Sian-Tsung Tan

Epigenome-wide association of DNA methylation markers in peripheral blood from Indian Asians and Europeans with incident type 2 diabetes: a nested case-control study.

BACKGROUNDnIndian Asians, who make up a quarter of the worlds population, are at high risk of developing type 2 diabetes. We investigated whether DNA methylation is associated with future type 2 diabetes incidence in Indian Asians and whether differences in methylation patterns between Indian Asians and Europeans are associated with, and could be used to predict, differences in the magnitude of risk of developing type 2 diabetes.nnnMETHODSnWe did a nested case-control study of DNA methylation in Indian Asians and Europeans with incident type 2 diabetes who were identified from the 8-year follow-up of 25 372 participants in the London Life Sciences Prospective Population (LOLIPOP) study. Patients were recruited between May 1, 2002, and Sept 12, 2008. We did epigenome-wide association analysis using samples from Indian Asians with incident type 2 diabetes and age-matched and sex-matched Indian Asian controls, followed by replication testing of top-ranking signals in Europeans. For both discovery and replication, DNA methylation was measured in the baseline blood sample, which was collected before the onset of type 2 diabetes. Epigenome-wide significance was set at p<1 × 10(-7). We compared methylation levels between Indian Asian and European controls without type 2 diabetes at baseline to estimate the potential contribution of DNA methylation to increased risk of future type 2 diabetes incidence among Indian Asians.nnnFINDINGSn1608 (11·9%) of 13u2008535 Indian Asians and 306 (4·3%) of 7066 Europeans developed type 2 diabetes over a mean of 8·5 years (SD 1·8) of follow-up. The age-adjusted and sex-adjusted incidence of type 2 diabetes was 3·1 times (95% CI 2·8-3·6; p<0·0001) higher among Indian Asians than among Europeans, and remained 2·5 times (2·1-2·9; p<0·0001) higher after adjustment for adiposity, physical activity, family history of type 2 diabetes, and baseline glycaemic measures. The mean absolute difference in methylation level between type 2 diabetes cases and controls ranged from 0·5% (SD 0·1) to 1·1% (0·2). Methylation markers at five loci were associated with future type 2 diabetes incidence; the relative risk per 1% increase in methylation was 1·09 (95% CI 1·07-1·11; p=1·3 × 10(-17)) for ABCG1, 0·94 (0·92-0·95; p=4·2 × 10(-11)) for PHOSPHO1, 0·94 (0·92-0·96; p=1·4 × 10(-9)) for SOCS3, 1·07 (1·04-1·09; p=2·1 × 10(-10)) for SREBF1, and 0·92 (0·90-0·94; p=1·2 × 10(-17)) for TXNIP. A methylation score combining results for the five loci was associated with future type 2 diabetes incidence (relative risk quartile 4 vs quartile 1 3·51, 95% CI 2·79-4·42; p=1·3 × 10(-26)), and was independent of established risk factors. Methylation score was higher among Indian Asians than Europeans (p=1 × 10(-34)).nnnINTERPRETATIONnDNA methylation might provide new insights into the pathways underlying type 2 diabetes and offer new opportunities for risk stratification and prevention of type 2 diabetes among Indian Asians.nnnFUNDINGnThe European Union, the UK National Institute for Health Research, the Wellcome Trust, the UK Medical Research Council, Action on Hearing Loss, the UK Biotechnology and Biological Sciences Research Council, the Oak Foundation, the Economic and Social Research Council, Helmholtz Zentrum Munchen, the German Research Center for Environmental Health, the German Federal Ministry of Education and Research, the German Center for Diabetes Research, the Munich Center for Health Sciences, the Ministry of Science and Research of the State of North Rhine-Westphalia, and the German Federal Ministry of Health.

The South Asian genome.

The genetic sequence variation of people from the Indian subcontinent who comprise one-quarter of the worlds population, is not well described. We carried out whole genome sequencing of 168 South Asians, along with whole-exome sequencing of 147 South Asians to provide deeper characterisation of coding regions. We identify 12,962,155 autosomal sequence variants, including 2,946,861 new SNPs and 312,738 novel indels. This catalogue of SNPs and indels amongst South Asians provides the first comprehensive map of genetic variation in this major human population, and reveals evidence for selective pressures on genes involved in skin biology, metabolism, infection and immunity. Our results will accelerate the search for the genetic variants underlying susceptibility to disorders such as type-2 diabetes and cardiovascular disease which are highly prevalent amongst South Asians.

