Multiomics Data Analysis Identified CpG Sites That Mediate the Impact of Smoking on Cardiometabolic Traits.

Understanding the epigenome paths through which smoking contributes to cardiometabolic traits is important for downstream applications. In this study, an SNP-based analytical pipeline was used to integrate several publicly available datasets in order to identify CpG sites that mediate the impact of smoking on cardiometabolic traits and to investigate the underlying molecular mechanisms. After applying stringent statistical criteria, 11 CpG sites were detected that showed significant association (p < 5 × 10-8) with cardiometabolic traits at both the discovery and replication stages. By integrating eQTL data, I found genes behind a number of these associations. cg05228408 was hypomethylated in smokers and contributed to higher blood pressure by lowering the expression of the CLCN6 gene. cg08639339 was hypermethylated in smokers and lowered the metabolic rate by increasing the expression of RAB29; furthermore, I noted TMEM120A mediated the impact of smoking-cg17325771 on LDL, and LTBP3 mediated the smoking-cg07029024 effect on heart rate. The pathway analysis identified processes through which the identified genes impact their traits. This study provides a list of CpG sites that mediates the impact of smoking on cardiometabolic traits and a framework to investigate the underlying molecular paths using publicly available data.

Genome-wide search identified DNA methylation sites that regulate the metabolome.

Background: Identifying DNA methylation sites that regulate the metabolome is important for several purposes. In this study, publicly available GWAS data were integrated to find methylation sites that impact metabolome through a discovery and replication scheme and by using Mendelian randomization. Results: The outcome of analyses revealed 107 methylation sites associated with 84 metabolites at the genome-wide significance level (p<5e-8) at both the discovery and replication stages. A large percentage of the observed associations (85%) were with lipids, significantly higher than expected (p = 0.0003). A number of CpG (methylation) sites showed specificity e.g., cg20133200 within PFKP was associated with glucose only and cg10760299 within GATM impacted the level of creatinine; in contrast, there were sites associated with numerous metabolites e.g., cg20102877 on the 2p23.3 region was associated with 39 metabolites. Integrating transcriptome data enabled identifying genes (N = 82) mediating the impact of methylation sites on the metabolome and cardiometabolic traits. For example, PABPC4 mediated the impact of cg15123755-HDL on type-2 diabetes. KCNK7 mediated the impact of cg21033440-lipids on hypertension. POC5, ILRUN, FDFT1, and NEIL2 mediated the impact of CpG sites on obesity through metabolic pathways. Conclusion: This study provides a catalog of DNA methylation sites that regulate the metabolome for downstream applications.

Genome-wide screening identifies DNA methylation sites that regulate the blood proteome.

Background: Identifying DNA methylation sites that regulate the blood proteome is important for biomedical purposes. Materials & methods: Here the authors performed a genome-wide search to find DNA methylation sites that impact proteins. Results: The authors identified 165 methylation sites associated with 138 proteins. The authors noted hotspot genomic regions that control the levels of several proteins. For example, methylation of the ABO locus impacted 37 proteins and contributed to cardiometabolic comorbidities, including the severity of SARS-CoV-2 infection. The authors made these findings publicly available as a Unix software that identifies methylation sites that cause disease and reveals the underlying proteins. The authors underlined the software application by showing that components of innate immunity contribute to systolic blood pressure. Conclusion: This study provides a catalog of DNA methylation sites that regulate the proteome, and the results are available as freeware for biological insight.

Our lifestyle choices and interactions with the world around us are continuously printed in our DNA through a biochemical process known as epigenomic modification. Excessive epigenomic modification at a DNA site may cause disease. To prevent or treat disease, it is important to find such sites and remove the excessive epigenomic modification with medications or lifestyle changes. Here the authors searched for DNA sites that undergo epigenomic modification. The authors also investigated the mechanism whereby these sites cause disease. The authors found that there are DNA sites where reverting the epigenomic modification could have a big impact on the body. The authors have made these findings publicly available.

Does the Presence of Type 2 Diabetes or Metabolic Syndrome Impact Reduction in Waist Circumference During Weight Loss?

