Increased <i>MLH1</i> , <i>MGMT</i> , and <i>p16INK4a</i> methylation levels in colon mucosa potentially useful as early risk marker of colon cancer

The genes MutL Homolog 1 (<i>MLH1</i>), O6-methylguanine-DNA methyltransferase (<i>MGMT</i>), and cyclin-dependent kinase inhibitor <i>p16INK4a</i> are commonly downregulated by hypermethylation in colorectal cancer. Long interspersed nucleotide element 1 (LINE-1) can be used as marker for global hypomethylation. This study compared <i>MLH1</i>, <i>MGMT</i>, <i>p16INK4a</i>, and LINE-1 methylation with gene expression in colon tumors, matched non-cancerous mucosa, and control mucosa to identify signs of premalignancy. Tissues were obtained from 20 colon cancer patients and 40 controls. CpG site methylation was quantified by pyrosequencing, expression by qPCR, and MSI/<i>KRAS</i> status by fragment analysis and droplet digital PCR. <i>MLH1</i>, <i>MGMT</i>, and <i>p16INK4a</i> methylation was increasingly higher in control mucosa, non-cancerous mucosa, and tumors. <i>MLH1</i> expression was lower in tumors compared to non-cancerous mucosa but higher compared to control mucosa. Tumoral LINE-1 methylation correlated negatively with <i>MLH1</i> (<i>r</i> = -0.51, <i>p</i> = .021) and p16INK4a (<i>r</i> = -0.55, <i>p</i> = .012) methylation, but positively (<i>r</i> = 0.74, <i>p</i> = .0002) with <i>MLH1</i> expression. A <i>p16INK4a</i> SNP (rs3814960 C>T) was associated with methylation, expression, and MSI/<i>KRAS</i> status. Aberrant methylation of tumor suppressor genes in colon mucosa could be an early cancer risk marker. Control mucosa is a more reliable reference than non-cancerous mucosa when identifying premalignant changes. Extended studies will evaluate the possible association between rs3814960 and cancer susceptibility. <b>Trial registration</b>: NCT03072641.