N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a strong predictor of mortality in coronary artery disease and is widely employed as a prognostic biomarker. However, a causal relationship between NT-proBNP and clinical endpoints has not been established. We have performed a genome-wide association and Mendelian randomization study of NT-proBNP. We used a discovery set of 3740 patients from the PLATelet inhibition and patient Outcomes (PLATO) trial, which enrolled 18 624 patients with acute coronary syndrome (ACS). A further set of 5492 patients, from the same trial, was used for replication. Genetic variants at two novel loci (SLC39A8 and POC1B/GALNT4) were associated with NT-proBNP levels and replicated together with the previously known NPPB locus. The most significant SNP (rs198389, pooled P = 1.07 × 10(-15)) in NPPB interrupts an E-box consensus motif in the gene promoter. The association in SLC39A8 is driven by a deleterious variant (rs13107325, pooled P = 5.99 × 10(-10)), whereas the most significant SNP in POC1B/GALNT4 (rs11105306, pooled P = 1.02 × 10(-16)) is intronic. The SLC39A8 SNP was associated with higher risk of cardiovascular (CV) death (HR = 1.39, 95% CI: 1.08-1.79, P = 0.0095), but the other loci were not associated with clinical endpoints. We have identified two novel loci to be associated with NT-proBNP in patients with ACS. Only the SLC39A8 variant, but not the NPPB variant, was associated with a clinical endpoint. Due to pleotropic effects of SLC39A8, these results do not suggest that NT-proBNP levels have a direct effect on mortality in ACS patients. PLATO Clinical Trial Registration: www.clinicaltrials.gov; NCT00391872.

BACKGROUND:In addition to lowering low density lipoprotein cholesterol (LDL-C), statin therapy also raises high density lipoprotein cholesterol (HDL-C) levels. Inter-individual variation in HDL-C response to statins may be partially explained by genetic variation. METHODS AND RESULTS:We performed a meta-analysis of genome-wide association studies (GWAS) to identify variants with an effect on statin-induced high density lipoprotein cholesterol (HDL-C) changes. The 123 most promising signals with p<1×10-4 from the 16 769 statin-treated participants in the first analysis stage were followed up in an independent group of 10 951 statin-treated individuals, providing a total sample size of 27 720 individuals. The only associations of genome-wide significance (p<5×10-8) were between minor alleles at the CETP locus and greater HDL-C response to statin treatment. CONCLUSIONS:Based on results from this study that included a relatively large sample size, we suggest that CETP may be the only detectable locus with common genetic variants that influence HDL-C response to statins substantially in individuals of European descent. Although CETP is known to be associated with HDL-C, we provide evidence that this pharmacogenetic effect is independent of its association with baseline HDL-C levels.

Background— Statin therapy influences not only low-density lipoprotein (LDL) cholesterol levels but also LDL-related biomarkers, including non–high-density lipoprotein cholesterol (non–HDL-C), apolipoprotein B, total number of LDL particles, and mean LDL particle size. Recent studies have identified many genetic loci influencing circulating lipid levels and statin-induced LDL cholesterol reduction. However, it is unknown how these genetic variants influence statin-induced changes in LDL subfractions and non–HDL-C. Methods and Results— One hundred sixty candidate single-nucleotide polymorphisms for effects on circulating lipid levels or statin-induced LDL-cholesterol lowering were tested for association with response of LDL subfractions and non–HDL-C to rosuvastatin or placebo for 1 year among 7046 participants from the Justification for Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial. Of the 51 single-nucleotide polymorphisms associated with statin response for ≥1 of the LDL subfractions or non–HDL-C, 20 single-nucleotide polymorphisms could be clustered according to effects predominantly on LDL particle size, predominantly on LDL particle number, and on apolipoprotein B but not on LDL cholesterol or non–HDL-C. Conclusions— These differential associations point to pathways of LDL response to statin therapy and possibly to mechanisms of statin-dependent cardiovascular disease risk reduction. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00239681.

