DOI: 10.5061/dryad.qfttdz0d8
title: A multi-ethnic epigenome-wide association study of leukocyte DNA methylation and blood lipids

description: We examined the association between DNA methylation in circulating
 leukocytes and blood lipids in a multi-ethnic sample of 16,265 subjects.
 We identified 148, 35, and 4 novel associations among Europeans, African
 Americans, and Hispanics, respectively, and an additional 186 novel
 associations through a trans-ethnic meta-analysis. We observed a high
 concordance in the direction of effects across racial/ethnic groups, a
 high correlation of effect sizes between high-density lipoprotein and
 triglycerides, a modest overlap of associations with epigenome-wide
 association studies of other cardio-metabolic traits, and a low overlap
 with lipid loci identified to date through genome-wide association
 studies. Thirty CpGs reached significance in at least 2 racial/ethnic
 groups including 7 that showed association with the expression of an
 annotated gene. CpGs annotated to CPT1A showed evidence of being
 influenced by triglycerides levels. DNA methylation levels of circulating
 leukocytes show robust and consistent association with blood lipid levels
 across multiple racial/ethnic groups.
description: A total of 15 cohorts (N=16,265) from the epigenetics working
 group in the Cohorts for Heart and Aging Research in Genomic Epidemiology
 (CHARGE) consortium participated in this study. These included the Old
 Order Amish (OOA), Atherosclerosis Risk in Communities (ARIC), Bogalusa
 Heart Study (BHS), Cardiovascular Health Study (CHS), Framingham Heart
 Study (FHS), Genetic Epidemiology Network of Arteriopathy (GENOA),
 Genetics of Lipid Lowering Drugs and Diet Network (GOLDN), Hypertension
 Genetic Epidemiology Network (HyperGEN), Cooperative health research in
 the Region of Augsburg (KORA), Normative Aging Study (NAS), Prospective
 Investigation of Vascularity of Uppsala Elders Study (PIVUS), Rotterdam
 Study (RS), UK Adult Twin Registry (TwinsUK), Women’s Health Initiative
 Broad Agency Announcement 23 (WHI-BA23), and the Women’s Health Initiative
 Epigenetic Mechanisms of PM-Mediated CVD (WHI-EMPC) cohorts. Four cohorts,
 BHS, CHS, WHI-BA23, and WHI-EMPC, examined more than one racial/ethnic
 group. The total number of cohorts in the European (EA), African (AA), and
 Hispanic (HA) study populations is 12 (N=11,114), 9 (N=4,452), and 2
 (N=699), respectively.  High-density-lipoprotein (HDL,
 mg per dl) and triglycerides (TG, mg per dl) were directly measured in
 blood samples taken from participants after at least an 8 hour fast.
 Low-density-lipoprotein (LDL, mg per dl) was inferred using the
 Friedewald’s formula in all cohorts except for GOLDN, HyperGEN, and KORA
 where LDL was measured directly.  We did not infer LDL in subjects with
 triglycerides &gt; 400 mg per dL and we excluded lipid measure from
 subjects who did not fast for at least 8 hours. We also excluded outliers
 as defined by &gt;5 standard deviations from the mean of a blood lipid
 in each cohort. To reduce skewness, HDL and triglycerides were natural
 log-transformed. DNA methylation was produced by
 investigators from each cohort independently. Levels were measured from
 peripheral blood leukocytes isolated from whole blood in all studies
 except GOLDN where only CD4 + T cells were examined. The EZ DNA
 Methylation Gold Kit (Zymo Research, Orange CA) was used for bisulfite
 conversion. The Illumina® Infinium HumanMethylation450 BeadChip and the
 Illumina BeadXpress reader were used to perform the methylation assays.
