Multi-ancestry genome-wide study identifies effector genes and druggable pathways for coronary artery calcification.

Coronary artery calcification (CAC), a measure of subclinical atherosclerosis, predicts future symptomatic coronary artery disease (CAD). Identifying genetic risk factors for CAC may point to new therapeutic avenues for prevention. Currently, there are only four known risk loci for CAC identified from genome-wide association studies (GWAS) in the general population. Here we conducted the largest multi-ancestry GWAS meta-analysis of CAC to date, which comprised 26,909 individuals of European ancestry and 8,867 individuals of African ancestry. We identified 11 independent risk loci, of which eight were new for CAC and five had not been reported for CAD. These new CAC loci are related to bone mineralization, phosphate catabolism and hormone metabolic pathways. Several new loci harbor candidate causal genes supported by multiple lines of functional evidence and are regulators of smooth muscle cell-mediated calcification ex vivo and in vitro. Together, these findings help refine the genetic architecture of CAC and extend our understanding of the biological and potential druggable pathways underlying CAC.

WHOLE GENOME SEQUENCING ANALYSIS OF BODY MASS INDEX IDENTIFIES NOVEL AFRICAN ANCESTRY-SPECIFIC RISK ALLELE.

Obesity is a major public health crisis associated with high mortality rates. Previous genome-wide association studies (GWAS) investigating body mass index (BMI) have largely relied on imputed data from European individuals. This study leveraged whole-genome sequencing (WGS) data from 88,873 participants from the Trans-Omics for Precision Medicine (TOPMed) Program, of which 51% were of non-European population groups. We discovered 18 BMI-associated signals (P < 5 × 10-9). Notably, we identified and replicated a novel low frequency single nucleotide polymorphism (SNP) in MTMR3 that was common in individuals of African descent. Using a diverse study population, we further identified two novel secondary signals in known BMI loci and pinpointed two likely causal variants in the POC5 and DMD loci. Our work demonstrates the benefits of combining WGS and diverse cohorts in expanding current catalog of variants and genes confer risk for obesity, bringing us one step closer to personalized medicine.

Multi-ancestry transcriptome-wide association analyses yield insights into tobacco use biology and drug repurposing.

Most transcriptome-wide association studies (TWASs) so far focus on European ancestry and lack diversity. To overcome this limitation, we aggregated genome-wide association study (GWAS) summary statistics, whole-genome sequences and expression quantitative trait locus (eQTL) data from diverse ancestries. We developed a new approach, TESLA (multi-ancestry integrative study using an optimal linear combination of association statistics), to integrate an eQTL dataset with a multi-ancestry GWAS. By exploiting shared phenotypic effects between ancestries and accommodating potential effect heterogeneities, TESLA improves power over other TWAS methods. When applied to tobacco use phenotypes, TESLA identified 273 new genes, up to 55% more compared with alternative TWAS methods. These hits and subsequent fine mapping using TESLA point to target genes with biological relevance. In silico drug-repurposing analyses highlight several drugs with known efficacy, including dextromethorphan and galantamine, and new drugs such as muscle relaxants that may be repurposed for treating nicotine addiction.

Genetic diversity fuels gene discovery for tobacco and alcohol use.

Tobacco and alcohol use are heritable behaviours associated with 15% and 5.3% of worldwide deaths, respectively, due largely to broad increased risk for disease and injury1-4. These substances are used across the globe, yet genome-wide association studies have focused largely on individuals of European ancestries5. Here we leveraged global genetic diversity across 3.4 million individuals from four major clines of global ancestry (approximately 21% non-European) to power the discovery and fine-mapping of genomic loci associated with tobacco and alcohol use, to inform function of these loci via ancestry-aware transcriptome-wide association studies, and to evaluate the genetic architecture and predictive power of polygenic risk within and across populations. We found that increases in sample size and genetic diversity improved locus identification and fine-mapping resolution, and that a large majority of the 3,823 associated variants (from 2,143 loci) showed consistent effect sizes across ancestry dimensions. However, polygenic risk scores developed in one ancestry performed poorly in others, highlighting the continued need to increase sample sizes of diverse ancestries to realize any potential benefit of polygenic prediction.

