Inactivation Of DNA Mismatch Repair Research Articles

Short chain fatty acids prime colorectal cancer cells to activate antitumor immunity.

Colorectal cancer (CRC) is a leading cause of death worldwide and its growth can either be promoted or inhibited by the metabolic activities of intestinal microbiota. Short chain fatty acids (SCFAs) are microbial metabolites with potent immunoregulatory properties yet there is a poor understanding of how they directly regulate immune modulating pathways within the CRC cells. We used engineered CRC cell lines, primary organoid cultures, orthotopic in vivo models, and patient CRC samples to investigate how SCFA treatment of CRC cells regulates their ability to activate CD8+ T cells. CRC cells treated with SCFAs induced much greater activation of CD8+ T cells than untreated CRC cells. CRCs exhibiting microsatellite instability (MSI) due to inactivation of DNA mismatch repair were much more sensitive to SCFAs and induced much greater CD8+ T cell activation than chromosomally instable (CIN) CRCs with intact DNA repair, indicating a subtype-dependent response to SCFAs. This was due to SCFA-induced DNA damage that triggered upregulation of chemokine, MHCI, and antigen processing or presenting genes. This response was further potentiated by a positive feedback loop between the stimulated CRC cells and activated CD8+ T cells in the tumor microenvironment. The initiating mechanism in the CRCs was inhibition of histone deacetylation by the SCFAs that triggered genetic instability and led to an overall upregulation of genes associated with SCFA signaling and chromatin regulation. Similar gene expression patterns were found in human MSI CRC samples and in orthotopically grown MSI CRCs independent of the amount of SCFA producing bacteria in the intestine. MSI CRCs are widely known to be more immunogenic than CIN CRCs and have a much better prognosis. Our findings indicate that a greater sensitivity to microbially produced SCFAs contributes to the successful activation of CD8+ T cells by MSI CRCs, thereby identifying a mechanism that could be therapeutically targeted to improve antitumor immunity in CIN CRCs.

Molecular testing for colorectal cancer: Clinical applications.

Molecular genetic analysis is an integral part of colorectal cancer (CRC) management. The choice of systemic therapy for CRC is largely based on the results of tumor molecular testing. Evaluation of the KRAS and NRAS gene status is mandatory for consideration of anti-epidermal growth factor receptor (EGFR) therapy. Tumors with the BRAF V600E substitution are characterized by aggressive behaviour, may require intensified cytotoxic regimens and benefit from combined BRAF and EGFR inhibition. The inactivation of DNA mismatch repair (MMR), or MUTYH gene, or DNA polymerase epsilon results in excessive tumor mutational burden; these CRCs are highly antigenic and therefore sensitive to immune checkpoint inhibitors. Some CRCs are characterized by overexpression of the HER2 oncogene and respond to the appropriate targeted therapy. There are CRCs with clinical signs of hereditary predisposition to this disease, which require germline genetic testing. Liquid biopsy is an emerging technology that has the potential to assist CRC screening, control the efficacy of surgical intervention and guide disease monitoring. The landscape of CRC molecular diagnosis is currently undergoing profound changes due to the increasing use of next generation sequencing.

High-fidelity DNA ligation enforces accurate Okazaki fragment maturation during DNA replication

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.

The Human DNA Mismatch Repair Protein MSH3 Contains Nuclear Localization and Export Signals That Enable Nuclear-Cytosolic Shuttling in Response to Inflammation.

