The Bangladesh Risk of Acute Vascular Events (BRAVE) Study: objectives and design.

During recent decades, Bangladesh has experienced a rapid epidemiological transition from communicable to non-communicable diseases. Coronary heart disease (CHD), with myocardial infarction (MI) as its main manifestation, is a major cause of death in the country. However, there is limited reliable evidence about its determinants in this population. The Bangladesh Risk of Acute Vascular Events (BRAVE) study is an epidemiological bioresource established to examine environmental, genetic, lifestyle and biochemical determinants of CHD among the Bangladeshi population. By early 2015, the ongoing BRAVE study had recruited over 5000 confirmed first-ever MI cases, and over 5000 controls "frequency-matched" by age and sex. For each participant, information has been recorded on demographic factors, lifestyle, socioeconomic, clinical, and anthropometric characteristics. A 12-lead electrocardiogram has been recorded. Biological samples have been collected and stored, including extracted DNA, plasma, serum and whole blood. Additionally, for the 3000 cases and 3000 controls initially recruited, genotyping has been done using the CardioMetabochip+ and the Exome+ arrays. The mean age (standard deviation) of MI cases is 53 (10) years, with 88 % of cases being male and 46 % aged 50 years or younger. The median interval between reported onset of symptoms and hospital admission is 5 h. Initial analyses indicate that Bangladeshis are genetically distinct from major non-South Asian ethnicities, as well as distinct from other South Asian ethnicities. The BRAVE study is well-placed to serve as a powerful resource to investigate current and future hypotheses relating to environmental, biochemical and genetic causes of CHD in an important but under-studied South Asian population.The Gates Cambridge Trust has supported Dr Chowdhury. Epidemiological fieldwork in BRAVE has been supported by grants to investigators at the Cardiovascular Epidemiology Unit, University of Cambridge. The Cardiovascular Epidemiology Unit is underpinned by programme grants from the British Heart Foundation (RG/13/13/30194), the UK Medical Research Council (MR/L003120/1), and the UK National Institute of Health Research Cambridge Biomedical Research Centre. BRAVE has received support for genetic assays from the European Research Council (ERC-2010-AdG-20100317), European Commission Framework 7 (Grant Agreement number: 279233), and the Cambridge British Heart Foundation Centre for Excellence in Cardiovascular Science; We would like to acknowledge the contributions of the following individuals: Cardiology Research Group in Bangladesh Mohammad Afzalur Rahman, Mohammad Abdul Kader Akanda, M Atahar Ali, Mir Jamal Uddin, SM Siddiqur Rahman, Amal Kumar Choudhury, Md. Mamunur Rashid, Nazir Ahmed Chowdhury, Mohammad Abdullahel Baqui, Kajal Kumar Karmoker, Mohammad Golam Azam; Setting up/implementation of fieldwork in Bangladesh Abbas Bhuiya, Susmita Chowdhury, Kamrun Nahar, Neelima Das, Proshon Roy, Sumona Ferdous, Taposh Kumar Biswas, Abu Sadat Mohammad Sayed Sharif, Ranjit Shingha, Rose Jinnath Tomas, Babulal Parshei, Mabubur Rahman, Mohammad Emon Hossain, Akhirunnesa Mily, AK Mottashir Ahmed, Sati Chowdhury, Sushila Roy, Dipak Kanti Chowdhury, Swapan Kumar Roy; Epidemiological/statistical support in Cambridge Stephen Kaptoge, Simon Thompson, Angela Wood, Narinder Bansal, Anna Ramond, Clare Oliver-Williams, Marinka Steur, Linda O’Keeffe, Eleni Sofianopoulou, Setor Kunutsor, Donal Gorman, Oscar H Franco, Malcolm Legget, Pinal Patel, Marc Suhrcke, Sylvaine Bruggraber, Jonathan Powell; Data management Matthew Walker, Steve Ellis, Shawkat Jahangir, Habibur Rahman, Rifat Hasan Shammi, Shafqat Ullah, Mohammad Abdul Matin and Administration Beth Collins, Hannah Lombardi, Binder Kaur, Rachel Henry, Marilena Papanikolaou, Robert Smith, Abdul Wazed, Robert Williams, Julie Jenkins, Keith Hoddy.This is the final published version of the article. It was originally published in the European Journal of Epidemiology (Chowdhury R, et al., European Journal of Epidemiology, 2015, doi:10.1007/s10654-015-0037-2). The final version is available at http://dx.doi.org/10.1007/s10654-015-0037-

