Coronary Artery Disease Risk Loci Research Articles

Micro-capture-C in vessel wall cell types identifies mechanisms underlying coronary artery disease susceptibility loci

Abstract Genetics is estimated to account for 50% of Coronary Artery Disease (CAD) risk. Genome-Wide Association Studies (GWAS) have revealed 100s of genomic loci which are associated with CAD (1). However, moving from GWAS signals to causal genes has proved challenging, slowing progress in developing interventions for CAD and other diseases. The greatest challenge in interpreting GWAS data is that only 10% of variants are in protein-coding regions and 90% are in "non-coding" genomic regions. Many of these non-coding variants are found in regulatory elements such as enhancers. Enhancers are distal regulatory elements that bind transcription factors and regulate genes up to 1 million base pairs away. This is achieved by DNA looping bringing enhancers into physical proximity with target genes. However, it is not possible to predict which gene(s) a given enhancer will regulate based on genomic location alone. We aimed to develop a generalisable workflow to solve CAD GWAS signals accurately and sensitively, by identifying causal enhancer SNPs and linking them mechanistically to output genes in a cell-type specific manner. We combined cell-specific epigenomic mapping with machine learning to prioritise 21,959 SNPs from 861 candidate CAD loci, in endothelial cells (HUVECs) and smooth muscle cells (HCASMCs). Micro-Capture-C (MCC) was used to resolve enhancer-promoter interactions at base-pair resolution (2). MNase footprinting determined differential transcription factor binding at specific enhancer SNPs. In HCASMCs, 20 putative enhancers containing CAD SNPs were found to interact with 32 genes, and in HUVECs 25 enhancers interacted with 48 genes. These genes were enriched for pathways including TGFb signalling, RUNX3 activity, and MAPK6/MAPK4 signalling. To prove causality at specific loci, heterozygous SNPs were identified in the individual primary cell lines. The interaction frequency at these elements was statistically compared between risk and non-risk alleles. For example, at the 9p21.3 locus, an enhancer containing a non-risk T allele was found to interact more strongly with the CDKN2B gene, compared to the risk G allele. Interestingly, this imbalance between allele interaction was potentiated by stimulation with TGFb, a key mediator in atherosclerosis. Furthermore, the transcription factor footprint at the SNP location was altered by the risk allele, indicating the mechanistic cause of the CAD-associated change in CDKN2B expression. We demonstrate a combined computational and experimental approach to identify causal non-coding variants in CAD risk loci, at scale. Among the key outputs of this study is the identification of the risk variant (rs1537373), output gene (CDKN2B), and specific cell type (HCASMC) underlying the 9p21.3 locus association with CAD risk. Using this approach to translate GWAS signals into cell-type specific outputs genes, will lead to a better understanding of the pathogenesis of CAD, and new therapeutic opportunities.

Abstract Mo084: Stem Cell-Based Functional Genomics Unravel A Novel Vascular Smooth Muscle Cell State Induced By The 9p21 Coronary Artery Disease Risk Locus

Background: Genome-wide association studies have identified common genetic variants (Single Nucleotide Polymorphisms – SNPs) at ~300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. We have previously generated isogenic induced Pluripotent Stem Cells (iPSCs) lines from risk and non-risk donors at 9p21.3 and performed a complete deletion of the locus. By using iPSC-derived vascular smooth muscle cells (iPSC-VSMCs) we demonstrated that the risk haplotype at 9p21.3 causes altered expression of numerous genes essential for muscle function, including contraction and adhesion. Notably, deletion of the risk haplotype restores the non-risk phenotype, suggesting a gain of function effect. Here, we aimed to identify the transcriptomic signature and state trajectories of VSMCs induced by the 9p21.3 risk haplotype and validate them through functional assays and ex-vivo analysis of human coronary arteries. Methods: We have used iPSCs from individuals carrying the risk and non-risk haplotype at 9p21.3 and isogenic knockout lines. After VSMCs differentiation induction, mature VSMCs were used for single cell RNA sequencing using 10X Chromium platform and functional assays. Results: Our analysis revealed that the 9p21.3 risk haplotype prompts VSMCs to acquire a novel cellular state showing reduced plasticity and divergent from other VSMC states previously linked to CAD. We found markers of alternative lineages, including osteogenic and neuronal, coupled with altered expression of genes within other CAD loci, and functional defects. We identified a set of new molecular markers crucial to define risk-associated VSMCs and uncovered their upstream regulators. Conclusions: Our study provides insights into CAD pathogenesis driven by the 9p21.3 risk locus and identified new gene regulatory networks essential for maintaining the normal functionality of the muscle layer of the arteries. Leveraging the power of iPSCs we present novel concepts about the biology of VSMCs and shed light on the impact of the strongest CAD genetic risk factor in preserving a healthy cellular state within the vasculature.