Rare genetic variants explain missing heritability in smoking.

Common genetic variants explain less variation in complex phenotypes than inferred from family-based studies, and there is a debate on the source of this 'missing heritability'. We investigated the contribution of rare genetic variants to tobacco use with whole-genome sequences from up to 26,257 unrelated individuals of European ancestries and 11,743 individuals of African ancestries. Across four smoking traits, single-nucleotide-polymorphism-based heritability ([Formula: see text]) was estimated from 0.13 to 0.28 (s.e., 0.10-0.13) in European ancestries, with 35-74% of it attributable to rare variants with minor allele frequencies between 0.01% and 1%. These heritability estimates are 1.5-4 times higher than past estimates based on common variants alone and accounted for 60% to 100% of our pedigree-based estimates of narrow-sense heritability ([Formula: see text], 0.18-0.34). In the African ancestry samples, [Formula: see text] was estimated from 0.03 to 0.33 (s.e., 0.09-0.14) across the four smoking traits. These results suggest that rare variants are important contributors to the heritability of smoking.

Association of Vascular Properties With the Brain White Matter Hyperintensity in Middle-Aged Population.

Background The periventricular white matter is more sensitive to the systemic hemodynamic alterations than the deep white matter because of differences in its vascular structure and systemic circulation relationship. We hypothesize that periventricular white matter hyperintensity (PVWMH) volume shows greater association than deep white matter hyperintensity (DWMH) volume with vascular properties (VPs) reflecting arterial stiffness and cardiovascular remodeling, indicators of the systemic circulation. Methods and Results A total of 426 participants (age, 59.0±6.1 years; 57.5% women; and 39.7% Black race) in the Genetic Study of Atherosclerosis Risk who were aged ≥50 years and had brain magnetic resonance imaging were studied. VPs included pulse pressure, hypertensive response to exercise, diastolic brachial artery diameter, diastolic common carotid artery diameter, common carotid artery distensibility coefficient, and left ventricular function. The relative associations of VPs with PVWMH and DWMH as multiple measures within the same individual were determined using multilevel linear models. We also determined if age modified the differences in VPs associations with PVWMH and DWMH. Our findings indicated that, within the same subject, PVWMH volume had greater association than DWMH volume with pulse pressure (P=0.002), hypertensive response to exercise (P=0.04), diastolic brachial artery diameter (P=0.012), and diastolic common carotid artery diameter (P=0.04), independent of age and cardiovascular risk factors. The differences of PVWMH versus DWMH associations with VPs did not differ at any age threshold. Conclusions We show, for the first time, that PVWMH has greater association than DWMH, independent of age, with vascular measurements of arterial stiffness and cardiovascular remodeling suggesting that changes in the systemic circulation affect the PVWMH and DWMH differently.

Cerebral Small-Vessel Disease in Individuals with a Family History of Coronary Heart Disease: The Atherosclerosis Risk in Communities Study.

INTRODUCTION: The degree to which a family history of coronary heart disease (FHCHD) is associated with silent cerebral small-vessel disease (cSVD) among healthy adults, independent of prevalent CHD and traditional risk factors, is unknown. METHODS: The Atherosclerosis Risk in Communities (ARIC) study is a community-based cohort study with self-reported family history data and brain magnetic resonance imaging (ages 68-88). The association between markers of cSVD (lacunar infarcts and cerebral microbleeds), or log-transformed white matter hyperintensity (WMH) volume, and FHCHD, or the number of affected relatives was examined using separate adjusted logistic or linear regression models, respectively. Race interaction terms were evaluated. RESULTS: Of 1,639 participants without prevalent CHD (76 ± 5 years, 62% female, 29% black), 686 (42%) had FHCHD. There were higher odds of lacunar infarct (OR 1.40, 95% CI 1.07-1.84) among those with parental FHCHD and higher odds of microhemorrhages (lobar OR 1.86, 95% CI 1.13-3.06; subcortical OR 1.47, 95% CI 1.01-2.15) among those with sibling FHCHD. A greater number of any relative affected was associated with higher odds of lacunar infarct (OR 1.24, 95% CI 1.04-1.47) and lobar microhemorrhages (OR 1.31, 95% CI 1.05-1.64) but not subcortical microhemorrhages (OR 1.09, 95% CI 0.92-1.28). Odds of having a lacunar infarct were higher among blacks (p-interaction 0.04) with paternal FHCHD (OR 2.20, CI 1.35-3.58) than whites with paternal FHCHD (OR 1.17, CI 0.87-1.56). There was no association with WMH. DISCUSSION/CONCLUSION: Markers of cSVD, specifically lacunar infarcts and microhemorrhages, appear to be associated with FHCHD, potentially representing shared mechanisms in different vascular beds, and perhaps a genetic propensity for vascular disease.

