A 6-CpG Validated Methylation Risk Score Model for Metabolic Syndrome: The HyperGEN and GOLDN Studies

There has been great interest in genetic risk prediction using risk scores in recent years, however, the utility of scores developed in European populations and later applied to non-European populations has not been successful. The goal of this study was to create a methylation risk score (MRS) for metabolic syndrome (MetS), demonstrating the utility of MRS across race groups using cross-sectional data from the Hypertension Genetic Epidemiology Network (HyperGEN, N = 614 African Americans (AA)) and the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN, N = 995 European Americans (EA)). To demonstrate this, we first selected cytosine-guanine dinucleotides (CpG) sites measured on Illumina Methyl450 arrays previously reported to be significantly associated with MetS and/or component conditions in more than one race/ethnic group (CPT1A cg00574958, PHOSPHO1 cg02650017, ABCG1 cg06500161, SREBF1 cg11024682, SOCS3 cg18181703, TXNIP cg19693031). Second, we calculated the parameter estimates for the 6 CpGs in the HyperGEN data (AA) and used the beta estimates as weights to construct a MRS in HyperGEN (AA), which was validated in GOLDN (EA). We performed association analyses using logistic mixed models to test the association between the MRS and MetS, adjusting for covariates. Results showed the MRS was significantly associated with MetS in both populations. In summary, a MRS for MetS was a strong predictor for the condition across two race groups, suggesting MRS may be useful to examine metabolic disease risk or related complications across race/ethnic groups

Epigenome-wide association study of triglyceride postprandial responses to a high-fat dietary challenge

Postprandial lipemia (PPL), the increased plasma TG concentration after consuming a high-fat meal, is an independent risk factor for CVD. Individual responses to a meal high in fat vary greatly, depending on genetic and lifestyle factors. However, only a few loci have been associated with TG-PPL response. Heritable epigenomic changes may be significant contributors to the unexplained inter-individual PPL variability. We conducted an epigenome-wide association study on 979 subjects with DNA methylation measured from CD4(+) T cells, who were challenged with a high-fat meal as a part of the Genetics of Lipid Lowering Drugs and Diet Network study. Eight methylation sites encompassing five genes, LPP, CPT1A, APOA5, SREBF1, and ABCG1, were significantly associated with PPL response at an epigenome-wide level (P < 1.1 × 10(−7)), but no methylation site reached epigenome-wide significance after adjusting for baseline TG levels. Higher methylation at LPP, APOA5, SREBF1, and ABCG1, and lower methylation at CPT1A methylation were correlated with an increased TG-PPL response. These PPL-associated methylation sites, also correlated with fasting TG, account for a substantially greater amount of phenotypic variance (14.9%) in PPL and fasting TG (16.3%) when compared with the genetic contribution of loci identified by our previous genome-wide association study (4.5%). In summary, the epigenome is a large contributor to the variation in PPL, and this has the potential to be used to modulate PPL and reduce CVD

Genetic Contributors of Incident Stroke in 10,700 African Americans with Hypertension: A Meta-Analysis from the Genetics of Hypertension Associated Treatments and Reasons for Geographic and Racial Differences in Stroke Studies

Background: African Americans (AAs) suffer a higher stroke burden due to hypertension. Identifying genetic contributors to stroke among AAs with hypertension is critical to understanding the genetic basis of the disease, as well as detecting at-risk individuals. Methods: In a population comprising over 10,700 AAs treated for hypertension from the Genetics of Hypertension Associated Treatments (GenHAT) and Reasons for Geographic and Racial Differences in Stroke (REGARDS) studies, we performed an inverse variance-weighted meta-analysis of incident stroke. Additionally, we tested the predictive accuracy of a polygenic risk score (PRS) derived from a European ancestral population in both GenHAT and REGARDS AAs aiming to evaluate cross-ethnic performance. Results: We identified 10 statistically significant (p \u3c 5.00E-08) and 90 additional suggestive (p \u3c 1.00E-06) variants associated with incident stroke in the meta-analysis. Six of the top 10 variants were located in an intergenic region on chromosome 18 (LINC01443-LOC644669). Additional variants of interest were located in or near the COL12A1, SNTG1, PCDH7, TMTC1, and NTM genes. Replication was conducted in the Warfarin Pharmacogenomics Cohort (WPC), and while none of the variants were directly validated, seven intronic variants of NTM proximal to our target variants, had a p-value \u3c5.00E-04 in the WPC. The inclusion of the PRS did not improve the prediction accuracy compared to a reference model adjusting for age, sex, and genetic ancestry in either study and had lower predictive accuracy compared to models accounting for established stroke risk factors. These results demonstrate the necessity for PRS derivation in AAs, particularly for diseases that affect AAs disproportionately. Conclusion: This study highlights biologically plausible genetic determinants for incident stroke in hypertensive AAs. Ultimately, a better understanding of genetic risk factors for stroke in AAs may give new insight into stroke burden and potential clinical tools for those among the highest at risk

Metabolic and Inflammatory Biomarkers are Associated with Epigenetic Aging Acceleration Estimates in the GOLDN Study

Background: Recently, epigenetic age acceleration-or older epigenetic age in comparison to chronological age-has been robustly associated with mortality and various morbidities. However, accelerated epigenetic aging has not been widely investigated in relation to inflammatory or metabolic markers, including postprandial lipids. Methods: We estimated measures of epigenetic age acceleration in 830 Caucasian participants from the Genetics Of Lipid Lowering Drugs and diet Network (GOLDN) considering two epigenetic age calculations based on differing sets of 5′-Cytosine-phosphate-guanine-3′ genomic site, derived from the Horvath and Hannum DNA methylation age calculators, respectively. GOLDN participants underwent a standardized high-fat meal challenge after fasting for at least 8 h followed by timed blood draws, the last being 6 h postmeal. We used adjusted linear mixed models to examine the association of the epigenetic age acceleration estimate with fasting and postprandial (0- and 6-h time points) low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglyceride (TG) levels as well as five fasting inflammatory markers plus adiponectin. Results: Both DNA methylation age estimates were highly correlated with chronological age (r \u3e 0.90). We found that the Horvath and Hannum measures of epigenetic age acceleration were moderately correlated (r = 0.50). The regression models revealed that the Horvath age acceleration measure exhibited marginal associations with increased postprandial HDL (p = 0.05), increased postprandial total cholesterol (p = 0.06), and decreased soluble interleukin 2 receptor subunit alpha (IL2sRα, p = 0.02). The Hannum measure of epigenetic age acceleration was inversely associated with fasting HDL (p = 0.02) and positively associated with postprandial TG (p = 0.02), interleukin-6 (IL6, p = 0.007), C-reactive protein (C-reactive protein, p = 0.0001), and tumor necrosis factor alpha (TNFα, p = 0.0001). Overall, the observed effect sizes were small and the association of the Hannum residual with inflammatory markers was attenuated by adjustment for estimated T cell type percentages. Conclusions: Our study demonstrates that epigenetic age acceleration in blood relates to inflammatory biomarkers and certain lipid classes in Caucasian individuals of the GOLDN study. Future studies should consider epigenetic age acceleration in other tissues and extend the analysis to other ethnic groups

Epigenomics and metabolomics reveal the mechanism of the APOA2-saturated fat intake interaction affecting obesity

Background: The putative functional variant -265T\u3eC (rs5082) within the APOA2 promoter has shown consistent interactions with saturated fatty acid (SFA) intake to influence the risk of obesity. Objective: The aim of this study was to implement an integrative approach to characterize the molecular basis of this interaction. Design: We conducted an epigenome-wide scan on 80 participants carrying either the rs5082 CC or TT genotypes and consuming either a low-SFA (\u3c22 g/d) or high-SFA diet (≥22 g/d), matched for age, sex, BMI, and diabetes status in the Boston Puerto Rican Health Study (BPRHS). We then validated the findings in selected participants in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) Study (n = 379) and the Framingham Heart Study (FHS) (n = 243). Transcription and metabolomics analyses were conducted to determine the relation between epigenetic status, APOA2 mRNA expression, and blood metabolites. Results: In the BPRHS, we identified methylation site cg04436964 as exhibiting significant differences between CC and TT participants consuming a high-SFA diet, but not among those consuming low-SFA. Similar results were observed in the GOLDN Study and the FHS. Additionally, in the FHS, cg04436964 methylation was negatively correlated with APOA2 expression in the blood of participants consuming a high-SFA diet. Furthermore, when consuming a high-SFA diet, CC carriers had lower APOA2 expression than those with the TT genotype. Lastly, metabolomic analysis identified 4 pathways as overrepresented by metabolite differences between CC and TT genotypes with high-SFA intake, including tryptophan and branched-chain amino acid (BCAA) pathways. Interestingly, these pathways were linked to rs5082-specific cg04436964 methylation differences in high-SFA consumers. Conclusions: The epigenetic status of the APOA2 regulatory region is associated with SFA intake and APOA2 -265T\u3eC genotype, promoting an APOA2 expression difference between APOA2 genotypes on a high-SFA diet, and modulating BCAA and tryptophan metabolic pathways. These findings identify potential mechanisms by which this highly reproducible gene-diet interaction influences obesity risk, and contribute new insights to ongoing investigations of the relation between SFA and human health. This study was registered at clinicaltrials.gov as NCT03452787

Epigenomics and Metabolomics Reveal the Mechanism of the \u3cem\u3eAPOA2\u3c/em\u3e-Saturated Fat Intake Interaction Affecting Obesity

Background: The putative functional variant −265T \u3e C (rs5082) within the APOA2 promoter has shown consistent interactions with saturated fatty acid (SFA) intake to influence the risk of obesity. Objective: The aim of this study was to implement an integrative approach to characterize the molecular basis of this interaction. Design: We conducted an epigenome-wide scan on 80 participants carrying either the rs5082 CC or TT genotypes and consuming either a low-SFA (\u3c 22 g/d) or high-SFA diet (≥ 22 g/d), matched for age, sex, BMI, and diabetes status in the Boston Puerto Rican Health Study (BPRHS). We then validated the findings in selected participants in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) Study (n = 379) and the Framingham Heart Study (FHS) (n = 243). Transcription and metabolomics analyses were conducted to determine the relation between epigenetic status, APOA2 mRNA expression, and blood metabolites. Results: In the BPRHS, we identified methylation site cg04436964 as exhibiting significant differences between CC and TT participants consuming a high-SFA diet, but not among those consuming low-SFA. Similar results were observed in the GOLDN Study and the FHS. Additionally, in the FHS, cg04436964 methylation was negatively correlated with APOA2 expression in the blood of participants consuming a high-SFA diet. Furthermore, when consuming a high-SFA diet, CC carriers had lower APOA2 expression than those with the TT genotype. Lastly, metabolomic analysis identified 4 pathways as overrepresented by metabolite differences between CC and TT genotypes with high-SFA intake, including tryptophan and branched-chain amino acid (BCAA) pathways. Interestingly, these pathways were linked to rs5082-specific cg04436964 methylation differences in high-SFA consumers. Conclusions: The epigenetic status of the APOA2 regulatory region is associated with SFA intake and APOA2 -265T \u3e C genotype, promoting an APOA2 expression difference between APOA2 genotypes on a high-SFA diet, and modulating BCAA and tryptophan metabolic pathways. These findings identify potential mechanisms by which this highly reproducible gene-diet interaction influences obesity risk, and contribute new insights to ongoing investigations of the relation between SFA and human health. This study was registered at clinicaltrials.gov as NCT03452787

Genome- and CD4\u3csup\u3e+\u3c/sup\u3e T-Cell Methylome-Wide Association Study of Circulating Trimethylamine-N-Oxide in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN)

Background: Trimethylamine-N-oxide (TMAO), an atherogenic metabolite species, has emerged as a possible new risk factor for cardiovascular disease. Animal studies have shown that circulating TMAO levels are regulated by genetic and environmental factors. However, large-scale human studies have failed to replicate the observed genetic associations, and epigenetic factors such as DNA methylation have never been examined in relation to TMAO levels. Methods and results: We used data from the family-based Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) to investigate the heritable determinants of plasma TMAO in humans. TMAO was not associated with other plasma markers of cardiovascular disease, e.g. lipids or inflammatory cytokines. We first estimated TMAO heritability at 27%, indicating a moderate genetic influence. We used 1000 Genomes imputed data (n = 626) to estimate genome-wide associations with TMAO levels, adjusting for age, sex, family relationships, and study site. The genome-wide study yielded one significant hit at the genome-wide level, located in an intergenic region on chromosome 4. We subsequently quantified epigenome-wide DNA methylation using the Illumina Infinium array on CD4þ Tcells. We tested for association of methylation loci with circulating TMAO (n = 847), adjusting for age, sex, family relationships, and study site as the genome-wide study plus principal components capturing CD4þ T-cell purity. Upon adjusting for multiple testing, none of the epigenetic findings were statistically significant. Conclusions: Our findings contribute to the growing body of evidence suggesting that neither genetic nor epigenetic factors play a critical role in establishing circulating TMAO levels in humans

Associations Between \u3cem\u3eSLC16A11\u3c/em\u3e Variants and Diabetes in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL)

Five sequence variants in SLC16A11 (rs117767867, rs13342692, rs13342232, rs75418188, and rs75493593), which occur in two non-reference haplotypes, were recently shown to be associated with diabetes in Mexicans from the SIGMA consortium. We aimed to determine whether these previous findings would replicate in the HCHS/SOL Mexican origin group and whether genotypic effects were similar in other HCHS/SOL groups. We analyzed these five variants in 2492 diabetes cases and 5236 controls from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), which includes U.S. participants from six diverse background groups (Mainland groups: Mexican, Central American, and South American; and Caribbean groups: Puerto Rican, Cuban, and Dominican). We estimated the SNP-diabetes association in the six groups and in the combined sample. We found that the risk alleles occur in two non-reference haplotypes in HCHS/SOL, as in the SIGMA Mexicans. The haplotype frequencies were very similar between SIGMA Mexicans and the HCHS/SOL Mainland groups, but different in the Caribbean groups. The SLC16A11 sequence variants were significantly associated with risk for diabetes in the Mexican origin group (P = 0.025), replicating the SIGMA findings. However, these variants were not significantly associated with diabetes in a combined analysis of all groups, although the power to detect such effects was 85% (assuming homogeneity of effects among the groups). Additional analyses performed separately in each of the five non-Mexican origin groups were not significant. We also analyzed (1) exclusion of young controls and, (2) SNP by BMI interactions, but neither was significant in the HCHS/SOL data. The previously reported effects of SLC16A11 variants on diabetes in Mexican samples was replicated in a large Mexican-American sample, but these effects were not significant in five non-Mexican Hispanic/Latino groups sampled from U.S. populations. Lack of replication in the HCHS/SOL non-Mexicans, and in the entire HCHS/SOL sample combined may represent underlying genetic heterogeneity. These results indicate a need for future genetic research to consider heterogeneity of the Hispanic/Latino population in the assessment of disease risk, but add to the evidence suggesting SLC16A11 as a potential therapeutic target for type 2 diabetes

An Exome-Wide Sequencing Study of Lipid Response to High-Fat Meal and Fenofibrate in Caucasians from the GOLDN Cohort

Our understanding of genetic influences on the response of lipids to specific interventions is limited. In this study, we sought to elucidate effects of rare genetic variants on lipid response to a high-fat meal challenge and fenofibrate (FFB) therapy in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) cohort using an exome-wide sequencing-based association study. Our results showed that the rare coding variants in ITGA7, SIPA1L2, and CEP72 are significantly associated with fasting LDL cholesterol response to FFB (P = 1.24E-07), triglyceride postprandial area under the increase (AUI) (P = 2.31E-06), and triglyceride postprandial AUI response to FFB (P = 1.88E-06), respectively. We sought to replicate the association for SIPA1L2 in the Heredity and Phenotype Intervention (HAPI) Heart Study, which included a high-fat meal challenge but not FFB treatment. The associated rare variants in GOLDN were not observed in the HAPI Heart study, and thus the gene-based result was not replicated. For functional validation, we found that gene transcript level of SIPA1L2 is associated with triglyceride postprandial AUI (P \u3c 0.05) in GOLDN. Our study suggests unique genetic mechanisms contributing to the lipid response to the high-fat meal challenge and FFB therapy

A genome-wide study of lipid response to fenofibrate in Caucasians: a combined analysis of the GOLDN and ACCORD studies

Fibrates are commonly prescribed for hypertriglyceridemia but also lower low-density lipoprotein cholesterol (LDL-C) and raise high-density lipoprotein cholesterol (HDL-C). Large inter-individual variation in lipid response suggests that some persons may benefit more than others and genetic studies could help identify those persons