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Genome-wide association studies (GWAS) have found few common variants that influence fasting measures of insulin sensitivity. We hypothesized that a GWAS of an integrated assessment of fasting and dynamic measures of insulin sensitivity would detect novel common variants. We performed a GWAS of the modified Stumvoll Insulin Sensitivity Index (ISI) within the Meta-Analyses of Glucose and Insulin-Related Traits Consortium. Discovery for genetic association was performed in 16,753 individuals, and replication was attempted for the 23 most significant novel loci in 13,354 independent individuals. Association with ISI was tested in models adjusted for age, sex, and BMI and in a model analyzing the combined influence of the genotype effect adjusted for BMI and the interaction effect between the genotype and BMI on ISI (model 3). In model 3, three variants reached genome-wide significance: rs13422522 (NYAP2; P = 8.87 × 10−11), rs12454712 (BCL2; P = 2.7 × 10−8), and rs10506418 (FAM19A2; P = 1.9 × 10−8). The association at NYAP2 was eliminated by conditioning on the known IRS1 insulin sensitivity locus; the BCL2 and FAM19A2 associations were independent of known cardiometabolic loci. In conclusion, we identified two novel loci and replicated known variants associated with insulin sensitivity. Further studies are needed to clarify the causal variant and function at the BCL2 and FAM19A2 loci.

Many common and rare variants associated with hematologic traits have been discovered through imputation on large-scale reference panels. However, the majority of genome-wide association studies (GWASs) have been conducted in Europeans, and determining causal variants has proved challenging. We performed a GWAS of total leukocyte, neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts generated from 109,563,748 variants in the autosomes and the X chromosome in the Trans-Omics for Precision Medicine (TOPMed) program, which included data from 61,802 individuals of diverse ancestry. We discovered and replicated 7 leukocyte trait associations, including (1) the association between a chromosome X, pseudo-autosomal region (PAR), noncoding variant located between cytokine receptor genes (CSF2RA and CLRF2) and lower eosinophil count; and (2) associations between single variants found predominantly among African Americans at the S1PR3 (9q22.1) and HBB (11p15.4) loci and monocyte and lymphocyte counts, respectively. We further provide evidence indicating that the newly discovered eosinophil-lowering chromosome X PAR variant might be associated with reduced susceptibility to common allergic diseases such as atopic dermatitis and asthma. Additionally, we found a burden of very rare FLT3 (13q12.2) variants associated with monocyte counts. Together, these results emphasize the utility of whole-genome sequencing in diverse samples in identifying associations missed by European-ancestry-driven GWASs.

Platelets play a key role in thrombosis and hemostasis. Platelet count (PLT) and mean platelet volume (MPV) are highly heritable quantitative traits, with hundreds of genetic signals previously identified, mostly in European ancestry populations. We here utilize whole genome sequencing (WGS) from NHLBI’s Trans-Omics for Precision Medicine initiative (TOPMed) in a large multi-ethnic sample to further explore common and rare variation contributing to PLT (n = 61 200) and MPV (n = 23 485). We identified and replicated secondary signals at MPL (rs532784633) and PECAM1 (rs73345162), both more common in African ancestry populations. We also observed rare variation in Mendelian platelet-related disorder genes influencing variation in platelet traits in TOPMed cohorts (not enriched for blood disorders). For example, association of GP9 with lower PLT and higher MPV was partly driven by a pathogenic Bernard-Soulier syndrome variant (rs5030764, p.Asn61Ser), and the signals at TUBB1 and CD36 were partly driven by loss of function variants not annotated as pathogenic in ClinVar (rs199948010 and rs571975065). However, residual signal remained for these gene-based signals after adjusting for lead variants, suggesting that additional variants in Mendelian genes with impacts in general population cohorts remain to be identified. Gene-based signals were also identified at several genome-wide association study identified loci for genes not annotated for Mendelian platelet disorders (PTPRH, TET2, CHEK2), with somatic variation driving the result at TET2. These results highlight the value of WGS in populations of diverse genetic ancestry to identify novel regulatory and coding signals, even for well-studied traits like platelet traits.

Mosaic mutations in blood are common with increasing age and are prognostic markers for cancer, cardiovascular dysfunction and other diseases. This group of acquired mutations include megabase-scale mosaic chromosomal alterations (mCAs). These large mutations have mainly been surveyed using SNP array data from individuals of European (EA) or Japanese genetic ancestry. To gain a better understanding of mCA rates and associated risk factors in genetically diverse populations, we surveyed whole genome sequencing data from 67,390 individuals, including 20,132 individuals of African ancestry (AA), and 7,608 of Hispanic ancestry (HA) with deep (30X) whole genome sequencing data from the NHLBI Trans Omics for Precision Medicine (TOPMed) program. We adapted an existing mCA calling algorithm for application to WGS data, and observed higher sensitivity with WGS data, compared with array-based data, in uncovering mCAs at low mutant cell fractions. As in previous reports, we observed a strong association with age and a non-uniform distribution of mCAs across the genome. The presence of autosomal (but not chromosome X) mCAs was associated with an increased risk of both lymphoid and myeloid malignancies. After adjusting for age, we found that individuals of European ancestry have the highest rates of autosomal mCAs, mirroring the higher rate of leukemia in this group. Our analysis also uncovered higher rates of chromosome X mCAs in AA and HA compared to EA, again after adjusting for age. Germline variants inATMandMPLshowed strong associations with mCAs incis, including ancestry specific variants. And rare variant gene-burden analysis confirmed the association of putatively protein altering variants inATMandMPLwith mCAs incis. Individual rare variants inDCPS, ADM17, PPP1R16B, andTET2were all associated with autosomal mCAs and rare variants inOR4C16were associated with chromosome X mCAs in females. There was significant enrichment of co-occurrence of CHIP mutations and mCAs both altering cancer associated genesTET2, DNMT3A, JAK2, CUX1, andTP53. Overall, our study demonstrates that rates of mCAs differ across populations and that rare inherited germline variants are strongly associated with mCAs across genetically diverse populations. These results strongly motivate further studies of mCAs in under-represented populations to better understand the causes and consequences of this class of somatic variation.

