Our study shows that individual differences in repetitive element DNA methylation predict the risk of developing ischemic heart disease and stroke in elderly men. The associations of LINE-1 hypomethylation with ischemic heart disease and stroke, both in cross-sectional and longitudinal analyses, suggest that DNA hypomethylation anticipates disease diagnosis. Hypomethylation may help identify individuals at risk well before the onset of clinical disease. This conclusion is supported by our observation of no changes in average methylation in a subset of subjects for whom blood DNA samples were collected before and after the diagnosis of heart disease or stroke. Epigenetic marks can be inherited and are largely established in utero or during early life. Thus, DNA hypomethylation may be part of the processes that determine transgenerational risks, as well as of the effects of in utero and early life conditions on adult disease.1,28
Hypomethylated DNA has been shown to be prone to mutations or aberrant gene expression patterns in vascular tissue, leading to the transition from normal phenotype to vascular fibrocellular lesions by increasing proliferation of vascular smooth cells and lipid deposition.29
Although the correlation of DNA methylation in blood and in vascular tissue is undetermined, blood DNA methylation content and repetitive element methylation both undergo progressive decline as people age,15,30
which may be related with the risk of common age-related disease.
In our data, lower LINE-1 methylation predicted the risk of cardiovascular disease independent of established risk factors. Epigenetic modifications—in contrast with genetic changes—are potentially reversible. Our results, if confirmed, would indicate the potential for lifestyle or pharmacological interventions to reverse deleterious epigenetic features.1
Although DNA methylation analysis of LINE-1 sequences has been frequently used to estimate global genomic methylation content,2,3
the observed association with heart disease and stroke may result directly from altered LINE-1 organization and function. Hypomethylation of repetitive DNA sequences is expected to lead to the transcriptional activation of those repetitive sequences that still contain active promoters.4-6,31
Repetitive elements have been demonstrated to be activated during conditions of cellular stress,5,32
and LINE-1 expression has been recently identified as a mediator of ischemic heart damage.33
However, it remains to be determined whether LINE-1 hypomethylation is associated with specific functions that mediate increased cardiovascular risk, and what the nature of these functions might be. Because we measured LINE-1 methylation in peripheral blood leukocytes, the potential functions of LINE-1 repetitive elements associated with hypomethylation in this cell type will be likely related to mechanisms different from those operating in the heart. In our data, LINE-1 methylation showed very few associations with the baseline characteristics we evaluated, including several established risk factors for cardiovascular disease. In particular, we did not find any association with plasma homocysteine, which has been inversely related with global or LINE-1 methylation in experimental models and human studies.9,11,13
Specific characteristics of this population, including older age, might have contributed to discrepancies with other studies.
Hypomethylation of repetitive elements has been found in association with hypermethylation of specific genes, particularly in studies conducted on cancer tissues.1
Further research is warranted to evaluate whether blood LINE-1 hypomethylation is associated with increased methylation of genes that are hypermethylated in atherosclerotic tissues.34,35
In previous work on 1,097 blood DNA samples from this same cohort, including multiple measures of LINE-1 methylation from a subset of the study subjects, we found that LINE-1 methylation showed a moderate cross-sectional association with age.15
At the examination taken as baseline, a 10-year increase in age was associated with −0.23 % 5mC lower LINE-1 methylation. All multivariable models we fitted included age as an independent variable. Therefore, the associations between LINE-1 methylation and cardiovascular risks appear independent from age.
We also recently found that participants in the Normative Aging Study exposed to higher levels of particulate air pollution from vehicular traffic in the week before the blood drawing exhibited lower LINE-1 methylation.18
This finding was replicated in a study of foundry workers exposed to airborne metal-rich particulate.36
Whereas air-pollution exposure is an established risk factor for atherosclerotic cardiovascular disease, the short time scale in our air pollution analysis points to rapid and transient changes that are unlikely to be responsible for cardiovascular events occurring several years later, such as those investigated in the current study.
Our study has the advantage of being based on a population of aging men unselected on the basis of disease status. However, our results can be generalized only to older white men. Future studies should address the role of repetitive-element hypomethylation among women, as well as in various age and ethnic groups. Results on hypomethylation-related risk of stroke were based on a limited number of cases, and should be replicated in larger samples. Because we measured methylation in blood DNA—a source of DNA that is easily obtainable and does not require processing before DNA extraction—our results might have reflected shifts in the proportions of white-blood-cell subsets caused by alterations related to impending disease onset. This scenario, however, predicts that the strength of the association of lower DNA methylation with higher incidence and mortality of ischemic heart disease and stroke should be maximal immediately after DNA methylation measurement and decline afterwards.37
In contrast, we did not see such a decline, and adjustment in multivariable analysis for percent neutrophils and lymphocytes (the major white blood cell types) did not cause major changes in the risk estimates. A limitation of the present study is that the ischemic heart disease and stroke diagnoses did not differentiate underlying disease. The diagnoses of ischemic heart disease and stroke might have included events occurring in subjects with little or no atherosclerosis. Because previous animal and human studies have specifically related DNA hypomethylation to atherosclerosis,7,22
this might have lead to a dilution of the hypomethylation-related risk estimates.
In summary, we showed that epigenetic changes measured in blood DNA predict the risk of common age-related diseases, such as ischemic heart disease and stroke. Our results indicate blood repetitive-element hypomethylation is a novel risk factor for cardiovascular risk and survival, and may contribute to better cardiovascular risk stratification as simple, standardized assays of DNA methylation become available. At the same time, our results give further substance to ongoing endeavors to develop interventions and treatments that act through epigenetic mechanisms.