OBJECTIVES: Our aim in this study was to compare the change in waist circumference given the same degree of weight loss in patients who meet the criteria for metabolic syndrome or type 2 diabetes and those who do not meet these criteria. Because visceral adiposity is a key feature of both conditions and intra-abdominal adipocytes show higher lipolytic activity, we sought to determine whether changes in waist circumference differed in individuals with and without these conditions. METHODS: The Ottawa Hospital Weight Management Clinic offers a course in lifestyle modification and uses 12 weeks of total meal replacement. We compared the decrease in waist circumference between patients with metabolic syndrome or diabetes and those without these conditions who had lost a similar amount of weight using measurements from the first 6 weeks of meal replacement. RESULTS: We evaluated 3,559 patients who attended the program between September 1992 and April 2015. The patient population was largely Caucasian and of European descent and all meetings were face to face. The mean weight loss for men was 15.1±20.2 kg, and the mean weight loss for women was 9.7±2.4 kg. There were no significant differences in decrease in waist circumference between those with and without metabolic syndrome in both men (11.7±3.9 cm vs 11.4±3.8 cm, p=0.48) and women (9.0±3.6 cm vs 9.1±3.7 cm, p=0.26). CONCLUSIONS: Our results show that, given the same degree of weight loss, patients with and without diabetes or metabolic syndrome experience a similar change in waist circumference.

Common Polymorphism That Protects From Cardiovascular Disease Increases Fibronectin Processing and Secretion.

BACKGROUND: Fibronectin (FN1) is an essential regulator of homodynamic processes and tissue remodeling that have been proposed to contribute to atherosclerosis. Moreover, recent large-scale genome-wide association studies (GWAS) have linked common genetic variants within the FN1 gene to coronary artery disease risk. METHODS: Public databases were analyzed by 2-Sample Mendelian Randomization. Expression constructs encoding short FN1 reporter constructs and full-length plasma FN1 variants were introduced in various cell models. Secreted and cellular levels were then analyzed and quantified by SDS-PAGE and fluorescence microscopy. Mass spectrometry and glycosylation analyses were performed to probe possible posttranscriptional differences. RESULTS: Bioinformatic analyses revealed that common coronary artery disease risk single nucleotide polymorphisms in the FN1 locus associate with circulating levels of FN1 and that higher FN1 (fibronectin 1) protein levels in plasma are linked to lower coronary artery disease risk. The coronary artery disease-associated FN1 locus encompasses a common polymorphism that translates a L15Q variant situated within the FN1 signal peptide. Introduction of FN1 reporter constructs, differing at position 15, revealed differences in secretion, with the FN1 Q15 variant being less well secreted. Moreover, the L15Q polymorphism was found to alter glycosylation in some cell models but not in human plasma. CONCLUSIONS: In addition to providing novel functional evidence implicating FN1 in cardioprotection, these findings demonstrate that a common variant within a secretion signal peptide regulates protein function.

Epigenome-Wide Study Identified Methylation Sites Associated with the Risk of Obesity.

Here, we performed a genome-wide search for methylation sites that contribute to the risk of obesity. We integrated methylation quantitative trait locus (mQTL) data with BMI GWAS information through a SNP-based multiomics approach to identify genomic regions where mQTLs for a methylation site co-localize with obesity risk SNPs. We then tested whether the identified site contributed to BMI through Mendelian randomization. We identified multiple methylation sites causally contributing to the risk of obesity. We validated these findings through a replication stage. By integrating expression quantitative trait locus (eQTL) data, we noted that lower methylation at cg21178254 site upstream of CCNL1 contributes to obesity by increasing the expression of this gene. Higher methylation at cg02814054 increases the risk of obesity by lowering the expression of MAST3, whereas lower methylation at cg06028605 contributes to obesity by decreasing the expression of SLC5A11. Finally, we noted that rare variants within 2p23.3 impact obesity by making the cg01884057 site more susceptible to methylation, which consequently lowers the expression of POMC, ADCY3 and DNAJC27. In this study, we identify methylation sites associated with the risk of obesity and reveal the mechanism whereby a number of these sites exert their effects. This study provides a framework to perform an omics-wide association study for a phenotype and to understand the mechanism whereby a rare variant causes a disease.