BACKGROUND:Interleukin 18 (IL-18) promotes atherosclerotic plaque formation and is increased in patients with acute coronary syndromes. However the relative contribution of genetic variants to the IL-18 levels has not been fully determined. METHODS AND RESULTS:Baseline plasma IL-18 levels were measured in 16 633 patients with acute coronary syndrome, of whom 9340 had genetic data that passed genotype quality control. A 2-stage genome-wide association study was performed, followed by combined analyses using >10 million genotyped or imputed genetic markers. Single nucleotide polymorphisms at 3 loci (IL18, NLRC4, and MROH6) were identified (P<3.15×10(-8)) in the discovery cohort (n=3777) and replicated in the remaining patients (n=5563). In the pooled data (discovery+replication cohort), 7 independent associations, in 5 chromosomal regions, were associated with IL-18 levels (minimum P=6.99×10(-72)). Six single nucleotide polymorphisms are located in predicted promoter regions of which one disrupts a transcription factor binding site. One single nucleotide polymorphism in NLRC4 is a rare missense variant, predicted to be deleterious to the protein. Altogether, the identified genetic variants explained 8% of the total variation in IL-18 levels in the cohort. CONCLUSIONS:Our results show that genetic variants play an important role in determining IL-18 levels in patients with acute coronary syndrome and we have identified genetic variants located in the IL-18 gene (IL18) or close to genes that are involved in procaspase-1 activation (NLRC4 and CARD16, CARD17, and CARD18). These associations also highlight the importance of the NLRC4 inflammasome for IL-18 production in acute coronary syndrome patients.

AIMS Ticagrelor, a direct-acting P2Y12-receptor antagonist, is rapidly absorbed and partly metabolized to the major metabolite AR-C124910XX (ARC). To identify single-nucleotide polymorphisms (SNPs) associated with pharmacokinetics of ticagrelor and clinical outcomes, we performed a genome-wide association study (GWAS) in patients treated with ticagrelor in the PLATO trial. METHODS AND RESULTS A two-stage design was used for the GWAS with discovery (discovery phase: n = 1812) and replication cohorts (replication phase: n = 1941). The steady-state area under the curve (AUCss) values, estimated by the population pharmacokinetic (PK) models, were log transformed and analysed on a genome-wide scale using linear regression. SNPs were analysed against clinical events using Cox-regression in 4990 patients. An SNP (rs113681054) in SLCO1B1 was associated with levels of ticagrelor (P = 1.1 × 10(-6)) and ARC (P = 4.6 × 10(-13)). This SNP is in linkage disequilibrium with a functional variant (rs4149056) that results in decreased OATP1B1 transporter activity. Ticagrelor levels were also associated with two independent SNPs (rs62471956, P = 7.7 × 10(-15) and rs56324128, P = 9.7 × 10(-12)) in the CYP3A4 region. Further, ARC levels were associated with rs61361928 (P = 3.0 × 10(-14)) in UGT2B7. At all loci, the effects were small. None of the identified SNPs that affected ticagrelor PK were associated with the primary composite outcome (cardiovascular death myocardial infarction, and stroke), non-CABG-related bleeds or investigator-reported dyspnoea. CONCLUSION In patients with ACS, ticagrelor pharmacokinetics is influenced by three genetic loci (SLCO1B1, UGT2B7, and CYP3A4). However, the modest genetic effects on ticagrelor plasma levels did not translate into any detectable effect on efficacy or safety during ticagrelor treatment. CLINICAL TRIAL REGISTRATION NCT00391872.

Statins effectively lower LDL cholesterol levels in large studies and the observed interindividual response variability may be partially explained by genetic variation. Here we perform a pharmacogenetic meta-analysis of genome-wide association studies (GWAS) in studies addressing the LDL cholesterol response to statins, including up to 18,596 statin-treated subjects. We validate the most promising signals in a further 22,318 statin recipients and identify two loci, SORT1/CELSR2/PSRC1 and SLCO1B1, not previously identified in GWAS. Moreover, we confirm the previously described associations with APOE and LPA. Our findings advance the understanding of the pharmacogenetic architecture of statin response.

