Title page Title: The CpG island methylator phenotype correlates with long-range epigenetic silencing in colorectal cancer Running title: CIMP correlates with LRES in colorectal cancer

The CpG island methylator phenotype (CIMP), characterized by an exceptionally high frequency of methylation of discrete CpG islands, is observed in 18% to 25% of sporadic colorectal cancers (CRCs). Another hypermethylation pattern found in CRCs, termed LRES (long-range epigenetic silencing), is associated with DNA/histone methylation in three distinct gene clusters at chromosome 2q14.2, demonstrating that DNA hypermethylation can span larger chromosomal domains and lead to the silencing of flanking, unmethylated genes. We investigated whether these two phenotypes are interrelated in CRCs. The CIMP status of 148 sporadic CRCs was determined by methylation-specific PCR (MSP). We determined the BRAF V600E mutation by mutant allele-specific PCR amplification (MASA). The methylation status of the MLH1 gene and of three CpG islands (EN1, SCTR and INHBB), corresponding to three distinct clusters along 2q14.2, was determined by MS-PCR. The average number of sites showing methylation in CIMP+ tumors was 2.21 compared to 1.22 for CIMPindividuals, and this difference was highly significant (P = 3.6×10-8, MannWhitney test). Moreover, all CIMP+ tumors showed hypermethylation of at least one of these loci in contrast to CIMPtumors, where 18 samples (16%) remained unmethylated. The mean number of simultaneously hypermethylated CpG islands at 2q14.2 differs significantly for CIMPand CIMP+ tumors suggesting varying effects of domain silencing in this region. Given that the number of hypermethylated loci at 2q14.2 likely affects the range of silenced flanking genes, high frequency of simultaneous hypermethylation of three CpG islands (EN1, SCTR and INHBB) may have potential influence on specific characteristics of CIMP+ CRCs.