Maria Samuel

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.

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.

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.

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.

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.

Physical activity, smoking, and genetic predisposition to obesity in people from Pakistan: the PROMIS study.

BackgroundMultiple genetic variants have been reliably associated with obesity-related traits in Europeans, but little is known about their associations and interactions with lifestyle factors in South Asians.MethodsIn 16,157 Pakistani adults (8232 controls; 7925 diagnosed with myocardial infarction [MI]) enrolled in the PROMIS Study, we tested whether: a) BMI-associated loci, individually or in aggregate (as a genetic risk score - GRS), are associated with BMI; b) physical activity and smoking modify the association of these loci with BMI. Analyses were adjusted for age, age2, sex, MI (yes/no), and population substructure.ResultsOf 95 SNPs studied here, 73 showed directionally consistent effects on BMI as reported in Europeans. Each additional BMI-raising allele of the GRS was associated with 0.04 (SE = 0.01) kg/m2 higher BMI (P = 4.5 × 10−14). We observed nominal evidence of interactions of CLIP1 rs11583200 (Pinteraction = 0.014), CADM2 rs13078960 (Pinteraction = 0.037) and GALNT10 rs7715256 (Pinteraction = 0.048) with physical activity, and PTBP2 rs11165643 (Pinteraction = 0.045), HIP1 rs1167827 (Pinteraction = 0.015), C6orf106 rs205262 (Pinteraction = 0.032) and GRID1 rs7899106 (Pinteraction = 0.043) with smoking on BMI.ConclusionsMost BMI-associated loci have directionally consistent effects on BMI in Pakistanis and Europeans. There were suggestive interactions of established BMI-related SNPs with smoking or physical activity.

A novel interaction between the FLJ33534 locus and smoking in obesity: a genome-wide study of 14 131 Pakistani adults.

Background:Obesity is a complex disease caused by the interplay of genetic and lifestyle factors, but identification of gene–lifestyle interactions in obesity has remained challenging. Few large-scale studies have reported use of genome-wide approaches to investigate gene–lifestyle interactions in obesity.Methods:In the Pakistan Risk of Myocardial Infraction Study, a cross-sectional study based in Pakistan, we calculated body mass index (BMI) variance estimates (square of the residual of inverse-normal transformed BMI z-score) in 14 131 participants and conducted genome-wide heterogeneity of variance analyses (GWHVA) for this outcome. All analyses were adjusted for age, age2, sex and genetic ancestry.Results:The GWHVA analyses identified an intronic variant, rs140133294, in the FLJ33544 gene in association with BMI variance (P-value=3.1 × 10−8). In explicit tests of gene × lifestyle interaction, smoking was found to significantly modify the effect of rs140133294 on BMI (Pinteraction=0.0005), whereby the minor allele (T) was associated with lower BMI in current smokers, while positively associated with BMI in never smokers. Analyses of ENCODE data at the FLJ33534 locus revealed features indicative of open chromatin and high confidence DNA-binding motifs for several transcription factors, providing suggestive biological support for a mechanism of interaction.Conclusions:In summary, we have identified a novel interaction between smoking and variation at the FLJ33534 locus in relation to BMI in people from Pakistan.

Stroke Radiology and Distinguishing Characteristics of Intracranial Atherosclerotic Disease in Native South Asian Pakistanis

