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.

HNPCC mutation MLH1 P648S makes the functional protein unstable, and homozygosity predisposes to mild neurofibromatosis type 1.

Heterozygous germ-line mutations in DNA mismatch repair (MMR) genes predispose individuals to hereditary nonpolyposis colorectal cancer (HNPCC), whereas with homozygous MMR gene mutations children are diagnosed at an early age with de novo neurofibromatosis type 1 (NF1) and/or hematological malignancies. Here, we describe a mutation, MLH1 P648S, which was found in a typical HNPCC family, with one homozygous child displaying mild features of NF1 and no hematological cancers. To evaluate the pathogenicity of the mutation, we studied both the expression and the function of the mutated protein. It generally has been assumed that the predisposing mutations prevent the production of a functional protein. The mutated MLH1 P648S protein was found to be unstable but still functional in mismatch repair, suggesting that the cancer susceptibility in the family and possibly also the mild disease phenotype in the homozygous individual are linked to shortage of the functional protein.

Pathogenicity of the hereditary colorectal cancer mutation hMLH1 del616 linked to shortage of the functional protein.

BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer is associated with mismatch repair deficiency. Most predisposing mutations prevent the production of functional mismatch repair protein. Thus, when the wild-type copy is also inactivated, the cell becomes mismatch repair deficient, and this leads to a high degree of microsatellite instability in tumors. However, tumors linked to nontruncating mutations may display positive or partly positive immunohistochemical staining of the mutated protein and low or atypical microsatellite instability status, which suggests impaired functional activity but not a total lack of mismatch repair. We found human mutL homology (hMLH) 1 del616, one of the most widespread recurring mutations in hereditary nonpolyposis colorectal cancer, segregating in a large hereditary nonpolyposis colorectal cancer family. Because the predicted coding change is a deletion of only 1 amino acid, the pathogenicity of the mutation was evaluated. METHODS: Many analyses were performed to assess the pathogenicity of hMLH1 del616 and to study the expression and function of the mutated messenger RNA and protein. RESULTS: Genetic and immunohistochemical evidence supported hMLH1-linked cancer predisposition in this family. Microsatellite instability varied from low to high, and the hMLH1 protein was lost in 2 tumors but was partly detectable in 1 tumor. Whereas similar optimal amounts of mutated hMLH1 del616 and wild-type hMLH1 proteins were equally functional in an in vitro mismatch repair assay, the amount of in vivo-expressed hMLH1 del616 was much lower than the amount of wild-type protein; this suggests that the deletion imparts instability to the mutant protein. CONCLUSIONS: Our results suggest that the pathogenicity of hMLH1 del616 is not linked to nonfunctionality, but to shortage of the functional protein.

Two mismatch repair gene mutations found in a colon cancer patient--which one is pathogenic?

Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome. Germline mutations in five different mismatch repair (MMR) genes, MSH2, MSH6, MLH1, MLH3, and PMS2 are linked to HNPCC. Here, we describe two colon cancer families in which the index patients carry missense mutations in both MSH2 and MSH6. The MSH2 mutation, I145M, is the same in both families, whereas the MSH6 mutations are different (R1095H and L1354Q). The families do not fulfil the international criteria for HNPCC, one family comprising two and the other family four colon cancer patients, all in one generation, resembling a recessive rather than dominant inheritance characteristic of HNPCC. The tumors of the index patients showed microsatellite instability. Functional analysis was performed to determine which one of the mutations could primarily underlie the cancer susceptibility in the families. MSH2 and MSH6 are known to form a heterodimeric complex (MutSalpha) responsible for mismatch recognition. The interaction of each mutated protein with its wild-type partner and with its mutated partner present in the colon cancer patient, and the MMR function of the mutated MutSalpha complexes were determined. Since none of the three mutations affected the MSH2-MSH6 interaction or the function of MutSalpha in an in-vitro MMR assay, our results suggest that alone the mutations do not cause MMR deficiency typical of HNPCC. However, our results do not exclude the possible compound pathogenicity of the two mutations.

Functional analysis of MSH6 mutations linked to kindreds with putative hereditary non-polyposis colorectal cancer syndrome.

To date, five mismatch-repair (MMR) genes, MLH1, MSH2, MSH6, MSH3 and PMS2, are known to be involved in human MMR function. Two of those, MLH1 and MSH2, are further the most common susceptibility genes for hereditary non-polyposis colorectal cancer (HNPCC), while MSH3 and PMS2 are seldom (PMS2) or not at all (MSH3 ) reported to be involved in HNPCC. Despite the increasing number of MSH6 germline mutations, their pathogenicity remains questionable, because the mutations are mainly linked to putative HNPCC families lacking the typical clinical and molecular characteristics of the syndrome, such as early age at onset and high microsatellite instability (MSI). High MSI is a consequence of MMR defect, and the pathogenicity of germline mutations in HNPCC is thus linked to malfunction of MMR. To address the question of whether and how MSH6 mutations cause susceptibility to HNPCC, we studied heterodimerization of four MSH6 variants with MSH2, and the functionality of these MutSalpha complexes in an in vitro MMR assay. All mutations occurred in putative HNPCC patients. Irrespective of the type or the site of the amino acid substitutions, all the variants repaired G.T mismatches to A.T as wild-type MSH6 protein. However, the MSH6 protein carrying a mutation in the MSH2/MSH6 interaction region was poorly expressed, suggesting problems in its stability. Our results are clinically relevant, since they demonstrate that under the stable in vitro conditions, when the amounts of the proteins are adequate for repair, the tested MSH6 mutations do not affect repair function. Consequently, while the typical HNPCC syndrome is associated with problems in repair reaction, the pathogenicity of mutations in putative HNPCC families may be linked to other biochemical events.