SummaryOverweight and obesity affect ~1.5 billion people worldwide, and are major risk factors for type-2 diabetes (T2D), cardiovascular disease and related metabolic and inflammatory disturbances.1,2 Although the mechanisms linking adiposity to its clinical sequelae are poorly understood, recent studies suggest that adiposity may influence DNA methylation,3–6 a key regulator of gene expression and molecular phenotype.7 Here we use epigenome-wide association to show that body mass index (BMI, a key measure of adiposity) is associated with widespread changes in DNA methylation (187 genetic loci at P<1x10-7, range P=9.2x10-8 to 6.0x10-46; N=10,261 samples). Genetic association analyses demonstrate that the alterations in DNA methylation are predominantly the consequence of adiposity, rather than the cause. We find the methylation loci are enriched for functional genomic features in multiple tissues (P<0.05), and show that sentinel methylation markers identify gene expression signatures at 38 loci (P<9.0x10-6, range P=5.5x10-6 to 6.1x10-35, N=1,785 samples). The methylation loci identified highlight genes involved in lipid and lipoprotein metabolism, substrate transport, and inflammatory pathways. Finally, we show that the disturbances in DNA methylation predict future type-2 diabetes (relative risk per 1SD increase in Methylation Risk Score: 2.3 [2.07-2.56]; P=1.1x10-54). Our results provide new insights into the biologic pathways influenced by adiposity, and may enable development of new strategies for prediction and prevention of type-2 diabetes and other adverse clinical consequences of obesity.

Rationale: Exposure to particulate air pollution has been related to increased hospitalization and death, particularly from cardiovascular disease. Lower blood DNA methylation content is found in processes related to cardiovascular outcomes, such as oxidative stress, aging, and atherosclerosis. Objectives: We evaluated whether particulate pollution modifies DNA methylation in heavily methylated sequences with high representation throughout the human genome. Methods: We measured DNA methylation of long interspersed nucleotide element (LINE)-1 and Alu repetitive elements by quantitative polymerase chain reaction-pyrosequencing of 1,097 blood samples from 718 elderly participants in the Boston area Normative Aging Study. We used covariate-adjusted mixed models to account for within-subject correlation in repeated measures. We estimated the effects on DNA methylation of ambient particulate pollutants (black carbon, particulate matter with aerodynamic diameter < 2.5 mm [PM 2.5 ], or sulfate) in multiple time windows (4 h to 7 d) before the examination. We estimated standardized regression coefficients (b) expressing the fraction of a standard deviation change in DNA methylation associated with a standard deviation increase in exposure. Measurements and Main Results: Repetitive element DNA methylation varied in association with time-related variables, such as day of the week and season. LINE-1 methylation decreased after recent exposure to higher black carbon (b 5 20.11; 95% confidence interval [CI], 20.18 to 20.04; P 5 0.002) and PM 2.5 (b 5 20.13; 95% CI, 20.19 to 20.06; P , 0.001 for the 7-d moving average). In two-pollutant models, only black carbon, a tracer of traffic particles, was significantly associated with LINE-1 methylation (b 5 20.09; 95% CI, 20.17 to 20.01; P 5 0.03). No association was found with Alu methylation (P . 0.12). Conclusions: We found decreased repeated-element methylation after exposure to traffic particles. Whether decreased methylation mediates exposure-related health effects remains to be determined.

The European Union, the UK National Institute for Health Research, the Wellcome Trust, the UK Medical Research Council, Action on Hearing Loss, the UK Biotechnology and Biological Sciences Research Council, the Oak Foundation, the Economic and Social Research Council, Helmholtz Zentrum Munchen, the German Research Center for Environmental Health, the German Federal Ministry of Education and Research, the German Center for Diabetes Research, the Munich Center for Health Sciences, the Ministry of Science and Research of the State of North Rhine-Westphalia, and the German Federal Ministry of Health.

Loss of genomic DNA methylation has been found in a variety of common human age-related diseases. Whether DNA methylation decreases over time as individuals age is unresolved. We measured DNA methylation in 1,097 blood DNA samples from 718 elderly subjects between 55-92 years of age (1-3 samples/subjects), who have been repeatedly evaluated over an 8-year time span in the Boston area Normative Aging Study. DNA methylation was measured using quantitative PCRPyrosequencing analysis in Alu and LINE-1 repetitive elements, heavily methylated sequences with high representation throughout the human genome. Age at the visit was negatively associated with Alu element methylation (β=−.12 %5mC/year, p=0.0005). A weaker association was observed with LINE-1 elements (β=−.06 %5mC/year, p=0.049). We observed a significant decrease in average Alu methylation over time, with a −0.2 %5mc change (p=0.012) compared to blood samples collected up to 8 years earlier. The longitudinal decline in Alu methylation was linear and highly correlated with time since the first measurement (β=−.089 %5mC/year, p<0.0001). In contrast, average LINE-1 methylation did not vary over time [p=0.51]. Our results demonstrate a progressive loss of DNA methylation in repetitive elements dispersed throughout the genome.