A Common Polymorphism in the FADS1 Locus Links miR1908 to Low-Density Lipoprotein Cholesterol Through BMP1.

OBJECTIVE: Leveraging microRNA-Seq data and the 1000 Genomes imputed genotypes, we identified rs174561 as a strong microRNA quantitative trait loci for circulating microRNA-1908-5p with higher miR-1908-5p and reduced LDL (lowdensity lipoprotein)-cholesterol, fasting glucose and A1c concentrations in carriers of the rs-174561-C allele. Here, we have investigated the molecular mechanism(s) linking miR-1908-5p to LDL-C concentrations. APPROACH AND RESULTS: Transfection experiments demonstrate that the presence of the C allele significantly increases miR- 1908-5p abundance relative to the T allele. LDLR mRNA and low-density lipoprotein receptor (LDLR) total protein were unchanged in response to differential miR-1908-5p expression. However, the ratio of the cleaved to full-length form of LDLR decreased with miR-1908-5p mimic and increased with miR-1908-5p inhibitor treatment. BMP1 (bone morphogenetic protein 1) is a protease responsible for LDLR cleavage, and we show that miR-1908-5p mimic reduces BMP1 mRNA. Using a reporter array, we identified the TGF-ß (transforming growth factor-beta) signaling pathway activity to be reduced by miR- 1908-5p mimic treatment, and this was associated with reduced TGFB1 expression. TGF-ß signaling increases BMP1, and we further demonstrate that the effect of miR-1908-5p on LDLR cleavage is abolished by exogenous TGF-ß treatment. CONCLUSIONS: These findings uncover a mechanism whereby miR-1908-5p reduces TGFB1 abundance resulting in lower expression of BMP1, ultimately leading to reduced LDLR cleavage. Cleavage of the mature LDLR is known to reduce cell surface affinity for LDL, thereby linking miR-1908-5p to lower circulating LDL-cholesterol levels.

Convergence of biomarkers and risk factor trait loci of coronary artery disease at 3p21.31 and HLA region.

Here we seek to identify molecular biomarkers that mediate the effect of risk factors on coronary artery disease (CAD). We perform a SNP-based multiomics data analysis to find biomarkers (probes) causally associated with the risk of CAD within known genomic loci for its risk factors. We identify 78 biomarkers, the majority (64%) of which are methylation probes. We detect the convergence of several CNS and lifestyle trait loci and their biomarkers at the 3p21.31 and human leukocyte antigen (HLA) regions. The 3p21.31 locus was the most populated region in the convergence of biomarkers and risk factors. In this region, we noted as the BSN gene becomes methylated the level of stomatin (STOM) in blood increases and this contributes to higher risk of CAD. In the HLA locus, we identify several methylation biomarkers associated with various CAD risk factors. SNPs in the CFB gene display a trans-regulatory impact on the GRIA4 protein level. A methylation site upstream of the APOE gene is associated with a higher protein level of S100A13 which in turn leads to higher LDL-C and greater CAD risk. We find UHRF1BP1 and ILRUN mediate the effect of obesity on CAD whereas methylation sites within NOS3 and CKM mediate the effect of their associated-risk factors on CAD. This study provides further insight into the biology of CAD and identifies a list of biomarkers that mediate the impact of risk factors on CAD. A SNP-based initiative can unite data from various fields of omics into a single network of knowledge.

Publisher Correction: RIPK1 gene variants associate with obesity in humans and can be therapeutically silenced to reduce obesity in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multiomics Screening Identifies Molecular Biomarkers Causally Associated With the Risk of Coronary Artery Disease.