Inactivation of DNA mismatch repair propels colorectal cancer (CRC) tumorigenesis. CRCs exhibiting elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) show reduced nuclear MutS homolog 3 (MSH3) expression with surrounding inflammation and portend poor patient outcomes. MSH3 reversibly exits from the nucleus to the cytosol in response to the proinflammatory cytokine interleukin-6 (IL-6), suggesting that MSH3 may be a shuttling protein. In this study, we manipulated three putative nuclear localization (NLS1 to -3) and two potential nuclear export signals (NES1 and -2) within MSH3. We found that both NLS1 and NLS2 possess nuclear import function, with NLS1 responsible for nuclear localization within full-length MSH3. We also found that NES1 and NES2 work synergistically to maximize nuclear export, with both being required for IL-6-induced MSH3 export. We examined a 27-bp deletion (Δ27bp) within the polymorphic exon 1 that occurs frequently in human CRC cells and neighbors NLS1. With oxidative stress, MSH3 with this deletion (Δ27bp MSH3) localizes to the cytoplasm, suggesting that NLS1 function in Δ27bp MSH3 is compromised. Overall, MSH3's shuttling in response to inflammation enables accumulation in the cytoplasm; reduced nuclear MSH3 increases EMAST and DNA damage. We suggest that polymorphic sequences adjacent to NLS1 may enhance cytosolic retention, which has clinical implications for inflammation-associated neoplastic processes.

Two main mutational processes operate in the absence of DNA mismatch repair

The analysis of tumour genome sequences has demonstrated high rates of base substitution mutagenesis upon the inactivation of DNA mismatch repair (MMR), and the resulting somatic mutations in MMR deficient tumours appear to significantly enhance the response to immune therapy. A handful of different algorithmically derived base substitution mutation signatures have been attributed to MMR deficiency in tumour somatic mutation datasets. In contrast, mutation data obtained from whole genome sequences of isogenic wild type and MMR deficient cell lines in this study, as well as from published sources, show a more uniform experimental mutation spectrum of MMR deficiency. In order to resolve this discrepancy, we reanalysed mutation data from MMR deficient tumour whole exome and whole genome sequences. We derived two base substitution signatures using non-negative matrix factorisation, which together adequately describe mutagenesis in all tumour and cell line samples. The two new signatures broadly resemble COSMIC signatures 6 and 20, but perform better than existing COSMIC signatures at identifying MMR deficient tumours in mutation signature deconstruction. We show that the contribution of the two identified signatures, one of which is dominated by C to T mutations at CpG sites, is biased by the different sequence composition of the exome and the whole genome. We further show that the identity of the inactivated MMR gene, the tissue type, the mutational burden or the patient’s age does not influence the mutation spectrum, but that a tendency for a greater contribution by the CpG mutational process is observed in tumours as compared to cultured cells. Our analysis suggest that two separable mutational processes operate in the genomes of MMR deficient cells.

Microsatellite Instability Occurs in a Subset of Follicular Thyroid Cancers.

Inactivation of DNA mismatch repair (MMR) and the resulting microsatellite instability (MSI) are frequently observed in endometrial, stomach, and colorectal cancers, as well as more rarely in other solid tumor types. The prevalence of MSI in thyroid cancer has not been explored in depth, although recent studies utilizing data from large cancer sequencing efforts such as The Cancer Genome Atlas indicate that MSI is absent or at least very rare in the most common and most well studied histologic subtype, papillary thyroid carcinoma. This study aimed to determine the prevalence of MSI in thyroid cancer by using a large series comprising all major histological subtypes. A total of 485 thyroid cancer patients were screened for MSI/MMR deficiency, including all major histologic subtypes (195 papillary thyroid carcinoma, 156 follicular thyroid carcinoma [FTC], 50 anaplastic thyroid carcinoma, 65 medullary thyroid carcinoma, and 17 poorly differentiated thyroid carcinomas) by using a combination of polymerase chain reaction-based detection, immunohistochemistry, and next-generation sequencing. A total of four tumors were MSI-high and had loss of MMR protein expression, all of which were from FTC patients. Whole-exome sequencing was performed on two MSI-high FTCs and revealed a hemizygous loss of function mutation in MSH2 in one tumor. Based on these data, it is estimated that the overall prevalence of MSI in FTC is 2.5%, and MSI is either entirely absent or rare in other histology subtypes of thyroid carcinoma. These findings highlight the importance of testing for MSI in FTC.

Mismatch repair protein expression in patients with stage II and III sporadic colorectal cancer.