In the PLATO trial ticagrelor was superior to clopidogrel in reducing ischemic outcome and death in patients with acute coronary syndromes (ACS). Ticagrelor is a direct-acting P2Y12 receptor antagonist that is rapidly absorbed and partly metabolized to the major metabolite AR-C124910XX (ARC). ARC is

Evaluation of the Effect of Interleukin 18 Associated Genetic Polymorphisms on Risk of Cardiovascular Events in Patients With Acute Coronary Syndrome

Are There Any Causal Relations Between Growth Differentiation Factor 15 and Outcomes in Patients With Acute Coronary Syndrome? : - A Report From the Plato Gwas Study

BACKGROUND:Carriers of the rs4363657C and rs4149056C alleles in SLCO1B1 have increased myopathic complaints when taking simvastatin. Whether rosuvastatin has a similar effect is uncertain. This study assesses whether SLCO1B1 polymorphisms relate to clinical myalgia after rosuvastatin therapy. METHODS:In the JUPITER trial, participants without prior cardiovascular disease or diabetes who had low-density lipoprotein cholesterol <130 mg/dL and C-reactive protein ≥2 mg/L were randomly allocated to rosuvastatin 20 mg or placebo and followed for the first cardiovascular disease events and adverse effects. We evaluated the effect of rs4363657 and rs4149056 in SLCO1B1, which encodes organic anion-transporting polypeptide OATP1B1, a regulator of hepatic statin uptake, on clinically reported myalgia. RESULTS:Among 4,404 participants allocated to rosuvastatin, clinical myalgia occurred with a rate of 4.1 events per 100 person-years as compared with 3.7 events per 100 person-years among 4,378 participants allocated to placebo (hazard ratio [HR] 1.13, 95% CI 0.98-1.30). Among those on rosuvastatin, there were no differences in the rate of myalgia among those with the rs4363657C (HR 0.95, 95% CI 0.79-1.14 per allele) or the rs4149056C allele (HR 0.95, 95% CI 0.79-1.15 per allele) compared with those without the C allele. Similar null data were observed when the myalgia definition was broadened to include muscle weakness, stiffness, or pain. None of the 3 participants on rosuvastatin or the 3 participants on placebo with frank myopathy had the minor allele at either polymorphism. CONCLUSION:There appears to be no increased risk of myalgia among users of rosuvastatin who carry the rs4363657C or the rs4149056C allele in SLCO1B1.

Center for Cardiovascular Disease Prevention, the Division of Preventive Medicine, JUPITER Trial Coordinating Center, Division of Genetics, Division of Cardiology, Brigham and Women’s Hospital, Boston, MA; Helmholtz Zentrum München, German Research Center for Environmental Health, Research Unit of Molecular Epidemiology, Neuherberg, Germany; National Heart, Lung, and Blood Institute’s & Boston University's Framingham Heart Study, Framingham; Dept of Biostatistics, Boston University School of Public Health, Boston, MA; Division of Atherosclerosis & Vascular Medicine, Dept of Medicine, Baylor College of Medicine, Methodist DeBaakey Heart and Vascular Center, Houston, TX; Personalised Healthcare & Biomarkers, AstraZeneca Research & Development, Alderley Park, UK; Global Epidemiology, AstraZeneca Research and Development, Mölndal; Unit of Occupational and Environmental Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; Harvard Medical School, Boston, MA †contributed equally

OBJECTIVE: To investigate genes that may be important in determining lung function in subjects with asthma. METHODS : A genome-wide association study has been performed in subjects with asthma from the COMPASS study (Bleecker et al, Lancet 370: 2118-25, 2007). To reduce population stratification, homogenous populations were identified based on geographic region, self-reported race, and genetic ancestry using HapMap reference data. Standard quality control measures were applied to the resulting genotype data. Genome-wide association studies (GWAS) for percent predicted FEV1 and FEV1/FVC was performed on the genetically homogenous population from Eastern Europe using PLINK. RESULTS : To date, GWAS has been performed on 587,020 SNPs in Eastern Europeans (n = 885). For percent predicted FEV1, the top three SNPs were in or near SLITRK5 (SLIT and NTRK-like family, member 5) on chromosome 13 (p=1.2-2.3x10-06). Additional genes of interest for percent predicted FEV1 include ADAM7 (A Disintegrin And Metalloprotease 7) (p=9.1x10-06) and ADRA1D (alpha-1D-adrenergic receptor) (p=1.1x10-05). For FEV1/FVC ratio, the top three genes are FGF1 (fibroblast growth factor 1) (p=2.2x10-06), EPHA5 (ephrin type-A receptor 5) (p=2.8x10-06) and WNT3A (wingless-type MMTV integration site family, member 3A) (p=6.3x10-06). CONCLUSIONS : Evidence was found to suggest that genes involved in regulation of cellular growth ( ADRA1D and FGF1 ) and asthma susceptibility ( EPHA5 ) are associated with lung function in subjects with asthma.

Aims: Lipoprotein-associated phospholipase A2 (Lp-PLA 2 ) is a pro-inflammatory enzyme bound to LDL-C and other lipoproteins in circulation that is associated with increased cardiovascular risk. There is little data regarding genetic determinants of Lp-PLA 2 mass and activity, and no prior data are available addressing genetic determinants of statin-induced changes for this inflammatory biomarker. Methods and Results: We performed a genome-wide association study of Lp-PLA 2 mass and activity at baseline and after 12 months of rosuvastatin therapy (20 mg/day) among 6,851 participants of European ancestry from the JUPITER trial. Genome-wide associations (p<5x10 -8 ) were identified in GCKR and CETP for baseline Lp-PLA 2 mass, and at MS4A4E and the APOC1 - APOE locus for baseline Lp-PLA 2 activity. A variant in PLA2G7 , the gene encoding the Lp-PLA 2 enzyme was associated with Lp-PLA2 activity (p=3.1x10 -7 ) at a sub-genome-wide significance (p<5x10 -6 ); this association attained genome-wide significance after adjusting for either baseline apoB (p=2.9x10 -8 ) or HDL-C levels (p=2.5x10 -9 ). Two other sub-genome-wide associations with Lp-PLA 2 activity attained genome-wide significance after adjusting for baseline lipoproteins: at the PSRC1 - SORT1 locus (before adjustment, p=8.3x10 -7 ; adjusting for triglycerides, p=3.4x10 -8 ) and the TMEM49 locus (before adjustment p=3.8x10 -6 , adjusting for HDL-C p=1.4x10 -8 ). Among 2,673 statin-allocated participants, median reductions were approximately 30% for Lp-PLA 2 mass and activity (versus ~50% for LDL-C) after 12 months of statin therapy (all p-values <0.001). Variants in ABCG2 and LPA were associated with statin-induced change in Lp-PLA 2 activity at genome-wide significance, and associations at sub-genome-wide significance (p<5x10 -6 ) were observed for EDG7 , C14orf177 , NOX5 , AP3B2 and RNF213 . All associations, with the exception of AP3B2 , were substantially attenuated after adjustment for statin induced changes in LDL-C or apoB. Conclusion: Genome-wide associations at the MS4A4E and TMEM49 loci may reflect novel influences on circulating levels of Lp-PLA 2 activity. In addition, genome-wide determinants of rosuvastatin-induced change in Lp-PLA 2 activity were observed in ABCG2 and LPA , likely due to their impact on statin-induced LDL-C reduction.

Background—Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a proinflammatory enzyme bound to low-density lipoprotein cholesterol and other circulating lipoproteins. Two measures of Lp-PLA2, mass and activity, are associated with increased cardiovascular risk. Data are sparse regarding genetic determinants of Lp-PLA2 mass and activity, and no prior data are available addressing genetic determinants of statin-induced changes for this proinflammatory biomarker. Methods and Results—We performed a genome-wide association study of Lp-PLA2 mass and activity at baseline and after 12 months of rosuvastatin therapy (20 mg/d) among 6851 participants of European ancestry from the Justification for Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) and performed replication in a meta-analysis of 13 664 participants from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. Novel associations were identified and replicated at MS4A4E and TMEM49 for baseline Lp-PLA2 activity with genome-wide significant joint P values (P=2.0×10−11 and P=2.9×10−9, respectively). In addition, genome-wide associations (P<5×10−8) were identified and replicated for baseline Lp-PLA2 mass at CETP and for Lp-PLA2 activity at the APOC1-APOE and PLA2G7 loci. Among 2673 statin-allocated participants, both Lp-PLA2 mass and activity were reduced by >30% and low-density lipoprotein cholesterol by 50% after 12 months of statin therapy (P<0.001 for both). Variants in ABCG2 and LPA were associated with change in statin-induced Lp-PLA2 activity at genome-wide significance but were substantially attenuated after adjustment for statin-induced changes in lipid levels. Conclusions—Genome-wide significant associations at MS4A4E and TMEM49 may reflect novel influences on circulating levels of Lp-PLA2 activity. In addition, genome-wide significant associations with rosuvastatin-induced change in Lp-PLA2 activity were observed in ABCG2 and LPA, likely because of their impact on statin-induced low-density lipoprotein cholesterol lowering.

Greater efficacy of ticagrelor compared to clopidogrel in acute coronary syndrome is not driven by outcomes in poor metabolizers of clopidogrel

Background— In randomized trials, statins reduce plasma levels of C-reactive protein (CRP) and low-density lipoprotein cholesterol (LDL-C), and the magnitude of event reduction relates to on-treatment levels of both. However, whether different mechanisms underlie statin-induced CRP and LDL-C reduction is unknown. Methods and Results— We performed a study to evaluate potential genetic determinants of CRP response using genome-wide genetic data from a total of 6766 participants of European ancestry randomly allocated to 20 mg/d of rosuvastatin or placebo in the JUPITER trial. Among 3386 rosuvastatin-allocated individuals, both CRP and LDL-C levels were reduced by 50% after 12 months of therapy (P<0.001 for both) and essentially uncorrelated (r 2<0.03). No variants in the 3 genes (ABCG2, LPA, and APOE) that previously showed genome-wide association with LDL-C reduction in this cohort and none of the candidate single-nucleotide polymorphisms associated with LDL-C reduction were associated with rosuvastatin-induced CRP change after multiple testing correction. Among candidate single-nucleotide polymorphisms selected from prior genetic analyses of baseline CRP, CRP reduction was associated with rs2794520 in CRP (mean, −3.5% [SE, 2.0%] change in CRP per minor allele; P=6.4×10−4) and with rs2847281 in PTPN2 (mean, 3.7% [SE, 1.9%] change in CRP per minor allele; P=7.4×10−4). These associations remained significant after multiple testing correction but were not significant in a formal test of interaction. Neither variant was associated with rosuvastatin-induced LDL-C reduction or with CRP reduction among 3380 placebo-allocated JUPITER participants. Conclusions— The genetic determinants of rosuvastatin-induced CRP reduction differ from, and are largely independent of, the major pharmacogenetic determinants of rosuvastatin-induced LDL-C reduction. This supports the hypothesis that differing pathways may mediate the anti-inflammatory and lipid-lowering properties of statin therapy.

AIM:To investigate potential relationships between SNPs and acute interstitial lung disease (ILD) events in Japanese non-small-cell lung cancer patients receiving gefitinib.MATERIALS & METHODS:Japanese non-small-cell lung cancer patients treated with gefitinib from a prospective pharmacoepidemiological cohort with a nested case-control study component ('CCS'; 52 ILD cases, 139 controls) and a retrospective study (28 ILD cases, 55 controls) were genotyped for nearly 500,000 SNPs. Associations between genotype and ILD were evaluated using Fisher's exact test and logistic regression modeling, and false discovery rate analysis was used to adjust for the large number of statistical tests.RESULTS:The CCS data provided some false discovery rate evidence that the significance of top-ranking SNPs exceeded levels expected by chance, suggesting some genuine associations. However, replication analyses using retrospective study data were not supportive and there was little evidence of strong genetic associations from a combined analysis. Adjustment of CCS analyses for clinical variables provided little additional convincing evidence. Significant gene-gene interactions between SNP pairs using CCS data were not confirmed in retrospective study replication analyses.CONCLUSION:Although it is not possible to exclude genetic influences in ILD etiology, common sequence variation is unlikely to explain a major component of ILD risk. Our top results may provide a useful hypothesis-generating starting point for further research.

Genome‐wide association (GWA) studies have proved extremely successful in identifying novel genetic loci contributing effects to complex human diseases. In doing so, they have highlighted the fact that many potential loci of modest effect remain undetected, partly due to the need for samples consisting of many thousands of individuals. Large‐scale international initiatives, such as the Wellcome Trust Case Control Consortium, the Genetic Association Information Network, and the database of genetic and phenotypic information, aim to facilitate discovery of modest‐effect genes by making genome‐wide data publicly available, allowing information to be combined for the purpose of pooled analysis. In principle, disease or control samples from these studies could be used to increase the power of any GWA study via judicious use as “genetically matched controls” for other traits. Here, we present the biological motivation for the problem and the theoretical potential for expanding the control group with publicly available disease or reference samples. We demonstrate that a naïve application of this strategy can greatly inflate the false‐positive error rate in the presence of population structure. As a remedy, we make use of genome‐wide data and model selection techniques to identify “axes” of genetic variation which are associated with disease. These axes are then included as covariates in association analysis to correct for population structure, which can result in increases in power over standard analysis of genetic information from the samples in the original GWA study. Genet. Epidemiol. 34: 319–326, 2010. © 2010 Wiley‐Liss, Inc.

Background In the PLATO trial of ticagrelor versus clopidogrel for treatment of acute coronary syndromes, ticagrelor reduced the composite outcome of cardiovascular death, myocardial infarction, and stroke, but increased events of major bleeding related to non-coronary artery bypass graft (CABG). CYP2C19 and ABCB1 genotypes are known to influence the effects of clopidogrel. In this substudy, we investigated the effects of these genotypes on outcomes between and within treatment groups.Methods DNA samples obtained from patients in the PLATO trial were genotyped for CYP2C19 loss-of-function alleles (*2, *3, *4, *5, *6, *7, and *8), the CYP2C19 gain-of-function allele *17, and the ABCB1 single nucleotide polymorphism 3435C -> T. For the CYP2C19 genotype, patients were stratified by the presence or absence of any loss-of-function allele, and for the ABCB1 genotype, patients were stratified by predicted gene expression (high, intermediate, or low). The primary efficacy endpoint was the composite of cardiovascular death, myocardial infarction, or stroke after up to 12 months' treatment with ticagrelor or clopidogrel.Findings 10 285 patients provided samples for genetic analysis. The primary outcome occurred less often with ticagrelor versus clopidogrel, irrespective of CYP2C19 genotype: 8.6% versus 11.2% (hazard ratio 0.77, 95% CI 0.60-0.99, p=0.0380) in patients with any loss-of-function allele; and 8.8% versus 10.0% (0.86, 0.74-1.01, p=0.0608) in those without any loss-of-function allele (interaction p=0.46). For the ABCB1 genotype, event rates for the primary outcome were also consistently lower in the ticagrelor than in the clopidogrel group for all genotype groups (interaction p=0.39; 8.8% vs 11.9%; 0.71, 0.55-0.92 for the high-expression genotype). In the clopidogrel group, the event rate at 30 days was higher in patients with than in those without any loss-of-function CYP2C19 alleles (5.7% vs 3.8%, p=0.028), leading to earlier separation of event rates between treatment groups in patients with loss-of-function alleles. Patients on clopidogrel who had any gain-of-function CYP2C19 allele had a higher frequency of major bleeding (11.9%) than did those without any gain-of-function or loss-of-function alleles (9.5%; p=0.022), but interaction between treatment and genotype groups was not significant for any type of major bleeding.Interpretation Ticagrelor is a more efficacious treatment for acute coronary syndromes than is dopidogrel, irrespective of CYP2C19 and ABCB1 polymorphisms. Use of ticagrelor instead of clopidogrel eliminates the need for presently recommended genetic testing before dual antiplatelet treatment.