Mismatch repair analysis of inherited MSH2 and/or MSH6 variation pairs found in cancer patients.

Mismatch repair (MMR) malfunction causes the accumulation of mismatches in the genome leading to genomic instability and cancer. The inactivation of an MMR gene (MSH2, MSH6, MLH1, or PMS2) with an inherited mutation causes Lynch syndrome (LS), a dominant susceptibility to cancer. MMR gene variants of uncertain significance (VUS) may be pathogenic mutations, which cause LS, may result in moderately increased cancer risks, or may be harmless polymorphisms. Our study suggests that an inherited MMR VUS individually assessed as proficient may, however, in a pair with another MMR VUS found in the same colorectal cancer (CRC) patient have a concomitant contribution to the MMR deficiency. Here, eight pairs of MMR gene variants found in cancer patients were functionally analyzed in an in vitro MMR assay. Although the other pairs do not suggest a compound deficiency, the MSH2 VUS pair c.380A>G/c.982G>C (p.Asn127Ser/p.Ala328Pro), which nearly halves the repair capability of the wild-type MSH2 protein, is presumed to increase the cancer risk considerably. Moreover, two MSH6 variants, c.1304T>C (p.Leu435Pro) and c.1754T>C (p.Leu585Pro), were shown to be MMR deficient. The role of one of the most frequently reported MMR gene VUS, MSH2 c.380A>G (p.Asn127Ser), is especially interesting because its concomitant defect with another variant could finally explain its recurrent occurrence in CRC patients.

Verification of the three-step model in assessing the pathogenicity of mismatch repair gene variants.

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.

Comprehensive characterization of HNPCC-related colorectal cancers reveals striking molecular features in families with no germline mismatch repair gene mutations.

A considerable fraction of families with HNPCC shows no germline mismatch repair (MMR) gene mutations. We previously detected 'hidden' MMR gene defects in 42% of such families, leaving the remaining 58% 'truly' mutation negative. Here, we characterized 50 colorectal carcinomas and five adenomas arising in HNPCC families; 24 truly MMR gene mutation negative and 31 MMR gene mutation positive. Among 31 tumors from MMR gene mutation positive families, 25 (81%) had active Wnt signaling as indicated by aberrant beta-catenin localization with or without CTNNB1 mutations, compared to only 7/18 tumors from MMR gene mutation negative families (39%; P=0.005). CGH studies revealed stable profiles in 9/16 (56%) of MMR gene mutation negative tumors, which was significantly associated with membranous beta-catenin (P=0.005). Tumors with membranous beta-catenin from the MMR gene mutation negative group also showed low frequency of TP53 mutations compared to those with nuclear beta-catenin. Thus, a majority of the MMR gene mutation negative cases exhibited a novel molecular pattern characterized by the paucity of changes in common pathways to colorectal carcinogenesis. This feature distinguishes the MMR gene mutation negative families from both HNPCC families linked to MMR defects and sporadic cases, suggesting the involvement of novel predisposition genes and pathways in such families.

Molecular analysis of familial endometrial carcinoma: a manifestation of hereditary nonpolyposis colorectal cancer or a separate syndrome?

PURPOSE: Familial clustering of endometrial carcinoma (EC) may occur as part of hereditary nonpolyposis colorectal cancer (HNPCC), a multiorgan cancer syndrome with mismatch repair (MMR) deficiency. Clustering of EC alone, termed as familial site-specific EC, may constitute a separate entity. Because its genetic basis is unknown, our purpose was to characterize such families molecularly. MATERIALS AND METHODS: Twenty-three families with site-specific EC were identified among 519 consecutive patients diagnosed with EC during 1986 to 1997. Tumor tissues were examined for MMR protein expression by immunohistochemical (IHC) analysis, and MMR genes pinpointed by IHC changes were screened for germline mutations by exon-by-exon sequencing, multiplex ligation-dependent probe amplification, and direct tests for mutations common in the population. RESULTS: Among 33 ECs from 23 families, MLH1 protein was lost in seven tumors (21%), MSH2 together with MSH6 was lost in four tumors (12%), and MSH6 alone was lost in five tumors (15%). A truncating germline mutation in MSH6 (3261insC) was identified in one family and a likely pathogenic missense mutation in MSH2 (D603N) was identified in another family. Among the original 519 patients, nine (all with colon cancer in the family) were diagnosed with HNPCC at the outset-six with MLH1 and three with MSH2 mutations. CONCLUSION: Our study gives a minimum overall frequency of 2.1% (11 of 519) for germline MMR defects ascertained through EC in the index patients. The fact that only two of 23 families with site-specific EC (8.7%) had germline mutations in MMR genes suggests another as yet unknown etiology in most families with site-specific EC.

