Microsatellite instability analysis in hereditary non-polyposis colon cancer using the Bethesda consensus panel of microsatellite markers in the absence of proband normal tissue

BACKGROUND: Hereditary non-polyposis colon cancer (HNPCC) is an autosomal dominant syndrome predisposing to the early development of various cancers including those of colon, rectum, endometrium, ovarium, small bowel, stomach and urinary tract. HNPCC is caused by germline mutations in the DNA mismatch repair genes, mostly hMSH2 or hMLH1. In this study, we report the analysis for genetic counseling of three first-degree relatives (the mother and two sisters) of a male who died of colorectal adenocarcinoma at the age of 23. The family fulfilled strict Amsterdam-I criteria (AC-I) with the presence of extracolonic tumors in the extended pedigree. We overcame the difficulty of having a proband post-mortem non-tumor tissue sample for MSI testing by studying the alleles carried by his progenitors. METHODS: Tumor MSI testing is described as initial screening in both primary and metastasis tumor tissue blocks, using the reference panel of 5 microsatellite markers standardized by the National Cancer Institute (NCI) for the screening of HNPCC (BAT-25, BAT-26, D2S123, D5S346 and D17S250). Subsequent mutation analysis of the hMLH1 and hMSH2 genes was performed. RESULTS: Three of five microsatellite markers (BAT-25, BAT-26 and D5S346) presented different alleles in the proband's tumor as compared to those inherited from his parents. The tumor was classified as high frequency microsatellite instability (MSI-H). We identified in the HNPCC family a novel germline missense (c.1864C>A) mutation in exon 12 of hMSH2 gene, leading to a proline 622 to threonine (p.Pro622Thr) amino acid substitution. CONCLUSION: This approach allowed us to establish the tumor MSI status using the NCI recommended panel in the absence of proband's non-tumor tissue and before sequencing the obligate carrier. According to the Human Gene Mutation Database (HGMD) and the International Society for Gastrointestinal Hereditary Tumors (InSiGHT) Database this is the first report of this mutation

Single-Molecule Analysis Reveals the Kinetics and Physiological Relevance of MutL-ssDNA Binding

DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (KD = 29 nM) while it dramatically decreases above 100 mM (KD>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR

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

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

<p>Abstract</p> <p>Background</p> <p>Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the <it>hMLH1 </it>gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all <it>MLH1 </it>alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, <it>in vivo </it>assays for functional characterization of <it>MLH1 </it>mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of <it>hMLH1 </it>mutations <it>in vivo</it>, based on co-expression of human MLH1 and PMS2 in yeast cells.</p> <p>Methods</p> <p>Yeast <it>MLH1 </it>and <it>PMS1 </it>genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.</p> <p>Results</p> <p>The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related <it>MLH1 </it>variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.</p> <p>Conclusion</p> <p>Results of our <it>in vivo </it>yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of <it>MLH1 </it>variants in cancer patients found throughout the entire coding region of the gene.</p

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

The Escherichia coli MutL protein stimulates binding of Vsr and MutS to heteroduplex DNA.

Vsr DNA mismatch endonuclease is the key enzyme of very short patch (VSP) DNA mismatch repair and nicks the T-containing strand at the site of a T-G mismatch in a sequence-dependent manner. MutS is part of the mutHLS repair system and binds to diverse mismatches in DNA. The function of the mutL gene product is currently unclear but mutations in the gene abolish mutHLS -dependent repair. The absence of MutL severely reduces VSP repair but does not abolish it. Purified MutL appears to act catalytically to bind Vsr to its substrate; one-hundredth of an equivalent of MutL is sufficient to bring about a significant effect. MutL enhances binding of MutS to its substrate 6-fold but does so in a stoichiometric manner. Mutational studies indicate that the MutL interaction region lies within the N-terminal 330 amino acids and that the MutL multimerization region is at the C-terminal end. MutL mutant monomeric forms can stimulate MutS binding

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

Interaction of MutS and Vsr: Some Dominant-Negative mutS Mutations That Disable Methyladenine-Directed Mismatch Repair Are Active in Very-Short-Patch Repair

In Escherichia coli and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T · G mismatches that result from the deamination of 5-methylcytosines in DNA to C · G. The products of mutS and mutL, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair. Multicopy plasmids carrying mutS alleles that are dominant negative for MMR were tested for their effects on VSP repair. Some mutS mutations (class I) did not lower VSP repair in a mutS(+) background, and most class I mutations increased VSP repair in mutS cells more than plasmids containing mutS(+). Other plasmid-borne mutS mutations (class II) and mutS(+) decreased VSP repair in the mutS(+) background. Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr

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