Philippe Frossard

Genome-wide association study in individuals of South Asian ancestry identifies six new type 2 diabetes susceptibility loci

We carried out a genome-wide association study of type-2 diabetes (T2D) in individuals of South Asian ancestry. Our discovery set included 5,561 individuals with T2D (cases) and 14,458 controls drawn from studies in London, Pakistan and Singapore. We identified 20 independent SNPs associated with T2D at P < 10−4 for testing in a replication sample of 13,170 cases and 25,398 controls, also all of South Asian ancestry. In the combined analysis, we identified common genetic variants at six loci (GRB14, ST6GAL1, VPS26A, HMG20A, AP3S2 and HNF4A) newly associated with T2D (P = 4.1 × 10−8 to P = 1.9 × 10−11). SNPs at GRB14 were also associated with insulin sensitivity (P = 5.0 × 10−4), and SNPs at ST6GAL1 and HNF4A were also associated with pancreatic beta-cell function (P = 0.02 and P = 0.001, respectively). Our findings provide additional insight into mechanisms underlying T2D and show the potential for new discovery from genetic association studies in South Asians, a population with increased susceptibility to T2D.

Clonal Hematopoiesis and Risk of Atherosclerotic Cardiovascular Disease

BACKGROUND Clonal hematopoiesis of indeterminate potential (CHIP), which is defined as the presence of an expanded somatic blood‐cell clone in persons without other hematologic abnormalities, is common among older persons and is associated with an increased risk of hematologic cancer. We previously found preliminary evidence for an association between CHIP and atherosclerotic cardiovascular disease, but the nature of this association was unclear. METHODS We used whole‐exome sequencing to detect the presence of CHIP in peripheral‐blood cells and associated such presence with coronary heart disease using samples from four case–control studies that together enrolled 4726 participants with coronary heart disease and 3529 controls. To assess causality, we perturbed the function of Tet2, the second most commonly mutated gene linked to clonal hematopoiesis, in the hematopoietic cells of atherosclerosis‐prone mice. RESULTS In nested case–control analyses from two prospective cohorts, carriers of CHIP had a risk of coronary heart disease that was 1.9 times as great as in noncarriers (95% confidence interval [CI], 1.4 to 2.7). In two retrospective case–control cohorts for the evaluation of early‐onset myocardial infarction, participants with CHIP had a risk of myocardial infarction that was 4.0 times as great as in noncarriers (95% CI, 2.4 to 6.7). Mutations in DNMT3A, TET2, ASXL1, and JAK2 were each individually associated with coronary heart disease. CHIP carriers with these mutations also had increased coronary‐artery calcification, a marker of coronary atherosclerosis burden. Hypercholesterolemia‐prone mice that were engrafted with bone marrow obtained from homozygous or heterozygous Tet2 knockout mice had larger atherosclerotic lesions in the aortic root and aorta than did mice that had received control bone marrow. Analyses of macrophages from Tet2 knockout mice showed elevated expression of several chemokine and cytokine genes that contribute to atherosclerosis. CONCLUSIONS The presence of CHIP in peripheral‐blood cells was associated with nearly a doubling in the risk of coronary heart disease in humans and with accelerated atherosclerosis in mice. (Funded by the National Institutes of Health and others.)

Genome-Wide Association Study Identifies a Novel Locus Contributing to Type 2 Diabetes Susceptibility in Sikhs of Punjabi Origin From India