 Either the SWAN method in the <i>minfi</i> R package, the Beta
 Mixture Quantile method (BMIQ), the DASEN method in the wateRmelon R
 package, or the GenomeStudio® Methylation Module was used for
 pre-processing and normalization of the data in each cohort. For each CpG
 site, a beta-value was calculated representing the percent methylation at
 that CpG site.   Any single value with a detection p-value &gt; 0.01
 was set to missing. In each cohort, we excluded probes with a detection
 p-value &gt; 0.01 in greater than 5% of samples. In addition, we
 excluded samples with a detection p-value &gt; 0.01 in greater than 5%
 of probes. To avoid spurious signals in DNA methylation data, we excluded
 29,233 CpGs that co-hybridize to alternate genomic sequences (highly
 homologous to the intended targets)
 Epigenome-wide association analyses (EWAS) were
 performed in each cohort stratified by racial/ethnic group (European,
 African, and Hispanic). For Model 1, a linear mixed effects model was used
 to study the association between the DNA methylation level of a CpG
 (dependent variable) and each of the lipid measures (independent variable;
 HDL, LDL, or TG), adjusting for age, sex (reference = male), smoking
 variable (never/previous/current, reference = never), lipid medication
 (Yes or No, reference = No), the top four principal components from
 genotypes (SNPs), and the proportion of 5 types of cells estimated with
 the Houseman method (CD8 T lymphocytes, CD4 T lymphocytes, natural killer
 cells, B cells, and monocytes). We added random effects for plate, row,
 and column. We also included family structure as a random effect among
 family-based studies. For Model 2, we further adjusted for BMI in addition
 to Model 1 covariates. We also ran Model 3 and Model 4 which were
 analogous to Model 1 and 2, respectively, in the subset of individuals not
 taking lipid lowering medication. We
 performed meta-analyses of all the participating cohorts (N=16,265) and
 also stratified by racial/ethnic group: European Americans (12 cohorts,
 N=11,114), African Americans (7 cohorts, N=4,452), and Hispanics (2
 cohorts, N=699). These meta-analyses were performed for each of the 4
 models, respectively. We used a random effects meta-analysis implemented
 in METASOFT <sup>33</sup> to take into account the
 heterogeneity of the effect sizes of different cohorts while achieving a
 higher or comparable statistical power compared to fixed effects
 meta-analysis. To avoid spurious findings from population substructure, we
 applied genomic control. We investigated the
 association between either imputed or genotyped SNPs located within 25kb
 upstream or downstream of each CpG and DNA methylation levels to identify
 cis-acting methylation quantitative trait loci (mQTL). For imputed SNP
 data, we restricted the mQTL analysis to SNPs with a good quality
 imputation (IMPUTE info&gt;=0.4 or MACH r^2&gt;=0.3). Subjects
 taking lipid lowering medications were excluded from this analysis.
 Beta-values of DNA methylation levels were inverse-normal transformed and
 regressed on age, sex, smoking (current/former/never), BMI, at least 4 SNP
 PCs, cell proportions (WBC count and/or estimated WBC proportions
 (granulocytes as a reference)), and technical covariates (plate, row, and
 column as random effects) (two-sided test). Family information was also
 included as a random effect if a cohort was a family-based study. We then
 regressed the residuals on each SNP of interests stratified by
 racial/ethnic group.  Five out of the 15 cohorts provided genetic data for
 this analysis including ARIC (N<sub>AA</sub>=1,717), GOLDN
 (N<sub>EA</sub>=713), KORA (N<sub>EA</sub>=1,379),
 WHI-BA23 (N<sub>EA</sub>=790, N<sub>AA</sub>=540,
 and N<sub>HISP</sub>=324), and WHI-EMPC
 (N<sub>EA</sub>=494, N<sub>AA</sub>=424, and
 N<sub>HISP</sub>=221). We restricted our analysis to SNPs
 located within 25 kilobases up- or downstream of the CpGs with a minor
 allele frequency (MAF) &gt; 0.01 in each cohort and implemented a
 fixed effects meta-analysis within each of the three racial/ethnic
 groups.  
description: Two sets of results files are included.  Set
 1. Txt files provide the results of the epigenomide association study for
 each of 3 lipid traits, 3 racial/ethnic subgroups followed by a
 trans-ethnic meta-analysis, and 4 models.   Files are labelled as
 lipid.model.racial/ethnic_group.GC. The three lipid trais are HDL:
 high-density lipoprotein; LDL: low-density lipoprotein; TG:
 triglycerides.  The three racial/ethnic groups are EA: European ancestry;
 AA: African ancestry; HA: Hispanic ancestry; ALL_wGOLDN: trans-ethnic. 
 The suffix "GC" indicates that a genomic-control correction has
 been applied.   Each file includes results for fixed effect meta-analysis
 as well as random effect meta-analysis where more than 1 cohort is
 available for analysis (p-value, beta, standard error).  Each file also
 includes heterogeneity statitics where more than 1 cohort is
 meta-analyzed.  Lastly, P values for each study included in the
 meta-analysis are provided.  Please note these P values are followed by M
 values labelled as NA. Set 2.  CSV files provide the
 results of the methylation QTL association meta-analyses for 30 CpGs found
 to be associated with one of three lipid traits in more than one
 racial/ethnic group.  SNPs tested are those within 25kb upstream or
 downstream of each CpG.  Both fixed efffect and random effect
 meta-analysis results are provided as well as heterogeneity results when
 more than 1 study contributes to the meta-analysis.  Each file is labelled
 by CpG followed by the racial/ethnic group analysed: EA: European
 ancestry; AA: African ancestry; HA: Hispanic ancestry.