Association of Coronary Artery Atherosclerosis With Brain White Matter Hyperintensity.

Background and Purpose: We aim to determine, in healthy high-risk adults, the association between subclinical coronary artery disease and white matter hyperintensity (WMH) volume and location, independent of atherosclerotic risk factors. Methods: Seven hundred eighty-two asymptomatic first-degree relatives of index cases with early-onset coronary artery disease (<60 years old) from GeneSTAR (Genetic Study of Atherosclerosis Risk) with contemporaneous coronary computed tomography angiography and brain magnetic resonance imaging were analyzed. Multilevel mixed-effects linear regression models, accounting for family structure, evaluated the association of total WMH volume and 3 regions (deep WMH, periventricular WMH [PVWMH], or borderzone [cuff]) with markers of coronary artery disease. Separate models were created for total WMH, deep WMH, PVWMH, and cuff volumes, each, as dependent variables, across coronary computed tomography angiography variables, adjusted for covariates. Results: Mean age was 51 years ±10, with 58% women and 39% African American people. Participants with any coronary plaque had 52% larger WMH volumes than those without plaque (95% CI, 0.24­0.59). Per 1% greater coronary plaque volume, total WMH volumes were 0.07% larger (95% CI, 0.04­0.10). Every 1% higher total coronary plaque volume was associated with 5.03% larger deep WMH volume (95% CI, 4.67­5.38), 5.10% PVWMH larger volume (95% CI, 4.72­5.48), and 2.74% larger cuff volume (95% CI, 2.38­3.09) with differences in this association when comparing deep WMH to PVWMH (P interaction, 0.001) or cuff (P interaction, <0.001), respectively. Conclusions: In healthy, high-risk individuals, the presence and volume of coronary artery plaque are associated with larger WMH volumes, appearing the strongest for PVWMH. These findings in high-risk families suggest a disease relationship in 2 different vascular beds, beyond traditional risk factors, possibly due to genetic predisposition.

FGL1 as a modulator of plasma D-dimer levels: Exome-wide marker analysis of plasma tPA, PAI-1, and D-dimer.

BACKGROUND: Use of targeted exome-arrays with common, rare variants and functionally enriched variation has led to discovery of new genes contributing to population variation in risk factors. Plasminogen activator-inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), and the plasma product D-dimer are important components of the fibrinolytic system. There have been few large-scale genome-wide or exome-wide studies of PAI-1, tPA, and D-dimer. OBJECTIVES: We sought to discover new genetic loci contributing to variation in these traits using an exome-array approach. METHODS: Cohort-level analyses and fixed effects meta-analyses of PAI-1 (n = 15 603), tPA (n = 6876,) and D-dimer (n = 19 306) from 12 cohorts of European ancestry with diverse study design were conducted, including single-variant analyses and gene-based burden testing. RESULTS: Five variants located in NME7, FGL1, and the fibrinogen locus, all associated with D-dimer levels, achieved genome-wide significance (P < 5 × 10-8 ). Replication was sought for these 5 variants, as well as 45 well-imputed variants with P < 1 × 10-4 in the discovery using an independent cohort. Replication was observed for three out of the five significant associations, including a novel and uncommon (0.013 allele frequency) coding variant p.Trp256Leu in FGL1 (fibrinogen-like-1) with increased plasma D-dimer levels. Additionally, a candidate-gene approach revealed a suggestive association for a coding variant (rs143202684-C) in SERPINB2, and suggestive associations with consistent effect in the replication analysis include an intronic variant (rs11057830-A) in SCARB1 associated with increased D-dimer levels. CONCLUSION: This work provides new evidence for a role of FGL1 in hemostasis.