We performed a genome-wide association study (GWAS) and a multistage meta-analysis of type 2 diabetes (T2D) in Punjabi Sikhs from India. Our discovery GWAS in 1,616 individuals (842 case subjects) was followed by in silico replication of the top 513 independent single nucleotide polymorphisms (SNPs) (P < 10−3) in Punjabi Sikhs (n = 2,819; 801 case subjects). We further replicated 66 SNPs (P < 10−4) through genotyping in a Punjabi Sikh sample (n = 2,894; 1,711 case subjects). On combined meta-analysis in Sikh populations (n = 7,329; 3,354 case subjects), we identified a novel locus in association with T2D at 13q12 represented by a directly genotyped intronic SNP (rs9552911, P = 1.82 × 10−8) in the SGCG gene. Next, we undertook in silico replication (stage 2b) of the top 513 signals (P < 10−3) in 29,157 non-Sikh South Asians (10,971 case subjects) and de novo genotyping of up to 31 top signals (P < 10−4) in 10,817 South Asians (5,157 case subjects) (stage 3b). In combined South Asian meta-analysis, we observed six suggestive associations (P < 10−5 to < 10−7), including SNPs at HMG1L1/CTCFL, PLXNA4, SCAP, and chr5p11. Further evaluation of 31 top SNPs in 33,707 East Asians (16,746 case subjects) (stage 3c) and 47,117 Europeans (8,130 case subjects) (stage 3d), and joint meta-analysis of 128,127 individuals (44,358 case subjects) from 27 multiethnic studies, did not reveal any additional loci nor was there any evidence of replication for the new variant. Our findings provide new evidence on the presence of a population-specific signal in relation to T2D, which may provide additional insights into T2D pathogenesis.

Factors Associated with Adherence to Anti-Hypertensive Treatment in Pakistan

Objectives Poor adherence is one of the biggest obstacles in therapeutic control of high blood pressure. The objectives of this study were (i) to measure adherence to antihypertensive therapy in a representative sample of the hypertensive Pakistani population and (ii) to investigate the factors associated with adherence in the studied population. Methods and Results A cross-sectional study was conducted on a simple random sample of 460 patients at the Aga Khan University Hospital (AKUH) and National Institute of Cardiovascular Diseases, Karachi, from September 2005–May 2006. Adherence was assessed using the Morisky Medication Adherence Scale (MMAS), with scores ranging from 0 (non-adherent) to 4 (adherent). In addition to MMAS, patient self-reports about the number of pills taken over a prescribed period were used to estimate adherence as a percentage. AKU Anxiety and Depression Scale (AKU-ADS) was incorporated to find any association between depression and adherence. At a cut-off value of 80%, 77% of the cases were adherent. Upon univariate analyses, increasing age, better awareness and increasing number of pills prescribed significantly improved adherence, while depression showed no association. Significant associations, upon multivariate analyses, included number of drugs that a patient was taking (P<0.02) and whether he/she was taking medication regularly or only for symptomatic relief (P<0.00001). Conclusions Similar to what has been reported worldwide, younger age, poor awareness, and symptomatic treatment adversely affected adherence to antihypertensive medication in our population. In contrast, monotherapy reduced adherence, whereas psychosocial factors such as depression showed no association. These findings may be used to identify the subset of population at risk of low adherence who should be targeted for interventions to achieve better blood pressure control and hence prevent complications.

Association of Phosphodiesterase 4D Gene With Ischemic Stroke in a Pakistani Population

Background and Objectives— Identification of STRK1 locus by the deCODE group followed by the discovery of phosphodiesterase 4D (PDE4D) gene in strong association with ischemic stroke patients has provided useful insights toward understanding the genetic etiology of the disease. In this study, we aimed at investigating the association between 3 polymorphisms of the PDE4D gene and ischemic stroke in the Pakistani population. Methods— Three polymorphisms in PDE4D gene were analyzed in 200 patients of ischemic stroke and 250 controls of Pakistani origin using polymerase chain reaction-restriction fragment length polymorphism method. Data were coded and entered in SPSS Windows (version 12.0). Odds ratios and 95% CIs were calculated using multivariate logistic regression analysis. Results— Marker SNP83(rs966221) was found significantly associated with ischemic stroke on univariate and multivariate analysis (P<0.005; odds ratio, 1.64 [1.13 to 2.40]). Haplotype analysis for markers in linkage disequilibrium failed to show any association with the disease. Conclusion— The association of PDE4D variation with ischemic stroke extends to the Pakistani population and supports a role for phosphodiesterases in stroke pathogenesis.