BACKGROUND: In this study, we aimed to investigate functional mechanisms underlying coronary artery disease (CAD) loci and find molecular biomarkers for CAD. METHODS: We devised a multiomics data analysis approach based on Mendelian randomization and utilized it to search for molecular biomarkers causally associated with the risk of CAD within genomic regions known to be associated with CAD. RESULTS: Through our CAD-centered multiomics data analysis approach, we identified 33 molecular biomarkers (probes) that were causally associated with the risk of CAD. The majority of these (N=19) were methylation probes; moreover, methylation was often behind the causal effect of expression/protein probes. We identified a number of novel loci that have a causal impact on CAD including C5orf38, SF3A3, DHX36, and MRPL33. Furthermore, by integrating the risk factors of CAD in our analysis, we were able to investigate the clinical pathways whereby several of our probes exert their effect. We found that the SELE protein level in the blood is under the trans-regulatory impact of methylation sites within the ABO gene and that SELE exerts its effect on CAD through immune, glycemic, and lipid metabolism, making it a candidate of interest for therapeutic interventions. We found the methylation site, cg05126514 within the BSN gene exert its effect on CAD through central nervous system-lifestyle risk factors. Finally, genes with a transcriptional regulatory role (SF3A3, ILF3, and N4BP2L2) exert their effect on CAD through height. CONCLUSIONS: We demonstrate that multiomics data analysis is a powerful approach to unravel the functional mechanisms underlying CAD loci and to identify novel molecular biomarkers. Our results indicate epigenetic modifications are important in the pathogenesis of CAD and identifying and targeting these sites is of potential therapeutic interest to address the detrimental effects of both environmental and genetic factors.

RIPK1 gene variants associate with obesity in humans and can be therapeutically silenced to reduce obesity in mice.

Obesity is a major public health burden worldwide and is characterized by chronic low-grade inflammation driven by the cooperation of the innate immune system and dysregulated metabolism in adipose tissue and other metabolic organs. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a central regulator of inflammatory cell function that coordinates inflammation, apoptosis and necroptosis in response to inflammatory stimuli. Here we show that genetic polymorphisms near the human RIPK1 locus associate with increased RIPK1 gene expression and obesity. We show that one of these single nucleotide polymorphisms is within a binding site for E4BP4 and increases RIPK1 promoter activity and RIPK1 gene expression in adipose tissue. Therapeutic silencing of RIPK1 in vivo in a mouse model of diet-induced obesity dramatically reduces fat mass, total body weight and improves insulin sensitivity, while simultaneously reducing macrophage and promoting invariant natural killer T cell accumulation in adipose tissue. These findings demonstrate that RIPK1 is genetically associated with obesity, and reducing RIPK1 expression is a potential therapeutic approach to target obesity and related diseases.

CARMAL Is a Long Non-coding RNA Locus That Regulates MFGE8 Expression.

Genome-wide association studies have identified several genetic loci linked to coronary artery disease (CAD) most of them located in non-protein coding regions of the genome. One such locus is the CAD Associated Region between MFGE8 and ABHD2 (CARMA), a ∼18 kb haplotype that was recently shown to regulate vicinal protein coding genes. Here, we further investigate the region by examining a long non-coding RNA gene locus (CARMAL/RP11-326A19.4/AC013565) abutting the CARMA region. Expression-genotype correlation analyses of public databases indicate that CARMAL levels are influenced by CAD associated variants suggesting that it might have cardioprotective functions. We found CARMAL to be stably expressed at relatively low levels and enriched in the cytosol. CARMAL function was investigated by several gene targeting approaches in HEK293T: inactive CRISPR fusion proteins, antisense, overexpression and inactivation by CRISPR-mediated knock-out. Modest increases in CARMAL (3-4×) obtained via CRISPRa using distinct single-guided RNAs did not result in consistent transcriptome effects. By contrast, CARMAL deletion or reduced CARMAL expression via CRISPRi increased MFGE8 levels, suggesting that CARMAL is contributing to reduce MFGE8 expression under basal conditions. While future investigations are required to clarify the mechanism(s) by which CARMAL acts on MFGE8, integrative bioinformatic analyses of the transcriptome of CARMAL deleted cells suggest that this locus may also be involved in leucine metabolism, splicing, transcriptional regulation and Shwachman-Bodian-Diamond syndrome protein function.

SGCG rs679482 Associates With Weight Loss Success in Response to an Intensively Supervised Outpatient Program.