Gene and protein expression in human megakaryocytes derived from induced pluripotent stem cells.

BACKGROUND: There is interest in deriving megakaryocytes (MKs) from pluripotent stem cells (iPSC) for biological studies. We previously found that genomic structural integrity and genotype concordance is maintained in iPSC-derived MKs. OBJECTIVE: To establish a comprehensive dataset of genes and proteins expressed in iPSC-derived MKs. METHODS: iPSCs were reprogrammed from peripheral blood mononuclear cells (MNCs) and MKs were derived from the iPSCs in 194 healthy European American and African American subjects. mRNA was isolated and gene expression measured by RNA sequencing. Protein expression was measured in 62 of the subjects using mass spectrometry. RESULTS AND CONCLUSIONS: MKs expressed genes and proteins known to be important in MK and platelet function and demonstrated good agreement with previous studies in human MKs derived from CD34+ progenitor cells. The percent of cells expressing the MK markers CD41 and CD42a was consistent in biological replicates, but variable across subjects, suggesting that unidentified subject-specific factors determine differentiation of MKs from iPSCs. Gene and protein sets important in platelet function were associated with increasing expression of CD41/42a, while those related to more basic cellular functions were associated with lower CD41/42a expression. There was differential gene expression by the sex and race (but not age) of the subject. Numerous genes and proteins were highly expressed in MKs but not known to play a role in MK or platelet function; these represent excellent candidates for future study of hematopoiesis, platelet formation, and/or platelet function.

Discovery and fine-mapping of height loci via high-density imputation of GWASs in individuals of African ancestry.

Although many loci have been associated with height in European ancestry populations, very few have been identified in African ancestry individuals. Furthermore, many of the known loci have yet to be generalized to and fine-mapped within a large-scale African ancestry sample. We performed sex-combined and sex-stratified meta-analyses in up to 52,764 individuals with height and genome-wide genotyping data from the African Ancestry Anthropometry Genetics Consortium (AAAGC). We additionally combined our African ancestry meta-analysis results with published European genome-wide association study (GWAS) data. In the African ancestry analyses, we identified three novel loci (SLC4A3, NCOA2, ECD/FAM149B1) in sex-combined results and two loci (CRB1, KLF6) in women only. In the African plus European sex-combined GWAS, we identified an additional three novel loci (RCCD1, G6PC3, CEP95) which were equally driven by AAAGC and European results. Among 39 genome-wide significant signals at known loci, conditioning index SNPs from European studies identified 20 secondary signals. Two of the 20 new secondary signals and none of the 8 novel loci had minor allele frequencies (MAF) < 5%. Of 802 known European height signals, 643 displayed directionally consistent associations with height, of which 205 were nominally significant (p < 0.05) in the African ancestry sex-combined sample. Furthermore, 148 of 241 loci contained ≤20 variants in the credible sets that jointly account for 99% of the posterior probability of driving the associations. In summary, trans-ethnic meta-analyses revealed novel signals and further improved fine-mapping of putative causal variants in loci shared between African and European ancestry populations.

White Matter Injury Is Associated with Reduced Manual Dexterity and Elevated Serum Ceramides in Subjects with Cerebral Small Vessel Disease.

