Stella Aslibekyan

Epigenome-Wide Association Study of Fasting Blood Lipids in the Genetics of Lipid-Lowering Drugs and Diet Network Study

Background— Genetic research regarding blood lipids has largely focused on DNA sequence variation; few studies have explored epigenetic effects. Genome-wide surveys of DNA methylation may uncover epigenetic factors influencing lipid metabolism. Methods and Results— To identify whether differential methylation of cytosine-(phosphate)-guanine dinucleotides (CpGs) correlated with lipid phenotypes, we isolated DNA from CD4+ T cells and quantified the proportion of sample methylation at >450 000 CpGs by using the Illumina Infinium HumanMethylation450 Beadchip in 991 participants of the Genetics of Lipid Lowering Drugs and Diet Network. We modeled the percentage of methylation at individual CpGs as a function of fasting very-low-density lipoprotein cholesterol and triglycerides (TGs) by using mixed linear regression adjusted for age, sex, study site, cell purity, and family structure. Four CpGs (cg00574958, cg17058475, cg01082498, and cg09737197) in intron 1 of carnitine palmitoyltransferase 1A (CPT1A) were strongly associated with very-low low-density lipoprotein cholesterol (P=1.8×10–21 to 1.6×10–8) and TG (P=1.6×10–26 to 1.5×10–9). Array findings were validated by bisulfite sequencing. We performed quantitative polymerase chain reaction experiments demonstrating that methylation of the top CpG (cg00574958) was correlated with CPT1A expression. The association of cg00574958 with TG and CPT1A expression were replicated in the Framingham Heart Study (P=4.1×10–14 and 3.1×10–13, respectively). DNA methylation at CPT1A cg00574958 explained 11.6% and 5.5% of the variation in TG in the discovery and replication cohorts, respectively. Conclusions— This genome-wide epigenomic study identified CPT1A methylation as strongly and robustly associated with fasting very-low low-density lipoprotein cholesterol and TG. Identifying novel epigenetic contributions to lipid traits may inform future efforts to identify new treatment targets and biomarkers of disease risk.

Epigenetic Signatures of Cigarette Smoking

Background—DNA methylation leaves a long-term signature of smoking exposure and is one potential mechanism by which tobacco exposure predisposes to adverse health outcomes, such as cancers, osteoporosis, lung, and cardiovascular disorders. Methods and Results—To comprehensively determine the association between cigarette smoking and DNA methylation, we conducted a meta-analysis of genome-wide DNA methylation assessed using the Illumina BeadChip 450K array on 15 907 blood-derived DNA samples from participants in 16 cohorts (including 2433 current, 6518 former, and 6956 never smokers). Comparing current versus never smokers, 2623 cytosine–phosphate–guanine sites (CpGs), annotated to 1405 genes, were statistically significantly differentially methylated at Bonferroni threshold of P<1×10−7 (18 760 CpGs at false discovery rate <0.05). Genes annotated to these CpGs were enriched for associations with several smoking-related traits in genome-wide studies including pulmonary function, cancers, inflammatory diseases, and heart disease. Comparing former versus never smokers, 185 of the CpGs that differed between current and never smokers were significant P<1×10−7 (2623 CpGs at false discovery rate <0.05), indicating a pattern of persistent altered methylation, with attenuation, after smoking cessation. Transcriptomic integration identified effects on gene expression at many differentially methylated CpGs. Conclusions—Cigarette smoking has a broad impact on genome-wide methylation that, at many loci, persists many years after smoking cessation. Many of the differentially methylated genes were novel genes with respect to biological effects of smoking and might represent therapeutic targets for prevention or treatment of tobacco-related diseases. Methylation at these sites could also serve as sensitive and stable biomarkers of lifetime exposure to tobacco smoke.

