Causal effects of body mass index on cardiometabolic traits and events: a Mendelian randomization analysis.

Elevated body mass index (BMI) associates with cardiometabolic traits on observational analysis, yet the underlying causal relationships remain unclear. We conducted Mendelian randomization analyses by using a genetic score (GS) comprising 14 BMI-associated SNPs from a recent discovery analysis to investigate the causal role of BMI in cardiometabolic traits and events. We used eight population-based cohorts, including 34,538 European-descent individuals (4,407 type 2 diabetes (T2D), 6,073 coronary heart disease (CHD), and 3,813 stroke cases). A 1 kg/m(2) genetically elevated BMI increased fasting glucose (0.18 mmol/l; 95% confidence interval (CI) = 0.12-0.24), fasting insulin (8.5%; 95% CI = 5.9-11.1), interleukin-6 (7.0%; 95% CI = 4.0-10.1), and systolic blood pressure (0.70 mmHg; 95% CI = 0.24-1.16) and reduced high-density lipoprotein cholesterol (-0.02 mmol/l; 95% CI = -0.03 to -0.01) and low-density lipoprotein cholesterol (LDL-C; -0.04 mmol/l; 95% CI = -0.07 to -0.01). Observational and causal estimates were directionally concordant, except for LDL-C. A 1 kg/m(2) genetically elevated BMI increased the odds of T2D (odds ratio [OR] = 1.27; 95% CI = 1.18-1.36) but did not alter risk of CHD (OR 1.01; 95% CI = 0.94-1.08) or stroke (OR = 1.03; 95% CI = 0.95-1.12). A meta-analysis incorporating published studies reporting 27,465 CHD events in 219,423 individuals yielded a pooled OR of 1.04 (95% CI = 0.97-1.12) per 1 kg/m(2) increase in BMI. In conclusion, we identified causal effects of BMI on several cardiometabolic traits; however, whether BMI causally impacts CHD risk requires further evidence.

Gene-centric meta-analysis in 87,736 individuals of European ancestry identifies multiple blood-pressure-related loci.

Blood pressure (BP) is a heritable risk factor for cardiovascular disease. To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), and pulse pressure (PP), we genotyped ~50,000 SNPs in up to 87,736 individuals of European ancestry and combined these in a meta-analysis. We replicated findings in an independent set of 68,368 individuals of European ancestry. Our analyses identified 11 previously undescribed associations in independent loci containing 31 genes including PDE1A, HLA-DQB1, CDK6, PRKAG2, VCL, H19, NUCB2, RELA, HOXC@ complex, FBN1, and NFAT5 at the Bonferroni-corrected array-wide significance threshold (p < 6 × 10(-7)) and confirmed 27 previously reported associations. Bioinformatic analysis of the 11 loci provided support for a putative role in hypertension of several genes, such as CDK6 and NUCB2. Analysis of potential pharmacological targets in databases of small molecules showed that ten of the genes are predicted to be a target for small molecules. In summary, we identified previously unknown loci associated with BP. Our findings extend our understanding of genes involved in BP regulation, which may provide new targets for therapeutic intervention or drug response stratification.

Loci influencing blood pressure identified using a cardiovascular gene-centric array.

Blood pressure (BP) is a heritable determinant of risk for cardiovascular disease (CVD). To investigate genetic associations with systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP) and pulse pressure (PP), we genotyped ∼50 000 single-nucleotide polymorphisms (SNPs) that capture variation in ∼2100 candidate genes for cardiovascular phenotypes in 61 619 individuals of European ancestry from cohort studies in the USA and Europe. We identified novel associations between rs347591 and SBP (chromosome 3p25.3, in an intron of HRH1) and between rs2169137 and DBP (chromosome1q32.1 in an intron of MDM4) and between rs2014408 and SBP (chromosome 11p15 in an intron of SOX6), previously reported to be associated with MAP. We also confirmed 10 previously known loci associated with SBP, DBP, MAP or PP (ADRB1, ATP2B1, SH2B3/ATXN2, CSK, CYP17A1, FURIN, HFE, LSP1, MTHFR, SOX6) at array-wide significance (P < 2.4 × 10(-6)). We then replicated these associations in an independent set of 65 886 individuals of European ancestry. The findings from expression QTL (eQTL) analysis showed associations of SNPs in the MDM4 region with MDM4 expression. We did not find any evidence of association of the two novel SNPs in MDM4 and HRH1 with sequelae of high BP including coronary artery disease (CAD), left ventricular hypertrophy (LVH) or stroke. In summary, we identified two novel loci associated with BP and confirmed multiple previously reported associations. Our findings extend our understanding of genes involved in BP regulation, some of which may eventually provide new targets for therapeutic intervention.

Meta-analysis of Dense Genecentric Association Studies Reveals Common and Uncommon Variants Associated with Height.

Height is a classic complex trait with common variants in a growing list of genes known to contribute to the phenotype. Using a genecentric genotyping array targeted toward cardiovascular-related loci, comprising 49,320 SNPs across approximately 2000 loci, we evaluated the association of common and uncommon SNPs with adult height in 114,223 individuals from 47 studies and six ethnicities. A total of 64 loci contained a SNP associated with height at array-wide significance (p < 2.4 × 10(-6)), with 42 loci surpassing the conventional genome-wide significance threshold (p < 5 × 10(-8)). Common variants with minor allele frequencies greater than 5% were observed to be associated with height in 37 previously reported loci. In individuals of European ancestry, uncommon SNPs in IL11 and SMAD3, which would not be genotyped with the use of standard genome-wide genotyping arrays, were strongly associated with height (p < 3 × 10(-11)). Conditional analysis within associated regions revealed five additional variants associated with height independent of lead SNPs within the locus, suggesting allelic heterogeneity. Although underpowered to replicate findings from individuals of European ancestry, the direction of effect of associated variants was largely consistent in African American, South Asian, and Hispanic populations. Overall, we show that dense coverage of genes for uncommon SNPs, coupled with large-scale meta-analysis, can successfully identify additional variants associated with a common complex trait.

Network features of the mammalian circadian clock.

The mammalian circadian clock is a cell-autonomous system that drives oscillations in behavior and physiology in anticipation of daily environmental change. To assess the robustness of a human molecular clock, we systematically depleted known clock components and observed that circadian oscillations are maintained over a wide range of disruptions. We developed a novel strategy termed Gene Dosage Network Analysis (GDNA) in which small interfering RNA (siRNA)-induced dose-dependent changes in gene expression were used to build gene association networks consistent with known biochemical constraints. The use of multiple doses powered the analysis to uncover several novel network features of the circadian clock, including proportional responses and signal propagation through interacting genetic modules. We also observed several examples where a gene is up-regulated following knockdown of its paralog, suggesting the clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. We propose that these network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation.

Genetic components of the circadian clock regulate thrombogenesis in vivo.

BACKGROUND: Myocardial infarction, stroke, and sudden death undergo diurnal variation. Although genes relevant to hemostasis and vascular integrity undergo circadian oscillation, the role of the molecular clock in thrombotic events remains to be established. METHODS AND RESULTS: A diurnal variation in the time to thrombotic vascular occlusion (TTVO) subsequent to a photochemical injury was observed in wild-type mice: TTVO varied from 24.6+/-2.7 minutes at zeitgeber time (ZT) 2 to 40.3+/-4.3 minutes at ZT8, 24.3+/-2.3 minutes at ZT14, and 31.0+/-4.4 minutes at ZT20. This pattern was disrupted or altered when core clock genes-BMAL1, CLOCK, and NPAS2-were mutated or deleted. Mutation of CLOCK abolished the diurnal variation in TTVO, whereas deletion of NPAS2 altered its temporal pattern. NPAS2 deletion prolonged TTVO and reduced blood pressure irrespective of clock time. Global BMAL1 deletion shortened TTVO at ZT8, and the diurnal variation in TTVO, but not in systemic blood pressure, was disrupted in mice in which BMAL1 had been selectively deleted in endothelium. CONCLUSIONS: Key components of the molecular clock regulate the response to a thrombogenic stimulus in vivo. Such a phenomenon may interact with environmental variables, and together with the influence of these genes on blood pressure may contribute to the diurnal variation in cardiovascular events observed in humans.

WAVECLOCK: wavelet analysis of circadian oscillation.

UNLABELLED: Oscillations in mRNA and protein of circadian clock components can be continuously monitored in vitro using synchronized cell lines. These rhythms can be highly variable due to culture conditions and are non-stationary due to baseline trends, damping and drift in period length. We present a technique for characterizing the modal frequencies of oscillation using continuous wavelet decomposition to non-parametrically model changes in amplitude and period while removing baseline effects and noise. AVAILABILITY: The method has been implemented as the package waveclock for the free statistical software program R and is available for download from http://cran.r-project.org/

Prostaglandin E synthases in zebrafish.

OBJECTIVE: Prostaglandin E synthases (PGESs) are being explored as antiinflammatory drug targets as alternatives to cyclooxygenase (COX)-2. Located downstream of the cyclooxygenases, PGESs catalyze PGE(2) formation, and deletion of microsomal (m)-PGES-1 abrogates inflammation. We sought to characterize the developmental expression of COX and PGES in zebrafish. METHODS AND RESULTS: We cloned zebrafish cytosolic (c) and m-PGES orthologs and mapped them to syntenic regions of chromosomes 23 and 5. cPGES was widely expressed during development and was coordinately regulated with zCOX-1 in the inner ear, the pronephros, and intestine. COX-2 and mPGES-1 exhibited restricted expression, dominantly in the vasculature of the aortic arch. However, the enzymes were anatomically segregated within the vessel wall. Experiments with antisense morpholinos and with nonsteroidal antiinflammatory drugs suggest that these genes may not be critical for development. CONCLUSIONS: mPGES-1 is developmentally coregulated with COX-2 in vasculature. Given the high fecundidity and translucency of the zebrafish, this model may afford a high throughput system for characterization of novel PGES inhibitors. Microsomal prostaglandin E synthase (mPGES)-1, located downstream of COX-2, may represent a novel antiinflammatory drug target. Zebrafish cytosolic (c) PGES-1 and COX-1 were coordinately expressed; mPGES-1 and COX-2 were expressed particularly in the vasculature. Zebrafish may afford a high throughput system for detection of novel PGES inhibitors.

Circadian variation of blood pressure and the vascular response to asynchronous stress.

The diurnal variation in the incidence of myocardial infarction and stroke may reflect an influence of the molecular clock and/or the time dependence of exposure to environmental stress. The circadian variation in blood pressure and heart rate is disrupted in mice, Bmal1(-/-), Clock(mut), and Npas2(mut), in which core clock genes are deleted or mutated. Although Bmal1 deletion abolishes the 24-h frequency in cardiovascular rhythms, a shorter ultradian rhythm remains. Sympathoadrenal function is disrupted in these mice, which reflects control of enzymes relevant to both synthesis (phenylethanolamine N-methyl transferase) and disposition (monoamine oxidase B and catechol-O-methyl transferase) of catecholamines by the clock. Both timing and disruption or mutation of clock genes modulate the magnitude of both the sympathoadrenal and pressor but not the adrenocortical response to stress. Despite diurnal variation of catecholamines and corticosteroids, they are regulated differentially by the molecular clock. Furthermore, the clock may influence the time-dependent incidence of cardiovascular events by controlling the integration of selective asynchronous stress responses with an underlying circadian rhythm in cardiovascular function.

Development and evaluation of real competitive PCR for high-throughput quantitative applications.

Real competitive PCR (rcPCR) has been shown to have high sensitivity, reproducibility, and high-throughput potential. We describe further development and evaluation of this methodology as a tool for measuring nucleic acid abundance within a cell. Modifications to the original protocol allow analysis of gene expression levels using standard conditions regardless of mRNA abundance and assay type, thereby increasing throughput and ease of reaction setup while decreasing optimization time. In addition, we have developed a software package, TITAN, to automatically analyze the results. The details are relevant to researchers performing competitive PCR using any detection technique. The effectiveness of the described developments is demonstrated using 12 genes known to have differential expression in cell lines grown under normal and hypoxic conditions. Quantitative and qualitative comparisons to real-time PCR are presented. It is also demonstrated that the technique is capable of detecting submicroscopic chromosomal DNA deletions.