The 9p21.3 coronary artery disease risk locus drives vascular smooth muscle cells to an osteochondrogenic state.

Genome-wide association studies have identified common genetic variants at ~400 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. Despite nearly two decades since its discovery, the functional mechanism of this genomic region remains incompletely resolved. To investigate the transcriptional gene programs mediated by 9p21.3 risk locus, we applied a model of induced pluripotent stem cells (iPSCs) from risk and non-risk donors at 9p21.3, as well as isogenic lines with a full haplotype deletion. Upon differentiation to vascular smooth muscle cells (VSMC), single-cell transcriptomic profiling demonstrated iPSC-VSMC phenotypes resembling those from native human coronary arteries, confirming the robustness of this model. Remarkably, our analyses revealed that VSMCs harboring the 9p21.3 risk haplotype preferentially adopt an osteochondrogenic state. Importantly, we identified LIMCH1 and CRABP1 as signature genes critical for defining this transcriptional program. Our study provides new insights into the mechanism at the 9p21.3 risk locus and defines its role in driving a disease-prone transcriptional state in VSMCs.

Abstract 2072: The 9p21.3 Coronary Artery Disease Risk Locus Induces Cell State Transitions In Vascular Smooth Muscle Cells

Background: Genome-wide association studies have identified common genetic variants (Single Nucleotide Polymorphisms - SNPs) at ~300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. We have previously generated isogenic induced Pluripotent Stem Cells (iPSCs) lines from risk and non-risk donors at 9p21.3 and performed a complete deletion of the locus. By using iPSC-derived vascular smooth muscle cells (iPSC-VSMCs) we demonstrated that the risk haplotype at 9p21.3 causes altered expression of numerous genes essential for muscle function, including contraction and adhesion. Notably, deletion of the risk haplotype restores the non-risk phenotype, suggesting a gain of function effect. Hypothesis: The 9p21.3 risk haplotype alters VSMCs plasticity and induces dedifferentiation and alternative lineage acquisition. Aims: Identify transcriptomic signatures and trajectories of VSMCs induced by the 9p21.3 risk haplotype Methods: We have used iPSCs from individuals carrying the risk and non-risk haplotype at 9p21.3 and isogenic knockout lines. After VSMCs differentiation induction, mature VSMCs were used for single cell RNA sequencing using 10X Chromium platform. Results: Our analysis revealed that the 9p21.3 risk haplotype prompts VSMCs to acquire a novel cellular state showing reduced plasticity and divergent from other VSMC states previously linked to CAD. We found markers of alternative lineages, including osteogenic and neuronal, coupled with altered expression of genes within other CAD loci. We identified a set of new molecular markers crucial to define risk-associated VSMCs and uncovered their upstream regulators. Conclusions: Our study provides insights into CAD pathogenesis driven by the 9p21.3 risk locus and identified new gene regulatory networks essential for maintaining the normal functionality of the muscle layer of the arteries. Leveraging the power of iPSCs we present novel concepts about the biology of VSMCs and shed light on the impact of CAD genetic risk factors in preserving a healthy cellular state within the vasculature.