Epigenome-wide Association Study (EWAS) of BMI, BMI Change, and Waist Circumference in African American Adults Identifies Multiple Replicated Loci

Obesity is an important component of the pathophysiology of chronic diseases. Identifying epigenetic modifications associated with elevated adiposity, including DNA methylation variation, may point to genomic pathways that are dysregulated in numerous conditions. The Illumina 450K Bead Chip array was used to assay DNA methylation in leukocyte DNA obtained from 2097 African American adults in the Atherosclerosis Risk in Communities (ARIC) study. Mixed-effects regression models were used to test the association of methylation beta value with concurrent body mass index (BMI) and waist circumference (WC), and BMI change, adjusting for batch effects and potential confounders. Replication using whole-blood DNA from 2377 White adults in the Framingham Heart Study and CD4+ T cell DNA from 991 Whites in the Genetics of Lipid Lowering Drugs and Diet Network Study was followed by testing using adipose tissue DNA from 648 women in the Multiple Tissue Human Expression Resource cohort. Seventy-six BMI-related probes, 164 WC-related probes and 8 BMI change-related probes passed the threshold for significance in ARIC (P < 1 × 10(-7); Bonferroni), including probes in the recently reported HIF3A, CPT1A and ABCG1 regions. Replication using blood DNA was achieved for 37 BMI probes and 1 additional WC probe. Sixteen of these also replicated in adipose tissue, including 15 novel methylation findings near genes involved in lipid metabolism, immune response/cytokine signaling and other diverse pathways, including LGALS3BP, KDM2B, PBX1 and BBS2, among others. Adiposity traits are associated with DNA methylation at numerous CpG sites that replicate across studies despite variation in tissue type, ethnicity and analytic approaches.

ω-3 Polyunsaturated Fatty Acid Biomarkers and Coronary Heart Disease: Pooling Project of 19 Cohort Studies.

IMPORTANCE The role of ω-3 polyunsaturated fatty acids for primary prevention of coronary heart disease (CHD) remains controversial. Most prior longitudinal studies evaluated self-reported consumption rather than biomarkers. OBJECTIVE To evaluate biomarkers of seafood-derived eicosapentaenoic acid (EPA; 20:5ω-3), docosapentaenoic acid (DPA; 22:5ω-3), and docosahexaenoic acid (DHA; 22:6ω-3) and plant-derived α-linolenic acid (ALA; 18:3ω-3) for incident CHD. DATA SOURCES A global consortium of 19 studies identified by November 2014. STUDY SELECTION Available prospective (cohort, nested case-control) or retrospective studies with circulating or tissue ω-3 biomarkers and ascertained CHD. DATA EXTRACTION AND SYNTHESIS Each study conducted standardized, individual-level analysis using harmonized models, exposures, outcomes, and covariates. Findings were centrally pooled using random-effects meta-analysis. Heterogeneity was examined by age, sex, race, diabetes, statins, aspirin, ω-6 levels, and FADS desaturase genes. MAIN OUTCOMES AND MEASURES Incident total CHD, fatal CHD, and nonfatal myocardial infarction (MI). RESULTS The 19 studies comprised 16 countries, 45 637 unique individuals, and 7973 total CHD, 2781 fatal CHD, and 7157 nonfatal MI events, with ω-3 measures in total plasma, phospholipids, cholesterol esters, and adipose tissue. Median age at baseline was 59 years (range, 18-97 years), and 28 660 (62.8%) were male. In continuous (per 1-SD increase) multivariable-adjusted analyses, the ω-3 biomarkers ALA, DPA, and DHA were associated with a lower risk of fatal CHD, with relative risks (RRs) of 0.91 (95% CI, 0.84-0.98) for ALA, 0.90 (95% CI, 0.85-0.96) for DPA, and 0.90 (95% CI, 0.84-0.96) for DHA. Although DPA was associated with a lower risk of total CHD (RR, 0.94; 95% CI, 0.90-0.99), ALA (RR, 1.00; 95% CI, 0.95-1.05), EPA (RR, 0.94; 95% CI, 0.87-1.02), and DHA (RR, 0.95; 95% CI, 0.91-1.00) were not. Significant associations with nonfatal MI were not evident. Associations appeared generally stronger in phospholipids and total plasma. Restricted cubic splines did not identify evidence of nonlinearity in dose responses. CONCLUSIONS AND RELEVANCE On the basis of available studies of free-living populations globally, biomarker concentrations of seafood and plant-derived ω-3 fatty acids are associated with a modestly lower incidence of fatal CHD.

