DNA Methylation as a Biomarker for Cardiovascular Disease Risk

Abstract

Introduction

Cardiovascular disease (CVD) is the leading cause of death in most countries [1]. Risk factors known to predispose to the development of CVD include increasing age, male gender, diabetes mellitus, high blood cholesterol, tobacco smoking, high blood pressure (hypertension), obesity, physical inactivity, and family history [1]. Elevated plasma homocysteine is also an independent risk factor for CVD [2], [3]. The mechanism by which elevated homocysteine contributes to CVD risk is not well understood, but it is well-established that dietary folate and vitamin B supplementation can reduce serum homocysteine levels by facilitating remethylation of homocysteine to methionine [4], [5], [6]. Accumulation of homocysteine can lead to increased intracellular levels of S-adenosylhomocysteine, a transmethylation inhibitor [7], [8]. The remethylation cycle is essential for the systemic methyl donor supply, which is used for important biological processes, such as cytosine-5 methylation of genomic DNA, an epigenetic modification that plays an important role in maintaining genomic stability, chromatin structure, and in controlling transcriptional capacity [2], [9]. Global DNA hypomethylation has been observed in atherosclerotic lesions as a consequence of low dietary folate or elevated plasma homocysteine in humans and animal models [10], [11]. We have previously identified age, sex, plasma folate, vitamin B-12 and vitamin B-6, and methylenetetrahydrofolate reductase (MTHFR) genotype as independent predictors of plasma homocysteine in Singapore Chinese [12]. In this study, we examined the relationships between prevalence of CVD (myocardial infarction, stroke) or its predisposing conditions (hypertension, diabetes) and peripheral blood leukocytes (PBL) global genomic DNA methylation to verify the potential value of DNA methylation as a CVD biomarker, using a validated MethyLight-based assay for ALU and Satellite 2 repetitive element (AS) DNA methylation [13].

Methods

Study Population

The subjects were participants of the Singapore Chinese Health Study, a population-based prospective cohort study of Chinese men and women, aged 45–74 years at baseline. They belonged to the two major Chinese dialect groups in Singapore (Hokkien and Cantonese) and lived in government housing estates where 86% of all residents in Singapore resided during the period of enrollment [12]. A total of 63,257 individuals gave informed written consent and enrolled between April 1993 and December 1998. The study was approved by the Institutional Review Boards of the University of Southern California, and the National University of Singapore.

At recruitment, each participant completed an in-person interview using a structured questionnaire that requested information about demographic characteristics, height and weight, use of tobacco, usual physical activity, medical history, and family history of cancer. The questionnaire included a validated semi-quantitative food frequency section listing 165 food items commonly consumed in the study population, from which average daily intake of calories and roughly 100 nutrients and non-nutritive ingredients per subject were computed using the Singapore Food Composition Table [14] which we developed in conjunction with the cohort study.

Between 1994 and 1998, a random 3% sample of cohort participants was recontacted for donation of blood and urine specimens. The 286 subjects in the current study represented the first accrued participants of this biospecimen subcohort [15], [16] (Figure 1). The entire cohort has been continuously followed for the occurrence of incident cancers and deaths ever since. All surviving cohort participants were interviewed by telephone during 1999–2003 for an updated medical history. The mean time interval between the two interviews (baseline and follow-up) is 5·8 years (range, 2·6–11·0 years).

Open in a separate window

Figure 1

Study Overview.

Results

Discussion

We present here the results of a population-based prospective cohort study of risk factors for CVD. This study was initiated to ascertain whether PBL DNA methylation could serve as a stable measure of systemic methyl group supply, analogous to the use of glycated forms of hemoglobin to provide measures of long-term mean blood glucose levels. However, we did not observe the anticipated correlations between PBL DNA methylation and plasma folate and homocysteine, dietary folate and the B vitamins, and folate metabolizing genotypes such as TYMS. Furthermore, we noted a statistically significant, positive association between PBL DNA methylation and prevalence of CVD or its risk factors, primarily in men, when we had anticipated an inverse association between the two sets of factors. This suggests that, rather than folate insufficiency, a different mechanism, such as systemic inflammation, may lead to increased PBL DNA methylation. Our results differ from those of Castro et al. (2003), who found that vascular disease patients with elevated plasma tHcy and AdoHcy concentrations and low plasma AdoMet/AdoHcy ratios had lower levels of genomic DNA methylation [19]. However, this study was based on a very small sample size of 17 vascular disease cases and 15 controls. Although Castro et al. (2003) used the intracellular AdoMet/AdoHcy ratio as a predictor of cellular methylation capacity, they failed to observe such association in their study. The global DNA methylation status and homocysteine, both plasma tHcy and AdoHcy, also seemed to be less correlated each other (r = 0.47; r = 0.54, respectively). Moreover, it has been observed that imprinted gene H19 is hypermethylated, not hypomethylated, in brain and aorta of hyperhomocysteinemic mice, although the effect of hyperhomocysteinemia on H19 DMD methylation was tissue-specific in these mice [20]. The result of significantly higher AS in men is consistent with our recent finding of higher DNA methylation at unique genomic loci in men than in women [15]. It has been reported that global DNA methylation levels decrease with age [21], [22]. However, the relatively narrow elderly age range (55–77 years) of our study population at blood draw may account for the lack of association between age and AS in this study. Age-dependent decrease of global DNA methylation levels measured by HPLC was also fairly small in human PBL among age groups [22].

Although we did not find a link between lipid metabolites [18] and global DNA methylation, baseline BMI was positively associated with AS. High relative weight is considered a risk factor for CVD in both western [23] and Chinese [24] populations. It is worth noting that most diabetics among Chinese have normal BMI according to western standards [25] and the recognized cutpoint for at-risk Chinese is BMI of 24 kg/m² [26] or higher.

In summary, this is the first report of an association between global DNA methylation assessed by ALU/SAT2 methylation [13] and prevalence of CVD, in addition to its risk factors, including male gender and obesity in a population-based Singapore Chinese cohort, a relatively lean population. Our novel findings, derived from analysis that were exploratory in nature, require confirmation from studies on other Asians as well as more distinct ethnic groups, such as those in the West with higher BMI. If confirmed, this blood-based marker could offer exciting new opportunities for population-based CVD risk assessment and prevention.

Supporting Information

Footnotes

References