Abstract 13953: A Genome-Wide European Ancestry Study Identifies Coronary Artery Disease-Associated Loci Through Gene-Sex Hormone Interaction

Background: Genome-wide association studies (GWAS) have improved our understanding of the genetic basis of coronary artery disease (CAD). While sex-differences in CAD have been observed, little is known about the role of sex hormones in CAD genetics. Accounting for sex hormone levels may help identify CAD-risk loci and extend our knowledge of its genetic architecture. Methods: We performed a GWAS to identify CAD risk loci by incorporating gene-sex hormone interactions in 365,662 individuals of European ancestry enrolled in the UK Biobank (UKB). Genetic interactions of total testosterone (TT), bioavailable testosterone (BAT) and sex-hormone binding globulin (SHBG) were evaluated. GEM software was used to conduct sex-stratified genome-wide interaction analysis with prevalent CAD. Enrollment age and principal components 1-10 were model covariates. The online post-GWAS tool FUMA was used to annotate the biological functions of marginal, interaction, and joint effects summary statistics. Results: We identified 64 loci in males and 8 loci in females that reached genome-wide significance (p < 5х10 -8 ) for CAD ( Table 1 ). Among males, 3 significant loci were identified for BAT, one for TT and one for SHBG using joint effect model that did not overlap with those identified with marginal effect. Among females, 5 significant loci were identified, all for TT using joint effect that that did not overlap with those identified with marginal effect. Of these 5 loci, two loci were independently identified with significant SNP-TT interaction. These loci were mapped close to the TRDN and THADA genes that have been implicated in congenital ventricular tachycardia and polycystic ovarian syndrome, respectively. Conclusion: This Genome-wide gene-sex hormone interaction study identified genomic-risk loci that may contribute to the differential CAD risk between men and women, which would otherwise not have been discovered in traditional GWAS only including marginal genetic effect.

Polygenic Risk Score Associates With Atherosclerotic Plaque Characteristics at Autopsy.

Polygenic risk scores (PRSs) for coronary artery disease (CAD) potentially improve cardiovascular risk prediction. However, their relationship with histopathologic features of CAD has never been examined systematically. From 4327 subjects referred to CVPath by the State of Maryland Office Chief Medical Examiner for sudden death between 1994 and 2015, 2455 cases were randomly selected for genotyping. We generated PRS from 291 known CAD risk loci. Detailed histopathologic examination of the coronary arteries was performed in all subjects. The primary study outcome measurements were histopathologic plaque features determining severity of atherosclerosis, including %stenosis, calcification, thin-cap fibroatheromas, and thrombotic CAD. After exclusion of cases with insufficient DNA sample quality or with missing data, 954 cases (mean age, 48.8±14.7 years; 75.7% men) remained in the final study cohort. Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared with subjects in the lowest quintile, with greater %stenosis (80.3%±27.0% versus 50.4%±38.7%; adjusted P<0.001) and a higher frequency of calcification (69.6% versus 35.8%; adjusted P=0.004) and thin-cap fibroatheroma (26.7% versus 9.5%; adjusted P=0.007). Even after adjustment for traditional CAD risk factors, subjects within the highest PRS quintile had higher odds of severe atherosclerosis (ie, ≥75% stenosis; adjusted odds ratio, 3.77 [95% CI, 2.10-6.78]; P<0.001) and plaque rupture (adjusted odds ratio, 4.05 [95% CI, 2.26-7.24]; P<0.001). Moreover, subjects within the highest quintile had higher odds of CAD-associated cause of death, especially among those aged ≤50 years (adjusted odds ratio, 4.08 [95% CI, 2.01-8.30]; P<0.001). No statistically significant associations were observed with plaque erosion after adjusting for covariates. This is the first autopsy study investigating associations between PRS and atherosclerosis severity at the histopathologic level in subjects with sudden death. Our pathological analysis suggests PRS correlates with plaque burden and features of advanced atherosclerosis and may be useful as a method for CAD risk stratification, especially in younger subjects.

Discovery and systematic characterization of risk variants and genes for coronary artery disease in over a million participants

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR–Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD.