INTRODUCTION: We have demonstrated that asymptomatic cerebral small vessel disease (cSVD) measured by white matter hyperintensity volume is associated with reduced manipulative manual dexterity on the Grooved Peg Board Test (GPBT) in middle-aged healthy individuals with a family history of early coronary artery disease. In this current study, we aim to identify the association of subcortical white matter microstructural impairment measured by diffusion tensor imaging, manual dexterity measured by GPBT and circulating serums ceramide, another marker for white matter injury. We hypothesize that lower regional fractional anisotropy (rFA) is associated with worse performance on GPBT and elevated serum ceramides in the same study population. METHODS: rFA of 48 regions representing the subcortical white matters were analyzed in GeneSTAR participants in addition to serum ceramides and GPBT scores. Unadjusted univariable analyses with Bonferroni correction for multiple comparisons were completed using Spearman correlation for testing the associations between ceramides, rFA of subcortical white matter, and GPBT performance. Subsequently, sensitivity analyses were performed after excluding the participants that had any physical limitation that may influence their performance on GPBT. Finally, in the adjusted analysis using generalized estimating equation, linear regression models were performed for the areas that met significance threshold in the unadjusted analyses. RESULTS: 112 subjects (age [49 ± 11], 51% female, 39.3% African American) were included. Adjusted analyses for the significant correlations that met the Bonferroni correction threshold in the unadjusted univariable analyses identified significant negative associations between rFA of the right fornix (RF) and log-GPBT score (ß = -0.497, p = 0.037). In addition, rFA of RF negatively correlated with log serum ceramide levels (C18: ß = -0.03, p = 0.003, C20: ß = -0.0002, p = 0.004) and rFA of left genu of corpus callosum negatively correlated with log C18 level (ß = -0.0103, p = 0.027). CONCLUSIONS: These results demonstrate that subcortical microstructural white matter disruption is associated with elevated serum ceramides and reduced manual dexterity in a population with cSVD. These findings suggest that injury to white matter tracts undermines neural networks, with functional consequences in a middle-aged population with cardiovascular risk factors.

Genetic loci associated with prevalent and incident myocardial infarction and coronary heart disease in the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium.

BACKGROUND: Genome-wide association studies have identified multiple genomic loci associated with coronary artery disease, but most are common variants in non-coding regions that provide limited information on causal genes and etiology of the disease. To overcome the limited scope that common variants provide, we focused our investigation on low-frequency and rare sequence variations primarily residing in coding regions of the genome. METHODS AND RESULTS: Using samples of individuals of European ancestry from ten cohorts within the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, both cross-sectional and prospective analyses were conducted to examine associations between genetic variants and myocardial infarction (MI), coronary heart disease (CHD), and all-cause mortality following these events. For prevalent events, a total of 27,349 participants of European ancestry, including 1831 prevalent MI cases and 2518 prevalent CHD cases were used. For incident cases, a total of 55,736 participants of European ancestry were included (3,031 incident MI cases and 5,425 incident CHD cases). There were 1,860 all-cause deaths among the 3,751 MI and CHD cases from six cohorts that contributed to the analysis of all-cause mortality. Single variant and gene-based analyses were performed separately in each cohort and then meta-analyzed for each outcome. A low-frequency intronic variant (rs988583) in PLCL1 was significantly associated with prevalent MI (OR = 1.80, 95% confidence interval: 1.43, 2.27; P = 7.12 × 10-7). We conducted gene-based burden tests for genes with a cumulative minor allele count (cMAC) ≥ 5 and variants with minor allele frequency (MAF) < 5%. TMPRSS5 and LDLRAD1 were significantly associated with prevalent MI and CHD, respectively, and RC3H2 and ANGPTL4 were significantly associated with incident MI and CHD, respectively. No loci were significantly associated with all-cause mortality following a MI or CHD event. CONCLUSION: This study identified one known locus (ANGPTL4) and four new loci (PLCL1, RC3H2, TMPRSS5, and LDLRAD1) associated with cardiovascular disease risk that warrant further investigation.

A genome-wide association study identifies genetic loci associated with specific lobar brain volumes.

