Three novel germline mutations in MLH1 and MSH2 in families with Lynch syndrome living on Jeju island, Korea

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome characterized by predisposition to early-onset cancers. HNPCC is caused by heterozygous loss-of-function mutations within the mismatch repair genes MLH1, MSH2, MSH6, PMS1, and PMS2. We genotyped the MLH1 and MSH2 genes in patients suffering from Lynch syndrome and in 11 unrelated patients who were diagnosed with colorectal cancer and had subsequently undergone surgery. Five Lynch syndrome patients carried germline mutations in MLH1 or MSH2. Two of these were identified as known mutations in MLH1: deletion of exon 10 and a point mutation (V384D). The remaining three patients exhibited novel mutations: a duplication (937_942dupGAAGTT) in MLH1; deletion of exons 8, 9, and 10; and a point mutation in MLH1 (F396I) combined with multiple missense mutations in MSH2 (D295G, K808E, Q855P, and I884T). The findings underline the importance of efficient pre-screening of conspicuous cases.     

Hijacked DNA repair proteins and unchained DNA polymerases

Somatic hypermutation of immunoglobulin (Ig) genes occurs at a frequency that is a million times greater than the mutation in other genes. Mutations occur in variable genes to increase antibody affinity, and in switch regions before constant genes to cause switching from IgM to IgG. Hypermutation is initiated in activated B cells when the activation-induced deaminase protein deaminates cytosine in DNA to uracil. Uracils can be processed by either a mutagenic pathway to produce mutations or a non-mutagenic pathway to remove mutations. In the mutagenic pathway, we first studied the role of mismatch repair proteins, MSH2, MSH3, MSH6, PMS2 and MLH1, since they would recognize mismatches. The MSH2–MSH6 heterodimer is involved in hypermutation by binding to U:G and other mismatches generated during repair synthesis, but the other proteins are not necessary. Second, we analysed the role of low-fidelity DNA polymerases η, ι and θ in synthesizing mutations, and conclude that polymerase η is the dominant participant by generating mutations at A:T base pairs. In the non-mutagenic pathway, we examined the role of the Cockayne syndrome B protein that interacts with other repair proteins. Mice deficient in this protein had normal hypermutation and class switch recombination, showing that it is not involved.     

Bcl-2 rescues the germinal center response but does not alter the V gene somatic hypermutation spectrum in MSH2-deficient mice

Ab V genes in mice deficient for the postreplication mismatch repair factor MutS homolog (MSH2) have been reported to display an abnormal bias for hypermutations at G and C nucleotides and hotspots. We previously showed that the germinal center (GC) response is severely attenuated in MSH2-deficient mice. This suggested that premature death of GC B cells might preclude multiple rounds of hypermutation necessary to generate a normal spectrum of base changes. To test this hypothesis, we created MSH2-deficient mice in which Bcl-2 expression was driven in B cells from a transgene. In such mice, the elevated levels of intra-GC apoptosis and untimely GC dissolution characteristic of MSH2-deficient mice are suppressed. However, the spectrum of hypermutation is unchanged. These data indicate that the effects of MSH2 deficiency on GC B cell viability and the hypermutation process are distinct.     

Isolation and Characterization of Point Mutations in Mismatch Repair Genes That Destabilize Microsatellites in Yeast

The stability of simple repetitive DNA sequences (microsatellites) is a sensitive indicator of the ability of a cell to repair DNA mismatches. In a genetic screen for yeast mutants with elevated microsatellite instability, we identified strains containing point mutations in the yeast mismatch repair genes, MSH2, MSH3, MLH1, and PMS1. Some of these mutations conferred phenotypes significantly different from those of null mutations in these genes. One semidominant MSH2 mutation was identified. Finally we showed that strains heterozygous for null mutations of mismatch repair genes in diploid strains in yeast confer subtle defects in the repair of small DNA loops.     

exo1-Dependent Mutator Mutations: Model System for Studying Functional Interactions in Mismatch Repair