Epigenome-Wide Association Study of Fasting Measures of Glucose, Insulin, and HOMA-IR in the Genetics of Lipid Lowering Drugs and Diet Network Study

Known genetic susceptibility loci for type 2 diabetes (T2D) explain only a small proportion of heritable T2D risk. We hypothesize that DNA methylation patterns may contribute to variation in diabetes-related risk factors, and this epigenetic variation across the genome can contribute to the missing heritability in T2D and related metabolic traits. We conducted an epigenome-wide association study for fasting glucose, insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) among 837 nondiabetic participants in the Genetics of Lipid Lowering Drugs and Diet Network study, divided into discovery (N = 544) and replication (N = 293) stages. Cytosine guanine dinucleotide (CpG) methylation at ∼470,000 CpG sites was assayed in CD4+ T cells using the Illumina Infinium HumanMethylation 450 Beadchip. We fit a mixed model with the methylation status of each CpG as the dependent variable, adjusting for age, sex, study site, and T-cell purity as fixed-effects and family structure as a random-effect. A Bonferroni corrected P value of 1.1 × 10−7 was considered significant in the discovery stage. Significant associations were tested in the replication stage using identical models. Methylation of a CpG site in ABCG1 on chromosome 21 was significantly associated with insulin (P = 1.83 × 10−7) and HOMA-IR (P = 1.60 × 10−9). Another site in the same gene was significant for HOMA-IR and of borderline significance for insulin (P = 1.29 × 10−7 and P = 3.36 × 10−6, respectively). Associations with the top two signals replicated for insulin and HOMA-IR (P = 5.75 × 10−3 and P = 3.35 × 10−2, respectively). Our findings suggest that methylation of a CpG site within ABCG1 is associated with fasting insulin and merits further evaluation as a novel disease risk marker.

SNPs located at CpG sites modulate genome-epigenome interaction

DNA methylation is an important molecular-level phenotype that links genotypes and complex disease traits. Previous studies have found local correlation between genetic variants and DNA methylation levels (cis-meQTLs). However, general mechanisms underlying cis-meQTLs are unclear. We conducted a cis-meQTL analysis of the Genetics of Lipid Lowering Drugs and Diet Network data (n = 593). We found that over 80% of genetic variants at CpG sites (meSNPs) are meQTL loci (P-value < 10−9), and meSNPs account for over two thirds of the strongest meQTL signals (P-value < 10−200). Beyond direct effects on the methylation of the meSNP site, the CpG-disrupting allele of meSNPs were associated with lowered methylation of CpG sites located within 45 bp. The effect of meSNPs extends to as far as 10 kb and can contribute to the observed meQTL signals in the surrounding region, likely through correlated methylation patterns and linkage disequilibrium. Therefore, meSNPs are behind a large portion of observed meQTL signals and play a crucial role in the biological process linking genetic variation to epigenetic changes.

Epigenome-wide study identifies novel methylation loci associated with body mass index and waist circumference

To conduct an epigenome‐wide analysis of DNA methylation and obesity traits.

DNA methylation signatures of chronic low-grade inflammation are associated with complex diseases

BackgroundChronic low-grade inflammation reflects a subclinical immune response implicated in the pathogenesis of complex diseases. Identifying genetic loci where DNA methylation is associated with chronic low-grade inflammation may reveal novel pathways or therapeutic targets for inflammation.ResultsWe performed a meta-analysis of epigenome-wide association studies (EWAS) of serum C-reactive protein (CRP), which is a sensitive marker of low-grade inflammation, in a large European population (n = 8863) and trans-ethnic replication in African Americans (n = 4111). We found differential methylation at 218 CpG sites to be associated with CRP (P < 1.15 × 10–7) in the discovery panel of European ancestry and replicated (P < 2.29 × 10–4) 58 CpG sites (45 unique loci) among African Americans. To further characterize the molecular and clinical relevance of the findings, we examined the association with gene expression, genetic sequence variants, and clinical outcomes. DNA methylation at nine (16%) CpG sites was associated with whole blood gene expression in cis (P < 8.47 × 10–5), ten (17%) CpG sites were associated with a nearby genetic variant (P < 2.50 × 10–3), and 51 (88%) were also associated with at least one related cardiometabolic entity (P < 9.58 × 10–5). An additive weighted score of replicated CpG sites accounted for up to 6% inter-individual variation (R2) of age-adjusted and sex-adjusted CRP, independent of known CRP-related genetic variants.ConclusionWe have completed an EWAS of chronic low-grade inflammation and identified many novel genetic loci underlying inflammation that may serve as targets for the development of novel therapeutic interventions for inflammation.