A coherent approach for analysis of the Illumina HumanMethylation450 BeadChip improves data quality and performance in epigenome-wide association studies

DNA methylation plays a fundamental role in the regulation of the genome, but the optimal strategy for analysis of genome-wide DNA methylation data remains to be determined. We developed a comprehensive analysis pipeline for epigenome-wide association studies (EWAS) using the Illumina Infinium HumanMethylation450 BeadChip, based on 2,687 individuals, with 36 samples measured in duplicate. We propose new approaches to quality control, data normalisation and batch correction through control-probe adjustment and establish a null hypothesis for EWAS using permutation testing. Our analysis pipeline outperforms existing approaches, enabling accurate identification of methylation quantitative trait loci for hypothesis driven follow-up experiments.

Coronary heart disease in Indian Asians.

The Indian Asian population accounts for a fifth of all global deaths from coronary heart disease (CHD). CHD deaths on the Indian subcontinent have doubled since 1990, and are predicted to rise a further 50% by 2030. Reasons underlying the increased CHD mortality among Indian Asians remain unknown. Although conventional cardiovascular risk factors contribute to CHD in Indian Asians as in other populations, these do not account for their increased risk. Type-2 diabetes, insulin resistance and related metabolic disturbances are more prevalent amongst Indian Asians than Europeans, and have been proposed as major determinants of higher CHD risk among Indian Asians. However, this view is not supported by prospective data. Genome-wide association studies have not identified differences in allele frequencies or effect sizes in known loci to explain the increased CHD risk in Indian Asians. Limited knowledge of mechanisms underlying higher CHD risk amongst Indian Asians presents a major obstacle to reducing the burden of CHD in this population. Systems biology approaches such as genomics, epigenomics, metabolomics and transcriptomics, provide a non-biased approach for discovery of novel biomarkers and disease pathways underlying CHD. Incorporation of these ‘omic’ approaches in prospective Indian Asian cohorts such as the London Life Sciences Population Study (LOLIPOP) provide an exciting opportunity for the identification of new risk factors underlying CHD in this high risk population.

Investigation of Genetic Variation Underlying Central Obesity amongst South Asians.

South Asians are 1/4 of the world’s population and have increased susceptibility to central obesity and related cardiometabolic disease. Knowledge of genetic variants affecting risk of central obesity is largely based on genome-wide association studies of common SNPs in Europeans. To evaluate the contribution of DNA sequence variation to the higher levels of central obesity (defined as waist hip ratio adjusted for body mass index, WHR) among South Asians compared to Europeans we carried out: i) a genome-wide association analysis of >6M genetic variants in 10,318 South Asians with focused analysis of population-specific SNPs; ii) an exome-wide association analysis of ~250K SNPs in protein-coding regions in 2,637 South Asians; iii) a comparison of risk allele frequencies and effect sizes of 48 known WHR SNPs in 12,240 South Asians compared to Europeans. In genome-wide analyses, we found no novel associations between common genetic variants and WHR in South Asians at P<5x10-8; variants showing equivocal association with WHR (P<1x10-5) did not replicate at P<0.05 in an independent cohort of South Asians (N = 1,922) or in published, predominantly European meta-analysis data. In the targeted analyses of 122,391 population-specific SNPs we also found no associations with WHR in South Asians at P<0.05 after multiple testing correction. Exome-wide analyses showed no new associations between genetic variants and WHR in South Asians, either individually at P<1.5x10-6 or grouped by gene locus at P<2.5x10−6. At known WHR loci, risk allele frequencies were not higher in South Asians compared to Europeans (P = 0.77), while effect sizes were unexpectedly smaller in South Asians than Europeans (P<5.0x10-8). Our findings argue against an important contribution for population-specific or cosmopolitan genetic variants underlying the increased risk of central obesity in South Asians compared to Europeans.

