Evaluation of Mismatch Repair Gene Polymorphisms and their Contribution to Colorectal Cancer and its Subsets

Colorectal cancer (CRC) is a major source of morbidity and mortality in the Western world. Approximately 15% of all CRCs develop via the mutator pathway, which results from a deficiency of mismatch repair (MMR) system and leads to genome-wide microsatellite instability (MSI). MLH1 promoter hypermethylation accounts for the majority of MSI CRCs. Numerous single nucleotide polymorphisms have been identified in MMR genes, however their functional roles in affecting MMR system, and therefore susceptibility to MSI CRCs, are unknown. This study uses a multidisciplinary approach combining molecular genetics, epigenetics, and epidemiology to examine the contribution of MMR gene polymorphisms in CRC. Among a panel of MMR SNPs examined, the MLH1 (-93G>A) promoter polymorphism (rs1800734) was shown to be associated with increased risk of MSI CRCs in two Canadian populations, Ontario and Newfoundland. Functional studies of the MLH1-93G>A polymorphism indicate that it has weak effects on the core promoter activity, although it dramatically reduces activity of the shorter promoter constructs in a panel of cell lines. Furthermore, MLH1 gene shares a bi-directional promoter with EMP2AIP1 gene, and the MLH1-93G>A polymorphism increases the activity of the reverse, EPM2AIP1 promoter. Examination of alternative role of the MLH1-93G>A polymorphism in MSI-H CRCs led to evaluation of a 500-kilobase pair chromosome 3 region around the MLH1 gene and identification of two additional SNPs, rs749072 and rs13098279, which are in strong linkage disequilibrium with rs1800734. All three SNPs showed strong associations with MLH1 promoter methylation, loss of MLH1 protein expression, and MSI-H CRCs in three populations, Ontario, Newfoundland, and Seattle. Such findings potentially implicate genetic susceptibility to DNA methylation. Logistic regression models for MSI-H versus non-MSI-H CRCs demonstrate that models including MLH1 IHC status and MLH1 promoter methylation status fit the data most parsimoniously in all three populations combined, however, when rs1800734/rs749072/rs13098279 was added to this model, polymorphisms no longer remained significant indicating that the observed associations of these polymorphisms with the MSI-H CRCs occur through their effect on DNA methylation. This study identified a novel mechanism in which common missense alterations may contribute to complex disease.Ph

The dynamic DNA methylation landscape of the mutL homolog 1 shore is altered by MLH1-93G>A polymorphism in normal tissues and colorectal cancer

Abstract Background Colorectal cancers (CRCs) undergo distinct genetic and epigenetic alterations. Expression of mutL homolog 1 (MLH1), a mismatch repair gene that corrects DNA replication errors, is lost in up to 15% of sporadic tumours due to mutation or, more commonly, due to DNA methylation of its promoter CpG island. A single nucleotide polymorphism (SNP) in the CpG island of MLH1 (MLH1-93G>A or rs1800734) is associated with CpG island hypermethylation and decreased MLH1 expression in CRC tumours. Further, in peripheral blood mononuclear cell (PBMC) DNA of both CRC cases and non-cancer controls, the variant allele of rs1800734 is associated with hypomethylation at the MLH1 shore, a region upstream of its CpG island that is less dense in CpG sites. Results To determine whether this genotype-epigenotype association is present in other tissue types, including colorectal tumours, we assessed DNA methylation in matched normal colorectal tissue, tumour, and PBMC DNA from 349 population-based CRC cases recruited from the Ontario Familial Colorectal Cancer Registry. Using the semi-quantitative real-time PCR-based MethyLight assay, MLH1 shore methylation was significantly higher in tumour tissue than normal colon or PBMCs (P < 0.01). When shore methylation levels were stratified by SNP genotype, normal colorectal DNA and PBMC DNA were significantly hypomethylated in association with variant SNP genotype (P < 0.05). However, this association was lost in tumour DNA. Among distinct stages of CRC, metastatic stage IV CRC tumours incurred significant hypomethylation compared to stage I–III cases, irrespective of genotype status. Shore methylation of MLH1 was not associated with MSI status or promoter CpG island hypermethylation, regardless of genotype. To confirm these results, bisulfite sequencing was performed in matched tumour and normal colorectal specimens from six CRC cases, including two cases per genotype (wildtype, heterozygous, and homozygous variant). Bisulfite sequencing results corroborated the methylation patterns found by MethyLight, with significant hypomethylation in normal colorectal tissue of variant SNP allele carriers. Conclusions These results indicate that the normal tissue types tested (colorectum and PBMC) experience dynamic genotype-associated epigenetic alterations at the MLH1 shore, whereas tumour DNA incurs aberrant hypermethylation compared to normal DNA