Brain lobar volumes are heritable but genetic studies are limited. We performed genome-wide association studies of frontal, occipital, parietal and temporal lobe volumes in 16,016 individuals, and replicated our findings in 8,789 individuals. We identified six genetic loci associated with specific lobar volumes independent of intracranial volume. Two loci, associated with occipital (6q22.32) and temporal lobe volume (12q14.3), were previously reported to associate with intracranial and hippocampal volume, respectively. We identified four loci previously unknown to affect brain volumes: 3q24 for parietal lobe volume, and 1q22, 4p16.3 and 14q23.1 for occipital lobe volume. The associated variants were located in regions enriched for histone modifications (DAAM1 and THBS3), or close to genes causing Mendelian brain-related diseases (ZIC4 and FGFRL1). No genetic overlap between lobar volumes and neurological or psychiatric diseases was observed. Our findings reveal part of the complex genetics underlying brain development and suggest a role for regulatory regions in determining brain volumes.

Mendelian randomization evaluation of causal effects of fibrinogen on incident coronary heart disease.

BACKGROUND: Fibrinogen is an essential hemostatic factor and cardiovascular disease risk factor. Early attempts at evaluating the causal effect of fibrinogen on coronary heart disease (CHD) and myocardial infraction (MI) using Mendelian randomization (MR) used single variant approaches, and did not take advantage of recent genome-wide association studies (GWAS) or multi-variant, pleiotropy robust MR methodologies. METHODS AND FINDINGS: We evaluated evidence for a causal effect of fibrinogen on both CHD and MI using MR. We used both an allele score approach and pleiotropy robust MR models. The allele score was composed of 38 fibrinogen-associated variants from recent GWAS. Initial analyses using the allele score used a meta-analysis of 11 European-ancestry prospective cohorts, free of CHD and MI at baseline, to examine incidence CHD and MI. We also applied 2 sample MR methods with data from a prevalent CHD and MI GWAS. Results are given in terms of the hazard ratio (HR) or odds ratio (OR), depending on the study design, and associated 95% confidence interval (CI). In single variant analyses no causal effect of fibrinogen on CHD or MI was observed. In multi-variant analyses using incidence CHD cases and the allele score approach, the estimated causal effect (HR) of a 1 g/L higher fibrinogen concentration was 1.62 (CI = 1.12, 2.36) when using incident cases and the allele score approach. In 2 sample MR analyses that accounted for pleiotropy, the causal estimate (OR) was reduced to 1.18 (CI = 0.98, 1.42) and 1.09 (CI = 0.89, 1.33) in the 2 most precise (smallest CI) models, out of 4 models evaluated. In the 2 sample MR analyses for MI, there was only very weak evidence of a causal effect in only 1 out of 4 models. CONCLUSIONS: A small causal effect of fibrinogen on CHD is observed using multi-variant MR approaches which account for pleiotropy, but not single variant MR approaches. Taken together, results indicate that even with large sample sizes and multi-variant approaches MR analyses still cannot exclude the null when estimating the causal effect of fibrinogen on CHD, but that any potential causal effect is likely to be much smaller than observed in epidemiological studies.

Evolution of Hominin Polyunsaturated Fatty Acid Metabolism: From Africa to the New World.

The metabolic conversion of dietary omega-3 and omega-6 18 carbon (18C) to long chain (>20 carbon) polyunsaturated fatty acids (LC-PUFAs) is vital for human life. The rate-limiting steps of this process are catalyzed by fatty acid desaturase (FADS) 1 and 2. Therefore, understanding the evolutionary history of the FADS genes is essential to our understanding of hominin evolution. The FADS genes have two haplogroups, ancestral and derived, with the derived haplogroup being associated with more efficient LC-PUFA biosynthesis than the ancestral haplogroup. In addition, there is a complex global distribution of these haplogroups that is suggestive of Neanderthal introgression. We confirm that Native American ancestry is nearly fixed for the ancestral haplogroup, and replicate a positive selection signal in Native Americans. This positive selection potentially continued after the founding of the Americas, although simulations suggest that the timing is dependent on the allele frequency of the ancestral Beringian population. We also find that the Neanderthal FADS haplotype is more closely related to the derived haplogroup and the Denisovan clusters closer to the ancestral haplogroup. Furthermore, the derived haplogroup has a time to the most recent common ancestor of 688,474 years before present. These results support an ancient polymorphism, as opposed to Neanderthal introgression, forming in the FADS region during the Pleistocene with possibly differential selection pressures on both haplogroups. The near fixation of the ancestral haplogroup in Native American ancestry calls for future studies to explore the potential health risk of associated low LC-PUFA levels in these populations.

