In this large multi-center clinical polyp prevention study, we found that methylation levels of the ERα and SFRP1 CGIs in the normal colorectal mucosa differed by large bowel region, age, race, and RBC folate levels. Methylation levels were significantly higher in the rectum compared to the right colon, and increasing age was strongly associated with higher levels of methylation in both the right colon and rectum. For both ERα and SFRP1, we observed that Caucasians had higher levels of methylation than African Americans, while Hispanics had relatively low levels of ERα and relatively high levels of SFRP1. We found no significant relationship of the level of methylation for either CGI site with aspirin or folic acid treatment, dietary folate or any 1-carbon folate metabolism polymorphism. However, we did observe a relationship between higher levels of year three RBC folate and increasing levels of ERα and SFRP1 methylation.
Our findings of an age-related increase in CGI methylation in the normal mucosa parallel those of several other investigations (3
). Our study is one of two to document age-related increases in SFRP1
methylation within the normal mucosa (14
). Another investigation (31
) reported no evidence of SFRP1
methylation in the normal mucosa in a younger study population (ages 20–54). Other studies have reported low-level SFRP1
methylation in normal tissue adjacent to tumors but no effort was made to examine an association with age (11
). For ERα
, the evidence of an age-related increase in methylation in the normal colorectal tissue has been documented in most other investigations (4
) yet not all (14
). While the differences detected are small in some instances (2–4% methylation), they can be significant if they represent differences in the numbers of colorectal crypts with dense methylation. This has previously been shown to be the case (32
Investigators have become increasingly aware that there are clear physiological, morphological, and biochemical differences between the right and left colorectum (33
) and these differences may shed light on why we observed differences in methylation between the right colon and rectum (33
). Our results parallel an earlier study which also showed higher ERα
methylation levels in the left colorectum compared to the right (4
), although in that study the samples were selected from a mix of patients with colorectal cancer, adenomas, other disease, and sigmoidoscopy only ‘normal controls,’ which represents a subject population that may limit comparability with ours. Of the two other investigations (6
) which examined ERα
methylation in normal colorectal mucosa, one (35
) reported no difference by anatomical location, and in the other biopsies were selected from the rectum only (6
). In the first study to show no difference by region of the large bowel (35
), no description is provided about the number of right versus left biopsies.
Why methylation patterns may differ by large bowel region is not well understood. Theoretically, the differences in CGI methylation between the right and left colorectum may be driven by physiologic/biologic differences in the anatomic regions. For example, there are many known differences in the biology of the right and left colorectum such as different embryologic origin, crypt length and apoptotic index (33
). Alternatively, the higher methylation levels that we observed in the rectum may be specific to the ERα
genes, and other CGIs might show a different pattern.
In our study population, African Americans had lower levels of methylation compared to Caucasians and Hispanics. We believe that our study is the first to consider CGI methylation levels in normal colorectal mucosa of different racial groups. One other study reported no significant difference in ERα
methylation levels in colorectal tumor tissue when comparing Caucasian and non-Caucasian persons, yet the average ERα
CGI methylation level was higher in Caucasians (36
). There are several possible explanations for our findings, including lifestyle factors, genetic protection or predisposition to methylation and polymorphisms in the genes studied themselves. Still, the number of African Americans studied is small, and our findings could also be a result of chance.
Very few of the lifestyle or dietary variables we examined were related to CGI methylation levels. Previous research has suggested a relationship between dietary folate and genomic methylation in normal mucosa (37
), but our study is the first to examine the association between folic acid supplementation and CGI methylation in normal tissue. In findings consistent with ours, another study reported no association between serum folate levels and ERα
methylation in normal mucosa (6
). However, we did observe that RBC folate levels were positively associated with ERα
methylation levels. It is interesting and biologically relevant that RBC folate levels showed a stronger association than plasma folate. The former more closely reflect long term intake, and also reflect intracellular folate directly. In light of these data, it might seem surprising that randomization to the folate supplementation arm was not associated with a difference in DNA methylation. This is likely due in part to the fact that the difference in RBC folate between the placebo and folate treatment arms (249 ng/ml) would correspond to less than a 0.5% difference in ERα
methylation, something that would be difficult to detect with this technology and sample size. These data have important implications regarding the safety of supplementary folate administration in healthy adults, given the hypothesis that methylation in normal mucosa may be a predisposing phenomenon for colorectal neoplasia (discussed below).