Colorectal cancer (CRC) may be classified according to the level of microsatellite instability exhibited by the tumor. The malignant transformation of normal colonic mucosae to carcinomas may be accelerated by the loss or inactivation of DNA mismatch repair (MMR) genes. The present study examined the expression of certain MMR proteins [namely, MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6) and PMS1 homolog 2 (PMS2)] in patients with stage II and III sporadic CRC. The association between the expression of these proteins, and the clinicopathological characteristics of patients with CRC and their tumors, was investigated. MMR protein expression was examined using immunohistochemistry. MLH1, MSH2, MSH6 and PMS2 protein expression was detected in 78.4% (120/153), 75.2% (115/153), 44.4% (68/153) and 79.7% (122/153) of stage II and III sporadic CRCs, respectively. Additionally, the expression of MLH1 and MSH6 was revealed to be significantly higher in stage III tumors when compared with stage II tumors (P<0.05). MLH1 and MSH6 negative tumors were larger, poorly differentiated and exhibited extraserosal invasion with infrequent lymph node metastasis (P<0.05). Patients with defects in MLH2 and PMS2 also had large tumors that exhibited extraserosal invasion and infrequent lymph node metastasis (P<0.05). No statistically significant associations were observed between MLH1, MSH2, MSH6 or PMS2 protein expression and patient age, sex, tumor localization or angiolymphatic invasion status (P>0.05). From the present study, it was concluded that MMR protein expression status evaluation may increase the efficiency of MMR testing and be useful in improving the individualized approach to patient monitoring and therapy.

Helicobacter pylori infection modulates the expression of miRNAs associated with DNA mismatch repair pathway.

Genetic and epigenetic inactivation of DNA mismatch repair (MMR) genes might lead to modifications in cancer-related gene expression and cancer development. Recently, it has been shown that the infection by Helicobacter pylori, the major causative agent of gastric cancer, induces DNA damage and inhibits MMR DNA repair. Also, it has been reported that microRNAs (miRs) have an important role in regulating genomic stability and MMR DNA repair. Thus, the aim of this study was to identify miRs regulating MMR pathway in H. pylori-associated gastric carcinogenesis. To address this question, a gastric epithelial cell line and AGS cancer gastric cells were infected with several H. pylori strains. MMR gene expression and miRs correlating with H. pylori strain infection were evaluated. The results showed that H. pylori infection significantly down-regulated the expression of all selected MMR genes. Also, H. pylori infection modulated the expression of several miRs (including miR-150-5p, miR-155-5p, and miR-3163), after 4, 8, and 12 h of infection. Computational prediction of candidate miRs and their predicted MMR targeting sites were obtained from TargetScan, mirDB, and MetaCore. The generated data indicated that the selected miRs (miR-150-5p, miR-155-5p, and miR-3163) could possibly target and modulate MMR genes (POLD3, MSH2, and MSH3, respectively). The target validation was performed using mimics and luciferase gene reporter assays. Briefly, this study shows that H. pylori impairs MMR DNA repair pathway and identifies miRs that regulate MMR gene expression in gastric cancer. © 2016 Wiley Periodicals, Inc.

Abstract 200: Effects of H. pylori infection in regulation of miRNAs-associated with DNA mismatch repair

