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RationaleDNA methylation is dynamic, varies throughout the life course, and its levels are influenced by lifestyle and environmental factors, as well as by genetic variation. The leading genetic variants at stroke risk loci identified to date explain roughly 1–2% of stroke heritability. Most of these single nucleotide polymorphisms are situated within a regulatory sequence marked by DNase I hypersensitivity sites, which would indicate involvement of an epigenetic mechanism.ObjectiveTo detect epigenetic variants associated to stroke occurrence and stroke subtypes.Methods and ResultsA two-stage case-control epigenome-wide association study was designed. The discovery sample with 401 samples included 218 ischemic stroke (IS) patients, assessed at Hospital del Mar (Barcelona, Spain) and 183 controls from the REGICOR cohort. In two independent samples (N=226 and N=166), we replicated 22 CpG sites differentially methylated in IS in 21 loci, including 2 CpGs in locus ZFHX3, which includes known genetic variants associated with stroke. The pathways associated to these loci are inflammation and angiogenesis. The meta-analysis identified 384 differentially methylated CpGs, including loci of known stroke and vascular risk genetic variants, enriched by loci involved in lipid metabolism, adipogenesis, circadian clock, and glycolysis pathways. Stratification analysis by stroke subtypes revealed distinct methylation patterns.ConclusionsWe identified a set of 22 CpGs in 21 loci associated with IS. Our analysis suggests that DNA methylation changes may contribute to orchestrating gene expression that contributes to IS.

BackgroundFamilial sitosterolemia is a rare, recessive Mendelian disorder characterized by hyperabsorption and decreased biliary excretion of dietary sterols. Affected individuals typically have complete genetic deficiency – homozygous loss-of-function (LoF) variants — in the ATP-binding cassette transporter G5 (ABCG5) or G8 (ABCG8) genes, and have substantially elevated plasma sitosterol and low-density lipoprotein cholesterol (LDL-C) levels. The impact of partial genetic deficiency ofABCG5orABCG8, as occurs in heterozygous carriers of LoF variants, on LDL-C and risk of coronary artery disease (CAD) has remained uncertain.MethodsWe first recruited nine sitosterolemia families, identified causative LoF variants inABCG5orABCG8, and evaluated the associations of theseABCG5orABCG8LoF variants with plasma phytosterols and lipid levels. We next assessed for LoF variants inABCG5orABCG8in CAD cases (n=29,361) versus controls (n=357,326). We tested the association of rare LoF variants inABCG5orABCG8with blood lipids and risk for CAD. Rare LoF variants were defined as protein-truncating variants with minor allele frequency less than 0.1% inABCG5orABCG8.ResultsIn sitosterolemia families, seven pedigrees harbored causative LoF variants inABCG5and two pedigrees inABCG8. Homozygous LoF variants in eitherABCG5orABCG8led to marked elevations in sitosterol and LDL-C. Of those sitosterolemia families, heterozygous carriers ofABCG5LoF variants exhibited increased sitosterol and LDL-C levels compared to non-carriers. Within the large-scale CAD case-control cohorts, prevalence of rare LoF variants inABCG5and inABCG8were approximately 0.1% each.ABCG5heterozygous LoF variant carriers had significantly elevated LDL-C levels (24.7 mg/dL; 95% confidence interval [CI] 14 to 35; P=1.1×10−6), and were at two-fold increased risk of CAD (odds ratio 2.06, 95% CI 1.27 to 3.35; P=0.004). By contrast,ABCG8heterozygous LoF carrier status was not associated with increased LDL-C or risk of CAD.ConclusionsAlthough familial sitosterolemia is traditionally considered as a recessive disorder, we observed that heterozygous carriers of a LoF variant inABCG5had significantly increased sitosterol and LDL-C levels and a two-fold increase in risk of CAD.