Background Epigenetic features such as DNA hypomethylation have been associated with conditions related to cardiovascular risk. We evaluated whether lower blood DNA methylation in heavily methylated repetitive sequences predicts the risk of ischemic heart disease and stroke. Methods We quantified blood DNA methylation of LINE-1 repetitive elements through PCR-pyrosequencing in 712 elderly individuals from the Boston-area Normative Aging Study. We estimated risk-factor adjusted relative risks (RRs) for ischemic heart disease and stroke at baseline (242 prevalent cases); as well as in incidence (44 new cases; median follow-up, 63 months); and subsequent mortality from ischemic heart disease (86 deaths; median follow-up, 75 months). Results Blood LINE-1 hypomethylation was associated with baseline ischemic heart disease (RR=2.1 [95% confidence interval = 1.2 to 4.0] for lowest vs. highest methylation quartile) and for stroke (2.5 [0.9 to 7.5]). Among participants free of baseline disease, individuals with methylation below the median also had higher risk of developing ischemic heart disease (4.0 [1.8 to 8.9]) or stroke (5.7 [0.8 to 39.5]). In the entire cohort, persons with methylation below the median had higher mortality from ischemic heart disease (3.3 [1.3 to 8.4]) and stroke (2.8 [0.6 to 14.3]). Total mortality was also increased (2.0 [1.2 to 3.3]). These results were confirmed in additional regression models using LINE-1 methylation as a continuous variable. Conclusions Subjects with prevalent IHD and stroke exhibited lower LINE-1 methylation. In longitudinal analyses, persons with lower LINE-1 methylation were at higher risk for incident ischemic heart disease and stroke, and for total mortality.

BackgroundAltered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined.ObjectivesWe aimed at identifying short- and long-term effects of PM exposure on DNA methylation, a major genomic mechanism of gene expression control, in workers in an electric furnace steel plant with well-characterized exposure to PM with aerodynamic diameters < 10 μm (PM10).MethodsWe measured global genomic DNA methylation content estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeated elements, and promoter DNA methylation of iNOS (inducible nitric oxide synthase), a gene suppressed by DNA methylation and induced by PM exposure in blood leukocytes. Quantitative DNA methylation analysis was performed through bisulfite PCR pyrosequencing on blood DNA obtained from 63 workers on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (postexposure). Individual PM10 exposure was between 73.4 and 1,220 μg/m3.ResultsGlobal methylation content estimated in Alu and LINE-1 repeated elements did not show changes in postexposure measures compared with baseline. PM10 exposure levels were negatively associated with methylation in both Alu [β = −0.19 %5-methylcytosine (%5mC); p = 0.04] and LINE-1 [β = −0.34 %5mC; p = 0.04], likely reflecting long-term PM10 effects. iNOS promoter DNA methylation was significantly lower in postexposure blood samples compared with baseline (difference = −0.61 %5mC; p = 0.02).ConclusionsWe observed changes in global and gene specific methylation that should be further characterized in future investigations on the effects of PM.

BackgroundPersistent organic pollutants (POPs) may influence epigenetic mechanisms; therefore, they could affect chromosomal stability and gene expression. DNA methylation, an epigenetic mechanism, has been associated with cancer initiation and progression. Greenlandic Inuit have some of the highest reported POP levels worldwide.ObjectiveOur aim in this study was to evaluate the relationship between plasma POPs concentrations and global DNA methylation (percent 5-methylcytosine) in DNA extracted from blood samples from 70 Greenlandic Inuit. Blood samples were collected under the Arctic Monitoring and Assessment Program and previously analyzed for a battery of POPs.MethodsWe used pyrosequencing to estimate global DNA methylation via Alu and LINE-1 assays of bisulfite-treated DNA. We investigated correlations between plasma POP concentrations and global DNA methylation via correlation coefficients and linear regression analyses.ResultsWe found inverse correlations between percents methylcytosine and many of the POP concentrations measured. Linear regressions, adjusting for age and cigarette smoking, showed statistically significant inverse linear relationships mainly for the Alu assay for p,p′-DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; β = −0.26), p,p′-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene; β = −0.38], β-hexachlorocyclohexane (β = −0.48), oxychlordane (β = −0.32), α-chlordane (β = −0.75), mirex (β = −0.27), sum of polychlorinated biphenyls (β = −0.56), and sum of all POPs (β = −0.48). Linear regressions for the LINE-1 assay showed β estimates of similar magnitudes to those using the Alu assay, however, none was statistically significant.ConclusionsThis is the first study to investigate environmental exposure to POPs and DNA methylation levels in a human population. Global methylation levels were inversely associated with blood plasma levels for several POPs and merit further investigation.

Global methylation measures in blood DNA vary in relation with certain host and lifestyle characteristics, including age, gender, alcohol drinking and white blood cell counts. These findings need to be considered in designing epidemiological investigations aimed at identifying associations between DNA methylation and health outcomes.