Association of the 9p21.3 Locus With Risk of First-Ever Myocardial Infarction in Pakistanis Case-Control Study in South Asia and Updated Meta-Analysis of Europeans

Objective—To examine variants at the 9p21 locus in a case-control study of acute myocardial infarction (MI) in Pakistanis and to perform an updated meta-analysis of published studies in people of European ancestry. Methods and Results—A total of 1851 patients with first-ever confirmed MI and 1903 controls were genotyped for 89 tagging single-nucleotide polymorphisms at locus 9p21, including the lead variant (rs1333049) identified by the Wellcome Trust Case Control Consortium. Minor allele frequencies and extent of linkage disequilibrium observed in Pakistanis were broadly similar to those seen in Europeans. In the Pakistani study, 6 variants were associated with MI (P<10−2) in the initial sample set, and in an additional 741 cases and 674 controls in whom further genotyping was performed for these variants. For Pakistanis, the odds ratio for MI was 1.13 (95% CI, 1.05 to 1.22; P=2×10−3) for each copy of the C allele at rs1333049. In comparison, a meta-analysis of studies in Europeans yielded an odds ratio of 1.31 (95% CI, 1.26 to 1.37) for the same variant (P=1×10−3 for heterogeneity). Meta-analyses of 23 variants, in up to 38 250 cases and 84 820 controls generally yielded higher values in Europeans than in Pakistanis. Conclusion—To our knowledge, this study provides the first demonstration that variants at the 9p21 locus are significantly associated with MI risk in Pakistanis. However, association signals at this locus were weaker in Pakistanis than those in European studies.

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.

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity

A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such ‘human knockouts’ can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a ‘human knockout project’, a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.

CAD risk factors and acute myocardial infarction in Pakistan.

Objective — Acute myocardial infarction (AMI) in adult patients under the age of 45 is relatively unusual. Recent studies have shown a higher prevalence of AMI in young South Asian migrants. Data on South Asians in South Asia on cardiovascular disease (CVD) patients is lacking. The purpose of this study is to look at the classical risk factors of coronary artery disease (CAD) in young men and women and their older counterparts who presented to the emergency service of the Aga Khan university hospital (AKUH) and were diagnosed with AMI. Methods — 976 consecutive patients admitted to AKUH with AMI between January 2000- December 2002 were divided into two groups: < 45 years (young) and > 45 years (old). Demographic factors, clinical symptoms and presence of risk factors for CAD were recorded with the help of pre-tested data extraction forms. Results — Young patients represented 16.1% of all patients with AMI. 93.1% of the young patients were men.Young male patients were more likely to be smokers and have high cholesterol levels as compared to their young female counterparts. Young AMI patients when compared to old AMI patients, were more likely to have hypertension, positive family history of coronary artery disease, high cholesterol, high LDL and high triglycerides. Conclusion — In this study, the risk factor profile between young and old patients and between the two genders was found to be different. Thus adult management protocols for AMI should be different from the ones for older patients and gender difference should also be considered.

Causal Assessment of Serum Urate Levels in Cardiometabolic Diseases Through a Mendelian Randomization Study

BACKGROUND Although epidemiological studies have reported positive associations between circulating urate levels and cardiometabolic diseases, causality remains uncertain. OBJECTIVES Through a Mendelian randomization approach, we assessed whether serum urate levels are causally relevant in type 2 diabetes mellitus (T2DM), coronary heart disease (CHD), ischemic stroke, and heart failure (HF). METHODS This study investigated 28 single nucleotide polymorphisms known to regulate serum urate levels in association with various vascular and nonvascular risk factors to assess pleiotropy. To limit genetic confounding, 14 single nucleotide polymorphisms exclusively associated with serum urate levels were used in a genetic risk score to assess associations with the following cardiometabolic diseases (cases/controls): T2DM (26,488/83,964), CHD (54,501/68,275), ischemic stroke (14,779/67,312), and HF (4,526/18,400). As a positive control, this study also investigated our genetic instrument in 3,151 gout cases and 68,350 controls. RESULTS Serum urate levels, increased by 1 SD due to the genetic score, were not associated with T2DM, CHD, ischemic stroke, or HF. These results were in contrast with previous prospective studies that did observe increased risks of these 4 cardiometabolic diseases for an equivalent increase in circulating urate levels. However, a 1 SD increase in serum urate levels due to the genetic score was associated with increased risk of gout (odds ratio: 5.84; 95% confidence interval: 4.56 to 7.49), which was directionally consistent with previous observations. CONCLUSIONS Evidence from this study does not support a causal role of circulating serum urate levels in T2DM, CHD, ischemic stroke, or HF. Decreasing serum urate levels may not translate into risk reductions for cardiometabolic conditions.