Fifteen new risk loci for coronary artery disease highlight arterial-wall-specific mechanisms

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP-CAD associations (P < 5 × 10(-8), in fixed-effects meta-analysis) from 15 genomic regions, including SNPs in or near genes involved in cellular adhesion, leukocyte migration and atherosclerosis (PECAM1, rs1867624), coagulation and inflammation (PROCR, rs867186 (p.Ser219Gly)) and vascular smooth muscle cell differentiation (LMOD1, rs2820315). Correlation of these regions with cell-type-specific gene expression and plasma protein levels sheds light on potential disease mechanisms

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease.

Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a loss-of-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant)

Trans-ancestry meta-analyses identify rare and common variants associated with blood pressure and hypertension

High blood pressure is a major risk factor for cardiovascular disease and premature death. However, there is limited knowledge on specific causal genes and pathways. To better understand the genetics of blood pressure, we genotyped 242,296 rare, low-frequency and common genetic variants in up to ~192,000 individuals, and used ~155,063 samples for independent replication. We identified 31 novel blood pressure or hypertension associated genetic regions in the general population, including three rare missense variants in RBM47, COL21A1 and RRAS with larger effects (>1.5mmHg/allele) than common variants. Multiple rare, nonsense and missense variant associations were found in A2ML1 and a low-frequency nonsense variant in ENPEP was identified. Our data extend the spectrum of allelic variation underlying blood pressure traits and hypertension, provide new insights into the pathophysiology of hypertension and indicate new targets for clinical intervention

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease Item Type Article Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease

Abstract Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a loss-of-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. Zanoni et al. Page 2 P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant). The strong inverse association between amounts of high-density lipoprotein (HDL) cholesterol (HDL-C) and coronary heart disease (CHD) risk has generated interest in a potential causal relationship between HDL metabolism and CHD. However, clinical trials with drugs that raise HDL-C levels, niacin and cholesteryl ester transfer protein (CETP) inhibitors, have produced disappointing results ( 1 ). Furthermore, recent studies of human genetic variants that are associated with HDL-C levels have generally failed to show association with CHD ( 2 , 3 ). Most notably, a loss-of-function variant in LIPG, a gene encoding an endothelial lipase that, in the heterozygous state, raises HDL-C by ~5 mg/dl, was found to have no association with CHD ( 4 ). Although these previous studies suggest that higher HDL-C levels may not be causally protective against CHD, we reasoned that additional human genetic analyses might provide mechanistic insight into the complex relationship between HDL and CHD. The scavenger receptor class BI (SR-BI), encoded by the gene SCARB1, was discovered to be an HDL receptor two decades ago ( 5 ). SR-BI promotes the selective uptake of HDL cholesteryl esters (HDL-CEs) into cells, particularly hepatocytes and steroidogenic cells ( 5 , 6 ). In mice, overexpression of SR-BI in the liver reduces levels of HDL-C ( 7 -10 ), and genetic deletion of SR-BI results in higher HDL-C levels ( 11 -13 ). Remarkably, these genetic manipulations in mice have effects on atherosclerosis opposite to those predicted by human epidemiological data: Overexpression reduces atherosclerosis despite the lower HDL-C levels ( 14 -16 ), and gene deletion increases atherosclerosis despite the higher HDL-C levels ( 17 -20 ). One potential explanation relates to the flux of cholesterol from macrophages through the reverse cholesterol transport (RCT) pathway; SR-BI overexpression increases macrophage RCT, and SR-BI knockout reduces macrophage RCT ( 21 ). The human relevance of these observations has been unclear. Identification of SCARB1 P376L homozygote and association with extremely high HDL-C We hypothesized that humans with extremely high levels of HDL-C may harbor loss-offunction variants in SCARB1 and undertook a targeted resequencing discovery experiment in 328 participants with very high HDL-C (>95th percentile, mean HDL-C of 106.8 mg/dl) and a control group of 398 subjects with low HDL-C (<25th percentile, mean HDL-C of 30.4 mg/dl). In this cohort, we sequenced the exons of ~990 genes located within 300 kb of each of the 95 loci with significant associations (P < 5 × 10 −8 ) with plasma lipid levels identified by the Global Lipids Genetics Consortium as of 2010 C>T, p.P376L, rs74830677), a 67-year-old female with an HDL-C of 152 mg/dl, and confirmed this finding by Sanger sequencing. This subject harbored no mutations in other high HDL-C genes such as CETP and LIPG. In addition to this homozygote, four P376L heterozygotes were identified by targeted sequencing in the high HDL-C group; no heterozygotes were found in the low HDL-C group (P =0.008, Fisher's exact test). Zanoni et al. Page 3 To identify additional P376L carriers, we genotyped an expanded cohort of very high versus low HDL-C subjects. Among 524 additional subjects with very high HDL-C (mean HDL-C 95.0 mg/dl), we identified 11 heterozygotes for P376L; whereas among 758 subjects with low HDL-C (mean HDL-C 33.5 mg/dl), we identified 3 heterozygotes. In total, our combined sequencing and genotyping for discovery of the P376L variant showed that this variant is significantly overrepresented in subjects with high HDL-C [minor allele frequency (MAF) = 0.010 in high HDL-C versus 0.0013 in low HDL-C controls, P = 0.000127, Fisher's exact test, Because this variant is present on the exome array, we expanded our analysis to the Global Lipid Genetics Consortium exome array data in >300,000 individuals. The P376L variant was very rare in this population (MAF of ~0.0003). It was significantly associated with higher HDL-C levels with a relatively large effect size (beta = 8.4 mg/dl; P =1.4 × 10 −15 ). Notably, this variant was not associated with plasma levels of low-density lipoprotein cholesterol (LDL-C) or triglycerides (TGs) (table S1). Thus, we conclude that SCARB1 P376L is associated specifically with elevated HDL-C levels. HDL-related phenotypes of SCARB1 P376L homozygote and heterozygotes We next recruited the P376L homozygote, eight heterozygous carriers, and both high HDL-C and normal HDL-C noncarrier controls for deep phenotyping of HDL metabolism and related traits. All of the P376L study participants were of European ancestry, almost exclusively of Ashkenazi Jewish descent. Clinical characteristics and lipid profiles of the subjects are reported in SCARB1 P376L results in complete loss of function of SR-BI Given the profound HDL phenotype of the P376L carriers, we sought to understand the impact of the variant on SR-BI function. We generated induced pluripotent stem cells (iPSCs) using peripheral blood mononuclear cells from the P376L homozygote and a noncarrier control. We next differentiated these cells into hepatocyte-like cells (HLCs) to study HDL metabolism in the setting of endogenous cellular SCARB1 expression. HLCs differentiated through this protocol recapitulate phenotypes of cultured primary hepatocytes Zanoni et al. Page 4 such as albumin and VLDL (very low density lipoprotein) secretion ( 23 -26 ). The cell lines from the control donor and the P376L homozygous subject demonstrated expression of hepatocyte-specific genes ALB (albumin) and AFP (alpha-fetoprotein) and exhibited comparable SCARB1 gene expression ( To evaluate the physiological impact of the P376L variant on HDL-C levels and catabolism in vivo, we used adeno-associated virus (AAV) vectors to direct hepatic overexpression of WT SR-BI or the P376L variant in mice with depleted Scarb1 [Scarb1 knockout (KO) mice]. The two groups of mice demonstrated similar hepatic expression levels of Scarb1 mRNA ( We hypothesized