Epigenome-wide association studies for cardiometabolic risk factors have discovered multiple loci associated with incident cardiovascular disease (CVD). However, few studies have sought to directly optimize a predictor of CVD risk. Furthermore, it is challenging to train multivariate models across multiple studies in the presence of study- or batch effects. Here, we analyzed existing DNA methylation data collected using the Illumina HumanMethylation450 microarray to create a predictor of CVD risk across three cohorts: Women’s Health Initiative, Framingham Heart Study Offspring Cohort, and Lothian Birth Cohorts. We trained Cox proportional hazards-based elastic net regressions for incident CVD separately in each cohort, and used a recently-introduced cross-study learning approach to integrate these individual predictions into an ensemble predictor. The methylation-based risk score (MRS) predicted CVD time-to-event in a held-out fraction of the Framingham dataset (HR per SD = 1.28, p = 2e-3) and predicted myocardial infarction status in the independent REGICOR dataset (OR per SD = 2.14, p = 9e-7). These associations remained after adjustment for traditional cardiovascular risk factors and were similar to those from elastic net models trained on a directly merged dataset. Additionally, we investigated interactions between the MRS and both genetic and biochemical CVD risk, showing preliminary evidence of an enhanced predictive power in those with less traditional risk factor elevation. This investigation provides proof-of-concept for a genome-wide, CVD-specific epigenomic risk score and suggests that the DNA methylation data may enable the discovery of high-risk individuals that would be missed by alternative risk metrics.

AimsCurrent risk prediction tools are not accurate enough to identify most individuals at high coronary risk. On the other hand, oxidized low-density lipoproteins (ox-LDLs) and miRNAs are actively involved in atherosclerosis. Our aim was to examine the association of ox-LDL-induced miRNAs with coronary artery disease (CAD), and to assess their predictive capacity of future CAD.Methods and resultsHuman endothelial and vascular smooth muscle cells were treated with oxidized or native LDLs (nLDL), and their miRNA expression was measured with the miRNA 4.0 array, and analyzed with moderated t-tests. Differently expressed miRNAs and others known to be associated with CAD, were examined in serum samples of 500 acute myocardial infarction (AMI) patients and 500 healthy controls, and baseline serum of 117 incident CAD cases and c 485 randomly-selected cohort participants (case-cohort). Both were developed within the REGICOR AMI Registry and population cohorts from Girona. miRNAs expression in serum was measured with custom OpenArray plates, and analyzed with fold change (age and sex-paired case-control) and survival models (case-cohort). Improvement in discrimination and reclassification by miRNAs was assessed. Twenty-one miRNAs were up- or down-regulated with ox-LDL in cell cultures. One of them, 1 (has-miR-122-5p, fold change=4.85) was upregulated in AMI cases. Of the 28 known CAD-associated miRNAs, 11 were upregulated in AMI cases, and 1 (hsa-miR-143-3p, hazard ratio=0.56 [0.38-0.82]) was associated with CAD incidence and improved reclassification.ConclusionWe identified 2 novel miRNAs associated with ox-LDLs (hsa-miR-193b-5p and hsa-miR-1229-5p), and 1 miRNA that improved reclassification of healthy individuals (hsa-miR-143-3p).

BackgroundThe causal role of high-density lipoproteins (HDL) in coronary artery disease (CAD) has been questioned. Our aim was to analyze whether genetically determined quantitative and qualitative HDL characteristics were independently associated with CAD.MethodsWe designed a two-sample multivariate Mendelian randomization study with available genome-wide association summary data. We identified genetic variants associated with HDL cholesterol and apolipoprotein A-I quantity, HDL size, particle levels, and lipid content to define our genetic instrumental variables in one sample (Kettunen et al study,N=24,925) and analyzed their association with CAD risk in a different study (CARDIoGRAMplusC4D,N=184,305). We validated these results by defining our genetic variables in another database (METSIM,N=8,372) and studied their relationship with CAD in the CARDIoGRAMplusC4D dataset. To estimate the effect size of the associations of interest adjusted for other lipoprotein traits (potential pleiotropy) we used the Multi-trait-based Conditional & Joint analysis.ResultsGenetically determined HDL cholesterol and apolipoprotein A-I levels were not associated with CAD. HDL mean diameter (β=0.27 [95%CI=0.19; 0.35]), cholesterol levels in very large HDLs (β=0.29 [95%CI=0.17; 0.40]), and triglyceride content in very large HDLs (β=0.14 [95%CI=0.040; 0.25]) were directly associated with CAD risk, whereas the cholesterol content in medium-sized HDLs (β=-0.076 [95%CI=-0.10; −0.052]) was inversely related to this risk. These results were validated in the METSIM-CARDIoGRAMplusC4D data. Genetic variants linked to both HDL qualitative traits and CAD risk were located withinLIPC, theAPOE/C1/C4/C2cluster,APOB, PCIF1-PLTP, andTTC39B.ConclusionsSome HDL characteristics related to size, particle distribution, and triglyceride content are related to CAD risk whilst HDL cholesterol levels are not. This relationship could be mediated by the hepatic lipase; the apolipoproteins E, C-I, and B; the phospholipid transfer protein; and the tetratricopeptide repeat domain protein 39B, which arise as potential therapeutic targets in cardiovascular disease.

