The association between genetic variants in hMLH1 and hMSH2 and the development of sporadic colorectal cancer in the Danish population

<p>Abstract</p> <p>Background</p> <p>Mutations in the mismatch repair genes <it>hMLH1 </it>and <it>hMSH2 </it>predispose to hereditary non-polyposis colorectal cancer (HNPCC). Genetic screening of more than 350 Danish patients with colorectal cancer (CRC) has led to the identification of several new genetic variants (e.g. missense, silent and non-coding) in <it>hMLH1 </it>and <it>hMSH2</it>. The aim of the present study was to investigate the frequency of these variants in <it>hMLH1 </it>and <it>hMSH2 </it>in Danish patients with sporadic colorectal cancer and in the healthy background population. The purpose was to reveal if any of the common variants lead to increased susceptibility to colorectal cancer.</p> <p>Methods</p> <p>Associations between genetic variants in <it>hMLH1 </it>and <it>hMSH2 </it>and sporadic colorectal cancer were evaluated using a case-cohort design. The genotyping was performed on DNA isolated from blood from the 380 cases with sporadic colorectal cancer and a sub-cohort of 770 individuals. The DNA samples were analyzed using Single Base Extension (SBE) Tag-arrays. A Bonferroni corrected Fisher exact test was used to test for association between the genotypes of each variant and colorectal cancer. Linkage disequilibrium (LD) was investigated using HaploView (v3.31).</p> <p>Results</p> <p>Heterozygous and homozygous changes were detected in 13 of 35 analyzed variants. Two variants showed a borderline association with colorectal cancer, whereas the remaining variants demonstrated no association. Furthermore, the genomic regions covering <it>hMLH1 </it>and <it>hMSH2 </it>displayed high linkage disequilibrium in the Danish population. Twenty-two variants were neither detected in the cases with sporadic colorectal cancer nor in the sub-cohort. Some of these rare variants have been classified either as pathogenic mutations or as neutral variants in other populations and some are unclassified Danish variants.</p> <p>Conclusion</p> <p>None of the variants in <it>hMLH1 </it>and <it>hMSH2 </it>analyzed in the present study were highly associated with colorectal cancer in the Danish population. High linkage disequilibrium in the genomic regions covering <it>hMLH1 </it>and <it>hMSH2</it>, indicate that common genetic variants in the two genes in general are not involved in the development of sporadic colorectal cancer. Nevertheless, some of the rare unclassified variants in <it>hMLH1 </it>and <it>hMSH2 </it>might be involved in the development of colorectal cancer in the families where they were originally identified.</p

Ultradeep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability

Ultradeep sequencing of genomes permits the detection of very low-level genomic instability in non-neoplastic tissues of patients with the most common form of inherited colorectal cancer

Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers

<p>Abstract</p> <p>Background</p> <p>Depending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (<it>MMR</it>) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from <it>MLH1</it>/<it>MSH2 </it>deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts.</p> <p>Methods</p> <p>The main <it>MMR </it>genes responsible for HNPCC, <it>MLH1</it>, <it>MSH2</it>, <it>MSH6</it>, and <it>PMS2</it>, were analyzed by MLPA (multiplex ligation-dependent probe amplification) in a total of 37 unrelated HNPCC-suspected patients whose <it>MLH1/MSH2 </it>genes gave negative results in previous sequencing experiments. An LOH study was performed on six tumors from LGR carriers by combining MLPA to assess LOH at LGR regions and sequencing to examine LOH at 28 SNP markers from the <it>MLH1 </it>and <it>MSH2 </it>genes.</p> <p>Results</p> <p>We found six rearrangements in the <it>MSH2 </it>gene (five deletions and dup5-6), and one aberration in the <it>MLH1 </it>gene (del5-6). The <it>MSH2 </it>deletions were of three types (del1, del1-3, del1-7). We detected LOH at the LGR region in the single <it>MLH1 </it>case, which was determined in a previous study to be LOH-negative in the intragenic D3S1611 marker. Three tumors displayed LOH of at least one SNP marker, including two cases that were LOH-negative at the LGR region.</p> <p>Conclusion</p> <p>LGRs accounted for 25% of germline <it>MMR </it>mutations identified in 28 Slovakian HNPCC families. A high frequency of LGRs among the <it>MSH2 </it>mutations provides a rationale for a MLPA screening of the Slovakian HNPCC families prior scanning by DNA sequencing. LOH at part of the informative loci confined to the <it>MLH1 </it>or <it>MSH2 </it>gene (heterozygous LGR region, SNP, or microsatellite) is a novel finding and can be regarded as a partial LOH. The conversion begins within the gene, and the details of conversion tracts are discussed for each case.</p

Human mismatch repair genes and their association with hereditary non-polyposis colon cancer

Hereditary non-polyposis colon cancer (HNPCC) may affect up to 1 in 200 people in industrialized nations (Bishop and Thomas 1990; Lynch et al. 1991, 1993; Peltomaki et al. 1993b). Four genes have been identified in which inherited mutations appear to cause HNPCC. hMSH2 on chromosome 2p21-22 appears to account for up to 60% of HNPCC (Fishel et al. 1993; Leach et al. 1993; Sandkuijl and Bishop 1993; Nystrom-Lahti et al. 1994), hMLH1 on chromosome 3p21 appears to account for up to 30% of HNPCC (Bronner et al. 1994; Nystrom-Lahti et al. 1994; Papadopoulos et al. 1994), and hPMS1 on chromosome 2q31-33 and hPMS2 on chromosome 7p21 may account for 5% of HNPCC (Nicolaides et al. 1994)

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 δ and ɛ 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 μM, 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