Summary Whole-genome sequencing (WGS), a powerful tool for detecting novel coding and non-coding disease-causing variants, has largely been applied to clinical diagnosis of inherited disorders. Here we leveraged WGS data in up to 62,653 ethnically diverse participants from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program and assessed statistical association of variants with seven red blood cell (RBC) quantitative traits. We discovered 14 single variant-RBC trait associations at 12 genomic loci, which have not been reported previously. Several of the RBC trait-variant associations (RPN1, ELL2, MIDN, HBB, HBA1, PIEZO1, and G6PD) were replicated in independent GWAS datasets imputed to the TOPMed reference panel. Most of these discovered variants are rare/low frequency, and several are observed disproportionately among non-European Ancestry (African, Hispanic/Latino, or East Asian) populations. We identified a 3 bp indel p.Lys2169del (g.88717175_88717177TCT[4]) (common only in the Ashkenazi Jewish population) of PIEZO1, a gene responsible for the Mendelian red cell disorder hereditary xerocytosis (MIM: 194380 ), associated with higher mean corpuscular hemoglobin concentration (MCHC). In stepwise conditional analysis and in gene-based rare variant aggregated association analysis, we identified several of the variants in HBB, HBA1, TMPRSS6, and G6PD that represent the carrier state for known coding, promoter, or splice site loss-of-function variants that cause inherited RBC disorders. Finally, we applied base and nuclease editing to demonstrate that the sentinel variant rs112097551 (nearest gene RPN1) acts through a cis-regulatory element that exerts long-range control of the gene RUVBL1 which is essential for hematopoiesis. Together, these results demonstrate the utility of WGS in ethnically diverse population-based samples and gene editing for expanding knowledge of the genetic architecture of quantitative hematologic traits and suggest a continuum between complex trait and Mendelian red cell disorders.

The platelet endothelial aggregation receptor-1 (PEAR1) rs12041331 variant has been identified as a genetic determinant of platelet aggregation in response to antiplatelet therapies, including aspirin. However, association with atherothrombotic cardiovascular events is less clear, with limited evidence from large trials. Here, we tested association of rs12041331 with cardiovascular events and aspirin use in a randomized trial population of healthy older individuals. We undertook post hoc analysis of 13,547 participants of the ASPirin in Reducing Events in the Elderly (ASPREE) trial, median age 74 years. Participants had no previous diagnosis of atherothrombotic cardiovascular disease at enrollment, and were randomized to either 100 mg daily low-dose aspirin or placebo for median 4.7 years follow-up. We used Cox proportional hazard regression to model the relationship between rs12041331 and the ASPREE primary cardiovascular disease (CVD) end point, and composites of major adverse cardiovascular events (MACE) and ischemic stroke (STROKE); and bleeding events; major hemorrhage (MHEM) and intracranial bleeding (ICB). We performed whole-population analysis using additive and dominant inheritance models, then stratified by treatment group. Interaction effects between genotypes and treatment group were examined. We observed no statistically significant association (P < 0.05) in the population, or by treatment group, between rs12041331 and cardiovascular or bleeding events in either model. We also found no significant interaction effects between rs12041331-A and treatment group, for CVD (P = 0.65), MACE (P = 0.32), STROKE (P = 0.56), MHEM (P = 0.59), or ICB (P = 0.56). The genetic variant PEAR1 rs12041331 is not associated with cardiovascular events in response to low-dose aspirin in a healthy elderly population.

A diverse set of driver genes, such as regulators of DNA methylation, RNA splicing, and chromatin remodeling, have been associated with pre-malignant clonal expansion of hematopoietic stem cells (HSCs). The factors mediating expansion of these mutant clones remain largely unknown, partially due to a paucity of large cohorts with longitudinal blood sampling. To circumvent this limitation, we developed and validated a method to infer clonal expansion rate from single timepoint data called PACER (passenger-approximated clonal expansion rate). Applying PACER to 5,071 persons with clonal hematopoiesis accurately recapitulated the known fitness effects due to different driver mutations. A genome-wide association study of PACER revealed that a common inherited polymorphism in the TCL1A promoter was associated with slower clonal expansion. Those carrying two copies of this protective allele had up to 80% reduced odds of having driver mutations in TET2, ASXL1, SF3B1, SRSF2, and JAK2, but not DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 by CRISPR editing led to aberrant expression of TCL1A and expansion of HSCs in vitro. These effects were abrogated in HSCs from donors carrying the protective TCL1A allele. Our results indicate that the fitness advantage of multiple common driver genes in clonal hematopoiesis is mediated through TCL1A activation. PACER is an approach that can be widely applied to uncover genetic and environmental determinants of pre-malignant clonal expansion in blood and other tissues.

Aims Clopidogrel is prescribed for the prevention of atherothrombotic events. While investigations have identified genetic determinants of inter-individual variability in on-treatment platelet inhibition (e.g. CYP2C19*2), evidence that these variants have clinical utility to predict major adverse cardiovascular events (CVEs) remains controversial. Methods and results We assessed the impact of 31 candidate gene polymorphisms on adenosine diphosphate (ADP)-stimulated platelet reactivity in 3391 clopidogrel-treated coronary artery disease patients of the International Clopidogrel Pharmacogenomics Consortium (ICPC). The influence of these polymorphisms on CVEs was tested in 2134 ICPC patients (N = 129 events) in whom clinical event data were available. Several variants were associated with on-treatment ADP-stimulated platelet reactivity (CYP2C19*2, P = 8.8 × 10−54; CES1 G143E, P = 1.3 × 10−16; CYP2C19*17, P = 9.5 × 10−10; CYP2B6 1294 + 53 C > T, P = 3.0 × 10−4; CYP2B6 516 G > T, P = 1.0 × 10−3; CYP2C9*2, P = 1.2 × 10−3; and CYP2C9*3, P = 1.5 × 10−3). While no individual variant was associated with CVEs, generation of a pharmacogenomic polygenic response score (PgxRS) revealed that patients who carried a greater number of alleles that associated with increased on-treatment platelet reactivity were more likely to experience CVEs (β = 0.17, SE 0.06, P = 0.01) and cardiovascular-related death (β = 0.43, SE 0.16, P = 0.007). Patients who carried eight or more risk alleles were significantly more likely to experience CVEs [odds ratio (OR) = 1.78, 95% confidence interval (CI) 1.14–2.76, P = 0.01] and cardiovascular death (OR = 4.39, 95% CI 1.35–14.27, P = 0.01) compared to patients who carried six or fewer of these alleles. Conclusion Several polymorphisms impact clopidogrel response and PgxRS is a predictor of cardiovascular outcomes. Additional investigations that identify novel determinants of clopidogrel response and validating polygenic models may facilitate future precision medicine strategies.

Traditional tissue culture platforms have been around for several decades and have enabled key findings in the cardiovascular field. However, these platforms failed to recreate the mechanical and dynamic features found within the body. Organs-on-chips (OOCs) are cellularized microfluidic-based devices that can mimic the basic structure, function, and responses of organs. These systems have been successfully utilized in disease, development, and drug studies. OOCs are designed to recapitulate the mechanical, electrical, chemical, and structural features of the in vivo microenvironment. Here, we review cardiovascular-themed OOC studies, design considerations, and techniques used to generate these cellularized devices. Furthermore, we will highlight the advantages of OOC models over traditional cell culture vessels, discuss implementation challenges, and provide perspectives on the state of the field.

