Mismatch repair gene polymorphisms and survival in invasive ovarian cancer patients

AimsInherited genetic factors may help partially explain variability of survival length amongst ovarian cancer patients. Of particular interest are genes involved in DNA repair, specifically those involved in mismatch repair (MMR). The aim of this study was to investigate the possible association between the common variants in MMR genes and invasive ovarian cancer overall survival.Method/resultsWe examined associations between 44 variants that tag the known common variants (minor allele frequency ⩾0.05) in seven MMR genes (MLH1, MLH3, MSH2, MSH3, MSH6, PMS1 and PMS2) and survival of invasive ovarian cancer patients in three case–control studies from United Kingdom (UK), Denmark and California of United States of America (USA). DNA from up to 1495 women were genotyped. The genotypes of each polymorphism were tested for association with survival using Cox regression analysis stratified by study. A nominally significant association (P = 0.04) between genotype and ovarian cancer survival was observed for rs2228006 in PMS2. The per-rare allele hazard ratio (HR 95%CI) was 0.84 (0.71–0.99), however, it was not significant after adjusting for multiple covariants (P = 0.47). When the analyses were restricted to serous type ovarian cancer, two SNPs showed marginal significant associations; the per-rare allele HR was 1.3 (1.05–1.6) (P = 0.02) for rs1799977 in MLH1 and 1.4 (1.03–1.9) (P = 0.04) for rs6151662 in MSH3. Neither was significant after adjusting for multiple covariants.ConclusionIt is unlikely that common variants in the MMR pathways examined have moderate effects on survival after diagnosis with ovarian cancer. Much larger studies would be needed to exclude common variants with small effects.     

Common germline variation in mismatch repair genes and survival after a diagnosis of colorectal cancer

The mismatch repair (MMR) genes are involved in the maintenance of genomic integrity. Recently, we showed that common variants in these genes are unlikely to contribute significantly to colorectal cancer risk. The aim of this study was to investigate the role of common variants in the mismatch repair pathway as prognostic markers in colorectal cancer patients. We genotyped 2,060 patients for 68 SNPs in 7 mismatch repair genes (MLH1, MLH3, MSH2, MSH3, MSH6, PMS1 and PMS2), using a single nucleotide polymorphism (SNP) tagging approach. Genotypes at the tag SNPs and multi‐SNP haplotypes were tested for association with overall survival (OS) and disease specific survival (DSS) using a Cox regression model. Eight SNPs and 10 haplotypes were significant at a nominal p < 0.05 in the univariate analyses. Stepwise analysis showed that haplotype effects were mainly due to associated SNPs carried by these haplotypes. After adjustment for sex, age at diagnosis and stage when using overall survival and stage only when using disease specific survival, prognostic values were unattenuated. The most significant SNP associated with disease specific survival after adjustment was rs863221, located in MSH3 (HR: 0.59, 95% confidence interval (CI) 0.42–0.82, p‐value: 0.001). In conclusion, we find some evidence that common variants in mismatch repair genes may contribute to survival of patients with colorectal cancer. © 2008 Wiley‐Liss, Inc.     

Genetic investigation of DNA-repair pathway genes PMS2, MLH1, MSH2, MSH6, MUTYH, OGG1 and MTH1 in sporadic colon cancer

Mutations in DNA repair genes have previously been identified as causative factors for hereditary nonpolyposis colon cancer (HNPCC). Recent evidence also supports an association between DNA sequence variation in these genes and sporadic colorectal carcinoma (CRC). Genetic investigation of DNA repair genes PMS2, MLH1, MSH2, MSH6, MUTYH, OGG1 and MTH1, as possible susceptibility factors for sporadic CRC, was done using both a haplotype tagging and a candidate (i.e. coding) single nucleotide polymorphism (SNP) approach. Some 1,068 patients with operated CRC (median age at diagnosis: 59 years) were compared to 738 sex-matched control individuals (median age: 67 years). Haplotype tagging SNPs, previously reported risk variants and all known coding SNPs with a minor allele frequency >0.005 were genotyped in PMS2 (N = 10), MLH1 (N = 11), MSH2 (N = 18), MSH6 (N = 15), MUTYH (N = 7), OGG1 (N = 11) and MTH1 (N = 3). No evidence for an association between CRC and any of the 7 genes was detected, neither with the tagging or coding SNPs nor in a sliding window haplotype analysis (all nominal p-values >0.05). The previously reported risk variants D132H in MLH1 and R154H in OGG1 were not even observed in the German population. Genetic CRC risk factors so far identified in DNA repair genes seem to be rare and population-specific. Their association with the disease could not be replicated in German CRC samples. It remains to be elucidated by more systematic, large-scale experiments whether common variants in the same genes, but present across populations, represent risk factors for sporadic CRC.     

Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease with high penetrance, caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 and MLH3. Most reported pathogenic mutations are point mutations, comprising single base substitutions, small insertions and deletions. In addition, genomic rearrangements, such as large deletions and duplications not detectable by PCR and Sanger sequencing, have been identified in a significant proportion of HNPCC families, which do not carry a pathogenic MMR gene point mutation. To clarify whether genomic rearrangements in MLH1, MSH2 or MSH6 also occur in patients carrying a point mutation, we subjected normal tissue DNA of 137 colorectal cancer (CRC) patients to multiplex ligation-dependent probe amplification (MLPA) analysis. Patients fulfilled the following pre-requisites: all patients met at least one criterion of the Bethesda guidelines and their tumors exhibited high microsatellite instability (MSI-H) and/or showed loss of expression of MLH1, MSH2 or MSH6 proteins. PCR amplification and Sanger sequencing of all exons of at least one MMR gene, whose protein expression had been lost in the tumor tissue, identified 52 index patients without a point mutation (Group 1), 71 index patients with a pathogenic point mutation in MLH1 (n=38) or MSH2 (n=22) or MSH6 (n=11) (Group 2) and 14 patients with an unclassified variant in MLH1 (n=9) or MSH2 (n=3) or MSH6 (n=2) (Group 3). In 13 of 52 patients of group 1 deletions of at least one exon were identified. In addition, in group 3 one EX1_15del in MLH1 was found. No genomic rearrangement was identified in group 2 patients. Genomic rearrangements represent a significant proportion of pathogenic mutations of MMR genes in HNPCC patients. However, genomic rearrangements are rare in patients carrying point mutations in MMR genes. These findings suggest the use of genomic rearrangement tests in addition to Sanger sequencing in HNPCC patients.     

Prevalence of germline mutations of MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer families from Spain

HNPCC is an autosomal dominantly inherited cancer-susceptibility syndrome that confers an increased risk for colorectal cancer and endometrial cancer at a young age. It also entails an increased risk of a variety of other tumors, such as ovarian, gastric, uroepithelial and biliary tract cancers. The underlying pathogenic mutation lies in 1 of the 5 known DNA MMR genes (MSH2, MLH1, PMS1, PMS2 and MSH6). We screened a total of 140 individuals from 56 Spanish families with suspected HNPCC for mutations in the DNA mismatch repair genes MLH1 and MSH2, using DGGE and direct DNA sequencing. Families were selected on the basis of a history of HNPCC-related tumors or the occurrence of other associated tumors in members besides the index case affected with colorectal cancer. We detected 14 definite pathogenic germline mutations, 9 in MLH1 and 5 in MSH2 in 13 unrelated families selected by the Amsterdam criteria and Bethesda guidelines (1 family carries 2 mutations) and 3 missense mutations in 3 unrelated families selected by the Amsterdam criteria. Among the 17 germline mutations noted in the Spanish cohort, 10 are novel, 7 in MLH1 and 3 in MSH2, perhaps demonstrating different mutational spectra in the Spanish population, where no founder mutation has been identified. Based on our results, we suggest that in the Spanish population not only HNPCC families fulfilling the Amsterdam criteria but also those following Bethesda guidelines should undergo genetic testing for MSH2 and MLH1 mutations.     

The added value of PMS2 immunostaining in the diagnosis of hereditary nonpolyposis colorectal cancer

