mutation database. METHODS: Mutations constructed by site-directed mutagenesis were studied for protein expression/stability, subcellular localization, protein-protein interaction, and repair efficiency. The genetic and biochemical data were correlated with clinical data. Finally, comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. RESULTS: Altogether, 22 mutations were pathogenic in more than one assay, 2 variants were impaired in one assay, and 10 variants acted like wild-type protein. Twenty of 34 mutations affected the quantity of MLH1 protein, whereas only 15 mainly amino-terminal mutations were defective in an in vitro repair assay. Comparative sequence analysis correctly predicted functional studies for 82% of variants. CONCLUSIONS: Pathogenic nontruncating alterations in MLH1 may interfere with different biochemical mechanisms but generally more than one. The severe biochemical defects are mirrored by phenotypic characteristics such as early age at onset and high microsatellite instability, whereas variants with no or mild defects in functionality are associated with variable clinical phenotypes.