The importance of functional testing in the genetic assessment of Muir-Torre syndrome, a clinical subphenotype of HNPCC.

A majority of families with hereditary nonpolyposis colorectal cancer (HNPCC) are attributable to germline mutations in three DNA mismatch repair (MMR) genes, MLH1, MSH2 and MSH6. However, the clinical phenotype appears to reflect a complex interplay between the predisposing mutation and putative constitutional and somatic modifiers. Certain MMR gene mutations predispose to combined occurrence of cutaneous sebaceous gland neoplasms and visceral malignancies, which is known as Muir-Torre syndrome (MTS) and regarded as a phenotypic variant of HNPCC. The sebaceous tumors associated with MTS appear in many patients before visceral malignancies providing important predictability of HNPCC-related integral cancers in mutation carriers. Since most sebaceous skin tumors are, however, sporadic, the contribution of non-truncating mutations found in skin cancer patients is difficult to interpret and genetic assessment of MTS requires a functional test. Here, we studied the repair efficiency of the two MSH2 missense mutations, L187P and C697F, found in HNPCC families including a few mutation carriers with sebaceous skin tumors. Both mutations were completely deficient in an MMR assay, which together with tumor findings suggested their predisposing role in both internal and skin malignancies in the families.

APC and beta-catenin protein expression patterns in HNPCC-related endometrial and colorectal cancers.

OBJECTIVE: The adenomatous polyposis coli (APC) and beta-catenin (CTNNB1) genes are the two major components of the Wnt signaling pathway that has been shown to play an important role in the formation of certain cancers. The overactivation of the pathway, which results in abnormal accumulation of beta-catenin protein in nuclei, contributes to most colorectal cancers (CRCs), both sporadic and hereditary, as well as sporadic endometrial cancers (ECs). Here, we studied the involvement of APC and beta-catenin in hereditary nonpolyposis colorectal cancer (HNPCC)-related ECs, and compared the expression patterns to those in HNPCC-related CRCs. MATERIALS AND METHODS: Nineteen ECs and 31 CRCs derived from HNPCC patients were immunohistochemically stained with anti-APC- and anti-beta-catenin-antibodies. RESULTS: Tumor-specific loss of APC was observed in 16 of endometrial cancers (3 of 19) and in 39 of colorectal cancers (12 of 31). Consistently, the loss of APC expression was associated with nuclear beta-catenin staining. Altogether, aberrant beta-catenin localization was observed in 53 of ECs (10 of 19) as compared to 84 of CRCs (26 of 31) (P=0.02). CONCLUSION: Our results suggest a frequent overactivation of the Wnt signaling pathway in hereditary endometrial cancer. In accordance with studies on sporadic cancers, abnormal accumulation of beta-catenin protein in nuclei occurred much less frequently in HNPCC-related ECs than CRCs, which may reflect organ-specific differences in their pathogenesis.

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.

Pathogenicity of MSH2 missense mutations is typically associated with impaired repair capability of the mutated protein.

BACKGROUND & AIMS: Inherited deleterious mutations in mismatch repair genes MLH1, MSH2, and MSH6 predispose to hereditary nonpolyposis colorectal cancer. A major diagnostic challenge is the difficulty in evaluating the pathogenicity of missense mutations. Previously we showed that most missense variants in MSH6 do not impair MMR capability and are associated with no or low cancer susceptibility, whereas in MLH1, functional studies distinguished nontruncating mutations with severe defects from those not or slightly impaired in protein expression or function. The present study was undertaken to evaluate the pathogenicity of inherited missense mutations in MSH2. METHODS: Fifteen mutated MSH2 proteins including 14 amino acid substitutions and one in-frame deletion were tested for expression/stability, MSH2/MSH6 interaction, and repair efficiency. The genetic and biochemical data were correlated with the clinical data. Comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. RESULTS: None of the studied MSH2 mutations destroyed the protein or abolished MSH2/MSH6 interaction, whereas 12 mutations impaired the repair capability of the protein. Comparative sequence analysis correctly predicted functional studies for 13 of 14 amino acid substitutions. CONCLUSIONS: Interpretation was pathogenic for 12, nonpathogenic for 2, and contradictory for 1 mutation. The pathogenicity could not be distinguished unambiguously by phenotypic characteristics, although correlation between the absence of staining for MSH2 and pathogenicity of the missense mutation was notable. Unlike in MSH6 and MLH1, the pathogenicity of missense mutations in MSH2 was always associated with impaired repair capability of the mutated protein.

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.

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.

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.

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.