117 Validation of Accelerometers for Measurement of Physical Activity Energy Expenditure in South Asians and Europeans

Background South Asians are at ~2 fold higher risk of cardiovascular disease (CVD) than Europeans. Physical inactivity is an independent risk factor for CVD, and may be more common in South Asians than Europeans. However, published studies of physical activity in South Asians have all relied on self-report by questionnaire, an approach of limited accuracy, confounded by linguistic, cultural and reporting bias. Accelerometers are validated tools for measurement of physical activity energy expenditure (PAEE) in Europeans, but are influenced by body habitus and device positioning. The accuracy of accelerometers in South Asians is not known. Aims To identify the accelerometer and accelerometer position that most accurately quantifies physical activity amongst South Asians, and to compare the accuracy of the chosen accelerometer between South Asians and Europeans. Methods We investigated 57 South Asians and 29 Europeans undergoing an exercise treadmill test (ETT) for clinical indications. All participants completed the treadmill test while simultaneously wearing 3 commercially available accelerometers in manufacturer recommended positions: i. Actigraph GT3X (3 positions - ankle, waist and wrist); ii. Geneactiv (3 positions - ankle, waist and wrist) and iii. Actiheart (chest only). Each participant thus had 7 device-position combinations. Personal and medical history was collected by questionnaire, followed by anthropometric measurements (height, weight, waist and hip circumference). We used linear regression and Bland-Altman plots to investigate the relationships between PAEE measured by the accelerometers (Measured) and actual PAEE on the treadmill (Actual). Results The relationship between Measured and Actual PAEE was highest for the Actigraph GT3X in the ankle position (regression beta: 0.55+/-0.03, Table). Wrist-worn devices consistently displayed poor PAEE prediction. Measured and Actual PAEE were similar amongst South Asians and Europeans for all accelerometers and positions (P > 0.05), including the ankle-worn Actigraph GT3X. However, even the ankle-worn Actigraph GT3X underestimated PAEE, especially at high workload (Figure). Conclusion The Actigraph GT3X is the most accurate device for measuring PAEE amongst both South Asians and Europeans. PAEE is best measured by the ankle-worn accelerometers followed by waist-worn accelerometers. Our results will inform the design of future studies to investigate the relationship between physical activity and CVD in the two populations. Relationship between Measured and Actual energy expenditure [beta (SE), from regression] in South Asians and Europeans. Abstract 66 Table 2 Procedural characteristics Variable Radial (N = 182) Femoral (N = 72) P-value Guide catheter size (Fr) 6.4 ± 0.5 6.8 ± 0.7 0.00 Burr size (mm) 1.47 ± 0.15 1.52 ± 0.19 0.08 Bare metal stent (%) 14.9 11.4 0.63 Number of stents (mean ± SD) 1.8 ± 0.9 2.0 ± 1.0 0.39 Stent diameter (mm) 3.1 ± 0.5 3.2 ± 0.5 0.65 Stent length (mm) 40.7 ± 20.1 40.1 ± 22.4 0.67 IVUS/OCT use (%) 11.7 6.8 0.24 Procedural success (%) 96.7 94.6 0.44 Abstract 117 Figure 1

114 Whole genome sequencing to identify genetic variants underlying cardiovascular disease among Indian Asians

Introduction Indian Asians have a twofold higher risk of cardiovascular disease compared to Europeans which is not explained by conventional cardiovascular risk factors or known genetic variants. The genetic architecture of Indian Asians has not previously been described. We hypothesised that whole genome sequencing of Indian Asians may identify both common and rare variants specific to this population that contribute to their increased cardiovascular disease risk. Methods We carried out whole genome sequencing (mean depth 28.4×) in eight men of Indian Asian origin participating in the London Life Sciences Population (LOLIPOP) study. Sequencing was carried out using paired end and mate pair libraries on an Illumina GA2 machine. Read alignment was done by BWA, and variants called using GATK and SAMtools. Sensitivity for single nucleotide polymorphism (SNP) detection was assessed by comparison to whole genome data. Results We identified 6u2008602u2008840 autosomal variants, 436u2008823 of which are novel SNPs. Of these, 50u2008585 appear to be common (present at least twice, corresponding to minor allele frequency >10%). We found 21u2008659 autosomal SNPs that were expected to affect protein coding, of which 2174 are novel. Among the coding SNPs identified, 145 are in genes linked to human diseases, such as obesity (FTO, UCP1), diabetes mellitus (CDKAL1, GCGR, HNF1B), lipid metabolism (APOB), hypertension (NOS2), and renal disease (NPHP4, PKD1). We also found 65u2008613 novel autosomal indels of which 35u2008097 are present at least twice, and 2301 novel deletions >100u2005bp. We show that >50% of the novel genetic variants are not in high LD (r2¡Ý0.8) with tag SNPs and hence not captured on available high-density microarrays. Conclusions We identify more than 500u2008000 genetic variants not previously reported in 1000 genomes or dbSNP, and likely to be Indian Asian specific. The novel variants identified here are strong candidates for genetic factors underlying the increased risk of diabetes and cardiovascular disease among Indian Asians.

