Asif Rasheed

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.

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.

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.

The burden of stroke and transient ischemic attack in Pakistan: a community-based prevalence study

BackgroundThe burden of cerebrovascular disease in developing countries is rising sharply. The prevalence of established risk factors of stroke is exceptionally high in Pakistan. However, there is limited data on the burden of stroke and transient ischemic attack (TIA) in South Asia. We report the first such study conducted in an urban slum of Karachi, Pakistan.MethodsIndividuals 35 years of age or older were invited for participation in this investigation through simple random sampling. A structured face-to-face interview was conducted using a pre-tested stroke symptom questionnaire in each participant to screen for past stroke or TIA followed by neurological examination of suspected cases. Anthropometric measurements and random blood glucose levels were recorded. Multivariable logistic regression was used to determine the association of vascular risk factors with prevalence of stroke.ResultsFive hundred and forty five individuals (49.4% females) participated in the study with a response rate of 90.8%. One hundred and four individuals (19.1%) were observed to have a prior stroke while TIA was found in 53 individuals (9.7%). Overall, 119 individuals (21.8% with 66.4% females) had stroke and/or TIA. Female gender, old age, raised random blood glucose level and use of chewable tobacco were significantly associated with the prevalence of cerebrovascular disease.ConclusionThis is the first study demonstrating an alarmingly high life-time prevalence of cerebrovascular disease in Pakistan. Individual and public health interventions in Pakistan to increase awareness about stroke, its prevention and therapy are warranted.

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.

Genome-wide association studies in the Japanese population identify seven novel loci for type 2 diabetes

Genome-wide association studies (GWAS) have identified more than 80 susceptibility loci for type 2 diabetes (T2D), but most of its heritability still remains to be elucidated. In this study, we conducted a meta-analysis of GWAS for T2D in the Japanese population. Combined data from discovery and subsequent validation analyses (23,399 T2D cases and 31,722 controls) identify 7 new loci with genome-wide significance (P<5 × 10−8), rs1116357 near CCDC85A, rs147538848 in FAM60A, rs1575972 near DMRTA1, rs9309245 near ASB3, rs67156297 near ATP8B2, rs7107784 near MIR4686 and rs67839313 near INAFM2. Of these, the association of 4 loci with T2D is replicated in multi-ethnic populations other than Japanese (up to 65,936 T2Ds and 158,030 controls, P<0.007). These results indicate that expansion of single ethnic GWAS is still useful to identify novel susceptibility loci to complex traits not only for ethnicity-specific loci but also for common loci across different ethnicities.

Genetic determinants of major blood lipids in Pakistanis compared with Europeans.

Background—Evidence is sparse about the genetic determinants of major lipids in Pakistanis. Methods and Results—Variants (n=45 000) across 2000 genes were assessed in 3200 Pakistanis and compared with 2450 Germans using the same gene array and similar lipid assays. We also did a meta-analysis of selected lipid-related variants in Europeans. Pakistani genetic architecture was distinct from that of several ethnic groups represented in international reference samples. Forty-one variants at 14 loci were significantly associated with levels of HDL-C, triglyceride, or LDL-C. The most significant lipid-related variants identified among Pakistanis corresponded to genes previously shown to be relevant to Europeans, such as CETP associated with HDL-C levels (rs711752; P<10−13), APOA5/ZNF259 (rs651821; P<10−13) and GCKR (rs1260326; P<10−13) with triglyceride levels; and CELSR2 variants with LDL-C levels (rs646776; P<10−9). For Pakistanis, these 41 variants explained 6.2%, 7.1%, and 0.9% of the variation in HDL-C, triglyceride, and LDL-C, respectively. Compared with Europeans, the allele frequency of rs662799 in APOA5 among Pakistanis was higher and its impact on triglyceride concentration was greater (P-value for difference <10−4). Conclusions—Several lipid-related genetic variants are common to Pakistanis and Europeans, though they explain only a modest proportion of population variation in lipid concentration. Allelic frequencies and effect sizes of lipid-related variants can differ between Pakistanis and Europeans.

Apolipoprotein(a) isoform size, lipoprotein(a) concentration, and coronary artery disease: a mendelian randomisation analysis

Summary Background The lipoprotein(a) pathway is a causal factor in coronary heart disease. We used a genetic approach to distinguish the relevance of two distinct components of this pathway, apolipoprotein(a) isoform size and circulating lipoprotein(a) concentration, to coronary heart disease. Methods In this mendelian randomisation study, we measured lipoprotein(a) concentration and determined apolipoprotein(a) isoform size with a genetic method (kringle IV type 2 [KIV2] repeats in the LPA gene) and a serum-based electrophoretic assay in patients and controls (frequency matched for age and sex) from the Pakistan Risk of Myocardial Infarction Study (PROMIS). We calculated odds ratios (ORs) for myocardial infarction per 1-SD difference in either LPA KIV2 repeats or lipoprotein(a) concentration. In a genome-wide analysis of up to 17 503 participants in PROMIS, we identified genetic variants associated with either apolipoprotein(a) isoform size or lipoprotein(a) concentration. Using a mendelian randomisation study design and genetic data on 60 801 patients with coronary heart disease and 123 504 controls from the CARDIoGRAMplusC4D consortium, we calculated ORs for myocardial infarction with variants that produced similar differences in either apolipoprotein(a) isoform size in serum or lipoprotein(a) concentration. Finally, we compared phenotypic versus genotypic ORs to estimate whether apolipoprotein(a) isoform size, lipoprotein(a) concentration, or both were causally associated with coronary heart disease. Findings The PROMIS cohort included 9015 patients with acute myocardial infarction and 8629 matched controls. In participants for whom KIV2 repeat and lipoprotein(a) data were available, the OR for myocardial infarction was 0·93 (95% CI 0·90–0·97; p<0·0001) per 1-SD increment in LPA KIV2 repeats after adjustment for lipoprotein(a) concentration and conventional lipid concentrations. The OR for myocardial infarction was 1·10 (1·05–1·14; p<0·0001) per 1-SD increment in lipoprotein(a) concentration, after adjustment for LPA KIV2 repeats and conventional lipids. Genome-wide analysis identified rs2457564 as a variant associated with smaller apolipoprotein(a) isoform size, but not lipoprotein(a) concentration, and rs3777392 as a variant associated with lipoprotein(a) concentration, but not apolipoprotein(a) isoform size. In 60 801 patients with coronary heart disease and 123 504 controls, OR for myocardial infarction was 0·96 (0·94–0·98; p<0·0001) per 1-SD increment in apolipoprotein(a) protein isoform size in serum due to rs2457564, which was directionally concordant with the OR observed in PROMIS for a similar change. The OR for myocardial infarction was 1·27 (1·07–1·50; p=0·007) per 1-SD increment in lipoprotein(a) concentration due to rs3777392, which was directionally concordant with the OR observed for a similar change in PROMIS. Interpretation Human genetic data suggest that both smaller apolipoprotein(a) isoform size and increased lipoprotein(a) concentration are independent and causal risk factors for coronary heart disease. Lipoprotein(a)-lowering interventions could be preferentially effective in reducing the risk of coronary heart disease in individuals with smaller apolipoprotein(a) isoforms. Funding British Heart Foundation, US National Institutes of Health, Fogarty International Center, Wellcome Trust, UK Medical Research Council, UK National Institute for Health Research, and Pfizer.