Whole genome sequencing (WGS), a powerful tool for detecting novel coding and non-coding disease-causing variants, has largely been applied to clinical diagnosis of inherited disorders. Here we leveraged WGS data in up to 62,653 ethnically diverse participants from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program and assessed statistical association of variants with seven red blood cell (RBC) quantitative traits. We discovered 14 single variant-RBC trait associations at 12 genomic loci. Several of the RBC trait-variant associations (RPN1, ELL2, MIDN, HBB, HBA1, PIEZO1, G6PD) were replicated in independent GWAS datasets imputed to the TOPMed reference panel. Most of these newly discovered variants are rare/low frequency, and several are observed disproportionately among non-European Ancestry (African, Hispanic/Latino, or East Asian) populations. We identified a 3bp indel p.Lys2169del (common only in the Ashkenazi Jewish population) of PIEZO1, a gene responsible for the Mendelian red cell disorder hereditary xerocytosis [OMIM 194380], associated with higher MCHC. In stepwise conditional analysis and in gene-based rare variant aggregated association analysis, we identified several of the variants in HBB, HBA1, TMPRSS6, and G6PD that represent the carrier state for known coding, promoter, or splice site loss-of-function variants that cause inherited RBC disorders. Finally, we applied base and nuclease editing to demonstrate that the sentinel variant rs112097551 (nearest gene RPN1) acts through a cis-regulatory element that exerts long-range control of the gene RUVBL1 which is essential for hematopoiesis. Together, these results demonstrate the utility of WGS in ethnically-diverse population-based samples and gene editing for expanding knowledge of the genetic architecture of quantitative hematologic traits and suggest a continuum between complex trait and Mendelian red cell disorders.

MotivationWhole-genome DNA sequencing (WGS) enables the discovery of non-coding variants, but tools are lacking to prioritize the subset that functionally impacts human phenotypes. DNA sequence variants that disrupt or create transcription factor binding sites (TFBS) can modulate gene expression. find-tfbs efficiently scans phased WGS in large cohorts to identify and count TFBSs in regulatory sequences. This information can then be used in association testing to find putatively functional non-coding variants associated with complex human diseases or traits.ResultsWe applied find-tfbs to discover functional non-coding variants associated with hematological traits in the NHLBI Trans-Omics for Precision Medicine (TOPMed) WGS dataset (Nmax=44,709). We identified >2000 associations at P−9, implicating specific blood cell-types, transcription factors and causal genes. The vast majority of these associations are captured by variants identified in large genome-wide association studies (GWAS) for blood-cell traits. find-tfbs is computationally efficient and robust, allowing for the rapid identification of non-coding variants associated with multiple human phenotypes in very large sample size.Availabilityhttps://github.com/Helkafen/find-tfbs and https://github.com/Helkafen/find-tfbs-demoContactssebastian.meric.de.bellefon@umontreal.ca and guillaume.lettre@umontreal.caSupplementary informationSupplementary data are available.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans., Circulating lipoprotein(a) is an important risk factor for cardiovascular disease and shows variability between different ethnic groups. Here, Zekavat et al. perform whole-genome sequencing in individuals of European and African ancestries and find ancestry-specific genetic determinants for lipoprotein(a) levels.

Antiplatelet response to clopidogrel shows wide variation, and poor response is correlated with adverse clinical outcomes. CYP2C19 loss-of-function alleles play an important role in this response, but account for only a small proportion of variability in response to clopidogrel. An aim of the International Clopidogrel Pharmacogenomics Consortium (ICPC) is to identify other genetic determinants of clopidogrel pharmacodynamics and clinical response. A genomewide association study (GWAS) was performed using DNA from 2,750 European ancestry individuals, using adenosine diphosphate-induced platelet reactivity and major cardiovascular and cerebrovascular events as outcome parameters. GWAS for platelet reactivity revealed a strong signal for CYP2C19*2 (P value = 1.67e−33). After correction for CYP2C19*2 no other single-nucleotide polymorphism reached genomewide significance. GWAS for a combined clinical end point of cardiovascular death, myocardial infarction, or stroke (5.0% event rate), or a combined end point of cardiovascular death or myocardial infarction (4.7% event rate) showed no significant results, although in coronary artery disease, percutaneous coronary intervention, and acute coronary syndrome subgroups, mutations in SCOS5P1, CDC42BPA, and CTRAC1 showed genomewide significance (lowest P values: 1.07e−09, 4.53e−08, and 2.60e−10, respectively). CYP2C19*2 is the strongest genetic determinant of on-clopidogrel platelet reactivity. We identified three novel associations in clinical outcome subgroups, suggestive for each of these outcomes.

Whole-genome sequencing (WGS) can improve assessment of low-frequency and rare variants, particularly in non-European populations that have been underrepresented in existing genomic studies. The genetic determinants of C-reactive protein (CRP), a biomarker of chronic inflammation, have been extensively studied, with existing genome-wide association studies (GWASs) conducted in >200,000 individuals of European ancestry. In order to discover novel loci associated with CRP levels, we examined a multi-ancestry population (n = 23,279) with WGS (∼38× coverage) from the Trans-Omics for Precision Medicine (TOPMed) program. We found evidence for eight distinct associations at the CRP locus, including two variants that have not been identified previously (rs11265259 and rs181704186), both of which are non-coding and more common in individuals of African ancestry (∼10% and ∼1% minor allele frequency, respectively, and rare or monomorphic in 1000 Genomes populations of East Asian, South Asian, and European ancestry). We show that the minor (G) allele of rs181704186 is associated with lower CRP levels and decreased transcriptional activity and protein binding in vitro, providing a plausible molecular mechanism for this African ancestry-specific signal. The individuals homozygous for rs181704186-G have a mean CRP level of 0.23 mg/L, in contrast to individuals heterozygous for rs181704186 with mean CRP of 2.97 mg/L and major allele homozygotes with mean CRP of 4.11 mg/L. This study demonstrates the utility of WGS in multi-ethnic populations to drive discovery of complex trait associations of large effect and to identify functional alleles in noncoding regulatory regions.

Exaggerated platelet aggregation at the site of vascular injury is the underlying pathophysiology of thrombotic diseases. Here, we conduct the largest whole genome sequencing (WGS) effort to uncover the genetic determinants of platelet aggregation. Leveraging 3,855 NHLBI Trans-Omics for Precision Medicine (TOPMed) individuals deeply phenotyped for platelet aggregation, we identify 18 loci using single-variant approaches. This includes the novel RGS18 locus encoding a myeloerythroid lineage-specific regulator of G-protein signaling that co-localizes with eQTL signatures for RGS18 expression in platelets. A gene-based approach focusing on deleterious coding variants identifies the SVEP1 gene, previously shown to be associated with coronary artery disease, as a novel determinant of platelet aggregation. Finally, in an integrative approach leveraging epigenetic data on megakaryocytes, we find strong association between rare variants mapping to a super enhancer region for PEAR1. This is a novel finding implicating the importance of rare variants with regulatory potential in a previously documented GWAS-identified locus.

