Uzma Afzal

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.

BACKGROUND Indian 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. METHODS We 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. FINDINGS 1608 (11·9%) of 13 535 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)). INTERPRETATION DNA 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. FUNDING The 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.

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

日 本 語 要 約 Epigenome ­ wide association study of body mass index , and the adverse outcomes of adiposity

Approximately 1.5 billion people worldwide are overweight or affected by obesity, and are at risk of developing type 2 diabetes, cardiovascular disease and related metabolic and inflammatory disturbances. Although the mechanisms linking adiposity to associated clinical conditions are poorly understood, recent studies suggest that adiposity may influence DNA methylation, a key regulator of gene expression and molecular phenotype. Here we use epigenome-wide association to show that body mass index (BMI; a key measure of adiposity) is associated with widespread changes in DNA methylation (187 genetic loci with P < 1 × 10−7, range P = 9.2 × 10−8 to 6.0 × 10−46; n = 10,261 samples). Genetic association analyses demonstrate that the alterations in DNA methylation are predominantly the consequence of adiposity, rather than the cause. We find that methylation loci are enriched for functional genomic features in multiple tissues (P < 0.05), and show that sentinel methylation markers identify gene expression signatures at 38 loci (P < 9.0 × 10−6, range P = 5.5 × 10−6 to 6.1 × 10−35, n = 1,785 samples). The methylation loci identify genes involved in lipid and lipoprotein metabolism, substrate transport and inflammatory pathways. Finally, we show that the disturbances in DNA methylation predict future development of type 2 diabetes (relative risk per 1 standard deviation increase in methylation risk score: 2.3 (2.07–2.56); P = 1.1 × 10−54). Our results provide new insights into the biologic pathways influenced by adiposity, and may enable development of new strategies for prediction and prevention of type 2 diabetes and other adverse clinical consequences of obesity.

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.