Distinct patterns of DNA methylation in conventional adenomas involving the right and left colon

Abstract

Recent studies have shown two distinct non-CIMP methylation clusters in colorectal cancer, raising the possibility that DNA methylation, involving non-CIMP genes, may play a role in the conventional adenoma-carcinoma pathway. A total of 135 adenomas (65 left colon and 70 right colon) were profiled for epigenome-wide DNA methylation using the Illumina HumanMethylation450 BeadChip. A principal components analysis was performed to examine the association between variability in DNA methylation and adenoma location. Linear regression and linear mixed effects models were used to identify locus-specific differential DNA methylation in adenomas of right and left colon. A significant association was present between the first principal component and adenoma location (P=0.007), even after adjustment for subject age and gender (P=0.009). A total of 168 CpG sites were differentially methylated between right- and left-colon adenomas and these loci demonstrated enrichment of homeobox genes (P=3.0 × 10(-12)). None of the 168 probes were associated with CIMP genes. Among CpG loci with the largest difference in methylation between right- and left-colon adenomas, probes associated with PRAC (prostate cancer susceptibility candidate) gene showed hypermethylation in right-colon adenomas whereas those associated with CDX2 (caudal type homeobox transcription factor 2) showed hypermethylation in left-colon adenomas. A subgroup of left-colon adenomas enriched for current smokers (OR=6.1, P=0.004) exhibited a methylation profile similar to right-colon adenomas. In summary, our results indicate distinct patterns of DNA methylation, independent of CIMP genes, in adenomas of the right and left colon.

Figure 1

Results from the locus-by-locus examination of differential methylation between right- and left-colon adenomas. (a) Volcano plot based on the locus-by-locus analysis. Points represent the −log₁₀(P-value) obtained from testing the difference in methylation between right- and left-colon polyps, adjusted for subject age and gender. Dotted line indicates −log₁₀(0.05) and the dashed line represents the Bonferroni threshold (ie, −log₁₀(0.05/no. of tests)). (b) Distribution of significant probes (those significant at the Bonferroni threshold) across CpG islands, shores, shelves, and oceans.

Figure 2

Methylation status between right- and left-colon adenomas for PRAC(a) and CDX2(b). Points represent the median methylation status for left- and right-colon polyps and lines represent the locally weighted polynomial smoothed methylation status across the profiled CpGs associated with that gene.

Figure 3

Difference in percent methylation between right- and left-colon adenomas based on the two target probes, (a) cg01499197, associated with gene CDX2, and (b) cg12374721, associated with PRAC, selected for pyrosequencing. P-values were adjusted for subject age and gender and were obtained from linear regression models fit to percent methylation measurements obtained from pyrosequencing.

Figure 4

Heat map of the 168 significantly differentially methylated CpG loci between right- and left-colon adenomas. Methylation values are presented as unmethylated (yellow) to methylated (blue). Rows are represented by the 168 differentially methylated loci and columns represent subjects who are grouped based on adenoma side. On the right-hand side of the heat map is a color side bar, which indicates the island status for the 168 CpG loci. Above the heat map are color side bars that indicate smoking status, polyp size, gender, and age for the study subjects. Red and green dotted boxes indicate two subgroups of left-colon adenoma subjects, referred to as subgroups 1 and 2.