Background There are no descriptions of stroke mechanisms from intracranial atherosclerotic disease in native South Asian Pakistanis. Methods Men and women aged ≥18 years with acute stroke presenting to four tertiary care hospitals in Karachi, Pakistan were screened using magnetic resonance angiography/transcranial Doppler scans. Trial of ORG 10172 in Acute Stroke Treatment criteria were applied to identify strokes from intracranial atherosclerotic disease. Results We studied 245 patients with acute stroke due to intracranial atherosclerotic disease. Two hundred thirty scans were reviewed. Also, 206/230 (89·0%) showed acute ischaemia. The most frequent presentation was with cortically based strokes in 42·2% (87/206) followed by border-zone infarcts (52/206, 25·2%). Increasing degrees of stenosis correlated with the development of both cortical and border-zone strokes (P = 0·002). Important associated findings were frequent atrophy (166/230, 72·2%), silent brain infarcts (66/230, 28%) and a marked lack of severe leukoaraiosis identified in only 68/230 (29·6%). A total of 1870 arteries were studied individually. Middle cerebral artery was the symptomatic stroke vessel in half, presenting with complete occlusion in 66%. Evidence of biological disease, symptomatic or asymptomatic was identified in 753 (40·2%) vessels of which 543 (72%) were significantly (>50%) stenosed at presentation. Conclusion Intracranial atherosclerotic disease is a diffuse process in Pakistani south Asians, with involvement of multiple vessels in addition to the symptomatic vessel. The middle cerebral artery is the most frequent symptomatic vessel presenting with cortical embolic infarcts. There is a relative lack of leukoaraiosis. Concomitant atrophy, silent brain infarcts and recent ischaemia in the symptomatic territory are all frequently associated findings.

Human knockouts 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.1 Null mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such ‘human knockouts’ can provide insight into gene function. To date, comprehensive analysis of genes knocked out in humans has been limited by the fact that null mutations are infrequent in the general population and so, observing an individual homozygous null for a given gene is exceedingly rare.2,3 However, consanguineous unions are more likely to result in offspring who carry homozygous null mutations. In Pakistan, consanguinity rates are notably high.4 Here, we sequenced the protein-coding regions of 7,078 adult participants living in Pakistan and performed phenotypic analysis to identify homozygous null individuals and to understand consequences of complete gene disruption in humans. We enumerated 36,850 rare (<1 % minor allele frequency) null mutations. These homozygous null mutations led to complete inactivation of 961 genes in at least one participant. Homozygosity for null mutations at APOC3 was associated with absent plasma apolipoprotein C-III levels; at PLAG27, with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; and at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations. After physiologic challenge with oral fat, APOC3 knockouts displayed marked blunting of the usual post-prandial rise in plasma triglycerides compared to wild-type family members. These observations provide a roadmap to understand the consequences of complete disruption of a large fraction of genes in the human genome.

The Karachi intracranial stenosis study (KISS) Protocol: An urban multicenter case-control investigation reporting the clinical, radiologic and biochemical associations of intracranial stenosis in Pakistan

BackgroundIntracranial stenosis is the most common cause of stroke among Asians. It has a poor prognosis with a high rate of recurrence. No effective medical or surgical treatment modality has been developed for the treatment of stroke due to intracranial stenosis. We aim to identify risk factors and biomarkers for intracranial stenosis and to develop techniques such as use of transcranial doppler to help diagnose intracranial stenosis in a cost-effective manner.Methods/DesignThe Karachi Intracranial Stenosis Study (KISS) is a prospective, observational, case-control study to describe the clinical features and determine the risk factors of patients with stroke due to intracranial stenosis and compare them to those with stroke due to other etiologies as well as to unaffected individuals. We plan to recruit 200 patients with stroke due to intracranial stenosis and two control groups each of 150 matched individuals. The first set of controls will include patients with ischemic stroke that is due to other atherosclerotic mechanisms specifically lacunar and cardioembolic strokes. The second group will consist of stroke free individuals. Standardized interviews will be conducted to determine demographic, medical, social, and behavioral variables along with baseline medications. Mandatory procedures for inclusion in the study are clinical confirmation of stroke by a healthcare professional within 72 hours of onset, 12 lead electrocardiogram, and neuroimaging. In addition, lipid profile, serum glucose, creatinine and HbA1C will be measured in all participants. Ancillary tests will include carotid ultrasound, transcranial doppler and magnetic resonance or computed tomography angiogram to rule out concurrent carotid disease. Echocardiogram and other additional investigations will be performed at these centers at the discretion of the regional physicians.DiscussionThe results of this study will help inform locally relevant clinical guidelines and effective public health and individual interventions.