DNA Mismatch Repair Genes hMSH2 Research Articles

Muir-Torre syndrome.

Muir-Torre syndrome (MTS) is a rare autosomal-dominant genodermatosis characterized by sebaceous neoplasms and one or more visceral malignancies. Sebaceous tumors include sebaceous adenoma and carcinoma, which may be solitary or multiple. Visceral malignancies most often arise in the colorectum and endometrium. Because a subset of patients with phenotypic MTS will have germline mutations in the DNA mismatch repair genes hMSH2 and hMLH1, MTS is considered a phenotypic subtype of Lynch syndrome (also known as hereditary nonpolyposis colorectal cancer syndrome), in which inherited defects in DNA mismatch repair genes result in microsatellite instability. Pathologists have an important role in the early detection and initial diagnosis of MTS: identification of at-risk individuals allows appropriate screening and surveillance for visceral malignancies, thereby reducing morbidity and mortality. Herein, we describe the clinicopathologic features of MTS.

The role of the human DNA mismatch repair gene hMSH2 in DNA repair, cell cycle control and apoptosis: implications for pathogenesis, progression and therapy of cancer

The cellular DNA mismatch repair (MMR) pathway, involving the DNA mismatch repair genes MLH1 and MSH2, detects and repairs DNA replication errors. Defects in MSH2 and MLH1 account for most cases of hereditary non-polyposis colorectal cancer as well as for sporadic colorectal tumors. Additionally, increased expression of MSH2 RNA and/or protein has been reported in various malignancies. Loss of DNA MMR in mammalian cells has been linked to resistance to certain DNA damaging agents including clinically important cytotoxic chemotherapeutics. Due to other functions besides its role in DNA repair, that include regulation of cell proliferation and apoptosis, MSH2 has recently been shown to be of importance for pathogenesis and progression of cancer. This review summarizes our present understanding of the function of MSH2 for DNA repair, cell cycle control, and apoptosis and discusses its importance for pathogenesis, progression and therapy of cancer.

Polymorphisms of the DNA Mismatch Repair Gene HMSH2 in Breast Cancer Occurence and Progression

The response of the cell to DNA damage and its ability to maintain genomic stability by DNA repair are crucial in preventing cancer initiation and progression. Therefore, polymorphism of DNA repair genes may affect the process of carcinogenesis. The importance of genetic variability of the components of mismatch repair (MMR) genes is well documented in colorectal cancer, but little is known about its role in breast cancer. hMSH2 is one of the crucial proteins of MMR. We performed a case-control study to test the association between two polymorphisms in the hMSH2 gene: an A --> G transition at 127 position producing an Asn --> Ser substitution at codon 127 (the Asn127Ser polymorphism) and a G --> A transition at 1032 position resulting in a Gly --> Asp change at codon 322 (the Gly322Asp polymorphism) and breast cancer risk and cancer progression. Genotypes were determined in DNA from peripheral blood lymphocytes of 150 breast cancer patients and 150 age-matched women (controls) by restriction fragment length polymorphism and allele-specific PCR. We did not observe any correlation between studied polymorphisms and breast cancer progression evaluated by node-metastasis, tumor size and Bloom-Richardson grading. A strong association between breast cancer occurrence and the Gly/Gly phenotype of the Gly322Asp polymorphism (odds ratio 8.39; 95% confidence interval 1.44-48.8) was found. Therefore, MMR may play a role in the breast carcinogenesis and the Gly322Asp polymorphism of the hMSH2 gene may be considered as a potential marker in breast cancer.

The DNA mismatch repair gene hMSH2 is a potent coactivator of oestrogen receptor α

The DNA mismatch repair gene is a key regulator in the elimination of base–base mismatches and insertion/deletion loops (IDLs). Human MutS homologue 2 (hMSH2), originally identified as a human homologue of the bacterial MutS, is a tumour suppressor gene frequently mutated in hereditary nonpolyposis colorectal cancer. Hereditary nonpolyposis colorectal cancer is characterised by the early onset of colorectal cancer and the development of extracolonic cancers such as endometrial, ovarian, and urological cancers. Oestrogen receptor (ER) α and β are members of a nuclear receptor (NR) superfamily. Ligand-dependent transcription of ER is regulated by the p160 steroid receptor coactivator family, the thyroid hormone receptor-associated proteins/the vitamin D receptor-interacting proteins (TRAP/DRIP) mediator complex, and the TATA box-binding protein (TBP)-free TBP associated factor complex (TFTC) type histone acetyltransferase complex. Here, we report the interaction between ER α/β and hMSH2. Immunoprecipitation and glutathione-S-transferase pulldown assay revealed that ER α and hMSH2 interacted in a ligand-dependent manner, whereas ER β and hMSH2 interacted in a ligand-independent manner. Oestrogen receptor α/β bound to hMSH2 through the hMSH3/hMSH6 interaction domain of hMSH2. In a transient expression assay, hMSH2 potentiated the transactivation function of liganded ER α, but not that of ER β. These results suggest that hMSH2 may play an important role as a putative coactivator in ER α dependent gene expression.