EXO1 interacts with MSH2 and MLH1 and has been proposed to be a redundant exonuclease that functions in mismatch repair (MMR). To better understand the role of EXO1 in mismatch repair, a genetic screen was performed to identify mutations that increase the mutation rates caused by weak mutator mutations such as exo1Delta and pms1-A130V mutations. In a screen starting with an exo1 mutation, exo1-dependent mutator mutations were obtained in MLH1, PMS1, MSH2, MSH3, POL30 (PCNA), POL32, and RNR1, whereas starting with the weak pms1 allele pms1-A130V, pms1-dependent mutator mutations were identified in MLH1, MSH2, MSH3, MSH6, and EXO1. These mutations only cause weak MMR defects as single mutants but cause strong MMR defects when combined with each other. Most of the mutations obtained caused amino acid substitutions in MLH1 or PMS1, and these clustered in either the ATP-binding region or the MLH1-PMS1 interaction regions of these proteins. The mutations showed two other types of interactions: specific pairs of mutations showed unlinked noncomplementation in diploid strains, and the defect caused by pairs of mutations could be suppressed by high-copy-number expression of a third gene, an effect that showed allele and overexpressed gene specificity. These results support a model in which EXO1 plays a structural role in MMR and stabilizes multiprotein complexes containing a number of MMR proteins. A similar role is proposed for PCNA based on the data presented.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.     

Soft tissue sarcoma and the hereditary non-polyposis colorectal cancer (HNPCC) syndrome: formulation of an hypothesis

Hereditary non-polyposis colorectal cancer (HNPCC) is a genetic disorder caused by mutation in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) which predisposes to colorectal cancer and other malignances, that not yet include sarcomas. For sustaining that soft tissue sarcomas could be HNPCC related malignances, we report on a HNPCC patient with leiomyosarcoma and review the English literature. Overall, we report on eleven cases of soft tissue malignant tumors involving HNPCC patients, with a mean age of 34 years at diagnosis of sarcomas. In the majority of these tumors loss of MSH2 expression can be found at immunohistochemistry (IHC) and in 10 patients a germline mutation in one of the MMR genes was found (7 cases were MSH2 defective and 3 cases MLH1 defective). Data for supporting our hypothesis are also experimental, epidemiologic, histopathological: excess of sarcomas in PMS2 defective mice; sporadic soft tissue sarcomas are rare, with mean age at onset of 56 years and normal IHC for MMR proteins. In conclusion, the data collected support the hypothesis that soft tissue sarcomas could be included in the spectrum of tumors that, even if rarely, depend on MMR genes deficiency.     

Deletions account for 17% of pathogenic germline alterations in MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer (HNPCC) families

Hereditary nonpolyposis colorectal cancer (HNPCC) is due to defects in DNA mismatch repair (MMR) genes MSH2, MLH1, MSH6, and to a lesser extent PMS2. Of 466 suspected HNPCC families, we defined 54 index patients with either tumors of high microsatellite instability (MSI-H) and/or loss of expression for either MLH1, MSH2, and/or MSH6, but without a detectable pathogenic point mutation in these genes. This study cohort was augmented to 64 patients by 10 mutation-negative index patients from Amsterdam families where no tumors were available. Deletion/duplication screening using the multiplex ligation-dependent probe amplification (MLPA) revealed 12 deletions in MSH2 and two deletions in MLH1. These deletions constitute 17% of pathogenic germline alterations but elucidate the susceptibility to HNPCC in only 22% of the mutation-negative study cohort, pointing towards other mutation mechanisms for an inherited inactivation of MLH1 or MSH2. We describe here four novel deletions. One novel and one known type of deletion were found for three and two unrelated families, respectively. MLPA analysis proved a reliable method for the detection of genomic deletions in MLH1 and MSH2; however, sequence variations in the ligation-probe binding site can mimic single exon deletions.     

Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair

The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.     