MLH1 Region Polymorphisms Show a Significant Association with CpG Island Shore Methylation in a Large Cohort of Healthy Individuals

<div><p>Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We previously demonstrated that SNPs (rs1800734, rs749072, and rs13098279) in the <em>MLH1</em> gene region are associated with <em>MLH1</em> promoter island methylation, loss of MLH1 protein expression, and microsatellite instability (MSI) in colorectal cancer (CRC) patients. Recent studies have identified less CpG-dense “shore” regions flanking many CpG islands. These shores often exhibit distinct methylation profiles between different tissues and matched normal versus tumor cells of patients. To date, most epigenetic studies have focused on <em>somatic</em> methylation events occurring within solid tumors; less is known of the contributions of peripheral blood cell (PBC) methylation to processes such as aging and tumorigenesis. To address whether <em>MLH1</em> methylation in PBCs is correlated with tumorigenesis we utilized the Illumina 450 K microarrays to measure methylation in PBC DNA of 846 healthy controls and 252 CRC patients from Ontario, Canada. Analysis of a region of chromosome 3p21 spanning the <em>MLH1</em> locus in healthy controls revealed that a CpG island shore 1 kb upstream of the <em>MLH1</em> gene exhibits different methylation profiles when stratified by SNP genotypes (rs1800734, rs749072, and rs13098279). Individuals with wild-type genotypes incur significantly higher PBC shore methylation than heterozygous or homozygous variant carriers (p<1.1×10⁻⁶; ANOVA). This trend is also seen in CRC cases (p<0.096; ANOVA). Shore methylation also decreases significantly with increasing age in cases and controls. This is the first study of its kind to integrate PBC methylation at a CpG island shore with SNP genotype status in CRC cases and controls. These results indicate that CpG island shore methylation in PBCs may be influenced by genotype as well as the normal aging process.</p> </div

Logistic regression analysis for association with methylation between CRC cases and controls.

<p>Mean β value of CRC cases and controls is shown along with logistic regression analysis at seven CpG sites in the <i>MLH1</i> CpG island shore. Analysis of CRC cases versus controls is adjusted for age and sex. Effect size represents the increased risk of CRC per 1% reduction in methylation.</p

Correlation between age and methylation.

<p>Partial correlation, controlling for sex, between age and methylation at seven sites in the <i>MLH1</i> CpG island shore for CRC cases and controls. Significant results are bolded when p<0.001.</p

Characteristics of study population.

<p>Distribution of clinicopathological features in primary colorectal carcinomas and controls from Ontario. Age at study recruitment is indicated for CRC cases and controls. Blood was drawn an average of less than one year and no more than six years after study recruitment.</p><p>SD = standard deviation.</p

Locations of CpG sites and methylation between cases and controls.

<p>Pictured are the 70 CpG sites analyzed, with indicated chromosomal positions located on chromosome 3. The CpG sites are located within the <i>EPM2AIP1, MLH1,</i> and <i>LRRFIP2</i> genes, with gene exons and transcriptional directions indicated. CpG islands are indicated in green. The seven CpG sites of the <i>MLH1</i> shore are highlighted in red. Each vertical bar represents a CpG site, with control methylation, n = 846, displayed to the left and CRC case methylation, n = 252, displayed to the right of the white dotted line. Controls and CRC case samples are displayed layered horizontally from highest methylation to lowest methylation. The distribution of degree of methylation in cases and controls is represented by the colour variation, according to the scale.</p

Associations between gender and methylation by logistic regression.

<p>Mean β value of is shown for males and females along with logistic regression analysis at seven CpG sites in the <i>MLH1</i> CpG island shore. Analysis of male versus female methylation is adjusted for age. Significant results are bolded when p<0.001.</p

Methylation between SNP genotypes in CRC cases by ANOVA.

<p>Mean β value of each genotype of the SNPs rs1800734, rs749072, and rs13098279 in CRC patients from Ontario at seven sites in the <i>MLH1</i> CpG island shore. Significant results are bolded when p<0.001.</p