Polygenic risk score associates with atherosclerosis severity at autopsy

Abstract Background Polygenic risk scores (PRS) for coronary artery disease (CAD) are emerging as a potential method to improve cardiovascular risk prediction. Questions remain about their applicability to diverse populations as well as their correlation with coronary histopathology. Purpose To assess whether high genetic risk associates with histopathologic coronary plaque morphology. Methods We assessed 122 known CAD risk loci in 954 Black and White subjects within our sudden death registry to generate a PRS. The cohort was divided into quintiles according to z-score-standardized PRS, both in a race-stratified fashion and in the pooled sample. Detailed histopathologic examination of the coronary arteries was performed in all subjects. Results Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared to subjects in the lowest quintile, with greater mean cross-sectional luminal narrowing (71.5% (95% CI, 66.6%-76.5%) vs. 56.6% (95% CI, 51.1%-62.1%); adjusted p&amp;lt;0.001; Figure 1) and a higher frequency of calcification (adjusted OR 2.19; 95% CI 1.31–3.68; p=0.003) after adjustment for the first 10 principal components, age, sex, and race. Higher z-score-standardized PRS was predictive for the finding of severe atherosclerosis (i.e., ≥75% cross-sectional luminal narrowing) even after additional controlling for traditional CAD risk factors including hypertension, smoking, diabetes mellitus, and hyperlipidemia (adjusted OR 1.39; 95% CI 1.19–1.63; p&amp;lt;0.001; Figure 2). Among Black subjects, higher PRS was associated with higher odds of plaque rupture (adjusted OR 1.31; 95% CI 1.03–1.66; p=0.03) and predicted CAD-associated cause of death among subjects younger than 50 years old (adjusted OR 1.26; 95% CI 1.01–1.58; p=0.04). Conclusions This is the first autopsy study investigating associations between PRS and atherosclerotic plaque morphology at a histopathologic level. Our pathological analysis suggests PRS correlates with plaque burden and coronary artery calcification and may be useful as a method for CAD risk stratification, especially in younger subjects. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): R01 HL141425 Leducq Foundation Grant

Genomic prediction of cardiovascular events in a coronary Southern European population

Abstract Background Traditional and clinical risk factors are indicators of atherosclerosis over time and strong independent predictors of cardiovascular events, but it is unknown whether other genetic markers could provide information about the evolution of atherosclerotic coronary artery disease (CAD). Objective We propose identifying the genetic predisposition to atherosclerotic plaque progression and events occurrence, through a study cohort from GENEMACOR study population. Methods We performed a study with a cohort of 1,712 patients who underwent coronary angiography with more than 70% stenosis of at least one main coronary vessel, during a mean follow-up of 5 years (amplitude range 20 years). 33 SNPs associated with risk of CAD in previous GWAS, were genotyped by TaqMan assays methodology. The best model in the bivariate analysis at 95% CI with all genetic variants was generated, to investigate their association with prognostic and events occurrence. The hazard function at a set of confounding-variables was determined to evaluate their relationship with the event's incidence by the Cox survival analysis regression model. Finally, we constructed Kaplan–Meier cumulative-event curves for the significant variants. Results The analysis revealed two SNPs associated with the progression of atherosclerosis and events occurrence: rs12190287 G&amp;gt;C in the TCF21 gene on chromosome 10 (dominant model; OR=1.542; 95% CI 1.069 – 2.224; p=0.020) and the rs1333049 G&amp;gt;C in the CDKN2-AS1 gene on chromosome 9 (recessive model; OR=1.228; 95% CI 1.001 – 1.518; p=0.050). The Kaplan-Meier cumulative event curves in the TCF21 variant rs12190287 G&amp;gt; C showed that the GC+CC vs GG genotype was associated with a worse prognosis (log-rank test, p=0.016) and the CDKN2B-AS1 rs1333049 G&amp;gt; C revealed that the CC vs GG+GC genotype also presented severe prognosis and more events at the end of the follow-up period (log-rank test, p=0.046). Conclusion We have identified two SNPs associated with the prognosis of CAD, rs12190287 of TCF21 gene and rs1333049 of CDKN2-AS1 gene. Both are in non-coding enhancer regions and regulate transcriptional mechanisms shared among multiple CAD risk loci and could provide new insights into CAD's pathophysiology identifying core mechanisms for therapeutic intervention modulating the disease risk. Funding Acknowledgement Type of funding sources: None.

