Whole Blood DNA Methylation Signatures of Diet Are Associated With Cardiovascular Disease Risk Factors and All-Cause Mortality

Abstract

Background: DNA methylation patterns associated with habitual diet have not been well studied.

Methods: Diet quality was characterized using a Mediterranean-style diet score and the Alternative Healthy Eating Index score. We conducted ethnicity-specific and trans-ethnic epigenome-wide association analyses for diet quality and leukocyte-derived DNA methylation at over 400 000 CpGs (cytosine-guanine dinucleotides) in 5 population-based cohorts including 6662 European ancestry, 2702 African ancestry, and 360 Hispanic ancestry participants. For diet-associated CpGs identified in epigenome-wide analyses, we conducted Mendelian randomization (MR) analysis to examine their relations to cardiovascular disease risk factors and examined their longitudinal associations with all-cause mortality.

Results: We identified 30 CpGs associated with either Mediterranean-style diet score or Alternative Healthy Eating Index, or both, in European ancestry participants. Among these CpGs, 12 CpGs were significantly associated with all-cause mortality (Bonferroni corrected P<1.6×10^-3). Hypermethylation of cg18181703 (SOCS3) was associated with higher scores of both Mediterranean-style diet score and Alternative Healthy Eating Index and lower risk for all-cause mortality (P=5.7×10^-15). Ten additional diet-associated CpGs were nominally associated with all-cause mortality (P<0.05). MR analysis revealed 8 putatively causal associations for 6 CpGs with 4 cardiovascular disease risk factors (body mass index, triglycerides, high-density lipoprotein cholesterol concentrations, and type 2 diabetes mellitus; Bonferroni corrected MR P<4.5×10^-4). For example, hypermethylation of cg11250194 (FADS2) was associated with lower triglyceride concentrations (MR, P=1.5×10^-14).and hypermethylation of cg02079413 (SNORA54; NAP1L4) was associated with body mass index (corrected MR, P=1×10^-6).

Conclusions: Habitual diet quality was associated with differential peripheral leukocyte DNA methylation levels of 30 CpGs, most of which were also associated with multiple health outcomes, in European ancestry individuals. These findings demonstrate that integrative genomic analysis of dietary information may reveal molecular targets for disease prevention and treatment.

Keywords: DNA methylation; cardiovascular disease; diet; epigenome; triglycerides.

Figure 1.

Study design flow chart. AA: African ancestry. EA: European ancestry. HA: Hispanic ancestry. CpGs: cytosine-guanine dinucleotides (DNA methylation sites). AHEI: Alternative Healthy Eating Index. MDS: Mediterranean-style diet score. FDR: false discovery rate. GWAS: genome-wide association study. cis-meQTLs: cis methylation quantitative loci. EWAS: epigenome-wide association study. GEO: Gene Expression Omnibus. GTEx: Genotype-Tissue Expression database. Discovery cohort: Framingham Heart Study (FHS). Replication cohorts: Atherosclerosis Risk in Communities (ARIC) Study, Genetics of Lipid Lowering Drugs and Diet Network (GOLDN), Multi-Ethnic Study of Atherosclerosis (MESA), and Rotterdam Study (RS). Cohorts for all-cause mortality includes: ARIC, FHS, ESTHER study, InChianti Study, Lothian Birth Cohort (LBC) Study 1921 and 1936, Cardiovascular Health Study (CHS), KORA F4 Study, Normative Aging Study (NAS), and Women’s Health Initiative (WHI).

Figure 2.

Effect of additional adjustment for lifestyle factors (smoking and physical activity) and BMI in European ancestry participants. A and B are 14 CpGs identified using the Mediterranean-style diet score (MDS). C and D are 24 CpGs identified using the Alternative Healthy Eating Index (AHEI). CpGs highlighted in red-colored rectangle are those identified in the two-step analysis alone and CpGs highlighted in green-colored rectangle are those identified in both one-step and two-step analyses. Orange colored dash line represents -log10 of 0.05 and green colored dash line represents -log10 of Bonferroni corrected p-value threshold, i.e., 0.05/14 for MDS and 0.05/24 for AHEI. Four CpGs (cg05575921, cg06126421, cg12075928, and cg25189904) in MDS analysis became non-significant after Bonferroni correction in models with adjustment for lifestyle factors and BMI, whereas all 24 CpGs in AHEI analysis remained significant.

Figure 3.

Mendelian Randomization (MR) analyses for associations between cg11250194 (FADS2) and triglycerides (TG), between cg02079413 (SNORA54; NAP1L4) and BMI, and between cg26470501 (BCL3) and BMI. IVW: inverse variance weighted. Symbols and bars represent effects size and standard errors of instruments variables (cis-meQTL variants) used in MR analysis. Solid line is for MR-IVW analysis and dashed line is for MR-Egger analysis. No horizontal pleiotropy effect was detected for all MR analyses.

Figure 4.

Meta-analysis of association between 30 diet-associated CpGs and all-cause mortality in 10 cohorts of European ancestry participants (n≈10,000). A positive sign for diet indicates that a higher dietary scores (MDS or AHEI, or both) were associated with DNA hypermethylation, whereas, a hazard ratio of over 1.0 indicates that DNA hypermethylation was associated with increased all-cause mortality. Models were adjusted for baseline covariates including sex, age, smoking status, physical activity level, alcohol intake, BMI, and prevalence disease status of hypertension, type 2 diabetes, cardiovascular disease, and cancer. Estimated leukocyte counts, technical variables, and kinship (for related study samples) were also considered. Hazard ratios and 95% confidence interval were estimated using Cox proportional hazard models and meta-analyzed using random effect models. X-axis is in logarithmic scale.