Exome-chip meta-analysis identifies association between variation in ANKRD26 and platelet aggregation.

Previous genome-wide association studies (GWAS) have identified several variants associated with platelet function phenotypes; however, the proportion of variance explained by the identified variants is mostly small. Rare coding variants, particularly those with high potential for impact on protein structure/function, may have substantial impact on phenotype but are difficult to detect by GWAS. The main purpose of this study was to identify low frequency or rare variants associated with platelet function using genotype data from the Illumina HumanExome Bead Chip. Three family-based cohorts of European ancestry, including ~4,000 total subjects, comprised the discovery cohort and two independent cohorts, one of European and one of African American ancestry, were used for replication. Optical aggregometry in platelet-rich plasma was performed in all the discovery cohorts in response to adenosine diphosphate (ADP), epinephrine, and collagen. Meta-analyses were performed using both gene-based and single nucleotide variant association methods. The gene-based meta-analysis identified a significant association (P = 7.13 × 10-7) between rare genetic variants in ANKRD26 and ADP-induced platelet aggregation. One of the ANKRD26 SNVs - rs191015656, encoding a threonine to isoleucine substitution predicted to alter protein structure/function, was replicated in Europeans. Aggregation increases of ~20-50% were observed in heterozygotes in all cohorts. Novel genetic signals in ABCG1 and HCP5 were also associated with platelet aggregation to ADP in meta-analyses, although only results for HCP5 could be replicated. The SNV in HCP5 intersects epigenetic signatures in CD41+ megakaryocytes suggesting a new functional role in platelet biology for HCP5. This is the first study to use gene-based association methods from SNV array genotypes to identify rare variants related to platelet function. The molecular mechanisms and pathophysiological relevance for the identified genetic associations requires further study.

Targeted deep sequencing of the PEAR1 locus for platelet aggregation in European and African American families.

Coronary artery disease (CAD) remains a major cause of mortality and morbidity worldwide. The aggregation of activated platelets on a ruptured atherosclerotic plaque is a critical step in most acute cardiovascular events like myocardial infarction. Platelet aggregation both at baseline and after aspirin is highly heritable. Genome-wide association studies (GWAS) have identified a common variant within the first intron of the platelet endothelial aggregation receptor1 (PEAR1), to be robustly associated with platelet aggregation. In this study, we used targeted deep sequencing to fine-map the prior GWAS peak and identify additional rare variants of PEAR1 that account for missing heritability in platelet aggregation within the GeneSTAR families. In this study, 1709 subjects (1043 European Americans, EA and 666 African Americans, AA) from families in the GeneSTAR study were included. In vitro platelet aggregation in response to collagen, ADP and epinephrine was measured at baseline and 14 days after aspirin therapy (81 mg/day). Targeted deep sequencing of PEAR1 in addition to 2kb of upstream and downstream of the gene was performed. Under an additive genetic model, the association of single variants of PEAR1 with platelet aggregation phenotypes were examined. Additionally, we examined the association between the burden of PEAR1 rare non-synonymous variants and platelet aggregation phenotypes. Of 532 variants identified through sequencing, the intron 1 variant, rs12041331, was significantly associated with all platelet aggregation phenotypes at baseline and after platelet inhibition with aspirin therapy. rs12566888, which is in linkage disequilibrium with rs12041331, was associated with platelet aggregation phenotypes but to a lesser extent. In the EA families, the burden of PEAR1 missense variants was associated with platelet aggregation after aspirin therapy when the platelets were stimulated with epinephrine (p = 0.0009) and collagen (p = 0.03). In AAs, the burden of PEAR1 missense variants was associated, to a lesser degree, with platelet aggregation in response to epinephrine (p = 0.02) and ADP (p = 0.04). Our study confirmed that the GWAS-identified variant, rs12041331, is the strongest variant associated with platelet aggregation both at baseline and after aspirin therapy in our GeneSTAR families in both races. We identified additional association of rare missense variants in PEAR1 with platelet aggregation following aspirin therapy. However, we observed a racial difference in the contribution of these rare variants to the platelet aggregation, most likely due to higher residual missing heritability of platelet aggregation after accounting for rs12041331 in the EAs compared to AAs.