Genetic variation in the platelet endothelial aggregation receptor 1 (PEAR1) gene, most notably rs12041331, is implicated in altered on-aspirin platelet aggregation and increased cardiovascular event risk. We prospectively tested the effects of aspirin administration at commonly prescribed doses (81, 162, and 324 mg/day) on agonist-induced platelet aggregation by rs12041331 genotype in 67 healthy individuals. Prior to aspirin administration, rs12041331 minor allele carriers had significantly reduced adenosine diphosphate (ADP)-induced platelet aggregation compared with noncarriers (P = 0.03) but was not associated with other platelet pathways. In contrast, rs12041331 was significantly associated with on-aspirin platelet aggregation when collagen and epinephrine were used to stimulate platelet aggregation (P < 0.05 for all associations), but not ADP. The influence of PEAR1 rs12041331 on platelet aggregation is pathway-specific and is altered by aspirin at therapeutic doses, but not in a dose-dependent manner. Additional studies are needed to determine the impact of PEAR1 on cardiovascular events in aspirin-treated patients.

With advances in whole-genome sequencing (WGS) technology, more advanced statistical methods for testing genetic association with rare variants are being developed. Methods in which variants are grouped for analysis are also known as variant-set, gene-based, and aggregate unit tests. The burden test and sequence kernel association test (SKAT) are two widely used variant-set tests, which were originally developed for samples of unrelated individuals and later have been extended to family data with known pedigree structures. However, computationally efficient and powerful variant-set tests are needed to make analyses tractable in large-scale WGS studies with complex study samples. In this paper, we propose the variant-set mixed model association tests (SMMAT) for continuous and binary traits using the generalized linear mixed model framework. These tests can be applied to large-scale WGS studies involving samples with population structure and relatedness, such as in the National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine (TOPMed) program. SMMATs share the same null model for different variant sets, and a virtue of this null model, which includes covariates only, is that it needs to be fit only once for all tests in each genome-wide analysis. Simulation studies show that all the proposed SMMATs correctly control type I error rates for both continuous and binary traits in the presence of population structure and relatedness. We also illustrate our tests in a real data example of analysis of plasma fibrinogen levels in the TOPMed program (n = 23,763), using the Analysis Commons, a cloud-based computing platform.

BACKGROUND: Post-prandial lipemia (PPL), defined as a prolonged or elevated rise in triglycerides that accompanies fat feeding, is a significant risk factor for coronary heart disease (CHD) and associated comorbidities. The impact of PPL on CHD risk is underscored by the preponderance of each day spent in the post-prandial state. OBJECTIVE: In this study, we evaluated cross-sectionally the association between usual (i.e., non-interventional) physical activity and the 6-hour triglyceride response to a standardized high-fat meal. METHODS: The high-fat meal intervention was carried out in 671 apparently healthy individuals as part of the Hereditary and Phenotype Intervention (HAPI) Heart Study. Triglyceride levels were measured in the fasting state and during six hours following administration of a standardized fat challenge. We defined PPL response as the triglyceride area under the fat load curve (AUC) and measured physical activity using accelerometers that were worn continuously over a 7-day period. RESULTS: Physical activity levels decreased with increasing age and were higher in men than women (both p < 0.001). The triglyceride AUC increased with increasing age in both men and women (both p

See Article by Lee et al Dual antiplatelet therapy with aspirin and a P2Y12 receptor inhibitor is a cornerstone for prevention of recurrent cardiovascular events in patients with acute coronary syndromes or undergoing percutaneous coronary intervention (PCI). Although clopidogrel remains the most commonly used P2Y12 receptor inhibitor and is generally effective, a large body of retrospective evidence suggests that a subset of patients do not respond adequately to this medication resulting in increased on-treatment platelet reactivity and rates of atherothrombotic events. A major determinant of variable clopidogrel efficacy includes loss-of-function (LOF) genetic variation in the CYP2C19 gene, most notably CYP2C19 *2 and *3. CYP2C19 is responsible, in part, for converting the clopidogrel prodrug into a biologically active thiol metabolite that irreversibly binds to the P2Y12 receptor thus inhibiting ADP-induced platelet aggregation. In recent years, alternative P2Y12 receptor inhibitors (ie, prasugrel and ticagrelor) have been developed and are approved for use for most clinical indications in which clopidogrel is prescribed. Although large-scale trials have shown that these agents are superior to clopidogrel in terms of platelet inhibition and reduction of major adverse cardiovascular events, clopidogrel continues to be the most widely used agent because of its cost and lower propensity to cause pathological bleeding.1,2 Importantly, however, genetic variation in CYP2C19 does not alter the pharmacokinetics or antiplatelet potential of prasugrel and ticagrelor thereby providing a modality for more personalized antiplatelet therapy approaches. Indeed, the US Food and Drug Administration updated the clopidogrel label in 2010 advising clinicians to be aware that poor metabolizers of clopidogrel (eg, those that carry CYP2C19 LOF alleles) exhibit high cardiovascular event rates and that alternative therapy should be considered in these individuals. In addition, current American College of Cardiology Foundation/American Heart Association PCI guidelines state that genetic testing and …

Platelets are critical in inflammation, wound healing, and thrombosis. Platelet count (PLT) and mean platelet volume (MPV) are heritable, frequently assessed measures for which hundreds of genetic associations have been identified, predominantly in European populations. However, causal variants are unknown at most loci, and additional genetic determinants in diverse ancestral populations remain to be discovered. We performed a multi-ancestry whole genome sequencing analysis for PLT in three Phase 1 TOPMed cohorts, the European American Framingham Heart (FHS, n=2259) and Old Order Amish (OOA, n=1095) studies and the African American Jackson Heart Study (JHS, n=3523). MPV was additionally analyzed in JHS and FHS. Heritability in JHS and FHS was 45-73%. Single variant and aggregate burden and sequence kernel association (SKAT) tests [5 and 50 kilobase (kb) sliding windows] were performed using a mixed model approach in GENESIS, with aggregate tests for variants with a minor allele frequency (MAF) < 1% only. Inverse normalized residuals were adjusted for sex, age, study, and an empirical genetic relationship matrix, allowing for heterogeneous variance by study. For PLT, several associations had the same index SNP ( ARHGEF3 , TPM4 ) or were in moderate LD ( TAOK1 , r 2 =0.46) with a previously reported signal; two associations were not in LD (r 2 -8 , MAF=16%), an intergenic variant upstream of telomerase reverse transcriptase ( TERT ), is common only in African ancestry populations (~36%, 1% in Europeans) and was significant only in JHS (p=4.7 x 10 -9 , p=0.91 FHS, absent OOA). This variant and an LD proxy (rs10052815) overlap a GATA1 binding site in erythroblasts, the sister lineage of platelet producing megakaryocytes. An intronic TERT variant (rs2736100) 17 kb away (r 2 =0.025, D’=0.418 in included TOPMed cohorts) has been associated with PLT in Europeans, as well as with red blood cell traits, telomere length, pulmonary fibrosis, and multiple cancer subtypes. The second locus (rs73437176, p=1.5 x 10 -8 , MAF=12%), an intronic variant in progestin and adipoQ receptor family member V ( PAQR5 ), is also more frequent in African ancestry populations (~25%, 4% in Europeans) and significant only in JHS (p=2.5 x 10 -8 , FHS p=0.47, OOA p=0.12); the nearest previously identified signal is ~4 Mb away and not in significant LD (D’DNM3 , ARHGEF3 , CCDC71L , JMJD1C , RHOF , TAOK1 , TPM4 ), with either the same lead variant as previously reported or a variant in close LD (r 2 >0.75). Aggregated sliding window tests did not identify any additional signals. These results show suggestive evidence for association with PLT for two novel variants common only in African ancestry populations, but larger sample sizes of diverse ancestry will be necessary for replication and further discovery of novel loci.

While it is well established that genetic variation is a significant contributor to interindividual variability in clopidogrel efficacy, candidate gene and genome-wide approaches have failed to reproducibly identify genetic determinants of antiplatelet response, apart from variants in CYP2C19, prompting the need for more innovative study designs. Herein, we highlight the potential benefit of exome sequencing of patients at the extremes of clopidogrel responsivity through examination of data reported in this issue of Clinical Pharmacology & Therapeutics.

With advances in Whole Genome Sequencing (WGS) technology, more advanced statistical methods for testing genetic association with rare variants are being developed. Methods in which variants are grouped for analysis are also known as variant-set, gene-based, and aggregate unit tests. The burden test and Sequence Kernel Association Test (SKAT) are two widely used variant-set tests, which were originally developed for samples of unrelated individuals and later have been extended to family data with known pedigree structures. However, computationally-efficient and powerful variant-set tests are needed to make analyses tractable in large-scale WGS studies with complex study samples. In this paper, we propose the variant-Set Mixed Model Association Tests (SMMAT) for continuous and binary traits using the generalized linear mixed model framework. These tests can be applied to large-scale WGS studies involving samples with population structure and relatedness, such as in the National Heart, Lung, and Blood Institute’s Trans-Omics for Precision Medicine (TOPMed) program. SMMAT tests share the same null model for different variant sets, and a virtue of this null model, which includes covariates only, is that it needs to be only fit once for all tests in each genome-wide analysis. Simulation studies show that all the proposed SMMAT tests correctly control type I error rates for both continuous and binary traits in the presence of population structure and relatedness. We also illustrate our tests in a real data example of analysis of plasma fibrinogen levels in the TOPMed program (n = 23,763), using the Analysis Commons, a cloud-based computing platform.

The exploding volume of whole-genome sequence (WGS) and multi-omics data requires new approaches for analysis. As one solution, we have created a cloud-based Analysis Commons, which brings together genotype and phenotype data from multiple studies in a setting that is accessible by multiple investigators. This framework addresses many of the challenges of multi-center WGS analyses, including data sharing mechanisms, phenotype harmonization, integrated multi-omics analyses, annotation, and computational flexibility. In this setting, the computational pipeline facilitates a sequence-to-discovery analysis workflow illustrated here by an analysis of plasma fibrinogen levels in 3996 individuals from the National Heart, Lung, and Blood Institute (NHLBI) Trans-Omics for Precision Medicine (TOPMed) WGS program. The Analysis Commons represents a novel model for transforming WGS resources from a massive quantity of phenotypic and genomic data into knowledge of the determinants of health and disease risk in diverse human populations.

Electronic health records (EHRs) linked with biobanks have been recognized as valuable data sources for pharmacogenomic studies, which require identification of patients with certain adverse drug reactions (ADRs) from a large population. Since manual chart review is costly and time-consuming, automatic methods to accurately identify patients with ADRs have been called for. In this study, we developed and compared different informatics approaches to identify ADRs from EHRs, using clopidogrel-induced bleeding as our case study. Three different types of methods were investigated: 1) rule-based methods; 2) machine learning-based methods; and 3) scoring function-based methods. Our results show that both machine learning and scoring methods are effective and the scoring method can achieve a high precision with a reasonable recall. We also analyzed the contributions of different types of features and found that the temporality information between clopidogrel and bleeding events, as well as textual evidence from physicians’ assertion of the adverse events are helpful. We believe that our findings are valuable in advancing EHR-based pharmacogenomic studies.

RATIONALE: The P2Y(12) receptor inhibitor clopidogrel is widely used in patients with acute coronary syndrome, percutaneous coronary intervention or ischemic stroke. Platelet inhibition by clopidogrel shows wide inter-patient variability and high on-treatment platelet reactivity is a risk factor for atherothrombotic events, particularly in high-risk populations. CYP2C19 polymorphism plays an important role in this variability, but heritability estimates suggest that additional genetic variants remain unidentified. The aim of the International Clopidogrel Pharmacogenomics Consortium (ICPC) is to identify genetic determinants of clopidogrel pharmacodynamics and clinical response. STUDY DESIGN: Based on the data published on www.clinicaltrials.gov, clopidogrel intervention studies containing genetic and platelet function data were identified for participation. Lead investigators were invited to share DNA samples, platelet function test results, patient characteristics and cardiovascular outcomes, to perform candidate gene and genome-wide association studies. RESULTS: In total, 17 study sites from 13 countries participate in the ICPC, contributing individual patient data from 8,829 patients. Available adenosine diphosphate (ADP) stimulated platelet function tests included Vasodilator-Stimulated Phosphoprotein (VASP) assay, Light Transmittance Aggregometry (LTA) and the VerifyNow P2Y(12) assay. A proof of principle analysis based on genotype data provided by each group showed a strong and consistent association between CYP2C19*2 and platelet reactivity (p-value = 5.1×10(−40)). CONCLUSION: The ICPC aims to identify new loci influencing clopidogrel efficacy by using state-of-the-art genetic techniques in a large cohort of clopidogrel-treated patients in order to better understand the genetic basis of on-treatment response variability.

Clopidogrel is one of the most commonly used therapeutics for secondary prevention of cardiovascular events in patients with acute coronary syndromes. However, substantial inter-individual variation in clopidogrel response has been documented, resulting in suboptimal therapy and an increased risk of recurrent events for some patients. In this investigation, we conducted the first genome-wide association study of circulating clopidogrel active metabolite levels in 513 healthy participants in order to directly measure clopidogrel pharmacokinetics. We observed that the CYP2C19 locus was the strongest genetic determinant of active metabolite formation (P=9.5×10−15). In addition, we identified novel genome-wide significant variants on chromosomes 3p25 (rs187941554, P=3.3×10−11) and 17q11 (rs80343429, P=1.3×10−8), as well as 6 additional loci that showed suggestive evidence of association (P≤1.0×10−6). Four of these loci demonstrated nominal associations with on-clopidogrel ADP-stimulated platelet aggregation (P≤0.05). Evaluation of clopidogrel active metabolite concentration may help identify novel genetic determinants of clopidogrel response, which has implications for the development of novel therapeutics and improved anti-platelet treatment for at-risk patients in the future.

Background Platelet endothelial aggregation receptor 1 (PEAR1) may affect platelet-platelet contact and aggregation. The aim of this study was to assess the association between PEAR1 polymorphisms and risks of platelet aggregation. Methods We searched the PubMed, EmBase, and Cochrane Library electronic databases for articles published through November 30th. 2016. Meta-analysis was performed to examine the relationship between PEAR1 and platelet aggregation and sensitivity analysis by removing individual study from meta-analysis. We collected and analyzed the results of 5 trials involving 5466 patients. Results Our results demonstrated that the G allele of rs12041331 was associated with a greater platelet aggregation by multiple agonists, both in the presence and absence of antiplatelet drugs, in several separate cohorts of different ethnicities along with an apparent allelic dose-response effect. However, the results of studies on rs2768759 locus were inconsistent and further studies are required. In the presence or absence of antiplatelet drugs treatment, the lowest platelet aggregation was observed in rs2768759 wild-type (AA) patients, followed by heterozygous (AC) and homozygous mutant (CC). Conclusion PEAR1 rs12041331 is associated with platelet function and antiplatelet drug pharmacodynamics. Future studies on relationship between single nucleotide polymorphisms of PEAR1 and incidence of cardiovascular diseases are required.

Background Platelet activation can lead to enhanced oxidative stress, inflammatory response, and endothelial dysfunction. To quantify the effects of platelet inhibition on endothelial function, we assessed platelet activity of healthy persons before and after clopidogrel administration and evaluated its effects on endothelial function. We hypothesized that clopidogrel, by attenuating platelet activity, would result in enhanced endothelial function. Methods and Results Microcirculatory endothelial function was quantified by laser Doppler flowmetry ( LDF ) mediated by thermal hyperemia ( TH ) and postocclusive reactive hyperemia, respectively, in 287 and 241 relatively healthy and homogenous Old Order Amish persons. LDF and platelet aggregation measures were obtained at baseline and after 7 days of clopidogrel administration. Our primary outcome was percentage change in post‐ versus preclopidogrel LDF measures. Preclopidogrel TH ‐ LDF and platelet aggregation were higher in women than in men ( P TH ‐ LDF in participants with high versus low baseline platelet aggregation (39.4±10.1% versus 17.4±5.6%, P =0.03). Clopidogrel also increased absolute TH ‐ LDF measures in persons with high platelet aggregation (1757±766 to 2154±1055, P =0.03), with a more prominent effect in women (1909±846 to 2518±1048, P =0.001). There was no evidence that clopidogrel influenced postocclusive reactive hyperemia LDF measures. Conclusions The administration of clopidogrel in healthy persons with high baseline platelet aggregation results in improved TH ‐induced microcirculatory endothelial function. These data suggest that clopidogrel may have a beneficial effect on microcirculatory endothelial function, presumably through antiplatelet activity, and may confer additional vascular benefits. Clinical Trial Registration URL : https://www.clinicaltrials.gov . Unique identifier: NCT 00799396.

Clopidogrel with aspirin is standard of care for the secondary prevention of ischemic events in patients with acute coronary syndromes. However, substantial inter-individual variation in clopidogrel response results in sub-optimal therapy in some patients and an increased risk of recurrent events. Up to 73% of the variation in clopidogrel efficacy is found to be heritable, and while key genetic variants such as CYP2C19 *2 have been identified, a large portion of the genetic heritability remains unaccounted for. To date, few studies have used clopidogrel active metabolite to evaluate clopidogrel pharmacogenomics. The evaluation of this biomarker may be particularly informative as it enables us to focus almost exclusively on clopidogrel pharmacokinetics and may allow new discoveries leading to a more thorough understanding of clopidogrel activation. Serum clopidogrel active metabolite concentration was measured by HPLC-MS/MS in 506 healthy, Amish participants of the Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study after a 7-day 75-mg/day clopidogrel intervention. Genome-wide genotyping was performed using Affymetrix 5.0 and 6.0 arrays, and SNPs were imputed to the 1000 Genomes cosmopolitan panel. Mixed models analysis was conducted under an additive model and adjusted for age, sex, BMI, and relatedness among study participants with MMAP (http://edn.som.umaryland.edu/mmap). The most significant signal of our GWAS was the previously identified CYP2C19 / CYP2C9 locus (P= 9.2x10 -15 ), and was fully accounted for by the CYP2C19 *2 variant. In addition to this signal, novel genome-wide significant signals were identified in loci on Chr. 3 (rs187941554, P=4.7x10 -11 ), Chr. 7 (rs73407739, p=3.6x10 -8 ), and Chr. 17 (rs80343429, P=3.7x10 -8 ). In this study we conducted the first ever GWAS of serum clopidogrel active metabolite concentration. Novel associations for clopidogrel efficacy were identified, and further study is required to determine the impact of these variants on clinical care Continual analysis of clopidogrel active metabolite concentration will contribute to a better understanding of the clopidogrel metabolic pathway, and potentially lay the groundwork for improved anti-platelet treatment for CVD patients in the future.

High interindividual variability in clinical outcomes following clopidogrel's standard dosing regimen continues to be a challenge even two decades after its approval. CYP2C19 polymorphisms, obesity, older age, diabetes, and drug-drug interactions have been identified as risk factors for adverse events and treatment failure. We conducted a mechanism-based pharmacokinetic/pharmacodynamic analysis, where we integrated knowledge on in vitro enzyme kinetic, physiological, genetic, and demographic information to characterize changes in platelet reactivity from baseline following clopidogrel antiplatelet therapy. When considering the combined impact of these covariates, our analysis results indicate that higher maintenance doses are required for CYP2C19 intermediate metabolizers and poor metabolizers compared to extensive metabolizers and that respective maintenance doses have to be further increased for obese subjects for each of these CYP2C19 phenotypes. In addition, interindividual differences in the fraction absorbed and the CES1 activity were identified as sources of interindividual differences in clopidogrel's active metabolite concentrations and, thus, platelet reactivity.

Uncovering the causes of interpatient variability in drug response, and then using that information for the benefit of patients, is at the heart of clinical pharmacology. Although the term “pharmacogenetics” was coined in the 1950s, only in the past decade has an explosion occurred in research focused on discovering the genetic basis for variations in drug efficacy, toxicity, and dose requirements. Pharmacogenomic research on cardiovascular drugs has been among the more active areas of investigation within this field. The past decade has seen substantial advances in our understanding of the genetic determinants of response to two commonly used cardiovascular drugs—clopidogrel and warfarin—such that the data on these drugs can now be used in the clinical setting. We highlight the data surrounding these examples along with other areas of active research in cardiovascular pharmacogenomics that—although they have not yet reached the stage of translation into practice—hold promise. The data arising from research on cardiovascular pharmacogenomics have not only led to potential clinical applications but have advanced our understanding of the metabolism and/or pharmacological mechanisms of a number of drugs. We also highlight the potential for research on pharmacogenomics to influence discovery and development of new drugs.

A genome-wide association study was performed using the Affymetrix 6.0 chip to identify genes associated with diabetic nephropathy in African Americans. Association analysis was performed adjusting for admixture in 965 type 2 diabetic African American patients with end-stage renal disease (ESRD) and in 1029 African Americans without type 2 diabetes or kidney disease as controls. The top 724 single nucleotide polymorphisms (SNPs) with evidence of association to diabetic nephropathy were then genotyped in a replication sample of an additional 709 type 2 diabetes-ESRD patients and 690 controls. SNPs with evidence of association in both the original and replication studies were tested in additional African American cohorts consisting of 1246 patients with type 2 diabetes without kidney disease and 1216 with non-diabetic ESRD to differentiate candidate loci for type 2 diabetes-ESRD, type 2 diabetes, and/or all-cause ESRD. Twenty-five SNPs were significantly associated with type 2 diabetes-ESRD in the genome-wide association and initial replication. Although genome-wide significance with type 2 diabetes was not found for any of these 25 SNPs, several genes, including RPS12, LIMK2, and SFI1 are strong candidates for diabetic nephropathy. A combined analysis of all 2890 patients with ESRD showed significant association SNPs in LIMK2 and SFI1 suggesting that they also contribute to all-cause ESRD. Thus, our results suggest that multiple loci underlie susceptibility to kidney disease in African Americans with type 2 diabetes and some may also contribute to all-cause ESRD.

Chromosome 20q12-q13.1 has been linked to type 2 diabetes (T2D) in multiple populations. We examined the influence of genes in this region on T2D and BMI in two European American case-control populations. SNPs were genotyped in 300 diabetic patients and 310 controls. A subset of 72 SNPs were further genotyped in 470 cases and 442 controls. All genes examined showed evidence of association with T2D in the initial sample (additive P-value [P(a)]=0.00090-0.045). SNPs near PREX1 were also associated in the second case-control population (P(a)=0.017-0.042). The combined analysis resulted in the same SNPs, among others, associated with T2D (P(a)=0.0013-0.041). Stratification analysis by T2D status showed that association with BMI was observed solely in cases (P(a)=0.0018-0.041). Mediation testing revealed that 30-40% of the effects of these SNPs on T2D were significantly mediated by BMI. SNPs near PREX1 may contribute to T2D susceptibility mediated through effects of adiposity in European Americans.

OBJECTIVE— Several whole-genome association studies have reported identification of type 2 diabetes susceptibility genes in various European-derived study populations. Little investigation of these loci has been reported in other ethnic groups, specifically African Americans. Striking differences exist between these populations, suggesting they may not share identical genetic risk factors. Our objective was to examine the influence of type 2 diabetes genes identified in whole-genome association studies in a large African American case-control population. RESEARCH DESIGN AND METHODS— Single nucleotide polymorphisms (SNPs) in 12 loci (e.g., TCF7L2, IDE/KIF11/HHEX, SLC30A8, CDKAL1, PKN2, IGF2BP2, FLJ39370, and EXT2/ALX4) associated with type 2 diabetes in European-derived populations were genotyped in 993 African American type 2 diabetic and 1,054 African American control subjects. Additionally, 68 ancestry-informative markers were genotyped to account for the impact of admixture on association results. RESULTS— Little evidence of association was observed between SNPs, with the exception of those in TCF7L2, and type 2 diabetes in African Americans. One TCF7L2 SNP (rs7903146) showed compelling evidence of association with type 2 diabetes (admixture-adjusted additive P [Pa] = 1.59 × 10−6). Only the intragenic SNP on 11p12 (rs9300039, dominant P [Pd] = 0.029) was also associated with type 2 diabetes after admixture adjustments. Interestingly, four of the SNPs are monomorphic in the Yoruba population of the HAPMAP project, with only the risk allele from the populations of European descent present. CONCLUSIONS— Results suggest that these variants do not significantly contribute to interindividual susceptibility to type 2 diabetes in African Americans. Consequently, genes contributing to type 2 diabetes in African Americans may, in part, be different from those in European-derived study populations. High frequency of risk alleles in several of these genes may, however, contribute to the increased prevalence of type 2 diabetes in African Americans.

Clopidogrel (Plavix®), is a widely used antiplatelet agent, which shows high inter-individual variability in treat- ment response in patients following the standard dosing regimen. In this study, a physiology-directed population pharmacokinetic/pharmacodynamic (PK/PD) model was developed based on clopidogrel and clopidogrel active metabolite (clop-AM) data from the PAPI and the PGXB2B studies using a step-wise approach in NONMEM (ver- sion 7.2). The developed model characterized the in vivo disposition of clopidogrel, its bioactivation into clop-AM in the liver and subsequent platelet aggregation inhibition in the systemic circulation reasonably well. It further allowed the identification of covariates that significantly impact clopidogrel's dose–concentration–response re- lationship. In particular, CYP2C19 intermediate and poor metabolizers converted 26.2% and 39.5% less clopidogrel to clop-AM, respectively, compared to extensive metabolizers. In addition, CES1 G143E mutation carriers have a reduced CES1 activity (82.9%) compared to wild-type subjects, which results in a significant increase in clop-AM formation. An increase in BMI was found to significantly decrease clopidogrel's bioactivation, whereas increased age was associated with increased platelet reactivity. Our PK/PD model analysis suggests that, in order to optimize clopidogrel dosing on a patient-by-patient basis, all of these factors have to be considered simultaneously, e.g. by using quantitative clinical pharmacology tools.

International audience; Introduction: Aspirin, clopidogrel, prasugrel and ticagrelor are antiplatelet agents for the prevention of ischemic events in patients with acute coronary syndromes (ACS), percutaneous coronary intervention (PCI) and other indications. Variability in response is observed to different degrees with these agents, which can translate to increased risks for adverse cardiovascular events. As such, potential pharmacogenetic determinants of antiplatelet pharmacokinetics, pharmacodynamics and clinical outcomes have been actively studied.Areas covered: This article provides an overview of the available antiplatelet pharmacogenetics literature. Evidence supporting the significance of candidate genes and their potential influence on antiplatelet response and clinical outcomes are summarized and evaluated. Additional focus is directed at CYP2C19 and clopidogrel response, including the availability of clinical testing and genotype-directed antiplatelet therapy.Expert opinion: The reported aspirin response candidate genes have not been adequately replicated and few candidate genes have thus far been implicated in prasugrel or ticagrelor response. However, abundant data support the clinical validity of CYP2C19 and clopidogrel response variability among ACS/PCI patients. Although limited prospective trial data are available to support the utility of routine CYP2C19 testing, the increased risks for reduced clopidogrel efficacy among ACS/PCI patients that carry CYP2C19 loss-of-function alleles should be considered when genotype results are available.

Background While aspirin is a well‐established and generally effective anti‐platelet agent, considerable inter‐individual variation in drug response exists, for which mechanisms are not completely understood. Metabolomics allows for extensive measurement of small molecules in biological samples, enabling detailed mapping of pathways involved in drug response. Methods and Results We used a mass‐spectrometry‐based metabolomics platform to investigate the changes in the serum oxylipid metabolome induced by an aspirin intervention (14 days, 81 mg/day) in healthy subjects (n=156). We observed a global decrease in serum oxylipids in response to aspirin (25 metabolites decreased out of 30 measured) regardless of sex. This decrease was concomitant with a significant decrease in serum linoleic acid levels (−19%, P =1.3×10 −5 ), one of the main precursors for oxylipid synthesis. Interestingly, several linoleic acid‐derived oxylipids were not significantly associated with arachidonic‐induced ex vivo platelet aggregation, a widely accepted marker of aspirin response, but were significantly correlated with platelet reactivity in response to collagen. Conclusions Together, these results suggest that linoleic acid‐derived oxylipids may contribute to the non‐ COX 1 mediated variability in response to aspirin. Pharmacometabolomics allowed for more comprehensive interrogation of mechanisms of action of low dose aspirin and of variation in aspirin response.

Background The role of the CYP 2C19 genotype on clopidogrel efficacy has been studied widely, with data suggesting reduced clopidogrel efficacy in loss‐of‐function variant carriers taking clopidogrel after percutaneous coronary intervention; however, data are limited regarding the association between CYP 2C19 genetic variants and outcomes in stroke patients. We investigated whether CYP 2C19 metabolizer status affects the risk of recurrent stroke or major bleeding in subcortical stroke patients taking dual antiplatelet therapy with aspirin and clopidogrel. Methods and Results CYP 2C19*2 and CYP 2C19*17 were genotyped in 522 patients treated with dual antiplatelet therapy from the Secondary Prevention of Small Subcortical Strokes ( SPS 3) study. CYP 2C19 metabolizer status was inferred from genotype, and associations with the risk of recurrent stroke and major bleeding were assessed in the overall cohort and by race/ethnic group with logistic regression modeling. In the overall cohort, there were no differences in outcomes by CYP 2C19 metabolizer status (recurrent stroke, odds ratio 1.81 [95% CI 0.76 to 4.30]; major bleeding, odds ratio 0.67 [95% CI 0.22 to 2.03]). In white participants, those with CYP 2C19 intermediate or poor metabolizer status had higher odds of recurrent stroke (odds ratio 5.19 [95% CI 1.08 to 24.90]) than those with extensive or ultrarapid metabolizer status, but there was no evidence of difference in major bleeding. Conclusions There were significant differences in recurrent stroke by CYP2C19 genotype‐inferred metabolizer status in white subcortical stroke patients receiving dual antiplatelet therapy with aspirin and clopidogrel, consistent with cardiovascular studies on CYP 2C19 and clopidogrel; however, the bleeding risk that led to early termination of the antiplatelet arm of the SPS 3 trial does not appear to be explained by CYP 2C19 genotype. This study was relatively underpowered; therefore, these findings should be interpreted with caution and warrant replication. Clinical Trial Registration URL : www.clinicaltrials.gov . Unique identifier: NCT 00059306.

In recent years, substantial effort has been made to better understand the influence of genetic factors on the efficacy and safety of numerous medications. These investigations suggest that the use of pharmacogenetic data to inform physician decision-making has great potential to enhance patient care by reducing on-treatment clinical events, adverse drug reactions, and health care-related costs. In fact, integration of such information into the clinical setting may be particularly applicable for antiplatelet and anticoagulation therapeutics, given the increasing body of evidence implicating genetic variation in variable drug response. In this review, we summarize currently available pharmacogenetic information for the most commonly used antiplatelet (ie, clopidogrel and aspirin) and anticoagulation (ie, warfarin) medications. Furthermore, we highlight the currently known role of genetic variability in response to next-generation antiplatelet (prasugrel and ticagrelor) and anticoagulant (dabigatran) agents. While compelling evidence suggests that genetic variants are important determinants of antiplatelet and anticoagulation therapy response, significant barriers to clinical implementation of pharmacogenetic testing exist and are described herein. In addition, we briefly discuss development of new diagnostic targets and therapeutic strategies as well as implications for enhanced patient care. In conclusion, pharmacogenetic testing can provide important information to assist clinicians with prescribing the most personalized and effective antiplatelet and anticoagulation therapy. However, several factors may limit its usefulness and should be considered.

Clopidogrel, in combination with aspirin, is the standard of care for preventing ischemic cardiovascular events in patients with coronary artery disease, especially those who undergo percutaneous coronary intervention (PCI). Despite its widespread use, significant interindividual variability in clopidogrel response is consistently observed, and recent studies have suggested that as much as 70% of this variability can be attributed to genetic factors.1,2 Article see p 250 Clopidogrel is a thienopyridine prodrug that once activated, exerts its antiplatelet effect by irreversibly binding to the P2Y12 receptor on the surface of platelets inhibiting ADP-stimulated platelet aggregation. Clopidogrel activation requires a 2-step conversion by hepatic enzymes, most notably CYP2C19, whereas esterases (eg, carboxylesterase 1 and carboxylesterase 2) lead to the production of biologically inactive carboxylic acid derivatives.3 Common loss-of-function variants in CYP2C19 are associated with lower active metabolite levels, higher residual on-clopidogrel ADP-stimulated platelet aggregation, and poorer cardiovascular outcomes.3 However, this variant explains only ≈12% of the variation in platelet response to clopidogrel, leaving most of the heritable variation unknown. Recently, Bouman and colleagues4 observed through in vitro studies that paraoxonase 1 (PON1) was the major enzyme in converting 2-oxo-clopidogrel to the active thiol metabolite. Moreover, they reported that a common missense variant in PON1 , Q192R, was a major determinant of clopidogrel efficacy, accounting for nearly 70% of the variability in ADP-stimulated platelet aggregation postclopidogrel treatment. 192Q PON1 had lower hydrolysis efficiency for 2-oxo-clopidogrel compared to 192R PON1 in microsomal preparations, and clopidogrel-treated PCI patients homozygous for the 192Q allele had a significantly higher rate of stent thrombosis compared with patients carrying at least 1 copy of the 192R allele. Curiously, no effect of the well-described CYP2C19 *2 loss-of-function variant on clopidogrel response was observed in the same patient sample. In the past year, our group and …

Clopidogrel and aspirin are commonly prescribed anti-platelet medications indicated for patients who have experienced, or are at risk for, ischemic cardiovascular events. The Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study was designed to characterize determinants of clopidogrel and dual anti-platelet therapy (DAPT) response in a healthy cohort of Old Order Amish from Lancaster, PA. Following a loading dose, clopidogrel was taken once a day for 7 days. One hour after the last dose of clopidogrel, 325 mg of aspirin was given. Ex vivo platelet aggregometry was performed at baseline, post-clopidogrel, and post-DAPT. Platelet aggregation measurements were significantly lower after both interventions for all agonists tested (p