Abstract 91: Analysis of global and repeat sequence DNA methylation in the blood and normal colonic mucosa of colorectal cancer patients, healthy individuals and patients with folate deficiency

Abstract

Abstract Aberrant DNA methylation is an early event in colorectal tumorigenesis and in colorectal cancer (CRC) patients these changes are detected in their normal colonic mucosa. DNA methylation changes may arise from aging or environmental exposures e.g. folate deficiency. We aimed to compare global and repeat-sequence DNA methylation in the colonic mucosa and blood of three groups of individuals – CRC patients, healthy people and patients with folate deficiency. We conducted a multisite cohort study collecting mucosa and blood from 176 patients who had undergone CRC resection and 194 healthy individuals at screening colonoscopy. Blood was also collected from 30 individuals with megaloblastic anaemia secondary to folate deficiency (red cell folate <539nM). Global DNA methylation was measured using liquid chromatography tandem mass spectrometry adapted from Quinlivan & Gregory (2008)1. Alu repeat methylation was determined using a novel, ultra-sensitive real-time PCR assay based upon methylation-sensitive restriction enzyme digestion coupled with a fluorescence-based readout2. Genotyping for a common polymorphism in the folate metabolism enzyme MTHFR (C677T) was performed using pyrosequencing. The mean age of CRC patients (68 ± 14 years) was significantly higher than healthy subjects (55 ± 14 years; p<0.01) but not significantly different from low-folate subjects (72 ± 22 years). The gender distribution was similar in all 3 groups. The prevalence of the variant T allele of MTHFR in CRC subjects (58%) and healthy controls (64%) were comparable to population-based estimates. However, the T allele was over-represented in low folate patients (79%). In the analysis of the first subset of CRC (n=38) and healthy controls (n=38), mean global DNA methylation was significantly lower in the colonic mucosa (CRC n=20: 4.1% ± 0.3; controls n=36: 4.0% ± 0.3) compared to peripheral blood (CRC n=17: 4.4% ± 0.4; controls n=36: 4.4% ± 0.3, p<0.01). No differences in DNA methylation were found in CRC patients compared to healthy subjects. Consistent with global methylation findings, Alu repeats were 2-fold less methylated in colon compared to peripheral blood in healthy (n=38) and CRC cohorts(n=36). Low-folate subjects had a trend towards lower blood global (4.26% ± 0.2) and Alu methylation (1.2-fold hypomethylated) compared to healthy subjects. To date, our results suggest that Alu repeat methylation reflect global DNA methylation and that this holds true across tissue types irrespective of cancer status. Global and Alu repeat demethylation in individuals with folate deficiency and overrepresentation of the T MTHFR allele suggests a possible link between diet and the epigenome that warrants further study. 1. Quinlivan, E.P. & Gregory, J.F., 3rd. (2008) Nucleic acids research 36, e119. 2. Rand, K. & Molloy, P. (2010) BioTechniques 49, xiii-xvii. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 91. doi:10.1158/1538-7445.AM2011-91