Identification and characterization of the genetic background in patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is important since control programmes can in a cost-effective manner prevent cancer development in high-risk individuals. HNPCC is caused by germline mismatch repair (MMR) gene mutations and the genetic analysis of HNPCC therefore includes assessment of microsatellite instability (MSI) and immunohistochemical MMR protein expression in the tumor tissue. MSI is found in >95% of the HNPCC-associated tumors and immunostaining using antibodies against the MMR proteins MLH1, MSH2, and MSH6 has been found to correctly pinpoint the affected gene in about 90% of the cases. The PMS2 antibody was the most recently developed and we have in a clinical material assessed the added value of PMS2 immunostaining in 213 patients with suspected hereditary colorectal cancer. All 119 MSS tumors showed retained expression for all four antibodies and PMS2 did thus not identify any underlying MMR defect in these cases. However, PMS2 immunostaining contributed to the characterization of the MMR defect in a subset of the MSI tumors. Concomitant loss of MLH1 and PMS2, which functionally interact in the MutLα complex, was found in 98% of the tumors from patients with germline MLH1 mutations. Among the 12 MSI-high tumors with retained expression of MLH1, MSH2 and MSH6, 8 tumors showed loss of PMS2 staining, and mutations in MLH1 were identified in 2 and mutations in PMS2 in 3 of these individuals. In summary, isolated loss of PMS2 was found in 8% of the MSI-high tumors in our series, including 8/12 previously unexplained MSI-high tumors, in which mutations either in MLH1 or in PMS2 were identified in five cases.     

Endometrial cancer risk is associated with variants of the mismatch repair genes MLH1 and MSH2.

Women with germ-line mutations in the mismatch repair genes (responsible for hereditary nonpolyposis colorectal cancer) face an increased risk of colonic and endometrial cancer. However, these germ-line mutations are rare and are responsible for fewer than 1% of endometrial cancers. Therefore, we examined whether or not common variants of the hereditary nonpolyposis colorectal cancer-associated genes might also be associated with an increased risk of endometrial cancer. Three single-nucleotide polymorphisms were selected in the MLH1 and MSH2 mismatch repair genes. All the various 672 women with endometrial cancer and 880 controls were genotyped. Each of these three single-nucleotide polymorphisms was associated with an increased risk of endometrial cancer. Carriers of the MLH1 nt-93 A allele were at a 1.5-fold increased risk of developing endometrial cancer compared with controls [95% confidence interval (95% CI), 1.2-2.0; P = 0.001]. The risk was higher for homozygote carriers [odds ratio (OR), 1.9; 95% CI, 1.2-3.2; P = 0.009]. For carriers of the MSH2 rs2303428 C allele, the OR was 1.4 (95% CI, 1.0-1.9; P = 0.05), and for carriers of the MSH2 rs2059520 G allele, the OR was 1.3 (95% CI, 1.0-1.7; P = 0.03). More than 9% of endometrial cancer cases carried a variant allele in both MLH1 and MSH2. For these women, the risk of endometrial cancer was particularly high (OR, 2.1; 95% CI, 1.2-3.6; P = 0.005). For patients younger than 50 years at diagnosis who carried both variants, the risk was even higher (OR, 3.4; 95% CI, 1.7-6.6; P = 0.0005). In summary, two common variant alleles of the MLH1 and MSH2 genes make a substantial contribution to endometrial cancer incidence in Ontario.     

Lynch Syndrome Genes

Since the discovery of the major human genes with DNA mismatch repair (MMR) function in 1993--1995, mutations in four, MSH2, MLH1, MSH6, and PMS2, have been convincingly linked to susceptibility of hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome. Among these, PMS2 mutations are associated with diverse clinical features, including those of the Turcot syndrome. Two additional MMR genes, MLH3 and PMS1, have also been proposed to play a role in Lynch syndrome predisposition, but the clinical significance of mutations in these genes is less clear. According to the database maintained by the International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer (ICG-HNPCC), current InSiGHT (International Society for Gastrointestinal Hereditary Tumors), approximately 500 different HNPCC-associated MMR gene mutations are known that primarily involve MLH1 (~50%), MSH2 (~40%), and MSH6 (~10%). Examination of HNPCC/Lynch syndrome-associated MMR genes and their mutations has revealed several other important functions for their protein products beyond postreplicative mismatch repair as well as many alternative mechanisms of pathogenicity. Despite these advances, much is yet to be learned about the molecular basis of correlations between genetic changes and clinical features of the disease.     

N-acetyltransferase (NAT) 2 acetylator status and age of onset in patients with hereditary nonpolyposis colorectal cancer (HNPCC)

N-acetyltransferase (NAT) 2 is an essential polymorphic enzyme involved in the metabolism of various xenobiotics, including potential carcinogens. The individual differences in the NAT2 metabolic capacity are caused by allelic variants of the NAT2 gene which are determined by a pattern of single nucleotide polymorphisms (SNPs) resulting in slow (SA), intermediate (IA) or rapid acetylator (RA) phenotypes. Highly penetrant germline mutations in mismatch repair (MMR) genes are the cause of the disease in hereditary nonpolyposis colorectal cancer (HNPCC). There is no strict correlation between the type of germline mutation in MMR genes and the HNPCC phenotype, but age of tumor onset (AO) in HNPCC has been associated at least in part with different variants in apoptosis-related genes. To clarify the potential modifying role of the NAT2 acetylator status in HNPCC, we performed a multicenter study in 226 individuals with colorectal cancer carrying exclusively pathogenic germline mutations in MSH2 or MLH1. We did not observe any significant difference in the NAT2 acetylator status frequency between HNPCC patients and 107 healthy controls (P=0.156), and between MLH1 and MSH2 mutation carriers (P=0.198). Multivariate Cox regression analysis revealed that male patients had a significantly increased risk to develop CRC compared to females during any interval (P=0.043), while the NAT2 acetylator status (P=0.447) and the mutated gene (MLH1 or MSH2) (P=0.236) were not risk factors for AO. The median AO in HNPCC patients was 39 years in patients with RA as well as with SA status (P=0.347). In MLH1 mutation carriers, the median AO was 38 years in RA and 36 years in SA status patients (P=0.901), whereas in MSH2 mutation carriers, the median AO was 39 years in RA and 42 years in SA status patients (P=0.163). Log-rank test revealed a significantly lower age of CRC onset in male compared to female HNPCC patients (P=0.0442). These data do not support the hypothesis that the NAT2 acetylatorship acts as a modifying factor on AO in HNPCC-associated CRC.     

Immunohistochemistry and microsatellite instability testing for selecting MLH1, MSH2 and MSH6 mutation carriers in hereditary non-polyposis colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC) represents 1-3% of all colorectal cancers. HNPCC is caused by a constitutional defect in a mismatch repair (MMR) gene, most commonly affecting the genes MLH1, MSH2 and MSH6. The MMR defect results in an increased cancer risk, with the greatest lifetime risk for colorectal cancer and other cancers associated to HNPCC. The HNPCC-associated tumor phenotype is generally characterized by microsatellite instability (MSI) and immunohistochemical loss of expression of the affected MMR protein. The aim of this study was to determine the sensitivity of IHC for MLH1, MSH2 and MSH6, and MSI analysis in tumors from known MMR gene mutation carriers. Fifty-eight paired normal and tumor samples from HNPCC families enrolled in our high-risk colorectal cancer registry were studied for the presence of germline mutations in MLH1, MSH2 and MSH6 by DGGE and direct sequencing. MSI analysis and immunostaining for MLH1, MSH2 and MSH6 were evaluated. Of the 28 patients with a real pathogenic mutation, loss of immunohistochemical expression for at least 1 of these MMR proteins was found, and all except 1 have MSI-H. Sensitivity by MSI analysis was 96%. IHC analysis had a sensitivity of 100% in detecting MMR deficiency in carriers of a pathogenic MMR mutation, and can be used to predict which gene is expected to harbor the mutation for MLH1, MSH2 and MSH6. This study suggests that both analyses are useful for selecting high-risk patients because most MLH1, MSH2 and MSH6 gene carriers will be detected by this 2-step approach. This practical method should have immediate application in the clinical work of patients with inherited colorectal cancer syndromes.     

Little evidence for involvement of MLH3 in colorectal cancer predisposition

Mutations in the DNA MMR genes MSH2, MLH1, MSH6 and PMS2 underlie a large subset of HNPCC cases, and a hallmark of the tumors is MSI. In many HNPCC families, however, a causative mutation has not been found. Therefore, the involvement of additional, thus far unknown, genes in MSI as well as MSS colorectal tumor predisposition is possible. The role of a relatively recently cloned MMR gene, MLH3, in familial CRC has been studied; but the results appear somewhat conflicting. To further evaluate the role of MLH3 in CRC predisposition, we analyzed 30 Finnish CRC cases for germline mutations by sequencing. These cases were selected from a large series of Finnish CRC patients, to match features previously proposed to associate with MLH3 germline defects. We found 5 missense variants, 4 of which were also found in Finnish cancer-free controls. The only remaining variant does not appear to be an attractive candidate for a disease-associated mutation because the amino acid change is located outside the conserved residues. We also screened for the previously reported variants, including a frameshift change, the most likely pathogenic MLH3 mutation observed so far. The frameshift was not present in the 30 CRC cases or in 700 cancer-free controls. While it is a difficult task to exclude a role of MLH3 in HNPCC, our study could not confirm a role for MLH3 in CRC predisposition.     