All patients had detectable levels of ERα
methylation and no significant differences were observed among patients with and without recurrence of colorectal adenomas. This implies that ERα
methylation are not rate-limiting in the development of adenomas in this population, and is consistent with the idea that a mutation in APC
) is a critical first step in this regard. However, there may be other age-related methylation genes such as MYOD1, DKK1, HPP1
) that could be associated with adenoma occurrence. It has also been proposed that mutations of these genes may be more likely to occur in a “predisposed” mucosa, and that aberrant DNA methylation is one of the factors that results in this predisposition (40
To test whether DNA methylation does in fact lead to this predisposition, one would need to study a mixed population with patients at high and low risk of adenoma formation, something that was not done here, given that all patients had adenomas at study entry. Comparing results across studies is difficult for many reasons, including differences in: (1) the genes examined (2) the control for confounding variables such as age and sex or (3) the colorectal location(s) selected for mucosal sampling (18
). Despite these challenges, several studies have examined CGI methylation levels in the normal mucosa of persons with and without neoplastic lesions (14
). Of these, five have specifically examined ERα
). Four of these studies have reported lower ERα
) among those with advanced neoplasia compared to adenomas or disease-free controls, while one reported higher ERα
levels in adenoma patients (15
was associated with lower levels of methylation in mucosa of patients with neoplasia compared to those without in one study (18
), and was unrelated to pathology in another (14
Although not statistically significant, we observed that a higher level of ERα
methylation was associated with an increased risk of hyperplastic polyps, especially right-sided hyperplastic polyps. Interestingly, two other investigations (6
) also observed increased levels of ERα
CGI methylation in the normal mucosa in persons with hyperplastic polyps or proximal serrated polyps compared to controls. Historically, hyperplastic polyps have been considered indolent, non-neoplastic lesions (43
). However, currently it is understood that a subset of “hyperplastic” polyps are in fact sessile serrated adenomas, the precursor lesion to cancers evolving along the serrated adenoma pathway (45
). The lesions of the serrated pathway have been closely associated with the CpG island methylator phenotype (CIMP) which involves the methylation of multiple cancer-specific CGIs (1
). Future studies will be needed to determine if CGI methylation in the normal mucosa predisposes toward to serrated lesions, since these lesions appear to have a somewhat distinct epidemiologic profile (50
There are several strengths to our study. First, we examined methylation levels in a well characterized population, with a systematic protocol, using a quantitative technique capable of detecting low-level methylation. ERα was selected because of strong prior data connecting its methylation state with aging, and SFRP1 was studied because of suggestions that it is a gatekeeper for neoplasia in the colon. The fact that ERα and SFRP1 methylation was consistently correlated with age (and with each other) confirms the validity of the measurements. Additionally, subjects were part of a large, multi-center polyp prevention study with excellent follow-up and compliance with treatment. Ours is the first study with the magnitude and rigor to examine the effect of folic acid supplementation on methylation levels. Limitations to the study include the entry criteria: all subjects had at least one adenoma, and so may be different from patients with no history of polyps. Thus, it remains to be determined whether mucosal methylation levels are lower in individuals who never develop adenomas. Furthermore, we used a convenience sample of specimens, which may not reflect the entire population of 768 persons with biopsies. There was a relatively low intraclass correlation between biopsies in a given individual. Given the relatively low CV of the assay, the primary reason for the low correlation is likely due to the natural variation of the biopsies. It will be important in future studies to obtain duplicate biopsies from many segments of the colorectum given the known biologic and anatomical differences which occur by region.
Overall, our study results point to differences in methylation levels by age, race, different regions of the large bowel and RBC folate levels. This is one of the first large scale studies to document associations between epigenetic measures and dietary factors in adults.