that the markedly reduced HDL-CE uptake could be because of aberrant processing of the P376L SR-BI protein, which leads to impaired cell surface localization. To test this, we isolated cell surface proteins from COS7 cells transfected with WT and P376L SR-BI using biotinylation and found markedly reduced cell surface SR-BI in the P376L transfected cell lysates after streptavidin cell surface protein pull-down assays ( SCARB1 P376L is associated with increased risk of CHD in humans Despite a profound increase in HDL-C, SR-BI deficiency in mice causes accelerated atherosclerosis ( 17 -20 ). The relationship of reduced SR-BI function to atherosclerotic cardiovascular disease in humans has not been established. The P376L homozygous subject did not have clinical CHD, but her carotid intimal-medial thickness (cIMT) was 0.789 mm (left-right average), which is in the >75th percentile for females of her age; in addition, she had detectable plaque throughout the left internal carotid artery and at the bifurcation of her right internal carotid artery. cIMT measurements were not significantly different in the P376L heterozygotes compared with both groups of controls ( To achieve greater statistical power to address this question, we performed a meta-analysis of large exome array genotyping studies of CHD cases and healthy controls to determine the relationship of the P376L variant with risk of CHD ( Discussion Studies of mice have provided important insights into the effects of SR-BI on HDL metabolism, RCT, and atherosclerosis. These studies revealed that overexpression of SR-BI reduces HDL-C ( 7 -10 ) and reduces atherosclerosis ( 14 -16 ), whereas gene deletion of SR-BI increases HDL-C ( 11 -13 ) and accelerates atherosclerosis ( 17 -20 ). The clinical relevance of these findings has remained uncertain, however. Studies of injected labeled HDL-CE in humans suggested that the majority of the HDL-CE was transported to the liver via CETPmediated exchange to apoB-containing lipoproteins rather than by direct uptake from HDL by the liver ( 30 ), which brings into question the importance of hepatic SR-BI in human physiology. Common genetic variants near the SCARB1 locus were found to be significantly associated with plasma HDL-C levels, which suggests that SR-BI may play a role in HDL metabolism in humans ( 22 , 31 ). A family with a rare SCARB1 variant in which serine replaces proline 297 (P297S) was reported in which the heterozygous carriers of the variant had modestly elevated HDL-C levels ( 31 ). However, the variant retains substantial SR-BI activity, no homozygotes were identified, the apparent effect on HDL-C was modest, and there was insufficient power to address its effects on atherosclerosis. Through sequencing of subjects with extremely high plasma levels of HDL-C, we identified a homozygote for a P376L variant in SR-BI. Our complementary approaches consistently Zanoni et al. Page 6 demonstrated that this variant confers virtually complete loss of function of SR-BI. Our results demonstrate many similarities in the consequences of SR-BI deficiency on HDL composition between mice and humans, including a shift toward large, buoyant HDL particles and a significant increase in apoA-I, but not apoA-II, in plasma and HDL ( 12 , 32 , 33 ). The homozygote is a woman who had two healthy children without fertility issues or delivery complications, which suggests that, in humans, SR-BI deficiency may not impair reproductive function in the same manner as it does in mice ( 18 , 34 ). In mice, SR-BImediated CE uptake from HDL is a critical process underlying steroid hormone synthesis in adrenal and gonadal tissues, and SR-BI deficiency alters adrenal cholesterol content, impairs adrenal glucocorticoid response under stress, and can lead to fasting-induced hypoglycemia ( 6 , 35 , 36 ). We did not observe any differences in fasting glucose, serum cortisol, adrenocorticotropic hormone, or female gonadal hormones in P376L heterozygous subjects versus controls, and we saw only a modest increase in testosterone in male P376L heterozygotes relative to noncarriers. We postulate that differences in expression or capacity for up-regulation of apoB-containing lipoprotein receptors relative to SR-BI between mouse models and humans in steroidogenic tissues may account, at least partially, for the lack of recapitulation of some of the phenotypes of SR-BI deficiency in mice. We also observed no differences in platelet levels, cholesterol content, and activation from the P376L carriers, despite reports of thrombocytopenia and altered platelet activity in Scarb1 KO mice ( 31 ). These results suggest a relatively different contribution of SR-BI to platelet function between mice and humans. Note that the phenotypes of human SCARB1 P376L homozygote (elevated HDL-C and large HDL particles but relatively normal steroidogenesis, reproductive viability, and platelet function) are comparable to those observed in mice lacking PDZ domain containing 1 (PDZK1), an adaptor protein for SR-BI ( 37 ). Perhaps the most important finding of our study is that, despite the elevation in HDL-C, P376L carriers exhibit increased risk of CHD, as do Scarb1 KO mice. Our results are consistent with a growing theme in HDL biology that steady-state more important than absolute levels. Using an in vivo assay of macrophage RCT, we previously showed that Scarb1 KO mice have impaired macrophage RCT even though they have elevated HDL-C levels ( 21 ). Our results suggest that reduced hepatic SR-BI function in humans causes impaired RCT, which leads to increased risk of CHD despite elevation in HDL-C levels. However, SR-BI is also expressed in vascular cell types, including endothelial cells, vascular smooth muscle cells, and macrophages, where it could have protective effects against atherosclerosis as well ( 38 , 39 ). Our results are also consistent with the previously suggested concept ( 39 ) that up-regulation or enhancement of SR-BI could be a novel therapeutic approach to reducing CHD risk in the general population

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease

Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a lossof-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant)