Genome-wide association and Mendelian randomization study of NT-proBNP in patients with acute coronary syndrome.

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.

Meta-analysis of genome-wide association studies of HDL cholesterol response to statins.

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.

Effect of genetic variations on ticagrelor plasma levels and clinical outcomes.

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.

Polymorphism of the cystatin C gene in patients with acute coronary syndromes: Results from the PLATelet inhibition and patient Outcomes study.

PURPOSE: Elevated cystatin C concentration is an independent risk factor for cardiovascular (CV) events in patients with acute coronary syndromes. Genetic polymorphisms in CST3 influence cystatin C levels, but their relationship to outcomes is unclear. METHODS: We measured cystatin C concentrations in plasma, obtained within 24 hours of admission, in 16,279 acute coronary syndrome patients from the PLATO trial. In 9,978 patients, we performed a genome-wide association study with up to 2.5 million single nucleotide polymorphisms. Single nucleotide polymorphisms affecting cystatin C levels were evaluated in relation to the first occurrence of myocardial infarction (MI) or CV death within 1 year using Cox regression analysis. RESULTS: Several single nucleotide polymorphisms were associated with cystatin C levels, most significantly rs6048952 (P = 7.82 × 10(-16)) adjacent to CST3. Median cystatin C concentrations per genotype were 0.85 mg/L (A/A), 0.80 mg/L (A/G), and 0.73 mg/L (G/G). Modeled as additive, the allelic effect, multivariable adjusted, was -0.045 mg/L per G allele for rs6048952. The multivariable adjusted c-statistic regarding the combined end point (CV death or MI) was 0.6735. Adding cystatin C or genotype-adjusted cystatin C levels resulted in c-statistics of 0.6761 and 0.6758, respectively. The multivariable adjusted hazard ratios per G allele at rs6048952 in the entire population were 0.94 (95% CI 0.83-1.06) for CV death or MI and 0.88 (95% CI 0.71-1.08) for CV death. CONCLUSIONS: Genetic polymorphisms affect cystatin C concentrations independently of kidney function. However, the polymorphisms were not observed to be associated with outcome, nor did they improve risk prediction or discriminative models.

Lack of association between SLCO1B1 polymorphisms and clinical myalgia following rosuvastatin therapy.

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.

Optimizing the power of genome-wide association studies by using publicly available reference samples to expand the control group.

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.

Effect of CYP2C19 and ABCB1 single nucleotide polymorphisms on outcomes of treatment with ticagrelor versus clopidogrel for acute coronary syndromes: a genetic substudy of the PLATO trial.

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 clopidogrel, irrespective of CYP2C19 and ABCB1 polymorphisms. Use of ticagrelor instead of clopidogrel eliminates the need for presently recommended genetic testing before dual antiplatelet treatment. FUNDING: AstraZeneca.

Sequencing and association analysis of the type 1 diabetes-linked region on chromosome 10p12-q11.

BACKGROUND: In an effort to locate susceptibility genes for type 1 diabetes (T1D) several genome-wide linkage scans have been undertaken. A chromosomal region designated IDDM10 retained genome-wide significance in a combined analysis of the main linkage scans. Here, we studied sequence polymorphisms in 23 Mb on chromosome 10p12-q11, including the putative IDDM10 region, to identify genes associated with T1D. RESULTS: Initially, we resequenced the functional candidate genes, CREM and SDF1, located in this region, genotyped 13 tag single nucleotide polymorphisms (SNPs) and found no association with T1D. We then undertook analysis of the whole 23 Mb region. We constructed and sequenced a contig tile path from two bacterial artificial clone libraries. By comparison with a clone library from an unrelated person used in the Human Genome Project, we identified 12,058 SNPs. We genotyped 303 SNPs and 25 polymorphic microsatellite markers in 765 multiplex T1D families and followed up 22 associated polymorphisms in up to 2,857 families. We found nominal evidence of association in six loci (P = 0.05 - 0.0026), located near the PAPD1 gene. Therefore, we resequenced 38.8 kb in this region, found 147 SNPs and genotyped 84 of them in the T1D families. We also tested 13 polymorphisms in the PAPD1 gene and in five other loci in 1,612 T1D patients and 1,828 controls from the UK. Overall, only the D10S193 microsatellite marker located 28 kb downstream of PAPD1 showed nominal evidence of association in both T1D families and in the case-control sample (P = 0.037 and 0.03, respectively). CONCLUSION: We conclude that polymorphisms in the CREM and SDF1 genes have no major effect on T1D. The weak T1D association that we detected in the association scan near the PAPD1 gene may be either false or due to a small genuine effect, and cannot explain linkage at the IDDM10 region.

Gene mapping strategies for complex disease and drug response.

The identification of genetic variants involved in disease susceptibility and response to drugs through the use of statistical and epidemiological approaches is a potentially powerful methodology for uncovering causal relationships in human disease and its treatment. Here we introduce and compare the application of genetics in these two fields of research.:

Maternal-fetal interactions and birth order influence insulin variable number of tandem repeats allele class associations with head size at birth and childhood weight gain.

Polymorphism of the insulin gene (INS) variable number of tandem repeats (VNTR; class I or class III alleles) locus has been associated with adult diseases and with birth size. Therefore, this variant is a potential contributory factor to the reported fetal origins of adult disease. In the population-based Avon Longitudinal Study of Pregnancy and Childhood birth cohort, we have confirmed in the present study the association between the INS VNTR III/III genotype and larger head circumference at birth (odds ratio [OR] 1.92, 95% CI 1.23-3.07; P = 0.004) and identified an association with higher cord blood IGF-II levels (P = 0.05 to 0.0001). The genotype association with head circumference was influenced by maternal parity (birth order): the III/III OR for larger head circumference was stronger in second and subsequent pregnancies (OR 5.0, 95% CI 2.2-11.5; P = 0.00003) than in first pregnancies (1.2, 0.6-2.2; P = 0.8; interaction with birth order, P = 0.02). During childhood, the III/III genotype remained associated with larger head circumference (P = 0.004) and was also associated with greater BMI (P = 0.03), waist circumference (P = 0.03), and higher fasting insulin levels in girls (P = 0.02). In addition, there were interactions between INS VNTR genotype and early postnatal weight gain in determining childhood BMI (P = 0.001 for interaction), weight (P = 0.005), and waist circumference (P = 0.0005), such that in the approximately 25% of children (n = 286) with rapid early postnatal weight gain, class III genotype-negative children among this group gained weight more rapidly. Our results indicate that complex prenatal and postnatal gene-maternal/fetal interactions influence size at birth and childhood risk factors for adult disease.

Haplotype tag single nucleotide polymorphism analysis of the human orthologues of the rat type 1 diabetes genes Ian4 (Lyp/Iddm1) and Cblb.

The diabetes-prone BioBreeding (BB) and Komeda diabetes-prone (KDP) rats are both spontaneous animal models of human autoimmune, T-cell-associated type 1 diabetes. Both resemble the human disease, and consequently, susceptibility genes for diabetes found in these two strains can be considered as potential candidate genes in humans. Recently, a frameshift deletion in Ian4, a member of the immune-associated nucleotide (Ian)-related gene family, has been shown to map to BB rat Iddm1. In the KDP rat, a nonsense mutation in the T-cell regulatory gene, Cblb, has been described as a major susceptibility locus. Following a strategy of examining the human orthologues of susceptibility genes identified in animal models for association with type 1 diabetes, we identified single nucleotide polymorphisms (SNPs) from each gene by resequencing PCR product from at least 32 type 1 diabetic patients. Haplotype tag SNPs (htSNPs) were selected and genotyped in 754 affected sib-pair families from the U.K. and U.S. Evaluation of disease association by a multilocus transmission/disequilibrium test (TDT) gave a P value of 0.484 for IAN4L1 and 0.692 for CBLB, suggesting that neither gene influences susceptibility to common alleles of human type 1 diabetes in these populations.

Analysis of the type 2 diabetes-associated single nucleotide polymorphisms in the genes IRS1, KCNJ11, and PPARG2 in type 1 diabetes.

It has been proposed that type 1 and 2 diabetes might share common pathophysiological pathways and, to some extent, genetic background. However, to date there has been no convincing data to establish a molecular genetic link between them. We have genotyped three single nucleotide polymorphisms associated with type 2 diabetes in a large type 1 diabetic family collection of European descent: Gly972Arg in the insulin receptor substrate 1 (IRS1) gene, Glu23Lys in the potassium inwardly-rectifying channel gene (KCNJ11), and Pro12Ala in the peroxisome proliferative-activated receptor gamma2 gene (PPARG2). We were unable to confirm a recently published association of the IRS1 Gly972Arg variant with type 1 diabetes. Moreover, KCNJ11 Glu23Lys showed no association with type 1 diabetes (P > 0.05). However, the PPARG2 Pro12Ala variant showed evidence of association (RR 1.15, 95% CI 1.04-1.28, P = 0.008). Additional studies need to be conducted to confirm this result.

Lack of association of the Ala(45)Thr polymorphism and other common variants of the NeuroD gene with type 1 diabetes.

Variation in genes necessary for normal functioning and development of beta-cells, e.g., NEUROD1, which encodes a transcription factor for the insulin gene and is important in beta-cell development, causes maturity-onset diabetes of the young. Some studies have reported an association between a nonsynonymous Ala(45)Thr (+182G-->A) single nucleotide polymorphism (SNP) in NEUROD1 and type 1 diabetes, but this result has not been consistently found. To clarify this, we genotyped Ala(45)Thr in 2,434 type 1 diabetic families of European descent and Caucasian ethnicity from five different countries. Taking the allele frequency of 36% for Thr(45) and an odds ratio (OR) of 1.2, this sample provided >99% power to detect an association (P < 0.05). We could not confirm the association (P = 0.77). No evidence of population heterogeneity in the lack of association of Thr(45) with type 1 diabetes was observed. To evaluate the possibility that another NEUROD1 variant was associated with type 1 diabetes, we resequenced the gene in 32 U.K. affected individuals and identified and genotyped all common SNPs (minor allele frequency >10%; n = 5) in 786 families. We report no evidence of association of these common variants in NEUROD1 and type 1 diabetes in these samples.

Association analysis of the lymphocyte-specific protein tyrosine kinase (LCK) gene in type 1 diabetes.

Prior data associating the expression of lymphocyte-specific protein tyrosine kinase (LCK) with type 1 diabetes, its critical function in lymphocytes, and the linkage of the region to diabetes in the nonobese diabetic (NOD) mouse model make LCK a premier candidate for a susceptibility gene. Resequencing of LCK in 32 individuals detected seven single nucleotide polymorphisms (SNPs) with allele frequencies >3%, including four common SNPs previously reported. These and six other SNPs from dbSNP were genotyped in a two-stage strategy using 2,430 families and were all shown not to be significantly associated with type 1 diabetes. We conclude that a major role for the common LCK polymorphisms in type 1 diabetes is unlikely. However, we cannot rule out the possibility of there being a causal variant outside the exonic, intronic, and untranslated regions studied.

Replication of an association between the lymphoid tyrosine phosphatase locus (LYP/PTPN22) with type 1 diabetes, and evidence for its role as a general autoimmunity locus.

In the genetic analysis of common, multifactorial diseases, such as type 1 diabetes, true positive irrefutable linkage and association results have been rare to date. Recently, it has been reported that a single nucleotide polymorphism (SNP), 1858C>T, in the gene PTPN22, encoding Arg620Trp in the lymphoid protein tyrosine phosphatase (LYP), which has been shown to be a negative regulator of T-cell activation, is associated with an increased risk of type 1 diabetes. Here, we have replicated these findings in 1,388 type 1 diabetic families and in a collection of 1,599 case and 1,718 control subjects, confirming the association of the PTPN22 locus with type 1 diabetes (family-based relative risk (RR) 1.67 [95% CI 1.46-1.91], and case-control odds ratio (OR) 1.78 [95% CI 1.54-2.06]; overall P = 6.02 x 10(-27)). We also report evidence for an association of Trp(620) with another autoimmune disorder, Graves' disease, in 1,734 case and control subjects (P = 6.24 x 10(-4); OR 1.43 [95% CI 1.17-1.76]). Taken together, these results indicate a more general association of the PTPN22 locus with autoimmune disease.

Remapping the insulin gene/IDDM2 locus in type 1 diabetes.

Type 1 diabetes susceptibility at the IDDM2 locus was previously mapped to a variable number tandem repeat (VNTR) 5' of the insulin gene (INS). However, the observation of associated markers outside a 4.1-kb interval, previously considered to define the limits of IDDM2 association, raised the possibility that the VNTR association might result from linkage disequilibrium (LD) with an unknown polymorphism. We therefore identified a total of 177 polymorphisms and obtained genotypes for 75 of these in up to 434 pedigrees. We found that, whereas disease susceptibility did map to within the 4.1-kb region, there were two equally likely candidates for the causal variant, -23HphI and +1140A/C, in addition to the VNTR. Further analyses in 2,960 pedigrees did not support the difference in association between VNTR lineages that had previously enabled the exclusion of these two polymorphisms. Therefore, we were unable to rule out -23HphI and +1140A/C having an etiological effect. Our mapping results using robust regression methods show how precisely a variant for a common disease can be mapped, even within a region of strong LD, and specifically that IDDM2 maps to one or more of three common variants in a approximately 2-kb region of chromosome 11p15.

Common polymorphism in H19 associated with birthweight and cord blood IGF-II levels in humans.

BACKGROUND: Common genetic variation at genes that are imprinted and exclusively maternally expressed could explain the apparent maternal-specific inheritance of low birthweight reported in large family pedigrees. We identified ten single nucleotide polymorphisms (SNPs) in H19, and we genotyped three of these SNPs in families from the contemporary ALSPAC UK birth cohort (1,696 children, 822 mothers and 661 fathers) in order to explore associations with size at birth and cord blood IGF-II levels. RESULTS: Both offspring's and mother's H19 2992C>T SNP genotypes showed associations with offspring birthweight (P = 0.03 to P = 0.003) and mother's genotype was also associated with cord blood IGF-II levels (P = 0.0003 to P = 0.0001). The offspring genotype association with birthweight was independent of mother's genotype (P = 0.01 to P = 0.007). However, mother's untransmitted H19 2992T allele was also associated with larger birthweight (P = 0.04) and higher cord blood IGF-II levels (P = 0.002), suggesting a direct effect of mother's genotype on placental IGF-II expression and fetal growth. The association between mother's untransmitted allele and cord blood IGF-II levels was more apparent in offspring of first pregnancies than subsequent pregnancies (P-interaction = 0.03). Study of the independent Cambridge birth cohort with available DNA in mothers (N = 646) provided additional support for mother's H19 2992 genotype associations with birthweight (P = 0.04) and with mother's glucose levels (P = 0.01) in first pregnancies. CONCLUSION: The common H19 2992T allele, in the mother or offspring or both, may confer reduced fetal growth restraint, as indicated by associations with larger offspring birth size, higher cord blood IGF-II levels, and lower compensatory early postnatal catch-up weight gain, that are more evident among mother's smaller first-born infants.

Differential Genetic Effects on Statin-Induced Changes Across Low-Density Lipoprotein-Related Measures.

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.

NLRC4 Inflammasome Is an Important Regulator of Interleukin-18 Levels in Patients With Acute Coronary Syndromes: Genome-Wide Association Study in the PLATelet inhibition and patient Outcomes Trial (PLATO).

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.

Investigating the utility of combining phi29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray genotyping.

BACKGROUND: Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated phi29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates. RESULTS: Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA. CONCLUSIONS: We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples.