Clinical and Molecular Characterization of Brazilian Patients Suspected to Have Lynch Syndrome

Lynch syndrome (LS) accounts for 3–5% of all colorectal cancers (CRC) and is inherited in an autosomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous/metachronous tumors and extra-colonic tumors. Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five mismatch repair genes (MMR), mostly in MLH1 and MSH2, but also in MSH6, PMS1 and PMS2. To comprehensively characterize 116 Brazilian patients suspected for LS, we assessed the frequency of germline mutations in the three minor genes MSH6, PMS1 and PMS2 in 82 patients negative for point mutations in MLH1 and MSH2. We also assessed large genomic rearrangements by MLPA for detecting copy number variations (CNVs) in MLH1, MSH2 and MSH6 generating a broad characterization of MMR genes. The complete analysis of the five MMR genes revealed 45 carriers of pathogenic mutations, including 25 in MSH2, 15 in MLH1, four in MSH6 and one in PMS2. Eleven novel pathogenic mutations (6 in MSH2, 4 in MSH6 and one in PMS2), and 11 variants of unknown significance (VUS) were found. Mutations in the MLH1 and MSH2 genes represented 89% of all mutations (40/45), whereas the three MMR genes (MSH6, PMS1 and PMS2) accounted for 11% (5/45). We also investigated the MLH1 p.Leu676Pro VUS located in the PMS2 interaction domain and our results revealed that this variant displayed no defective function in terms of cellular location and heterodimer interaction. Additionally, we assessed the tumor phenotype of a subset of patients and also the frequency of CRC and extra-colonic tumors in 2,365 individuals of the 116 families, generating the first comprehensive portrait of the genetic and clinical aspects of patients suspected of LS in a Brazilian cohort.     

Mismatch repair (MMR) efficiency and <Emphasis Type="Italic">MSH2</Emphasis> gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLH1. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

Background

Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.

Methods

Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.

Results

Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.

Conclusions

Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.     

MSH2 c.1452-1455delAATG is a founder mutation and an important cause of hereditary nonpolyposis colorectal cancer in the southern Chinese population

Hereditary nonpolyposis colorectal cancer (HNPCC) accounts for approximately 2% of all colorectal cancer (CRC) cases and is the most common hereditary CRC syndrome. We have previously reported a high incidence of microsatellite instability (MSI) and germline mismatch repair (MMR) gene mutations in young Hong Kong Chinese with CRC. Ongoing studies at the Hereditary Gastrointestinal Cancer Registry in Hong Kong have revealed a unique germline MSH2 c.1452-1455delAATG mutation that has not been reported in other ethnic groups. Detailed analysis showed that this specific MSH2 mutation constituted 21% of all germline MMR gene mutations and 36% of all MSH2 germline mutations identified. We designed a specific PCR-based diagnostic test on paraffin-embedded tissues and identified this germline mutation in 2 (1.5%) of 138 consecutive patients with early-onset CRC (<46 years of age at diagnosis). Haplotype analysis was performed using 11 microsatellite markers located between D2S391 and D2S123. All 10 families had the same disease haplotype, suggesting a founder effect. These 10 families all originated from the Chinese province of Guangdong, which historically included Hong Kong. It is the most populous of the Chinese provinces, with a population of >93 million. Further analysis suggested that this founder mutation may date back to between 22 and 103 generations ago. The identification of this MSH2 founder mutation has important implications for the design of mutation-detection strategies for the southern Chinese population. Since there were major emigrations from Hong Kong and Guangdong province during the 19th and 20th centuries, this finding is also significant for Chinese communities worldwide.     

The Mutational Spectrum of Lynch Syndrome in Cyprus

Lynch syndrome is the most common form of hereditary colorectal cancer and is caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Mutation carriers have an increased lifetime risk of developing colorectal cancer as well as other extracolonic tumours. The aim of the current study was to evaluate the frequency and distribution of mutations in the MLH1, MSH2 and MSH6 genes within a cohort of Cypriot families that fulfilled the revised Bethesda guidelines. The study cohort included 77 patients who fulfilled at least one of the revised Bethesda guidelines. Mutational analysis revealed the presence of 4 pathogenic mutations, 3 in the MLH1 gene and 1 in the MSH2 gene, in 5 unrelated individuals. It is noted that out of the 4 pathogenic mutations detected, one is novel (c.1610delG in exon 14 of the MLH1) and has been detected for the first time in the Cypriot population. Overall, the pathogenic mutation detection rate in our patient cohort was 7%. This percentage is relatively low but could be explained by the fact that the sole criterion for genetic screening was compliance to the revised Bethesda guidelines. Larger numbers of Lynch syndrome families and screening of the two additional predisposition genes, PMS2 and EPCAM, are needed in order to decipher the full spectrum of mutations associated with Lynch syndrome predisposition in Cyprus.     

MUTYH and the mismatch repair system: partners in crime?

Biallelic germline mutations of MUTYH—a gene encoding a base excision repair protein—are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P=0.002) and the published controls (P=0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk.     

Mismatch recognition function of Arabidopsis thaliana MutSγ

Genetic stability depends in part on an efficient DNA lesion recognition and correction by the DNA mismatch repair (MMR) system. In eukaryotes, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes, MSH2–MSH6 and MSH2–MSH3, which recognize and bind mismatches and unpaired nucleotides. Plants encode another mismatch recognition protein, named MSH7. MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSγ. We here report the effect the expression of Arabidopsis MSH2 and MSH7 alone or in combination exert on the genomic stability of Saccharomyces cerevisiae. AtMSH2 and AtMutSγ proteins failed to complement the hypermutator phenotype of an msh2 deficient strain. However, overexpressing AtMutSγ in MMR proficient strains generated a 4-fold increase in CAN1 forward mutation rate, when compared to wild-type strains. Can^r mutation spectrum analysis of AtMutSγ overproducing strains revealed a substantial increase in the frequency of base substitution mutations, including an increased accumulation of base pair changes from G:C to A:T and T:A to C:G, G:C or A:T. Taken together, these results suggest that AtMutSγ affects yeast genomic stability by recognizing specific mismatches and preventing correction by yeast MutSα and MutSβ, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.     

Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.     

Phosphorylation of mismatch repair proteins MSH2 and MSH6 affecting MutSalpha mismatch-binding activity

Mismatch repair (MMR) is involved in the removal of mispaired bases from DNA and thus plays an important role in the maintenance of genomic stability and the prevention of mutations and cancer. Moreover, MMR triggers genotoxicity and apoptosis upon processing of DNA lesions such as O⁶-methylguanine. Whereas the enzymology of MMR has been elucidated in great detail, only limited data are available concerning its regulation. Here we show that the major mismatch-binding proteins MSH2 and MSH6, forming the MutSα complex, are phosphorylated in vitro by protein kinase C and casein kinase II, but not by protein kinase A. Phosphorylation of MSH2 and MSH6 was also found within the cell, with MSH6 being more extensively phosphorylated than MSH2. Lack of MSH2 and MSH6 phosphorylation in vivo due to phosphate depletion, kinase inhibition (by H7 and quercetin) and treatment with phosphatases (CIP, SAP and λ-PPase) significantly reduced mismatch-binding activity of MutSα. It also prevented methylation-induced nuclear translocation of the repair complex, indicating that nuclear translocation of MutSα upon mutagen treatment is dependent on protein phosphorylation. The finding that MSH2 and MSH6 are subject to phosphorylation resulting in increased mismatch binding by MutSα indicates a novel type of post-translational regulation of MMR which might be involved in the response of cells to genotoxic stress.     

Do MSH6 mutations contribute to double primary cancers of the colorectum and endometrium?

Mismatch repair (MMR) gene mutations cause hereditary nonpolyposis colorectal cancer (HNPCC), a common form of familial colorectal cancer. Among MMR genes, germline MSH6 mutations are often observed in HNPCC-like families with an increased frequency of endometrial cancer. We have previously shown that a proportion of women affected with double primary cancers of the colorectum and endometrium carry germline MSH2 or MLH1 mutations and, thus, belong to HNPCC families. In this study, we have investigated the specific contribution of MSH6 defects to such double primary patients. By sequence analysis of the entire coding region of MSH6, three putative missense mutations were identified in patients with atypical family histories that do not meet HNPCC criteria. Moreover, one of these mutations, a novel substitution Arg901 His, was found in a patient previously shown to carry a truncating germline MLH1 mutation. Thus, MSH6 mutations are likely to contribute to the etiology of double primary cancers of the colorectum and endometrium.     

Prevalence of germline mutations of hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in 75 French kindreds with nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is a syndrome characterized by familial predisposition to colorectal carcinoma and extracolonic cancers of the gastrointestinal, urological, and female reproductive tracts. This dominant disorder is caused by germline defects in one of at least five DNA mismatch repair (MMR) genes: hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 (GTBP). Germline mutations of hMSH2 and hMLH1 are also frequently identified in families not fulfilling all the Amsterdam criteria, thereby demonstrating that the involvement of these genes is not confined to typical HNPCC. To evaluate the respective involvement of the various MMR genes in typical and incomplete HNPCC syndromes, we have performed an analysis of the hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in a large series of French kindreds (n=75) with colorectal tumors and/or aggregation of extracolonic cancers belonging to the HNPCC spectrum. Mutational analysis has been performed in all families, without preselection for the tumor phenotype. We have detected 26 pathogenic germline mutations of the hMLH1 and hMSH2 genes and several novel variants of the hPMS1, hPMS2, and hMSH6 genes. Our data confirm that, regardless of the type of families and the tumor phenotype, hPMS1, hPMS2, and hMSH6 germline mutations are rare in familial aggregation of colorectal cancers. Furthermore, they suggest that the presence of multiple primary malignancies in a single individual and the observation of extracolonic tumors in relatives of a colorectal cancer patient should be included among the guidelines for referring patients for genetic testing. Electronic Publication     

Promoter Methylation of MLH1, PMS2, MSH2 and p16 Is a Phenomenon of Advanced-Stage HCCs

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future.     

Unstable DNA Repair Genes Shaped by Their Own Sequence Modifying Phenotypes

The question of whether natural selection favors genetic stability or genetic variability is a fundamental problem in evolutionary biology. Bioinformatic analyses demonstrate that selection favors genetic stability by avoiding unstable nucleotide sequences in protein encoding DNA. Yet, such unstable sequences are maintained in several DNA repair genes, thereby promoting breakdown of repair and destabilizing the genome. Several studies have therefore argued that selection favors genetic variability at the expense of stability. Here we propose a new evolutionary mechanism, with supporting bioinformatic evidence, that resolves this paradox. Combining the concepts of gene-dependent mutation biases and meiotic recombination, we argue that unstable sequences in the DNA mismatch repair (MMR) genes are maintained by their own phenotype. In particular, we predict that human MMR maintains an overrepresentation of mononucleotide repeats (monorepeats) within and around the MMR genes. In support of this hypothesis, we report a 31% excess in monorepeats in 250 kb regions surrounding the seven MMR genes compared to all other RefSeq genes (1.75 vs. 1.34%, P = 0.0047), with a particularly high content in PMS2 (2.41%, P = 0.0047) and MSH6 (2.07%, P = 0.043). Based on a mathematical model of monorepeat frequency, we argue that the proposed mechanism may suffice to explain the observed excess of repeats around MMR genes. Our findings thus indicate that unstable sequences in MMR genes are maintained through evolution by the MMR mechanism. The evolutionary paradox of genetically unstable DNA repair genes may thus be explained by an equilibrium in which the phenotype acts back on its own genotype.     

Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.     

Microsatellite instability in pediatric high grade glioma is associated with genomic profile and differential target gene inactivation

High grade gliomas (HGG) are one of the leading causes of cancer-related deaths in children, and there is increasing evidence that pediatric HGG may harbor distinct molecular characteristics compared to adult tumors. We have sought to clarify the role of microsatellite instability (MSI) in pediatric versus adult HGG. MSI status was determined in 144 patients (71 pediatric and 73 adults) using a well established panel of five quasimonomorphic mononucleotide repeat markers. Expression of MLH1, MSH2, MSH6 and PMS2 was determined by immunohistochemistry, MLH1 was assessed for mutations by direct sequencing and promoter methylation using MS-PCR. DNA copy number profiles were derived using array CGH, and mutations in eighteen MSI target genes studied by multiplex PCR and genotyping. MSI was found in 14/71 (19.7%) pediatric cases, significantly more than observed in adults (5/73, 6.8%; p = 0.02, Chi-square test). MLH1 expression was downregulated in 10/13 cases, however no mutations or promoter methylation were found. MSH6 was absent in one pediatric MSI-High tumor, consistent with an inherited mismatch repair deficiency associated with germline MSH6 mutation. MSI was classed as Type A, and associated with a remarkably stable genomic profile. Of the eighteen classic MSI target genes, we identified mutations only in MSH6 and DNAPKcs and described a polymorphism in MRE11 without apparent functional consequences in DNA double strand break detection and repair. This study thus provides evidence for a potential novel molecular pathway in a proportion of gliomas associated with the presence of MSI.     

Mismatch Repair-Deficient Crypt Foci in Lynch Syndrome – Molecular Alterations and Association with Clinical Parameters

Lynch syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes, most frequently MLH1 and MSH2. Recently, MMR-deficient crypt foci (MMR-DCF) have been identified as a novel lesion which occurs at high frequency in the intestinal mucosa from Lynch syndrome mutation carriers, but very rarely progress to cancer. To shed light on molecular alterations and clinical associations of MMR-DCF, we systematically searched the intestinal mucosa from Lynch syndrome patients for MMR-DCF by immunohistochemistry. The identified lesions were characterised for alterations in microsatellite-bearing genes with proven or suspected role in malignant transformation. We demonstrate that the prevalence of MMR-DCF (mean 0.84 MMR-DCF per 1 cm² mucosa in the colorectum of Lynch syndrome patients) was significantly associated with patients’ age, but not with patients’ gender. No MMR-DCF were detectable in the mucosa of patients with sporadic MSI-H colorectal cancer (n = 12). Microsatellite instability of at least one tested marker was detected in 89% of the MMR-DCF examined, indicating an immediate onset of microsatellite instability after MMR gene inactivation. Coding microsatellite mutations were most frequent in the genes HT001 (ASTE1) with 33%, followed by AIM2 (17%) and BAX (10%). Though MMR deficiency alone appears to be insufficient for malignant transformation, it leads to measurable microsatellite instability even in single MMR-deficient crypts. Our data indicate for the first time that the frequency of MMR-DCF increases with patients’ age. Similar patterns of coding microsatellite instability in MMR-DCF and MMR-deficient cancers suggest that certain combinations of coding microsatellite mutations, including mutations of the HT001, AIM2 and BAX gene, may contribute to the progression of MMR-deficient lesions into MMR-deficient cancers.     

Mismatch repair (MMR) efficiency and MSH2 gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLHL. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT 116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Multiple factors insulate Msh2-Msh6 mismatch repair activity from defects in Msh2 domain I

DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2-Msh6 binds mispairs and small insertion/deletion loops, and Msh2-Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2-Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2-Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2-Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1-Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.     

MSH2 deficiency abolishes the anticancer and pro-aging activity of short telomeres

Mutations in the mismatch repair (MMR) pathway occur in human colorectal cancers with microsatellite instability. Mounting evidence suggests that cell-cycle arrest in response to a number of cellular stresses, including telomere shortening, is a potent anticancer barrier. The telomerase-deficient mouse model illustrates the anticancer effect of cell-cycle arrest provoked by short telomeres. Here, we describe a role for the MMR protein, MSH2, in signaling cell-cycle arrest in a p21/p53-dependent manner in response to short telomeres in the context of telomerase-deficient mice. In particular, progressively shorter telomeres at successive generations of MSH2^−/–Terc^−/–- mice did not suppress cancer in these mice, indicating that MSH2 deficiency abolishes the tumor suppressor activity of short telomeres. Interestingly, MSH2 deficiency prevented degenerative pathologies in the gastrointestinal tract of MSH2^−/–Terc^−/– mice concomitant with a rescue of proliferative defects. The abolishment of the anticancer and pro-aging effects of short telomeres provoked by MSH2 abrogation was independent of changes in telomere length. These results highlight a role for MSH2 in the organismal response to dysfunctional telomeres, which in turn may be important in the pathobiology of human cancers bearing mutations in the MMR pathway.