Alterations of DNA mismatch repair proteins and microsatellite instability levels in gastric cancer cell lines

Alterations in DNA mismatch repair (MMR) proteins result in microsatellite instability (MSI), increased mutation accumulation at target genes and cancer development. About one-third of gastric cancers display high-level microsatellite instability (MSI-High) and low-level microsatellite instability (MSI-Low) is frequently detected. To determine whether variations in the levels of MMR proteins or mutations in the main DNA MMR genes are associated with MSI-Low and MSI-High in gastric cancer cell lines, the MSI status (MSI-High, MSI-Low or MS-Stable (MSS)) of 14 gastric cancer lines was determined using multiple clone analysis with a panel of five microsatellite markers. Protein levels of hMLH1, hMSH2, hMSH6, hPMS2 and hPMS1 were determined by Western blot. Sequence analysis of hMLH1 and hMSH2 was performed and the methylation status of the hMLH1 promoter was examined. The cell lines SNU1 and SNU638 showed MSI-High, decreased to essentially absent hMLH1 and hPMS2 and reduced hPMS1 and hMSH6 protein levels. The hMLH1 promoter region was hypermethylated in SNU638 cells. The MKN28, MKN87, KATOIII and SNU601 cell lines showed MSI-Low. The MMR protein levels of cells with MSI-Low status was similar to the levels detected in MSS cells. A marked decrease in the expression levels of MutL MMR proteins (hMLH1, hPMS2 and hPMS1) is associated with high levels of MSI mutations in gastric cancer cells. Gastric cancer cell lines with MSI-Low status do not show significant changes in the levels of the main DNA MMR proteins or mutations in the DNA mismatch repair genes hMSH2 and hMLH1. These well-characterized gastric cancer cell lines are a valuable resource to further our understanding of DNA MMR deficiency in cancer development, progression and prognosis.

High-Frequency Microsatellite Instability is Associated with Defective DNA Mismatch Repair in Human Melanoma

Hereditary nonpolyposis colorectal cancers and a steadily increasing number of sporadic tumors display microsatellite instability. In colorectal tumors, high-frequency microsatellite instability is strictly associated with inactivation of the DNA mismatch repair genes hMSH2, hMLH1, or hPMS2, whereas mutations in the mismatch repair gene hMSH6 have been identified in a subset of tumors with low-frequency microsatellite instability. In addition to epithelial tumors of the colon, endometrium, and ovary, microsatellite instability has been reported to occur also in sporadic melanoma. The relationship between microsatellite instability and mismatch repair in melanoma cells, however, has not been investigated so far. In this study, we analyzed microsatellite instability, mismatch repair activity, and expression of the hMSH2, hMSH6, hMLH1, and hPMS2 proteins in five melanoma cell lines and in tumor specimens from which the cells were derived. Four cell lines displayed normal levels of mismatch repair activity and expressed all the mismatch repair proteins. The extracts of the fifth cell line lacked the hMLH1 and hPMS2 proteins, and were correspondingly deficient in the repair of DNA mismatches. This line displayed high-frequency microsatellite instability, whereas the four mismatch-repair-proficient cell lines displayed either no or low-frequency microsatellite instability. These findings could be confirmed in the tumor specimens, in that only the tumor that did not express hMLH1 and hPMS2 displayed high-frequency microsatellite instability. Our data are consistent with the hypothesis that in melanoma, similarly to epithelial tumors, only the high-frequency microsatellite instability phenotype is strictly dependent on a defective mismatch repair system. Further studies on a large series of tumor specimens are required to establish the frequency of mismatch repair loss in human melanoma.

Cell type specificity in alternative splicing of the human mismatch repair gene hMSH2.

Human non-polyposis colorectal cancer is caused by germline mutations in the DNA mismatch repair genes hMSH2 and hMHL1. Several alternatively spliced mRNA species of these genes are present in peripheral blood lymphocytes of normal individuals, which can confound RT-PCR based techniques of mutation detection. Using RT-PCR, we compared the pattern of alternative splicing in whole peripheral blood lymphocytes (PBLs), separated T and B cells, lymphoblastoid cell lines (LCLs) from the same individuals, and a variety of tissues. Alternatively spliced forms of hMLH1 lacking exons 9/10, 10/11 and 9/10/11 were found to have similar patterns of expression in T cells, B cells, and LCLs. By contrast, a subset of hMSH2 transcripts, some of which were produced by utilisation of novel splicing motifs, were generally expressed in T but not in B cells. LCLs derived from the same blood samples showed no expression of any hMSH2 splicing variants. The hMSH2 delta ex13 transcript, while absent from LCLs, was expressed in whole PBLs and both T and B cell fractions. This transcript was furthermore largely undetectable in tissues other than mononuclear blood cells. These data provide evidence for tissue specificity in the regulation of alternative splicing in hMSH2. In particular we show that LCLs generally do not express alternatively spliced forms of hMSH2 mRNA and are thus suited for RT-PCR based mutation screening in that gene.

Various mutation screening techniques in the DNA mismatch repair genes hMSH2 and hMLH1.

Germline alterations in one of five human DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6) cause hereditary nonpolyposis colorectal cancer. Mutation analyses of these genes reveal gene carriers with a high risk for colorectal cancer, who benefit from surveillance to prevent disease. Equally important, presymptomatic testing allows nondisposed individuals to discontinue surveillance. We tested different mutation screening methods to optimize mutation detection in hMSH2 and hMLH1. Affected members from a total of 142 unrelated colorectal cancer families were analyzed. Denaturant gradient gel electrophoresis (DGGE), RT-PCR, and the protein truncation test (PTT) were used to screen for mutations on a DNA or RNA basis, respectively. In addition, a mutation-specific test on genomic DNA was used to find the Finnish mutation no. 1, a deletion of hMLH1 exon 16. DGGE identified most of the mutations in the mismatch repair genes hMLH1 and hMSH2. The RNA-based techniques were used to identify large deletions; however, these were rare in our materials. We describe our compiled results and experience from all our mutation screening studies, as well as unpublished data from our last DGGE screening of 58 patients and RT-PCR and PTT screening of 73 patients.

Mutator phenotypes of common polymorphisms and missense mutations in MSH2

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in the DNA mismatch repair gene hMSH2[1], the human homologue of the Escherichia coli MutS gene. These are mostly nonsense, frameshift or deletion mutations that result in loss of intact protein and complete inactivation of DNA mismatch repair. However, cancer is also associated with hMSH2 missense mutations that are merely inferred to be deleterious because they result in non-conservative substitutions of amino acids that are highly conserved among MutS family proteins. Moreover, sequence polymorphisms exist in hMSH2 that also change conserved amino acids but whose functional consequences and relationship to cancer are uncertain. Here, we show that yeast strains harboring putative equivalents of three hMSH2 polymorphisms have elevated mutation rates. Mutator effects were also observed for yeast equivalents of hMSH2 missense mutations found in HNPCC families and in an early onset colon tumor. Several distinct phenotypes were observed, indicating that these missense mutations have differential effects on MSH2 function(s). The results suggest that cancer may be associated with even partial loss of hMSH2 function and they are consistent with the hypothesis that polymorphisms in hMSH2 might predispose humans to disease.

A Novel Missense Mutation in the DNA Mismatch Repair Gene hMLH1 Present among East Asians but Not among Europeans

In a search for germline mutations in the DNA mismatch repair genes hMSH2 and hMLH1 in Chinese patients with colorectal cancer we identified a novel missense mutation (V384D) in exon 12 of the hMLH1 gene in 4 out of 26 individuals. This mutation had not been observed in any of 109 German patients who either fulfill the clinical criteria of hereditary nonpolyposis colorectal cancer or had developed colorectal cancer at an early age. In a second series of experiments, DNA samples of 80 healthy Japanese and 100 healthy Germans were examined for the presence of the V384D mutation. It was observed in 4 Japanese, but in none of the German individuals. Thus, the V384D mutation seems to be confined to the East-Asian population. Since this missense mutation occurs at a less conserved region of the hMLH1 gene, it may represent a nonfunctional polymorphism. However, a role in the pathogenesis of colorectal cancer cannot be ruled out from the present study.