A genome-wide association study of inflammatory biomarker changes in response to fenofibrate treatment in the Genetics of Lipid Lowering Drug and Diet Network.

Objective Despite the evidence in support of the anti-inflammatory and triglyceride-lowering effects of fenofibrate, little is known about genetic determinants of the observed heterogeneity in treatment response. This study provides the first genome-wide examination of fenofibrate effects on systemic inflammation. Methods Biomarkers of inflammation were measured in participants of the Genetics of Lipid Lowering Drugs and Diet Network (n=1092) before and after a 3-week daily treatment with 160 mg of fenofibrate. Two inflammatory patterns [high-sensitivity C-reactive protein-interleukin-6 and monocyte chemoattractant protein-1-tumor necrosis factor (MCP1-TNF-&agr;)] were derived using principal component analysis. Associations between single nucleotide polymorphisms on the Affymetrix 6.0 chip and phenotypes were assessed using mixed linear models, adjusted for age, sex, study center, and ancestry as fixed effects and pedigree as a random effect. Results Before fenofibrate treatment, the strongest evidence for association was observed for polymorphisms near or within the IL2RA gene with the high-sensitivity C-reactive protein-interleukin-6 (IL6) pattern (rs7911500, P=5×10−9 and rs12722605, P=5×10−8). Associations of the MCP1-TNF-&agr; pattern with loci in several biologically plausible genes [CYP4F8 (rs3764563), APBB1IP (rs1775246), COL13A1 (rs2683572), and COMMD10 (rs1396485)] approached genome-wide significance (P=3×10−7, 5×10−7, 6×10−7, and 7×10−7, respectively) before fenofibrate treatment. After fenofibrate treatment, the rs12722605 locus in IL2RA was also associated with the MCP1-TNF-&agr; pattern (P=3×10−7). The analyses of individual biomarker response to fenofibrate did not yield genome-wide significant results, but the rs6517147 locus near the immunologically relevant IFNAR2 gene was suggestively associated with IL6 (P=7×10−7). Conclusion We have identified several novel biologically relevant loci associated with systemic inflammation before and after fenofibrate treatment.

Association of body mass index with DNA methylation and gene expression in blood cells and relations to cardiometabolic disease: a Mendelian randomization approach

Background The link between DNA methylation, obesity, and adiposity-related diseases in the general population remains uncertain. Methods and Findings We conducted an association study of body mass index (BMI) and differential methylation for over 400,000 CpGs assayed by microarray in whole-blood-derived DNA from 3,743 participants in the Framingham Heart Study and the Lothian Birth Cohorts, with independent replication in three external cohorts of 4,055 participants. We examined variations in whole blood gene expression and conducted Mendelian randomization analyses to investigate the functional and clinical relevance of the findings. We identified novel and previously reported BMI-related differential methylation at 83 CpGs that replicated across cohorts; BMI-related differential methylation was associated with concurrent changes in the expression of genes in lipid metabolism pathways. Genetic instrumental variable analysis of alterations in methylation at one of the 83 replicated CpGs, cg11024682 (intronic to sterol regulatory element binding transcription factor 1 [SREBF1]), demonstrated links to BMI, adiposity-related traits, and coronary artery disease. Independent genetic instruments for expression of SREBF1 supported the findings linking methylation to adiposity and cardiometabolic disease. Methylation at a substantial proportion (16 of 83) of the identified loci was found to be secondary to differences in BMI. However, the cross-sectional nature of the data limits definitive causal determination. Conclusions We present robust associations of BMI with differential DNA methylation at numerous loci in blood cells. BMI-related DNA methylation and gene expression provide mechanistic insights into the relationship between DNA methylation, obesity, and adiposity-related diseases.