Abstract It is well known that genetic and epigenetic inactivation of DNA mismatch repair (MMR) genes might leads to mutations in cancer-related genes and to cancer development. In gastric carcinogenesis, it has been shown that the infection by H. pylori, the major causative agent of gastric cancer, induces DNA damage and mutation and/or inhibits MMR DNA repair. Recently, it has been reported that miRNAs (miRs) have an important role in regulating genomic stability and MMR DNA repair. Thus, the aim of this work was to identify miRs regulating MMR pathway in H. pylori-associated gastric carcinogenesis To evaluate the effects of H. pylori on miRs releated with MMR, AGS gastric carcinoma cell line and the H. pylori strains 26695, and P12 were used in co-culture experiments for 4, 8 and 12 h. As an initial screening, the AGS cells were co-cultured with H. pylori strain 26695 and subjected to Cancer PathwayFinder miRNA PCR Array (Qiagen) to determine the expression profile of 84 miRs. The expression levels of EXO1, MLH1, MSH2, MSH3, MSH6, POLD3, PMS1, and PMS2 were assessed by real-time PCR. Computational prediction of putative interaction of selected miRs to 3′UTR of MMR mRNAs were obtained from TargetScan, mirDB and MetaCore. The target validation was evaluated using mimics/inhibitors and luciferase gene reporter assays. The results from the Cancer PathwayFinder PCR miRNA Array showded that H. pylori infection (strain 26695) modulates the expression of 23% (19/84) of the miRs after 4 and 12 h of infection, of which 89% were up-regulated and 11% down-regulated. Our data show that H. pylori infection significantly down-regulates the expression of all selected genes. The data generated from computational miR target prediction enriched with the gene expression fold induction indicated that 4 miRs (miR-150-5p, miR-155-5p, miR-3163, and miR-4775) modulated by H. pylori has at least one MMR target gene. Regarding the target validation, our results indicated that MLH1 and MSH2, are regulated by miR-150-5p. The miR-155-5p is involved in down-regulation of MLH1, MSH2, MSH3 and POLD3 expression. MSH3 is regulated by both miR-3163 and miR-4775. Briefly, this study shows that H. pylori impairs MMR DNA repair pathway thought a modulation of miR expression. Citation Format: Juliana C. Santos, Mitsue Brianti, Victor de Almeida, Ander Matheu, Marcelo L. Ribeiro. Effects of H. pylori infection in regulation of miRNAs-associated with DNA mismatch repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 200. doi:10.1158/1538-7445.AM2015-200

Inactivation of DNA Mismatch Repair by Variants of Uncertain Significance in the PMS2 Gene

Lynch syndrome (LS) is a common cancer predisposition caused by an inactivating mutation in one of four DNA mismatch repair (MMR) genes. Frequently a variant of uncertain significance (VUS), rather than an obviously pathogenic mutation, is identified in one of these genes. The inability to define pathogenicity of such variants precludes targeted healthcare. Here, we have modified a cell-free assay to test VUS in the MMR gene PMS2 for functional activity. We have analyzed nearly all VUS in PMS2 found thus far and describe loss of MMR activity for five, suggesting the applicability of the assay for diagnosis of LS.

Abstract PL06-02: Inflammation, DNA mismatch repair, and colon cancer.

Abstract Colorectal neoplasia is driven by at least two pathogenic pathways for cancer development. Chromosomal instability, observed in ~80% of sporadic colorectal cancers, is named due to the rearrangement of chromosomes, resulting in aneuploid nuclei. The etiology for CIN is not known. A common finding is allelic mutation of a tumor suppressor gene, followed by loss of the remaining normal allele (termed loss of heterozygosity, LOH). Microsatellite instability (MSI), observed in up to 20% of sporadic colorectal cancers, is named for frameshifts (or instability) that occur at DNA microsatellite sequences. Cells remain diploid. The etiology for MSI is inactivation of DNA mismatch repair (MMR), an enzyme system functioning in the nucleus to repair interstrand nucleotide mispairs and loops of DNA that are formed on one strand at microsatellites. The specificity of mismatch recognition is directed by hMutSa and hMutSb; hMutSa (hMSH2-hMSH6 heterodimer) binds to single mispairs and single insertion-deletion loops, whereas hMutSb (hMSH2-hMSH3 heterodimer) binds larger insertion-deletion loops. MSI sporadic cancers are caused by hypermethylation of the MMR gene hMLH1's promoter, preventing its transcription and translation. With loss of hMLH1, MMR completely fails and mutations accumulate in the genome due to non-repair of DNA polymerase errors. Clinically, MSI tumors contain surrounding and intratumoral mononuclear inflammatory cells which are thought responsible in containing the tumor in place, leading to improved survival over same staged patients with non-MSI tumors. Although the types of inflammatory cells within MSI tumors have not been characterized, it is believed that neo-peptides from frameshifted proteins as a result of MMR failure are generated to attract mononuclear cells into the tumor, producing a protective immune response. Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colon cancers. Unlike MSI colorectal cancers in which hypermethylation of hMLH1 drives multiple target gene mutations, the cause of EMAST has been recently associated with reduced expression of hMSH3, which we have also observed in hamartomatous polyps and during the adenoma-to-carcinoma progression, and is hypothesized to be triggered as a consequence of inflammation with subsequent relaxation of MMR function. EMAST-positive human colorectal cancers are strongly associated with inflammation, particularly within tumor nests, but unlike MSI tumors, this inflammation is associated with advance staged colorectal cancer, implying reduced survival. Citation Format: John M. Carethers. Inflammation, DNA mismatch repair, and colon cancer. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL06-02.

