Global DNA methylation status was determined in 600 participants in the PDAY study to evaluate whether variation in hepatic DNA methylation was associated with subclinical atherosclerosis in young adults matched for age, gender, and ethnicity. The liver is the site of synthesis of endogenous cholesterol and lipoproteins as well as acute-phase response reactants to inflammation such as C-reactive protein that have been shown to predict the risk of cardiovascular disease when their levels are elevated [14
]. Experiments performed in mouse models of atherosclerosis suggest that gene expression changes in the liver with disease progression [16
], and modification of either global or locus-specific DNA methylation could serve as a possible regulatory mechanism.
In this context, global DNA methylation has been shown to decrease with age in human peripheral blood leukocytes [17
]. In addition, Bjornsson et al. have demonstrated that global DNA methylation status can change over time within a single individual. When peripheral blood leukocytes from participants in the population-based AGES (Aging Gene-Environment Susceptibility)-Reykjavik study were investigated, 29% showed a change of 10% or more over an 11-year period [18
]. Although to date all of the published reports of epigenetic differences between affected and unaffected individuals for complex diseases including diabetic neuropathy and preeclampsia have been confined to cross-sectional analyses, an important implication of this work is that longitudinal alterations in DNA methylation that depend on a specific environment or metabolic status may lead to disease-specific transcriptional changes [19
]. Alterations in gene-specific methylation have also been reported for human atherosclerotic aortas in vivo
. In an early study, Post et al. demonstrated that the estrogen receptor-α (ESR1
) CpG island was more highly methylated in coronary atherosclerotic plaques when compared to normal samples taken from the proximal aorta, possibly contributing to abnormal proliferation of smooth muscle cells within these lesions [21
]. In contrast, when the methylation status of 10,367 CpG islands in 45 arteries from individuals with atherosclerosis and 16 normal controls was interrogated using DNA methylation microarrays, a subset were found to be consistently hypermethylated in control arteries but largely unmethylated and expressed in atherosclerotic tissues, including regions adjacent to genes encoding transcription factors implicated in atherogenesis [22
In this study, variation in hepatic global DNA methylation was not associated with disease risk in the study population considered as a whole using conditional logistic regression models to analyze matched pairs. Since the estimation of the risk of atherosclerosis associated with inter-individual variation in DNA methylation was similar if unconditional logistic regression was used, subgroup analyses were carried out after adjusting for the matching variables. An association of borderline significance with methylation levels below the median for the study population was found in white but not in African-Americans study participants.
There have been inconsistent results reported in previous studies of global methylation in peripheral blood leukocytes using a variety of laboratory methods in ethnically diverse populations. Higher global methylation of Alu and satellite 2 repeats determined by real-time polymerase chain reaction was found in males but not females aged 45–74 years with a history of cardiovascular disease among 286 individuals enrolled in the Singapore Chinese Health Study [7
]. Similarly, in 287 Indian subjects who were 30–75 years of age, global DNA methylation was higher in blood from patients with angiographically confirmed coronary artery disease than in controls as assessed by a cytosine extension assay [6
]. In contrast, lower methylation of LINE-1 repeats measured by pyrosequencing has been shown to predict both baseline and incident ischemic heart disease and stroke in a cohort of 712 elderly individuals from the Boston-area Normative Aging Study [8
While the modest association between high global DNA methylation index and susceptibility to subclinical atherosclerosis in whites in the PDAY study may be due to chance, differences in the prevalence of cardiovascular risk factors between African-American and white children as well as differences in susceptibility to cardiovascular disease have been documented in other studies. For example, there was a significantly greater prevalence of high BMI-for-age at three different cut-points for non-Hispanic black girls when compared to non-Hispanic white girls among individuals included in the 2007–2008 Health and Nutrition Examination Survey, a representative sample of the United States population [23
]. In the multiethnic SEARCH for Diabetes in Youth Study, newly diagnosed type 2 diabetes was more common in African-American than in non-Hispanic white adolescents 10–19 years of age, and was combined with a higher incidence rate [24
]. Similarly, African-American children aged 5–14 years of age had higher blood pressure than did white children, and more extensive fatty streaks were found in the aortas from African-American than from white adolescents and young adults in the community-based Bogalusa Heart Study [25
There is precedent for differences in DNA methylation patterns between racial and ethnic groups in two recent studies that were not related to the etiology of cardiovascular disease. In an analysis of the association between global methylation levels measured in middle age and epidemiologic risk factors including early life exposures, DNA methylation in peripheral blood leukocytes was lower in African-Americans and higher in Hispanics when compared to non-Hispanic whites in a birth cohort of women born between 1959 and 1963 in New York City [27
]. There were also significant differences between African-Americans and whites in the level of methylation present at birth for 13.7% of 26,485 CpG dinucleotides interrogated by microarray in umbilical cord blood from 201 infants [28
]. In these studies as well as the PDAY study, variation in genetic or environmental factors such as diet or the level of homocysteine and other compounds involved in one-carbon metabolism that could contribute to differences in DNA methylation may underlie the observed racial disparities. DNA methyltransferases use S-adenosyl methionine (SAM) as a methyl group donor to modify CpG dinucleotides after a series of reactions that depend on the transmethylation of homocysteine to methionine. If the supply of methyl groups is inadequate, the abnormally high level of homocysteine leads to an elevation in the amount of S-adenosylhomocysteine (SAH), a direct inhibitor of methylatransferases. Hyperhomocysteinemia has been associated with an increased risk of cardiovascular disease in two independent meta-analyses [29
]. Though homocysteine levels have not been measured in PDAY study participants, evaluation of their impact on DNA methylation in other multiethnic cohorts may be warranted.
Although important caveats in interpreting the study results include the possibility that the methylation measurements in autopsied specimens may reflect changes immediately prior to death, and that the LUMA assay is limited to analysis of only a restricted set of enzyme cleavage sites across the genome, hepatic global DNA methylation does not appear to be a definitive determinant of subclinical atherosclerosis in this postmortem sample of young adults. Replication in other study populations will be required to confirm the modest association between global DNA methylation status and disease risk identified in a subgroup of white study participants.