24-OR: Time-to-Event GWAS for Incident Cardiovascular Disease in Type 2 Diabetes Patients

Patients with type 2 diabetes (T2D) are at two-fold increased risk of cardiovascular disease (CVD), which is the leading cause of T2D morbidity and mortality. Here, we conducted a time-to-event genome-wide association study (GWAS) to identify genetic variants associated with incident CVD among patients with T2D in the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium. Incident CVD was defined based on a composite of myocardial infarction, stroke, and cardiovascular death that occurred at least one year after the diagnosis of T2D. In each participating prospective cohort, we performed GWAS using a Cox proportional hazard survival model adjusting for age, and sex to identify genetic variants associated with incident CVD, and cohort-level estimated effect sizes were combined using inverse variance weighted fixed effects meta-analysis. A total of 37,327 participants were included in the analyses (26,928 European ancestry and 10,399 non-European ancestry) which comprised 6,012 primary event cases. We identified three distinct genetic loci associated with incident CVD among individuals with T2D that reached the threshold for genome-wide significance: rs231964 (intronic variant in NOX3, hazard ratio [HR] 1.4, P=8.3x10-9), rs139753656 (intronic variant in DCC, HR 2.0, P=2.4x10-8), and rs77202398 (intronic variant in TNS3, HR 1.4, P=4.4x10-8). Among 204 known coronary artery disease risk loci, 27 were associated with incident CVD with P&amp;lt;0.05 and rs6842241 upstream of EDNRA was associated with increased risk of CVD after Bonferroni correction (HR 1.1, P=1.2x10-4). The data provide evidence of novel and known genomic regions associated with incident CVD among individuals with T2D that require validation but point to novel targets. Disclosure S. Kwak: None. P. Wu: None. J. B. Meigs: Consultant; Self; Quest Diagnostics. On behalf of the charge diabetes working group: n/a. Funding National Institutes of Health (1R01HL151855-01)

Sortilin as a Biomarker for Cardiovascular Disease Revisited.

Genetic variants in the genomic region containing SORT1 (encoding the protein sortilin) are strongly associated with cholesterol levels and the risk of coronary artery disease (CAD). Circulating sortilin has therefore been proposed as a potential biomarker for cardiovascular disease. Multiple studies have reported association between plasma sortilin levels and cardiovascular outcomes. However, the findings are not consistent across studies, and most studies have small sample sizes. The aim of this study was to evaluate sortilin as a biomarker for CAD in a well-characterized cohort with symptoms suggestive of CAD. In total, we enrolled 1,173 patients with suspected stable CAD referred to coronary computed tomography angiography. Sortilin was measured in plasma using two different technologies for quantifying circulating sortilin: a custom-made enzyme-linked immunosorbent assay (ELISA) and OLINK Cardiovascular Panel II. We found a relative poor correlation between the two methods (correlation coefficient = 0.21). In addition, genotyping and whole-genome sequencing were performed on all patients. By whole-genome regression analysis of sortilin levels measured with ELISA and OLINK, two independent cis protein quantitative trait loci (pQTL) on chromosome 1p13.3 were identified, with one of them being a well-established risk locus for CAD. Incorporating rare genetic variants from whole-genome sequence data did not identify any additional pQTLs for plasma sortilin. None of the traditional CAD risk factors, such as sex, age, smoking, and statin use, were associated with plasma sortilin levels. Furthermore, there was no association between circulating sortilin levels and coronary artery calcium score (CACS) or disease severity. Sortilin did not improve discrimination of obstructive CAD, when added to a clinical pretest probability (PTP) model for CAD. Overall, our results indicate that studies using different methodologies for measuring circulating sortilin should be compared with caution. In conclusion, the well-known SORT1 risk locus for CAD is linked to lower sortilin levels in circulation, measured with ELISA; however, the effect sizes are too small for sortilin to be a useful biomarker for CAD in a clinical setting of low- to intermediate-risk chest-pain patients.

Genome-wide association study on coronary artery disease in type 1 diabetes suggests beta-defensin 127 as a risk locus.

