Patterns of DNA methylation in the normal colon vary by anatomical location, gender, and age

Abstract

Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon's normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.

Keywords: DNA methylation; colorectal cancer; field effect; methylation microarray; normal colon methylation.

Figures

Figure 1. DNA methylation in repetitive elements in the various anatomical colon segments from eight patients. Each panel shows the degree of methylation in two adjacent biopsies for each patient in LINE-1 (A–D) and SAT-α (E–H). There was minimal variability in methylation between adjacent biopsies from each patient, minimal patient-to-patient variability, and low variability between colon segments.

Figure 2. A single colon biopsy from each colon site from each individual was used to compare methylation of LINE-1 (A) and SAT-α (B). Each data point represents the percent methylation for one individual. The average methylation of LINE-1 did not differ between rectum, sigmoid, transverse, and ascending colon. The differences in methylation of SAT-α between colon sites did meet criteria for statistical significance but overall the variability between colon segments was minimal. *P < 0.001 for mean methylation level between each site.

Figure 3. Methylation status of cancer-related genes EVL, CDKN2A, MGMT, and ESR1 is shown by colon location for each of eight individuals. Each data point represents the methylation level for a single individual, and the average methylation level for each colon site is shown. EVL showed a statistically significant decrease in average methylation between rectum, sigmoid, transverse, and ascending colon samples (P < 0.01 using ANOVA test to compare means of four groups).

Figure 4. Relationship of ESR1 methylation to the age of each individual. In DNA isolated from all colon sites (A–D), there was a weak positive correlation (r2) between ESR1 methylation and age.

Figure 5. (A) Strip plot showing the top 50 (smallest FDR q value) differentially methylated loci between the ascending colon and rectal DNA samples. Each ascending colon sample is depicted as a blue dot, and each rectal sample as an orange dot. The magnitude of methylation difference (difference in β value) can be seen for each group of samples at each locus. The corresponding genes and probe locations of the top 20 loci are shown in Table 1. (B) The overall percentage of HM450 array probe locations (black bars) compared with the probe locations of the 8388 differentially methylated loci (gray bars) is shown. A greater proportion of the differentially methylated loci (DML) were located in intragenic and intergenic regions as opposed to promoter regions. The total percent of probe types is greater than 100% because a few probes were classified as belonging in more than one type of CpG class. (P < 0.001 using hypergeometric testing of expected vs. actual number of DML for promoter, intragenic, and intergenic probes.)

Figure 6. (A) Strip plot depicting the top 12 DML (FDR q value < 0.01) when comparing individuals aged >60 to those aged <60. Each sample is shown as a blue dot (age >60) or red dot (age <60). In 11 of 12 top DML, methylation was higher in the older age group. The corresponding genes and probe locations of the loci are listed in Table 3. (B) Regression curves for the same top 12 DML illustrating the linear relationship between methylation and age. For the same 11 out of 12 loci, the level of methylation increased along with increasing age; methylation decreased with age at one locus.