Hereditary nonpolyposis colorectal cancer in endometrial cancer patients

Endometrial cancer is the second most common cancer in hereditary nonpolyposis colorectal cancer (HNPCC). It has often been overlooked to explore the possibility of HNPCC in endometrial cancer patients. Our study was to investigate how many HNPCC patients existed among endometrial cancer patients. Among patients who underwent hysterectomy for endometrial cancer at Seoul National University Hospital from 1996 to 2004, 113 patients were included, whose family history and clinical data could be obtained and tumor specimens were available for microsatellite instability (MSI) testing and immunohistochemical (IHC) staining of MLH1, MSH2 and MSH6 proteins. There were 4 (3.5%) clinical HNPCC patients fulfilling the Amsterdam criteria II, and 2 (2/4, 50%) of them carried MSH2 germline mutations. There were also 8 (7.1%) suspected HNPCC (s-HNPCC) patients fulfilling the revised criteria for s-HNPCC, and one (1/8, 12.5%) of them revealed MLH1 germline mutation. In 101 patients, who were not clinical HNPCC or s-HNPCC, 11 patients showed both MSI-high and loss of expression of MLH1, MSH2 or MSH6 proteins, and 2 (2/11, 18.2%) of them showed MSH6 germline mutations. In 113 patients with endometrial cancer, we could find 5 (4.4%) HNPCC patients with MMR germline mutation and 2 (1.8%) clinical HNPCC patients without identified MMR gene mutation. Family history was critical in detecting 3 HNPCC patients with MMR germline mutation, and MSI testing with IHC staining for MLH1, MSH2 and MSH6 proteins was needed in the diagnosis of 2 HNPCC patients who were not clinical HNPCC or s-HNPCC, especially for MSH6 germline mutation.     

Polymorphisms in DNA repair genes,recreational physical activity and breast cancer risk

The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single‐strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Polymorphisms in MMR or other DNA repair gene variants may modify the association between RPA and breast cancer incidence. We investigated the individual and joint effects of variants in three MMR pathway genes (MSH3, MLH1 and MSH2) on breast cancer occurrence using resources from the Long Island Breast Cancer Study Project. We additionally characterized interactions between RPA and genetic polymorphisms in MMR, BER and NER pathways. We found statistically significant multiplicative interactions (p < 0.05) between MSH2 and MLH1, as well as between postmenopausal RPA and four variants in DNA repair (XPC‐Ala499Val, XPF‐Arg415Gln, XPG‐Asp1104His and MLH1‐lle219Val). Significant risk reductions were observed among highly active women with the common genotype for XPC (OR = 0.54; 95% CI, 0.36–0.81) and XPF (OR = 0.62; 95% CI, 0.44–0.87), as well as among active women who carried at least one variant allele in XPG (OR = 0.46; 95% CI, 0.29–0.77) and MLH1 (OR = 0.46; 95% CI, 0.30–0.71). Our data show that women with minor alleles in both MSH2 and MLH1 could be at increased breast cancer risk. RPA may be modified by genes in the DNA repair pathway, and merit further investigation.     

High-throughput gene sequencing assay development for hereditary nonpolyposis colon cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common hereditary colon cancer syndrome and is responsible for as many as 10% of all colorectal cancers. Hereditary nonpolyposis colorectal cancer is autosomally dominant with a prevalence of 1 in 200-2000 and exhibits incomplete penetrance. Affected individuals have an approximately 70% lifetime risk of colon cancer with a mean age of onset of 44 years and an approximately 40% lifetime risk of endometrial cancer. At least 5 mismatch repair genes (MLH1, MSH2, MSH6, PMS1, PMS2) have been implicated in HNPCC; however, no predominant mutations were found in these genes. Mutation detection by direct sequencing has proven to be the most sensitive method. We have developed high-throughput full-length sequencing assays of the MLH1, MSH2, and MSH6 genes. These 3 genes account for approximately 90% of all germline mutations found in HNPCC. In our assays, 19 exons of MLH1, 16 exons of MSH2, 10 exons of MSH6, and the adjacent splice sites were amplified using polymerase chain reaction and loaded onto a capillary sequencing machine. Results were analyzed using sequence analysis software and stored in a relational database. Our assay method was validated using 15 affected patients and normal controls. It is anticipated that our high-throughput assay technique will provide accurate diagnoses for patients at risk for HNPCC and thereby facilitate early curative intervention.     

Identification and characterization of a novel MLH1 genomic rearrangement as the cause of HNPCC in a Tunisian family: evidence for a homologous Alu-mediated recombination

High rates of early colorectal cancers are observed in Tunisia suggesting high genetic susceptibility. Nevertheless, up to now no molecular studies have been performed. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent cause of inherited colorectal cancer. It is caused by constitutional mutations in the DNA mismatch repair (MMR) genes. Here, we investigated a Tunisian family highly suspected of hereditary nonpolyposis colorectal cancer (HNPCC). Six patients were diagnosed with a colorectal or an endometrial cancer at an early age, including one young female who developed a colorectal cancer at 22 years and we tested for germline mutations in MMR genes. MMR genes were tested for rearrangements by MLPA (MLH1, MSH2) and the presence of point mutations by sequencing (MLH1, MSH2, MSH6). Moreover, tumors were analyzed for microsatellite instability and expression of MMR proteins, as well as for somatic rearrangements in MLH1 and MSH2 by MLPA. MMR gene analysis by MLPA revealed the presence of a large deletion in MLH1 removing exon 6. Sequence analysis of the breakpoint region showed that this rearrangement resulted from a homologous unequal recombination mediated by a repetitive Alu sequence. Moreover, tumors harbored biallelic deletion of MLH1 exon 6 and loss of heterozygosity at MLH1 intragenic markers, suggesting duplication of the rearranged allele in the tumor. This germline MLH1 rearrangement was associated to a severe phenotype in this family. This is the first report of a molecular analysis in a Tunisian family with HNPCC.     

Lower incidence of colorectal cancer and later age of disease onset in 27 families with pathogenic MSH6 germline mutations compared with families with MLH1 or MSH2 mutations: the German Hereditary Nonpolyposis Colorectal Cancer Consortium.

PURPOSE: The aim of the study was the analysis of the involvement and phenotypic manifestations of MSH6 germline mutations in families suspected of hereditary nonpolyposis colorectal cancer (HNPCC). PATIENTS AND METHODS: Patients were preselected among 706 families by microsatellite instability, immunohistochemistry, and/or exclusion of MLH1 or MSH2 mutations and were subjected to MSH6 mutation analysis. Clinical and molecular data of MSH6 mutation families were compared with data from families with MLH1 and MSH2 mutations. RESULTS: We identified 27 families with 24 different pathogenic MSH6 germline mutations, representing 3.8% of the total of the families, and 14.7% of all families with DNA mismatch repair (MMR) gene mutations (n = 183). The median age of onset of colorectal cancer in putative mutation carriers was 10 years higher for MSH6 (54 years; 95% CI, 51 to 56) compared with MLH1 and MSH2 (44 years; 95% CI, 43 to 45; log-rank test, P = .0038). Relative to other malignant tumors, colorectal cancer was less frequent in MSH6 families compared with MLH1 and MSH2 families (Fisher's exact test, P < .001). In contrast, the frequency of non-HNPCC-associated tumors was increased (Fisher's exact test, P < .001). CONCLUSION: Later age of disease onset and lower incidence of colorectal cancer may contribute to a lower proportion of identified MSH6 mutations in families suspected of HNPCC. However, in approximately half of these families, at least one patient developed colorectal or endometrial cancer in the fourth decade of life. Therefore, a surveillance program as stringent as that for families with MLH1 or MSH2 mutations is recommended.     

Mismatch repair polymorphisms and the risk of colorectal cancer

Rare germline variants in mismatch repair genes have been linked to hereditary nonpolyposis colorectal cancer; however, it is unknown whether common polymorphisms in these genes alter the risk of colorectal cancer. To examine the association between common variants in mismatch repair genes and colorectal cancer, we conducted a case-cohort study within the CLUE II cohort. Four single nucleotide polymorphisms in 3 mismatch repair genes (MSH3 R940Q, MSH3 T1036A, MSH6 G39E and MLH1 I219V) were genotyped in 237 colorectal cancer cases and a subcohort of 2,189 participants. Incidence rate ratios (RRs) and 95% confidence intervals (95% CIs) for each polymorphism were estimated. The MSH3 1036A variant was found to be associated with an increased risk of colorectal cancer (RR=1.28, 95% CI: 0.94-1.74 and RR=1.65, 95% CI: 1.01-2.70 for the AT and TT genotypes, respectively, with p(trend)=0.02), particularly proximal colon cancer. Although the MSH3 940Q variant was only weakly associated with colorectal cancer overall (p(trend)=0.07), it was associated with a significant increased risk of proximal colon cancer (RR=1.69, 95% CI: 1.10-2.61 and RR=2.68, 95% CI: 0.96-7.47 for the RQ and QQ genotypes, respectively with p(trend)=0.005). Processed meat intake appeared to modify the association between the MSH3 polymorphisms and colorectal cancer (p(interaction) < 0.10 for both). No association was observed with the MSH6 and MLH1 polymorphisms overall. This study suggests that common polymorphisms in the mismatch repair gene, MSH3, may increase the risk of colorectal cancer, especially proximal colon cancer.     

Association of low-risk MSH3 and MSH2 variant alleles with Lynch syndrome: probability of synergistic effects

Mutations in the MLH1 and MSH2 genes account for a majority of cases of families with Lynch Syndrome. Germ-line mutations in MSH6, PMS2 and MLH3 are responsible for disease in a minority of cases, usually associated with milder and variable phenotypes. No germ-line mutations in MSH3 have so far been associated with Lynch Syndrome, although it is known that impaired MSH3 activity leads to a partial defect in mismatch repair (MMR), with low levels of microsatellite instability at the loci with dinucleotide repeats in colorectal cancer (CRC), thus suggesting a role for MSH3 in carcinogenesis. To determine a possible role of MSH3 as predisposing to CRC in Lynch syndrome, we screened MSH3 for germ-line mutations in 79 unrelated Lynch patients who were negative for pathogenetic mutations in MLH1, MSH2 and MSH6. We found 13 mutant alleles, including silent, missense and intronic variants. These variants were identified through denaturing high performance liquid chromatography and subsequent DNA sequencing. In one Lynch family, the index case with early-onset colon cancer was a carrier of a polymorphism in the MSH2 gene and two variants in the MSH3 gene. These variants were associated with the disease in the family, thus suggesting the involvement of MSH3 in colon tumour progression. We hypothesise a model in which variants of the MSH3 gene behave as low-risk alleles that contribute to the risk of colon cancer in Lynch families, mostly with other low-risk alleles of MMR genes.     

Microsatellite instability and expression of MLH1 and MSH2 in carcinomas of the small intestine

BACKGROUND: Carcinomas of the small intestine are rare, but the risk is greatly increased in patients with hereditary nonpolyposis colorectal cancer (HNPCC) due to an inherited mismatch repair (MMR) gene mutation, most commonly affecting the genes MLH1 or MSH2. Defective MMR is characterized by microsatellite instability (MSI) and loss of MMR protein expression in the tumor tissue. However, a subset of several sporadic tumor types, including about 15% of colon cancers, also evolve through defective MMR. METHODS: The authors have assessed the frequency of MSI and analyzed the immunohistochemical expression of MLH1 and MSH2 in a population-based series of 89 adenocarcinomas of the small intestine. To study the contribution of MSI and defective MMR protein expression in young patients, 43 cancers of the small intestine from patients below age 60 years (including 24 tumors from the population-based series and an additional 19 tumors from young individuals) were also analyzed. RESULTS: MSI was detected in 16/89 tumors (18%) in the population-based series, and immunohistochemistry revealed loss of expression for MLH1 in 7/16 MSI tumors and in 2/73 MSS tumors, whereas all tumors showed normal expression for MSH2. Among the young patients, the authors identified MSI in 10/43 tumors (23%), and 6 of these 10 MSI tumors showed immunohistochemical loss of MMR protein expression, which affected MLH1 in 3 cases and MSH2 in 3 cases. CONCLUSIONS: The frequency of MSI (18%) in adenocarcinomas of the small intestine equals that of colon cancer. However, silencing of MLH1 seems to explain the MSI status in only about half of the MSI tumors. Among patients with cancer of the small intestine before age 60 years, MSI is found in 23% of the cases, with MLH1 and MSH2 being affected at equal frequencies, indicating that HNPCC may underly a subset of such cases.