Weight loss in response to energy restriction is highly variable, and identification of genetic contributors can provide insights into underlying biology. Leveraging 1000 Genomes imputed genotypes, we carried out genome-wide association study (GWAS) analysis in 551 unrelated obese subjects of European ancestry who participated in an intensively supervised weight loss program with replication of promising signals in an independent sample of 1,331 obese subjects who completed the program at a later date. By single nucleotide polymorphism-based and sib-pair analysis, we show that that weight loss is a heritable trait, with estimated heritability (h 2 = 0.49) within the range reported for obesity. We find rs679482, intronic to SGCG (sarcoglycan γ), highly expressed in skeletal muscle, to concordantly associate with weight loss in discovery and replication samples reaching GWAS significance in the combined meta-analysis (ß = -0.35, P = 1.7 × 10-12). Located in a region of open chromatin, rs679482 is predicted to bind DMRT2, and allele-specific transcription factor binding analysis indicates preferential binding of DMRT2 to rs679482-A. Concordantly, rs679482-A impairs native repressor activity and increases basal and DMRT2-mediated enhancer activity. These findings confirm that weight loss is a heritable trait and provide evidence by which a novel variant in SGCG, rs679482, leads to impaired diet response.

Phenome-wide screening for traits causally associated with the risk of coronary artery disease.

Using two independent approaches, Mendelian randomization and Polygenic risk score in a sample of 6194 CAD cases and 4287 controls of European ancestry, we did a comprehensive phenome-wide search (PheWAS) for traits that causally associated with the risk of CAD. We found 46 risk factors that represented diverse categories including cardiovascular, CNS (central nervous system), diabetes, lipids, immune, anthropometry, and life style features; moreover, we noted numerous evidences of genetic correlations and causal associations between risk factors from different categories. Among the identified risk factors, CAD showed highest genetic relatedness with thrombotic conditions. The most represented category was life style features (29%) with evidence of strong genetic overlap with CNS traits. Genetic variants associated with higher cognition were associated with life style characteristics and cardiometabolic features that lower the risk of CAD. Conditional analysis indicated this trend is in part attributed to higher age of first sexual intercourse (AFS) in those with higher cognition. Lower AFS was concordantly associated with higher risk of CAD in males, females, and the combined sample; furthermore, lower AFS was causally associated with several CAD-risk factors including, higher fasting insulin, fasting glucose, LDL, immature reticulocyte fraction, HbA1c levels, as well as, higher risk of T2D and pulmonary embolism but lower levels of HDL. These results indicate CAD is the outcome of several phenotypically distinct but genetically interrelated sources; moreover, we identified lower AFS as an independent causal risk factor of CAD and revealed its role in mediating the effect of other risk factors.

Regulation of MFGE8 by the intergenic coronary artery disease locus on 15q26.1.

BACKGROUND AND AIMS: A recently identified locus for coronary artery disease (CAD) tagged by rs8042271 is in a region of tight linkage disequilibrium (LD) between 2 genes (MFGE8, ABHD2) previously linked to atherosclerosis. Here we have explored the regulatory framework of this region to identify its functional relationship to CAD. METHODS: The CAD Associated Region between MFGE8 and ABHD2 (CARMA) was investigated by bioinformatic approaches and transcriptional reporter assays to prioritize target genes and identify putative causal variants. Findings were integrated with publicly available gene expression datasets. MFGE8 silencing was performed in cell models relevant to CAD. RESULTS: The regulatory potential of CARMA is disseminated sparsely over the entire region. CARMA contains multiple eQTL that regulate MFGE8 in coronary artery and coronary artery smooth muscle cell (CoSMC). SNPs that predict the expression of MFGE8 in artery are concordantly associated with higher risk of CAD (pval = 0.0014). Targeting CARMA by CRISPR/Cas9 in a cellular model increased MFGE8 expression. MFGE8 silencing was found to reduce CoSMC and monocyte (THP-1) but not endothelial cell proliferation. CONCLUSIONS: These findings support a mechanistic link between a GWAS identified CAD risk locus and atherosclerosis. The intergenic locus CARMA regulates MFGE8 in a haplotype dependent manner. Individuals genetically susceptible to increased MFGE8 expression exhibit greater CAD risk. Suppressing MFGE8 expression reduced SMC and THP-1 proliferation. These data support an atherogenic contribution of CARMA/MFGE8 that may be linked to cell proliferation and/or improved survival of CAD relevant cell types.

