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Epigenetic silencing of cancer-related genes by promoter methylation is a frequent event in sporadic colorectal cancer. The CpG island methylator phenotype (CIMP+), in which discrete genes throughout the genome are simultaneously methylated, and long-range epigenetic silencing, whereby multiple genes within contiguous chromosomal regions are methylated, have been described in subsets of colorectal cancer. We previously reported the concurrent methylation of the mismatch repair gene MLH1with a cluster of flanking genes in chromosome region 3p22 in sporadic colorectal carcinoma exhibiting microsatellite instability and the BRAF V600E mutation. Herein, we aimed to determine whether methylation of MLH1and neighbouring 3p22 genes, singly or concomitantly, correlate with the germline c.-93G>A SNP within the MLH1promoter, CIMP+ and other clinicopathological and molecular features of the tumours. By studying a cohort of 946 sporadic colorectal cancer cases, we show a strong association between concordant methylation of ≥3 of five 3p22 genes with CIMP+ and the BRAF V600E mutation (P<0.001). These associations were independent of microsatellite instability, as concomitant methylation of 3p22 genes other than MLH1was found in microsatellite stable cancers. These findings show that long-range epigenetic silencing across 3p22 occurs in the context of CIMP+ and the BRAF V600E mutation, and only gives rise to microsatellite instability when this process encompasses MLH1. Furthermore, the strong relationship between long-range epigenetic silencing of 3p22 and CIMP+ provides further evidence that these two purportedly distinct epigenetic phenotypes represent a single entity with a common aetiology. Low-level methylation of MLH1and flanking 3p22 genes, as well as the BRAF V600E mutation, were detected in the apparently normal colonic mucosa of a small number of cases whose tumours showed a similar molecular profile, suggesting that these concurring genetic and epigenetic events can occur as a field defect in neoplastic development.

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that restores mutagenic O6-methylguanine to guanine. MGMTmethylation is frequently observed in sporadic colorectal cancer and was recently correlated with the C>T allele at SNP rs16906252, within the transcriptional enhancer element of the promoter. MGMTmethylation has also been associated with KRASmutations, particularly G>A transitions. We studied 1123 colorectal carcinoma to define the molecular and clinicopathological profiles associated with MGMTmethylation. Furthermore, we assessed factors contributing to MGMTmethylation in the development of colorectal cancer by studying the allelic pattern of MGMTmethylation using SNP rs16906252, and the methylation status of neighbouring genes within 10q26 in selected tumours and matched normal colonic mucosa. MGMTmethylation was detected by combined bisulphite restriction analysis in 28% of tumours and was associated with a number of characteristics, including CDKN2Amethylation, absent lymphovascular space invasion and KRASmutations (but not specifically with KRASG>A transitions). In a multivariate analysis adjusted for age and sex, MGMTmethylation was associated with the T allele of SNP rs16906252 (P<0.0001, OR 5.5, 95% CI 3.8–7.9). Low-level methylation was detected by quantitative methylation-specific PCR in the normal colonic mucosa of cases, particularly those with a correspondingly methylated tumour, as well as controls without neoplasia, and this was also associated with the C>T SNP. We show that the T allele at SNP rs16906252 is a key determinant in the onset of MGMTmethylation in colorectal cancer, whereas the association of methylation at MGMTand CDKN2Asuggests that these loci may be targets of a common mechanism of epigenetic dysregulation.