A mutation in the putative MLH3 endonuclease domain confers a defect in both mismatch repair and meiosis in Saccharomyces cerevisiae

Interference-dependent crossing over in yeast and mammalian meioses involves the mismatch repair protein homologs MSH4-MSH5 and MLH1-MLH3. The MLH3 protein contains a highly conserved metal-binding motif DQHA(X)(2)E(X)(4)E that is found in a subset of MLH proteins predicted to have endonuclease activities (Kadyrov et al. 2006). Mutations within this motif in human PMS2 and Saccharomyces cerevisiae PMS1 disrupted the endonuclease and mismatch repair activities of MLH1-PMS2 and MLH1-PMS1, respectively (Kadyrov et al. 2006, 2007; Erdeniz et al. 2007). As a first step in determining whether such an activity is required during meiosis, we made mutations in the MLH3 putative endonuclease domain motif (-D523N, -E529K) and found that single and double mutations conferred mlh3-null-like defects with respect to meiotic spore viability and crossing over. Yeast two-hybrid and chromatography analyses showed that the interaction between MLH1 and mlh3-D523N was maintained, suggesting that the mlh3-D523N mutation did not disrupt the stability of MLH3. The mlh3-D523N mutant also displayed a mutator phenotype in vegetative growth that was similar to mlh3Delta. Overexpression of this allele conferred a dominant-negative phenotype with respect to mismatch repair. These studies suggest that the putative endonuclease domain of MLH3 plays an important role in facilitating mismatch repair and meiotic crossing over.     

Efficient repair of A/C mismatches in mouse cells deficient in long-patch mismatch repair

A previously unrecognized mismatch repair activity is described. Extracts of immortalized MSH2-deficient mouse fibroblasts did not correct most single base mispairs. The same extracts carried out efficient repair of A/C mismatches. A/G mispairs were less efficiently corrected and there was no significant repair of A/A. MLH1-defective mouse extracts also repaired an A/C mispair. A/C correction by Msh2(-/-) mouse cell extracts was not affected by antibodies against the PMS2 protein, which inhibited long-patch mismatch repair. A/C repair activity is thus independent of MutSalpha, MutSbeta and MutLalpha. A/C mismatches were corrected 5-fold more efficiently by extracts of Msh2 knockout mouse cells than by comparable extracts prepared from hMSH2- or hMLH1-deficient human cells. MSH2-independent A/C correction by mouse cell extracts did not require a nick in the circular duplex DNA substrate. Repair involved replacement of the A and was associated with the resynthesis of a limited stretch of 相似文献   

Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.     

A role for Mlh3 in somatic hypermutation

Somatic hypermutation (SHM) and class switch recombination (CSR) allow B cells to make high affinity antibodies of various isotypes. Both processes are initiated by activation-induced cytidine deaminase (AID) to generate dG:dU mismatches in the immunoglobulin genes that are resolved differently in SHM and CSR to introduce point mutations and recombination, respectively. The MutL homolog MLH3 has been implicated in meiosis and DNA mismatch repair (MMR). Since it interacts with MLH1, which plays a role in SHM and CSR, we examined these processes in Mlh3-deficient mice. Although deficiencies in other MMR proteins result in defects in SHM, Mlh3(-/-) mice exhibited an increased frequency of mutations in their immunoglobulin variable regions, compared to wild type littermates. Alterations of mutation spectra were observed in the Jh4 flanking region in Mlh3(-/-) mice. Nevertheless, Mlh3(-/-) mice were able to switch to IgG3 or IgG1 with similar frequencies to control mice. This is the first instance where a loss of a DNA repair protein has a positive impact on the rate of SHM, suggesting that Mlh3 normally inhibits the accumulation of mutations in SHM.     

Cloning of rat MLH1 and expression analysis of MSH2, MSH3, MSH6, and MLH1 during spermatogenesis

The mismatch repair system has been highly conserved in various species. In eukaryotic cells, the Mut S and Mut L homologues play crucial roles in both DNA mismatch repair and meiotic recombination. A full-length rat cDNA clone for rat MLH1 has been constructed using the RT-PCR method. The cDNA has an open reading frame of 2274 nucleotides for a protein of 757 amino acids. We have also obtained partial cDNA clones for MSH3 and MSH6. Northern blot analysis of rat MLH1, MSH2, MSH3, and MSH6 in the testes of rats of different ages showed differential expression of these genes as a function of developmental maturation of the testes. The expression analysis suggests that MSH3 may have a more predominant role in the meiotic recombination process.     

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.