Genome-wide identification of circulating-miRNA expression quantitative trait loci reveals the role of several miRNAs in the regulation of cardiometabolic phenotypes.

AIMS: To identify genetic variants that have a regulatory impact on circulating microRNAs (miRNAs) and to connect genetic risk to blood traits/biomarkers through the circulating miRNAs. METHODS AND RESULTS: Leveraging miRNA-Seq data and the 1000 Genomes imputed genotypes, we carried out genome-wide association analysis for SNPs that regulate the expression of circulating miRNAs in a sample of 710 unrelated subjects of European ancestry. Wherever possible, we used data from the Framingham and the Geuvadis studies to replicate our findings. We found at least one genome-wide significant (P < 5e-8) miRNA-eQTL (mirQTL) for 143 circulating miRNAs. Overall each mirQTL explained a small portion (<1%) of variation in miRNA levels; however, we identified a few mirQTLs that explained 4% to 20% of variation in miRNA levels in plasma. Unlike trans-mirQTLs (P = 0.7), cis-mirQTLs tend to be also associated with their counterpart mature miRNAs (P < 0.0001), this suggests trans-mirQTLs exert their effect through processes that affect the stability of mature miRNAs; whereas, cis-mirQTLs mainly regulate the expression of primary-miRNAs. Next, we used the identified mirQTLs to investigate the links between circulating miRNAs with blood traits/biomarkers through Mendelian randomization analysis. We found miR-1908-5p plays an important role in regulating low-density lipoprotein (LDL), total cholesterol (TC), fasting glucose, HbA1c, and several lipid-metabolites in blood, whereas, miR-10b-5p mediates the trans-regulatory effect of the ABO locus on several blood proteins, coronary artery disease, and TC. Moreover, we demonstrated that a higher plasma level of miR-199a is causally associated with lower levels of LDL and TC. Finally, we found miR-143-3p and miR-145-5p are functionally related and mediate the effect of ZFPM2 on a number of its protein targets in blood including VEGFA, SERPINE1, and PDGFs. CONCLUSIONS: This study identifies SNPs that have a regulatory impact on circulating miRNAs, and underlines the role of several circulating miRNAs in mediating the effect of a number of GWAS loci on cardiometabolic phenotypes.

Partitioning the Pleiotropy Between Coronary Artery Disease and Body Mass Index Reveals the Importance of Low Frequency Variants and Central Nervous System-Specific Functional Elements.

BACKGROUND: The objective of this study is to investigate the extent and nature of pleiotropy between coronary artery disease (CAD) and body mass index (BMI). METHODS: We examined the contribution of genome-wide single-nucleotide polymorphisms (minor allele frequency ≥0.01) to co-occurrence of CAD and BMI in a sample of genetically unrelated 8041 subjects (genetic resemblance ≤0.025) of European ancestry using mixed-linear-models. We further partitioned the estimated pleiotropy according to biological features to gain insight into the nature of pleiotropy between CAD and BMI. RESULTS: We found significant (P<0.0001) positive genetic correlation between CAD and BMI (rg =0.60). The estimated pleiotropy explained 68% of phenotypic correlation, and it was not proportionally distributed across the chromosomes; notably, chromosome 10 contributed more; whereas, chromosomes 11 and 14 contributed less to pleiotropy than expected given their chromosomal length. We noted that a large proportion (63%; P=0.002) of the pleiotropy is attributed to single-nucleotide polymorphisms with low allele frequency (minor allele frequency <0.05). Of note, pleiotropy was enriched among central nervous system genes and genes of metabolic pathways. Further analyses revealed that these effects are more pronounced in the proopiomelanocortin pathway and genes involved in carbohydrate metabolism. After genome-wide association study meta-analysis, only single-nucleotide polymorphisms downstream of the MC4R gene were found concordantly associated with (P<5×10-8) BMI and CAD with lead single-nucleotide polymorphism being rs663129 (combined P=2.7×10-65). Finally, partitioning the pleiotropy according to functional elements pointed to the importance of superenhancers and notably brain-specific superenhancers. CONCLUSIONS: Genome-wide pleiotropy substantially contributes to co-occurrence of CAD and obesity, and it is highly enriched among low frequency variants and central nervous system-specific functional elements.

Partitioning the heritability of coronary artery disease highlights the importance of immune-mediated processes and epigenetic sites associated with transcriptional activity.

AIMS: With the availability of genome-wide genotype data from GWAS studies, it is now possible to compute the genetic relatedness among individuals and estimate its contribution (SNP-based heritability) to phenotypic variance using Mixed-Linear-Models (MLMs). The estimated heritability can be partitioned according to biological features to gain insight into the genetic architecture of a disease. Here, we aimed to examine the genetic structure of coronary artery disease (CAD). METHODS AND RESULTS: We investigated the genetic structure of CAD using 3,163,082 autosomal genome-wide SNPs (MAF ≥ 0.01) and MLMs in a sample of genetically 'unrelated' 4535 cases and 2977 controls. We find that genome-wide SNPs explain 22% of liability to CAD (55% of narrow-sense heritability) and sex-differences in CAD is not due to common SNPs on autosomal chromosomes. Heritability was proportionally distributed across the allele frequency spectrum and notably enriched among genic SNPs. We identified a number of modules that are significantly associated with CAD including: Dendritic cells stimulation; Basigin interactions; and a Cancer module. Of note, genes involved in inflammation account for one-fifth of SNP-based heritability. Heritability-enrichment analysis showed significant enrichment in epigenetic sites associated with transcriptionally activity; namely, enhancers, H3K9ac/H3K27ac/H3K4me1/H3K4me3 histone modifications, and Fetal DNase I hypersensitivity sites whereas heritability was highly depleted in transcriptionally repressed regions. CONCLUSIONS: More individual SNP associations will be detected for CAD as sample size increases. The identified modules provide further biological insight for CAD and highlight the importance of immune-mediated processes in CAD pathogenesis. Finally, we showed that genetic liability to CAD is mainly attributed to epigenetic sites associated with transcriptional activity which encourage the design of custom sequencing/genotyping panels based on transcriptionally active regions.

Functional Validation of a Common Nonsynonymous Coding Variant in ZC3HC1 Associated With Protection From Coronary Artery Disease.

BACKGROUND: Although virtually all coronary artery disease associated single-nucleotide polymorphisms identified by genome-wide association studies (GWAS) are in noncoding regions of the genome, a common polymorphism in ZC3HC1 (rs11556924), resulting in an arginine (Arg) to histidine (His) substitution in its encoded protein, NIPA (Nuclear Interacting Partner of Anaplastic Lyphoma Kinase) is linked to a protection from coronary artery disease. NIPA plays a role in cell cycle progression, but the functional consequences of this polymorphism have not been established. METHODS AND RESULTS: Here we demonstrate that total ZC3HC1 expression in whole blood is similar across genotypes, despite expression being slightly biased toward the risk allele in heterozygotes. At the protein level, the protective His363 NIPA variant exhibits increased phosphorylation of a critical serine residue (Ser354) and higher protein expression as compared with the Arg363 variant. Binding experiments indicate that neither SKP1 (S-phase kinase-associated protein 1) nor CCNB1 binding were affected by the polymorphism. Despite similar nuclear distribution, NIPA His363 exhibits greater nuclear mobility. NIPA suppression results in a modest reduction of proliferation in vascular smooth muscle cells, but given low proliferative capacity, a significant effect of the variant was not noted. By contrast, we demonstrate that the protective variant reduces cell proliferation in HeLa cells. CONCLUSIONS: These findings extend the genetic association between rs11556924 and coronary artery disease risk by characterizing its effects on the encoded protein, NIPA. The resulting amino acid change Arg363His is associated with increased expression and nuclear mobility, as well as lower rates of cell growth in HeLa cells, further supporting a role for cell proliferation in atherosclerosis and its clinical consequences.