Analyzing 5770 all-cause cirrhosis cases and 572,850 controls from seven cohorts, we identify a missense variant in the Mitochondrial Amidoxime Reducing Component 1 gene (MARC1 p.A165T) that associates with protection from all-cause cirrhosis (OR 0.88, p=2.1*10−8). This same variant also associates with lower levels of hepatic fat on computed tomographic imaging and lower odds of physician-diagnosed fatty liver as well as lower blood levels of alanine transaminase (−0.012 SD, 1.4*10−8), alkaline phosphatase (−0.019 SD, 6.6*10−9), total cholesterol (−0.037 SD, p=1*10−18) and LDL cholesterol (−0.035 SD, p=7.3*10−16). Carriers of rare protein-truncating variants in MARC1 had lower liver enzyme levels, cholesterol levels, and reduced odds of liver disease (OR 0.19, p= 0.04) suggesting that deficiency of the MARC1 enzyme protects against cirrhosis.

INTRODUCTION: Limited evidence suggests that epigenetic mechanisms may partially mediate the adverse effects of air pollution on health. Our aims were to identify new genomic loci showing differential DNA methylation associated with long-term exposure to air pollution and to replicate loci previously identified in other studies. METHODS: A two-stage epigenome-wide association study was designed: 630 individuals from the REGICOR study were included in the discovery and 454 participants of the EPIC-Italy study in the validation stage. DNA methylation was assessed using the Infinium HumanMethylation450 BeadChip. NOX, NO2, PM10, PM2.5, PMcoarse, traffic intensity and traffic load exposure were measured according to the ESCAPE protocol. A systematic review was undertaken to identify those cytosine-phosphate-guanine (CpGs) associated with air pollution in previous studies and we screened for them in the discovery study. RESULTS: In the discovery stage of the epigenome-wide association study, 81 unique CpGs were associated with air pollution (p-value

High blood pressure is the foremost heritable global risk factor for cardiovascular disease. We report the largest genetic association study of blood pressure traits to date (systolic, diastolic, pulse pressure) in over one million people of European ancestry. We identify 535 novel blood pressure loci that not only offer new biological insights into blood pressure regulation but also reveal shared loci influencing lifestyle exposures. Our findings offer the potential for a precision medicine strategy for future cardiovascular disease prevention.

Elevated blood pressure is a major risk factor for cardiovascular disease and has a substantial genetic contribution. Genetic variation influencing blood pressure has the potential to identify new pharmacological targets for the treatment of hypertension. To discover additional novel blood pressure loci, we used 1000 Genomes Project-based imputation in 150,134 European ancestry individuals and sought significant evidence for independent replication in a further 228,245 individuals. We report 6 new signals of association in or near HSPB7, TNXB, LRP12, LOC283335, SEPT9 and AKT2, and provide new replication evidence for a further 2 signals in EBF2 and NFKBIA. Combining large whole-blood gene expression resources totaling 12,607 individuals, we investigated all novel and previously reported signals and identified 48 genes with evidence for involvement in BP regulation that are significant in multiple resources. Three novel kidney-specific signals were also detected. These robustly implicated genes may provide new leads for therapeutic innovation.