The relationship of family history and risk of type 2 diabetes differs by ancestry.

AIM: Type 2 diabetes (T2DM) in a first-degree relative is a risk factor for incident diabetes. Americans of African ancestry (AA) have higher rates of T2DM than Americans of European ancestry (EA). Thus, we aimed to determine whether the presence, number and kinship of affected relatives are associated with race-specific T2DM incidence in a prospective study of participants from the Genetic Study of Atherosclerosis Risk (GeneSTAR), who underwent baseline screening including a detailed family history. METHODS: Nondiabetic healthy siblings (n=1405) of patients with early-onset coronary artery disease (18-59 years) were enrolled (861 EA and 544 AA) and followed for incident T2DM (mean 14±6 years). RESULTS: Baseline age was 46.2±7.3 years and 56% were female. T2DM occurred in 12.3% of EA and 19.1% of AA. Among EA, 32.6% had ≥1 affected first-degree relatives versus 53.1% in AA, P<0.0001. In fully adjusted Cox proportional hazard analyses, any family history was related to incident T2DM in EA (HR=2.53, 95% CI: 1.58-4.06) but not in AA (HR=1.01, 0.67-1.53). The number of affected relatives conferred incremental risk of T2DM in EA with HR=1.82 (1.08-3.06), 4.83 (2.15-10.85) and 8.46 (3.09-23.91) for 1, 2, and ≥3 affected, respectively. In AA only ≥3 affected increased risk (HR=2.45, 1.44-4.19). Specific kinship patterns were associated with incident T2DM in EA but not in AA. CONCLUSIONS: The presence of any first-degree relative with T2DM does not discriminate risk in AA given the high race-specific prevalence of diabetes. Accounting for the number of affected relatives may more appropriately estimate risk for incident diabetes in both races.

Genome-Wide Association Transethnic Meta-Analyses Identifies Novel Associations Regulating Coagulation Factor VIII and von Willebrand Factor Plasma Levels.

BACKGROUND: Factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are associated with risk of arterial and venous thrombosis and with hemorrhagic disorders. We aimed to identify and functionally test novel genetic associations regulating plasma FVIII and VWF. METHODS: We meta-analyzed genome-wide association results from 46 354 individuals of European, African, East Asian, and Hispanic ancestry. All studies performed linear regression analysis using an additive genetic model and associated ≈35 million imputed variants with natural log-transformed phenotype levels. In vitro gene silencing in cultured endothelial cells was performed for candidate genes to provide additional evidence on association and function. Two-sample Mendelian randomization analyses were applied to test the causal role of FVIII and VWF plasma levels on the risk of arterial and venous thrombotic events. RESULTS: We identified 13 novel genome-wide significant ( P≤2.5×10-8) associations, 7 with FVIII levels ( FCHO2/TMEM171/TNPO1, HLA, SOX17/RP1, LINC00583/NFIB, RAB5C-KAT2A, RPL3/TAB1/SYNGR1, and ARSA) and 11 with VWF levels ( PDHB/PXK/KCTD6, SLC39A8, FCHO2/TMEM171/TNPO1, HLA, GIMAP7/GIMAP4, OR13C5/NIPSNAP, DAB2IP, C2CD4B, RAB5C-KAT2A, TAB1/SYNGR1, and ARSA), beyond 10 previously reported associations with these phenotypes. Functional validation provided further evidence of association for all loci on VWF except ARSA and DAB2IP. Mendelian randomization suggested causal effects of plasma FVIII activity levels on venous thrombosis and coronary artery disease risk and plasma VWF levels on ischemic stroke risk. CONCLUSIONS: The meta-analysis identified 13 novel genetic loci regulating FVIII and VWF plasma levels, 10 of which we validated functionally. We provide some evidence for a causal role of these proteins in thrombotic events.