111 Investigation of the Validity of Cardiovascular Death Certification Amongst Uk Indian Asians and Europeans

Background National mortality statistics report that UK Indian Asians are at approxiamately 2 fold higher risk of cardiovascular disease mortality compared with people of European ancestry. However, previous studies in North American and European populations suggest that up to 40% of death certificates are incorrect, leading to an overestimation of cardiovascular disease mortality. The validity of routine death certification amongst Indian Asians is unknown. Aims and methods We investigated the accuracy of routine death certification amongst UK Indian Asians, to determine whether incorrect certification contributes to the reported higher cardiovascular disease amongst Indian Asians. We reviewed all available medical records (hospital, primary care and coroner) for a representative sample of 315 Indian Asians and Europeans who died in London, UK between 2002 and 2011. Deaths were independently coded as cardiovascular or non-cardiovascular according to published international diagnostic criteria by 2 cardiologists; where there was disagreement a third adjudicated. Results Age of death was lower amongst Indian Asians than Europeans (66.6 ± 10.1 vs. 69.7 ± 8.2, P = 0.003). Compared to review of records, routine death certification overestimated cardiovascular disease as underlying cause of death amongst Indian Asians (33.6 vs. 29.6%, p < 0.001) and Europeans (32.5 vs. 27.0%, p < 0.001). However, routine death certification showed similar accuracy for the diagnosis of cardiovascular disease amongst both Indian Asians and Europeans (sensitivity: 88.9 vs. 93.2%, p = 0.714; specificity: 88.9 vs. 89.9%, p = 0.961; positive predictive value: 78.4 vs. 77.4%, p = 0.895, respectively). Conclusion Routine death certification overestimates cardiovascular deaths by ~20% amongst UK Indian Asians and Europeans, but the accuracy of death certification is similar in the two populations. Inaccurate death certification does not contribute to higher reported cardiovascular disease mortality amongst Indian Asians compared to Europeans.

151 Nuclear magnetic resonance profiling of serum identifies novel biomarker associated with coronary atherosclerosis

Introduction Coronary heart disease (CHD) is the leading cause of death worldwide. Current CHD risk prediction tools do not accurately identify risk of CHD in all populations. There is an urgent need for discovery of novel biomarkers to help understand mechanisms underlying CHD and improve risk functions to identify people at increased risk. We used serum metabonomic nuclear magnetic resonance (NMR) profiling to identify biomarkers associated with the presence of coronary artery calcification (CAC), a quantitative measure of coronary atherosclerosis. Methods We investigated 2358 men and women aged 35–75u2005years participating in the London Life Sciences Population (LOLIPOP) Study. Participants with prior history of CHD were excluded. All participants completed a structured health questionnaire, underwent physical assessment and had blood collected after an 8u2005h fast. CAC was quantified using electron beam CT. Quantitative NMR spectroscopy was used to characterise serum samples for lipoproteins, fatty acids and low molecular weight markers. Linear regression was used to determine the relationship between metabolites and Agatston score. Statistical significance was inferred at p<0.0013, corresponding to a Bonferroni correction for 39 primary NMR measures. Results CAC was associated with older age, male gender, cigarette smoking, raised systolic and diastolic blood pressure, obesity and hypercholesterolaemia. Among the 39 NMR measures, 11 were associated with CAC after adjustment for age, sex and ethnicity (p=0.0013 to p=5.0×10−12). In multivariate analysis, after further adjustment for conventional risk factors, we identified an association of the metabonomic biomarker (an abundant naturally occurring non-essential amino acid) with coronary artery calcium (p=7.9×10−4). The OR for the presence of significant coronary atherosclerosis (CAC>100) was 2.21 (95% CI 1.59 to 3.07, p=2.2×10−6) among people in the lowest quartile for the novel metabolite, compared to those in the highest quartile, implying that lower levels of the metabonomic biomarker confers increased CHD risk. Conclusions Using NMR spectroscopy, we have identified a novel metabonomic biomarker to be independently associated with CAC, a measure of coronary atherosclerosis. Further testing in large prospective cohorts is required to evaluate the predictive value of this novel metabonomic biomarker to CHD.