Diabetes is a known risk factor for coronary artery disease (CAD). There is accumulating evidence that CAD pathogenesis differs for individuals with type 1 diabetes (T1D). However, the genetic background has not been extensively studied. We aimed to discover genetic loci increasing CAD susceptibility, especially in T1D, to examine the function of these discoveries and to study the role of the known risk loci in T1D. We performed the largest genome-wide association study to date for CAD in T1D, comprising 4869 individuals with T1D (cases/controls: 941/3928). Two loci reached genome-wide significance, rs1970112 in CDKN2B-AS1 [odds ratio (OR) = 1.32, P = 1.50 × 10-8], and rs6055069 on DEFB127 promoter (OR = 4.17, P = 2.35 × 10-9), with consistent results in survival analysis. The CDKN2B-AS1 variant replicated (P = 0.04) when adjusted for diabetic kidney disease in three additional T1D cohorts (cases/controls: 434/3123). Furthermore, we explored the function of the lead discoveries with a cardio-phenome-wide analysis. Among the eight suggestive loci (P < 1 × 10-6), rs70962766 near B3GNT2 associated with central blood pressure, rs1344228 near CNTNAP5 with intima media thickness, and rs2112481 on GRAMD2B promoter with serum leucocyte concentration. Finally, we calculated genetic risk scores for individuals with T1D with the known susceptibility loci. General population risk variants were modestly but significantly associated with CAD also in T1D (P = 4.21 × 10-7). While general population CAD risk loci had limited effect on the risk in T1D, for the first time, variants at the CDKN2B-AS1 locus were robustly associated with CAD in individuals with T1D. The novel finding on β-defensin DEFB127 promoter provides a link between diabetes, infection susceptibility, and CAD, although pending on future confirmation.

9p21.3 coronary artery disease risk locus and interferon alpha 21: Association study in an Asian Indian population

IntroductionType I Interferons (INFαs and INF β) are known to be proinflammatory cytokines that promote atherosclerosis. IFNA21 is a member of alpha Interferon gene cluster on short arm of chromosome 9. We analyzed the potential link between 9p21 coronary artery disease (CAD) risk locus and IFNA21. Objectivesa) study of association between serum IFNA21 levels and 14 demographic/clinical variables, including age, gender, diabetes, hypertension, and duration of CAD, b) study of association between high serum IFNA21 levels and 30 9p21 SNP genotypes. MethodsTo estimate serum circulating levels of IFNA21, we performed sandwich ELISA in 184 controls and 167 CAD cases. The IFNA21 levels could be classified into two broad classes: a) Low-level group: ≤15.6 pg/ml b) High-level group: >15.6 pg/ml. We also performed SNP genotyping for 30 SNPs at 9p21 locus in all study subjects using Sequenom MassARRAY technology. Statistical software SPSS (Version 21) was used to analyze the data obtained. ResultsOur analysis indicates that there could be an association of high IFNA21 levels with variables – gender, age, and duration of CAD in the study population. SNPs rs10757272 (TT), rs10757274 (GG), rs10757283 (TT), rs1333045 (CC), rs1333048 (CC), rs1333049 (CC), and rs4977574 (GG) showed significant risk association with high-level IFNA21 group. ConclusionsIFNA21 may be involved in inflammatory processes in an age-dependent manner and in progression of CAD. This IFNA21-mediated mechanism may be more active in females. Several 9p21 SNPs may modulate inflammatory processes mediated by IFNA21 and may, therefore, contribute to pathophysiology of CAD.

Genetic variation at the coronary artery disease risk locus GUCY1A3 modifies cardiovascular disease prevention effects of aspirin.

Efficacy of aspirin in primary prevention of cardiovascular disease (CVD) may be influenced by a common allele in guanylate cyclase GUCY1A3, which has been shown to modify platelet function and increase CVD risk. We investigated whether homozygotes of the GUCY1A3 rs7692387 risk (G) allele benefited from aspirin in two long-term, randomized placebo-controlled trials of aspirin in primary CVD prevention: the Women's Genome Health Study (WGHS, N = 23294) and a myocardial infarction (MI, N = 550) and stroke (N = 382) case-control set from the Physician's Health Study (PHS, N = 22071). Bleeding risk was evaluated in the WGHS. In the placebo group of the WGHS, the GUCY1A3 risk (G) allele was confirmed to increase CVD risk [hazard ratio 1.38; 95% confidence interval (CI) 1.08-1.78; P = 0.01]. Random-effects meta-analysis of the WGHS and PHS revealed that aspirin reduced CVD events among risk allele homozygotes [G/G: odds ratio (OR) 0.79; 95% CI 0.65-0.97; P = 0.03] but increased CVD events among non-risk allele carriers (e.g. G/A: OR 1.39; 95% CI 1.03-1.87; P = 0.03) thus implying an interaction between genotype stratum and aspirin intake (Pinteraction = 0.01). Bleeding associated with aspirin increased in all genotype groups, with higher risks in heterozygotes. In two randomized placebo-controlled trials in the setting of primary prevention, aspirin reduced the incidence of CVD events in individuals homozygous for the GUCY1A3 risk (G) allele, whereas heterozygote individuals had more events when taking aspirin.

Genome-wide association study of white-coat effect in hypertensive patients

Background: White-coat effect (WCE) confounds diagnosis and treatment of hypertension. The prevalence of white-coat hypertension is higher in Europe and Asia compared to other continents suggesting that genetic factors could play a role.Methods: To study genetic variation affecting WCE, we conducted a two-stage genome-wide association study involving 1343 Finnish subjects. For the discovery stage, we used Genetics of Drug Responsiveness in Essential Hypertension (GENRES) cohort (n = 206), providing the mean WCE values from up to four separate office/ambulatory recordings conducted on placebo. Associations with p values <1 × 10−5 were included in the replication step in three independent cohorts: Haemodynamics in Primary and Secondary Hypertension (DYNAMIC) (n = 182), Finn-Home study (n = 773) and Dietary, Lifestyle and Genetic Determinants of Obesity and Metabolic Syndrome (DILGOM) (n = 182).Results: No single nucleotide polymorphisms reached genome-wide significance for association with either systolic or diastolic WCE. However, two loci provided suggestive evidence for association. A known coronary artery disease risk locus rs2292954 in SPG7 associated with systolic WCE (discovery p value = 2.2 × 10−6, replication p value = 0.03 in Finn-Home, meta-analysis p value 2.6 × 10−4), and rs10033652 in RASGEF1B with diastolic WCE (discovery p value = 4.9 × 10−6, replication p value = 0.04 in DILGOM, meta-analysis p value = 5.0 × 10−3).Conclusion: This study provides evidence for two novel candidate genes, SPG7 and RASGEF1B, associating with WCE. Our results need to be validated in even larger studies carried out in other populations.

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

Background and aimsA 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. MethodsThe 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. ResultsThe 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. ConclusionsThese 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.

Effects of the coronary artery disease associated LPA and 9p21 loci on risk of aortic valve stenosis

BackgroundAortic valve stenosis (AVS) and coronary artery disease (CAD) have a significant genetic contribution and commonly co-exist. To compare and contrast genetic determinants of the two diseases, we investigated associations of the LPA and 9p21 loci, i.e. the two strongest CAD risk loci, with risk of AVS. MethodsWe genotyped the CAD-associated variants at the LPA (rs10455872) and 9p21 loci (rs1333049) in the GeneCAST (Genetics of Calcific Aortic STenosis) Consortium and conducted a meta-analysis for their association with AVS. Cases and controls were stratified by CAD status. External validation of findings was undertaken in five cohorts including 7880 cases and 851,152 controls. ResultsIn the meta-analysis including 4651 cases and 8231 controls the CAD-associated allele at the LPA locus was associated with increased risk of AVS (OR 1.37; 95%CI 1.24–1.52, p = 6.9 × 10−10) with a larger effect size in those without CAD (OR 1.53; 95%CI 1.31–1.79) compared to those with CAD (OR 1.27; 95%CI 1.12–1.45). The CAD-associated allele at 9p21 was associated with a trend towards lower risk of AVS (OR 0.93; 95%CI 0.88–0.99, p = 0.014). External validation confirmed the association of the LPA risk allele with risk of AVS (OR 1.37; 95%CI 1.27–1.47), again with a higher effect size in those without CAD. The small protective effect of the 9p21 CAD risk allele could not be replicated (OR 0.98; 95%CI 0.95–1.02). ConclusionsOur study confirms the association of the LPA locus with risk of AVS, with a higher effect in those without concomitant CAD. Overall, 9p21 was not associated with AVS.

Genome-wide association meta-analysis of coronary artery disease and periodontitis reveals a novel shared risk locus

Evidence for a shared genetic basis of association between coronary artery disease (CAD) and periodontitis (PD) exists. To explore the joint genetic basis, we performed a GWAS meta-analysis. In the discovery stage, we used a German aggressive periodontitis sample (AgP-Ger; 680 cases vs 3,973 controls) and the CARDIoGRAMplusC4D CAD meta-analysis dataset (60,801 cases vs 123,504 controls). Two SNPs at the known CAD risk loci ADAMTS7 (rs11634042) and VAMP8 (rs1561198) passed the pre-assigned selection criteria (PAgP-Ger < 0.05; PCAD < 5 × 10−8; concordant effect direction) and were replicated in an independent GWAS meta-analysis dataset of PD (4,415 cases vs 5,935 controls). SNP rs1561198 showed significant association (PD[Replication]: P = 0.008 OR = 1.09, 95% CI = [1.02–1.16]; PD [Discovery + Replication]: P = 0.0002, OR = 1.11, 95% CI = [1.05–1.17]). For the associated haplotype block, allele specific cis-effects on VAMP8 expression were reported. Our data adds to the shared genetic basis of CAD and PD and indicate that the observed association of the two disease conditions cannot be solely explained by shared environmental risk factors. We conclude that the molecular pathway shared by CAD and PD involves VAMP8 function, which has a role in membrane vesicular trafficking, and is manipulated by pathogens to corrupt host immune defense.

Genetic Regulatory Mechanisms of Smooth Muscle Cells Map to Coronary Artery Disease Risk Loci

Coronary artery disease (CAD) is the leading cause of death globally. Genome-wide association studies (GWASs) have identified more than 95 independent loci that influence CAD risk, most of which reside in non-coding regions of the genome. To interpret these loci, we generated transcriptome and whole-genome datasets using human coronary artery smooth muscle cells (HCASMCs) from 52 unrelated donors, as well as epigenomic datasets using ATAC-seq on a subset of 8 donors. Through systematic comparison with publicly available datasets from GTEx and ENCODE projects, we identified transcriptomic, epigenetic, and genetic regulatory mechanisms specific to HCASMCs. We assessed the relevance of HCASMCs to CAD risk using transcriptomic and epigenomic level analyses. By jointly modeling eQTL and GWAS datasets, we identified five genes (SIPA1, TCF21, SMAD3, FES, and PDGFRA) that may modulate CAD risk through HCASMCs, all of which have relevant functional roles in vascular remodeling. Comparison with GTEx data suggests that SIPA1 and PDGFRA influence CAD risk predominantly through HCASMCs, while other annotated genes may have multiple cell and tissue targets. Together, these results provide tissue-specific and mechanistic insights into the regulation of a critical vascular cell type associated with CAD in human populations.

Druggability of Coronary Artery Disease Risk Loci.

Genome-wide association studies have identified multiple loci associated with coronary artery disease and myocardial infarction, but only a few of these loci are current targets for on-market medications. To identify drugs suitable for repurposing and their targets, we created 2 unique pipelines integrating public data on 49 coronary artery disease/myocardial infarction-genome-wide association studies loci, drug-gene interactions, side effects, and chemical interactions. We first used publicly available genome-wide association studies results on all phenotypes to predict relevant side effects, identified drug-gene interactions, and prioritized candidates for repurposing among existing drugs. Second, we prioritized gene product targets by calculating a druggability score to estimate how accessible pockets of coronary artery disease/myocardial infarction-associated gene products are, then used again the genome-wide association studies results to predict side effects, excluded loci with widespread cross-tissue expression to avoid housekeeping and genes involved in vital processes and accordingly ranked the remaining gene products. These pipelines ultimately led to 3 suggestions for drug repurposing: pentolinium, adenosine triphosphate, and riociguat (to target CHRNB4, ACSS2, and GUCY1A3, respectively); and 3 proteins for drug development: LMOD1 (leiomodin 1), HIP1 (huntingtin-interacting protein 1), and PPP2R3A (protein phosphatase 2, regulatory subunit b-double prime, α). Most current therapies for coronary artery disease/myocardial infarction treatment were also rediscovered. Integration of genomic and pharmacological data may prove beneficial for drug repurposing and development, as evidence from our pipelines suggests.