Coding microsatellite instability analysis in microsatellite unstable small intestinal adenocarcinomas identifies MARCKS as a common target of inactivation

Approximately 15% of small intestinal adenocarcinomas show inactivation of DNA-mismatch repair (MMR) and display high-level microsatellite instability (MSI-H). MSI-H tumors progress as a result of mutations affecting coding microsatellites (coding microsatellite instability, cMSI) that may result in a functional inactivation of the encoded proteins and provide a selective growth advantage for the affected cell. To investigate the cMSI selection in small intestinal carcinogenesis 56 adenocarcinomas were tested for MSI. Eleven MSI-H carcinomas (19.6%) were identified and subjected to cMSI analysis in 24 potentially tumor relevant genes. Mutation frequencies were similar to those observed in colorectal cancer (CRC). Beside high frequencies of cMSI in TGFbetaR2, ACVR2, and AIM2 we detected MARCKS mutations in 10 out of 11 (91%) tumors with a 30% share of biallelic mutations. Since little is known about MARCKS expression in the intestine, we analyzed MARCKS protein expression in 31 carcinomas. In non-neoplastic mucosa, MARCKS was found to be expressed with a concentration gradient along the crypt-villus axis. In line with cMSI induced functional inactivation of MARCKS, 8 out of 11 MSI-H adenocarcinomas showed regional or complete loss of the protein. In microsatellite stable (MSS) small bowel adenocarcinoma, loss of MARCKS expression was seen in 2 out of 20 tumors (10%). In conclusion, we herein present a cMSI profile of MSI-H small intestinal adenocarcinomas identifying MARCKS as a frequent target of mutation. Loss of MARCKS protein expression suggests a significant role of MARCKS inactivation in the pathogenesis of small intestinal adenocarcinomas.

Foxp3 and microsatellite stability phenotype in colorectal cancer

Colorectal tumourigenesis is in the majority of cases regarded as a multistep process resulting in chromosomal instability.1 High-frequency microsatellite instability (MSI-H) can be detected in ∼15–20% of all sporadic colorectal...

When does MMR loss occur during HNPCC progression?

Inactivation of DNA mismatch repair (MMR) is the hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) and sporadic colorectal cancers with microsatellite instability (MSI+). MMR loss results in a markedly elevated mutation rate, and many MS mutations are found in MSI+ cancers. In theory, it is possible to estimate the interval between MMR loss and cancer removal by counting numbers of cancer MS mutations--the more MS mutations, the longer the intervals since MMR loss. Using this somatic molecular clock approach, MMR loss is estimated to precede transformation (clonal expansion) and likely occurs in normal appearing colon. Surprising, ages at MMR loss are more consistent with MMR loss as a relatively late event during progression to MSI+ cancer.

Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas.

Genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair. Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells. Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/TTF (ARHH) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5' untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks. By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis.

Inactivation of DNA mismatch repair by increased expression of yeast MLH1.

Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177-3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)(+) mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1 overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1 overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3'-->5' exonuclease activity of replicative DNA polymerases delta and epsilon but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, or PMS1. This suggests that overexpression of MLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K(d) of 3.14 microM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressing MLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.

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.