Prevalence of MYH germline mutations in Swiss APC mutation-negative polyposis patients

In 10-30% of patients with classical familial adenomatous polyposis (FAP) and up to 90% of those with attenuated (<100 colorectal adenomas; AFAP) polyposis, no pathogenic germline mutation in the adenomatous polyposis coli (APC) gene can be identified (APC mutation-negative). Recently, biallelic mutations in the base excision repair gene MYH have been shown to predispose to a multiple adenoma and carcinoma phenotype. This study aimed to (i) assess the MYH mutation carrier frequency among Swiss APC mutation-negative patients and (ii) identify phenotypic differences between MYH mutation carriers and APC/MYH mutation-negative polyposis patients. Seventy-nine unrelated APC mutation-negative Swiss patients with either classical (n=18) or attenuated (n=61) polyposis were screened for germline mutations in MYH by dHPLC and direct genomic DNA sequencing. Overall, 7 (8.9%) biallelic and 9 (11.4%) monoallelic MYH germline mutation carriers were identified. Among patients with a family history compatible with autosomal recessive inheritance (n=45), 1 (10.0%) out of 10 classical polyposis and 6 (17.1%) out of 35 attenuated polyposis patients carried biallelic MYH alterations, 2 of which represent novel gene variants (p.R171Q and p.R231H). Colorectal cancer was significantly (p<0.007) more frequent in biallelic mutation carriers (71.4%) compared with that of monoallelic and MYH mutation-negative polyposis patients (0 and 13.8%, respectively). On the basis of our findings and earlier reports, MYH mutation screening should be considered if all of the following criteria are fulfilled: (i) presence of classical or attenuated polyposis coli, (ii) absence of a pathogenic APC mutation, and (iii) a family history compatible with an autosomal recessive mode of inheritance.     

Mutation analysis of the MYH gene in unrelated Czech APC mutation-negative polyposis patients

Some of the APC negative FAP and AFAP cases have recently been found to be attributable to MYH associated polyposis (MAP). MAP is an autosomal recessive syndrome associated with 5-100 colorectal adenomas and caused by mutation in the MYH gene. Here, we screened for germline MYH mutations in 82 APC-mutation-negative probands with classical and attenuated familial adenomatous polyposis using the denaturing high performance liquid chromatography (DHPLC) method in combination with sequencing. Altogether 12 previously reported changes and four novel genetic alterations, mostly in intronic sequences, were identified. The results revealed the presence of biallelic germline MYH mutations in two patients. These patients were compound heterozygotes for two of the most common germline mutations c.494 A>G (p.Y165C); c.1,145 G>A (p.G382D). These variants are established to be associated with adenomatous polyposis and colorectal cancer. No novel pathogenic mutation has been identified in our study.     

APC gene mutations and colorectal adenomatosis in familial adenomatous polyposis

Correlations between germline APC mutation sites and colorectal pathophenotypes, as evaluated by the direct count of adenomas at colectomy, were investigated analysing colectomy specimens from 29 FAP patients carrying one mis-sense (codon 208) and 14 frame-shift or non-sense APC mutations (codons 232, 367, 437, 623, 876, 995, 1061, 1068, 1075, 1112, 1114, 1309, 1324, 1556). The mis-sense mutation at codon 208 was associated with a relatively mild colorectal pathophenotype. The mutation at codon 367, subject to alternative splicing, was associated with attenuated FAP. The mutation at codon 1309 was associated with the profuse colorectal adenomatosis. For 13 mutations, predicted to result in null alleles or truncated APC proteins, we correlated density and distribution of colorectal adenomas with the predicted functional effects of the mutation. The most severe colorectal pathophenotype was significantly associated with the truncating mutation at codon 1309, which is located downstream to the I beta-catenin binding domain but upstream II beta-catenin-binding domain. Mutations between codons 867 and 1114, which affect the I beta-catenin binding domain, as well as mutations occurring in exons 6 and 9, predicted to result in null alleles, were associated with a less severe colorectal pathophenotype. Overall, the highest number of adenomas was detected in the right colon, followed by the left colon, transverse colon sigma and rectum. However, the highest density of adenomas was observed in the left colon, followed by the right colon, sigma, transverse colon and rectum. Colorectal carcinomas, observed in only five patients, were all in the left colon.     

BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency

Genes from the RAF family are Ras-regulated kinases involved in growth cellular responses. Recently, a V599E hotspot mutation within the BRAF gene was reported in a high percentage of colorectal tumors and significantly associated to defective mismatch repair (MMR). Additionally, BRAF mutations were described only in K-Ras-negative colon carcinomas, suggesting that BRAF/K-Ras activating mutations might be alternative genetic events in colon cancer. We have addressed to what extent the tumorigenic-positive selection exerted by BRAF mutations seen in colorectal MMR-deficient tumors was also involved in the tumorigenesis of gastric cancer. Accordingly, BRAF mutations were detected in 34% (25/74) of colorectal MMR-deficient tumors and in 5% (7/142) of MMR-proficient colorectal cases (P=0.0001). All mutations found in the MSI cases corresponded to the previously reported hotspot V599E. Two D593K and a K600E additional mutations were also detected in three MSS cases. However, only one mutation of BRAF was found within 124 MSS gastric tumors and none in 37 MSI gastric tumors, clearly suggesting that BRAF mutations are not involved in gastric tumorigenesis. Nonetheless, a high incidence of mutations of K-Ras was found within the MSI gastric group of tumors (P=0.0005), suggesting that the activation of K-Ras-dependent pathways contributes to the tumorigenesis of gastric cancers with MMR deficiency. Accordingly, our results show evidences that BRAF mutations characterize colon but not gastric tumors with MMR deficiency and are not involved in the tumorigenesis of gastric cancer of the mutator phenotype pathway.     

Microsatellite instability markers for identifying early-onset colorectal cancers caused by germ-line mutations in DNA mismatch repair genes.

PURPOSE: Microsatellite instability (MSI) testing of colorectal cancer tumors is used as a screening tool to identify patients most likely to be mismatch repair (MMR) gene mutation carriers. We wanted to examine which microsatellite markers currently used to detect MSI best predict early-onset colorectal cancer caused by germ-line mutations in MMR genes. EXPERIMENTAL DESIGN: Invasive primary tumors from a population-based sample of 107 cases of colorectal cancer diagnosed before age 45 years and tested for germ-line mutations in MLH1, MSH2, MSH6, and PMS2 and MMR protein expression were screened for MSI using the National Cancer Institute panel and an expanded 10-microsatellite marker panel. RESULTS: The National Cancer Institute five-marker panel system scored 31 (29%) as (NCI)MSI-High, 13 (12%) as (NCI)MSI-Low, and 63 (59%) as (NCI)MS-Stable. The 10-marker panel classified 18 (17%) as (10)MSI-High, 17 (16%) as (10)MSI-Low, and 72 (67%) as (10)MS-Stable. Of the 26 cancers that lacked the expression of at least one MMR gene, 24 (92%) were positive for some level of MSI (using either microsatellite panel). The mononucleotide repeats Bat26, Bat40, and Myb were unstable in all (10)MSI-High cancers and all MLH1 and MSH2 mutation carriers (100% sensitive). Bat40 and Bat25 were unstable in all tumors of MSH6 mutation carriers (100% sensitive). Bat40 was unstable in all MMR gene mutation carriers (100% sensitive). By incorporating seven mononucleotide repeats markers into the 10-marker panel, we were able to distinguish the carriers of MSH6 mutations (all scored (10)MSI-Low) from the MLH1 and MSH2 mutation carriers (all scored (10)MSI-High). CONCLUSIONS: In early-onset colorectal cancer, a microsatellite panel containing a high proportion of mononuclear repeats can distinguish between tumors caused by MLH1 and MSH2 mutations from those caused by MSH6 mutations.     

Correlation between the location of germ-line mutations in the APC gene and the number of colorectal polyps in familial adenomatous polyposis patients.

Recently we have isolated the adenomatous polyposis coli (APC) gene which causes familial adenomatous polyposis (FAP), and its germ-line mutations in a substantial number of FAP patients have been identified. On the basis of this information, we compared the location of germ-line mutations in the APC gene in 22 unrelated patients (12 of whom have been reported previously) with the number of colorectal polyps developed in FAP patients; 17 were sparse types and five were profuse types. All but one of the mutations were considered to cause truncation of the gene product by frame-shift due to deletion (14 cases) or nonsense mutation (seven cases). The location of the germ-line mutations seems to correlate with the two clinical types; germ-line mutations in five FAP patients with profuse polyps were observed between codon 1250 and codon 1464, whereas mutations in 17 FAP patients with fewer polyps were observed in the other regions of the APC gene. The result suggests that the number of colorectal polyps in FAP patients may be associated with a difference in the stability or biological function of the truncated APC protein.     

Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: Report from the constitutional mismatch repair deficiency consortium

BackgroundConstitutional mismatch repair deficiency (CMMRD) is a devastating cancer predisposition syndrome for which data regarding clinical manifestations, molecular screening tools and management are limited.MethodsWe established an international CMMRD consortium and collected comprehensive clinical and genetic data. Molecular diagnosis of tumour and germline biospecimens was performed. A surveillance protocol was developed and implemented.ResultsOverall, 22/23 (96%) of children with CMMRD developed 40 different tumours. While childhood CMMRD related tumours were observed in all families, Lynch related tumours in adults were observed in only 2/14 families (p = 0.0007). All children with CMMRD had café-au-lait spots and 11/14 came from consanguineous families. Brain tumours were the most common cancers reported (48%) followed by gastrointestinal (32%) and haematological malignancies (15%). Importantly, 12 (30%) of these were low grade and resectable cancers. Tumour immunohistochemistry was 100% sensitive and specific in diagnosing mismatch repair (MMR) deficiency of the corresponding gene while microsatellite instability was neither sensitive nor specific as a diagnostic tool (p < 0.0001). Furthermore, screening of normal tissue by immunohistochemistry correlated with genetic confirmation of CMMRD. The surveillance protocol detected 39 lesions which included asymptomatic malignant gliomas and gastrointestinal carcinomas. All tumours were amenable to complete resection and all patients undergoing surveillance are alive.DiscussionCMMRD is a highly penetrant syndrome where family history of cancer may not be contributory. Screening tumours and normal tissues using immunohistochemistry for abnormal expression of MMR gene products may help in diagnosis and early implementation of surveillance for these children.     

Somatic frameshift mutations in the MBD4 gene of sporadic colon cancers with mismatch repair deficiency

Defects of mismatch repair are thought to be responsible for carcinogenesis in hereditary non-polyposis colorectal cancer and about 15% of sporadic colon cancers. The phenotype is seen as microsatellite instability and is known to be caused either by mutations in mismatch repair genes or by aberrant methylation of these genes stabilizing their downregulation. Lack of repair of microsatellite sequence errors, created during replication, leads to a mutation-prone phenotype. Where mutations occur within mononucleotide tracts within exons they cause translation frameshifts, premature cessation of translation and abnormal protein expression. Such mutations have been observed in the TGFbetaRII, BAX, IGFIIR, MSH3 and MSH6 genes in colon and other cancers. We describe here frameshift mutations affecting the gene for the methyl-CpG binding thymine glycosylase, MBD4, in over 40% of microsatellite unstable sporadic colon cancers. The mutations all appear heterozygous but their location would ensure truncation of the protein between the methyl-CpG binding and glycosylase domains, thus potentially generating a dominant negative effect. It is thus possible that such mutations enhance mutation frequency at other sites in these tumours. A suggestion has been made that MBD4 (MED1) mutations may lead to an increased rate of microsatellite instability but this mechanism appears unlikely due to the nature of mutations we have found.     

DNA mismatch repair deficiency stimulates N-ethyl-N-nitrosourea-induced mutagenesis and lymphomagenesis

The primary role of the mismatch repair (MMR) system is the avoidance of mutations caused by replication and recombination errors. Furthermore, the lethality of methylating agents has been attributed to the processing of O(6)-methylguanine lesions in DNA by MMR. Loss of the MSH2 protein completely abolishes repair function and results in reduced cell killing by methylating agents and accelerated accumulation of methylation-damage-induced mutations. This has raised the question as to whether MMR is also involved in the cellular response to other genotoxic insults. Here we describe that in mice deficient for Msh2, lymphomagenesis was strongly accelerated by an ethylating agent, N-ethyl-N-nitrosourea (ENU), given at a dose that did not induce lymphomas in wild-type mice. This suggests that MMR deficiency and ENU-induced mutagenesis synergistically collaborate in inducing tumorigenesis. To study the interaction between MMR and ENU-induced DNA damage, we compared the lethality and mutagenicity of ENU in MSH2-proficient and -deficient mouse embryonic stem cells. Although MSH2-deficiency only slightly reduced the lethality of ENU, it strongly enhanced the mutagenicity of ENU. Mutation analysis of ENU-induced Hprt mutants revealed that base substitutions occurred predominantly at A-T base-pairs. These results suggest that MMR modulates the processing of ethylation damage at AT base-pairs.     

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.     

Analysis of the human APC mutation spectrum in a saccharomyces cerevisiae strain with a mismatch repair defect

Somatic APC mutations in colorectal tumors with an RER phenotype reflect excessive frameshift mutations, especially in simple repetition tracts within the coding sequence. Because this type of mutation is characteristic of cells with a deficient DNA MMR system, the APC mutation signature of RER tumors may be attributable to a defect in the MMR system. However, there is little experimental evidence to prove that the spectrum of mutations and the APC gene distribution are directly influenced by MMR system defects. We therefore examined the mutation spectrum of the MCR of the APC gene after transfection into both MMR-proficient and MMR-deficient yeast strains and compared it with a previously reported human APC mutation database. Small insertions or deletions in mono- or dinucleotide repeats were more common in the MMR-deficient than in the MMR-proficient strain (91.2% vs. 38.1%, Fisher's exact test p < 0.0001). Furthermore, the 2 mutation hot spots, 4385-4394(AG)(5) and 4661-4666(A)(6), found in the yeast system corresponded with those in human tumors. Combining our data with those from human tumors, there appears to be hypermutable mutations in specific simple repetitive sequences within the MCR, which are more prevalent in MMR-deficient cells and RER tumors than in MMR-proficient cells and non-RER tumors. We therefore consider that the differences in the spectra of RER and non-RER tumors are attributable at least in part to the MMR system of the host cells.     

Analysis of mismatch repair gene mutations in Turkish HNPCC patients

Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome) is caused by the inheritance of a mutant allele of a DNA mismatch repair gene. We aimed to investigate types and frequencies of mismatch repair (MMR) gene mutations in Turkish patients with HNPCC and to identify specific biomarkers for early diagnosis of their non-symptomatic kindred’s. The molecular characteristics of 28 Turkish colorectal cancer patients at high-risk for HNPCC were investigated by analysis of microsatellite instability (MSI), immunohistochemistry and methylation-specific PCR in order to select tumors for mutation analysis. Ten cases (35.7%) were classified as MSI (+). Lack of expression of the main MMR proteins was observed in MSI (+) tumors. Hypermethylation of the MLH1 promoter region was observed in one tumor. Nine Lynch syndrome cases showed novel germ-line alterations of the MMR gene: two frame-shifts (MLH1 c.1843dupC and MLH1 c.1743delG) and three missense mutations (MLH1 c.293G>C, MLH1 c.954_955delinsTA and MSH2 c.2210G>A). Unclassified variants were evaluated as likely to be pathogenic by using the in-silico analyses. In addition, the MSH2 c.2210G>A alteration could be considered as a founder mutation for the Turkish population due to its identification in five different Lynch syndrome families and absence in control group. The present study adds new information about MMR gene mutation types and their role in Lynch syndrome. This is the first detailed research on Turkish Lynch syndrome families.     

Working through a diagnostic challenge: colonic polyposis,Amsterdam criteria,and a mismatch repair mutation

The two most common causes of hereditary colorectal cancer are Lynch syndrome and familial adenomatous polyposis (FAP). The phenotype of Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is differentiated in part from FAP by the lack of profuse colonic polyposis. Here we describe a proband who presented with greater than 50 adenomatous colonic polyps prior to developing cancer of the colon and urinary bladder, and a family history that fulfills the Amsterdam criteria. Germline analyses of APC and MYH in the proband did not reveal any mutations. Comprehensive analysis of the mismatch repair genes associated with Lynch syndrome revealed a germline hMSH6 missense mutation 2314C>T (arg772trp) and normal sequencing for hMSH2 and hMLH1. We outline evidence supporting the pathogenicity of the identified hMSH6 mutation (arg772trp) and suggest possible etiologies for the unexplained colonic adenomatous polyposis.     

Zebrafish with mutations in mismatch repair genes develop neurofibromas and other tumors

Defective mismatch repair (MMR) in humans causes hereditary nonpolyposis colorectal cancer. This genetic predisposition to colon cancer is linked to heterozygous familial mutations, and loss-of-heterozygosity is necessary for tumor development. In contrast, the rare cases with biallelic MMR mutations are juvenile patients with brain tumors, skin neurofibromas, and café-au-lait spots, resembling the neurofibromatosis syndrome. Many of them also display lymphomas and leukemias, which phenotypically resembles the frequent lymphoma development in mouse MMR knockouts. Here, we describe the identification and characterization of novel knockout mutants of the three major MMR genes, mlh1, msh2, and msh6, in zebrafish and show that they develop tumors at low frequencies. Predominantly, neurofibromas/malignant peripheral nerve sheath tumors were observed; however, a range of other tumor types was also observed. Our findings indicate that zebrafish mimic distinct features of the human disease and are complementary to mouse models.     

The role of mismatch repair in DNA damage-induced apoptosis

DNA mismatch repair plays a critical role in maintaining genomic integrity. Defects in human mismatch repair are the primary cause of certain types of cancer, including hereditary nonpolyposis colorectal cancer. In the past, the ability of mismatch repair proteins to correct DNA mismatches that occur during DNA replication, repair, and recombination was considered the primary mechanism by which it contributes to genomic stability. However, increasing evidence supports the idea that the mismatch repair system also contributes to genome stability by stimulating DNA damage-induced apoptosis as part of the cytotoxic response to physical and chemical agents. MutS/MutL homologues mediate the process of apoptosis by binding to DNA adducts and either provoking futile repair events or blocking steps in DNA metabolism (i.e., DNA replication and/or repair). This damage recognition step by mismatch repair (MMR) proteins stimulates a signaling cascade for apoptosis, resulting in activation of protein kinase(s) that phosphorylate p53 and/or the related protein p73. Activated p53 and p73 in turn transmit a signal to the apoptotic machinery to execute cell death. The goal of this commentary is to discuss the molecular mechanism(s) by which mismatch repair proteins stimulate apoptosis.