An Environment-Wide Association Study (EWAS) on type 2 diabetes mellitus

Abstract

Background: Type 2 Diabetes (T2D) and other chronic diseases are caused by a complex combination of many genetic and environmental factors. Few methods are available to comprehensively associate specific physical environmental factors with disease. We conducted a pilot Environmental-Wide Association Study (EWAS), in which epidemiological data are comprehensively and systematically interpreted in a manner analogous to a Genome Wide Association Study (GWAS).

Methods and findings: We performed multiple cross-sectional analyses associating 266 unique environmental factors with clinical status for T2D defined by fasting blood sugar (FBG) concentration > or =126 mg/dL. We utilized available Centers for Disease Control (CDC) National Health and Nutrition Examination Survey (NHANES) cohorts from years 1999 to 2006. Within cohort sample numbers ranged from 503 to 3,318. Logistic regression models were adjusted for age, sex, body mass index (BMI), ethnicity, and an estimate of socioeconomic status (SES). As in GWAS, multiple comparisons were controlled and significant findings were validated with other cohorts. We discovered significant associations for the pesticide-derivative heptachlor epoxide (adjusted OR in three combined cohorts of 1.7 for a 1 SD change in exposure amount; p<0.001), and the vitamin gamma-tocopherol (adjusted OR 1.5; p<0.001). Higher concentrations of polychlorinated biphenyls (PCBs) such as PCB170 (adjusted OR 2.2; p<0.001) were also found. Protective factors associated with T2D included beta-carotenes (adjusted OR 0.6; p<0.001).

Conclusions and significance: Despite difficulty in ascertaining causality, the potential for novel factors of large effect associated with T2D justify the use of EWAS to create hypotheses regarding the broad contribution of the environment to disease. Even in this study based on prior collected epidemiological measures, environmental factors can be found with effect sizes comparable to the best loci yet found by GWAS.

Figure 1. Summary of EWAS Environment Factors and Analytic Method.

A.) Summary of the 21 factor classes and the number of factors within them for each NHANES cohort. B.) Individuals were stratified into T2D status (“cases” and “controls”) through the ADA FBS threshold for diabetes diagnosis. 6–7% of the unweighted observations in all cohorts had T2D under this rule. C.) Each of these 75 to 211 factors was tested for association with T2D status with a logistic regression model (coefficient labeled “FACTOR”) adjusted for age, sex, BMI, ethnicity, and SES. Statistical significance (α = 0.02) was determined by controlling the FDR between 10 to 30%. Between 4 and 23 factors were found to be significant using this threshold α = 0.02. D.) “Multi-stage” validation. For factors that were deemed significant in C, we deemed a factor validated if the factor was significant to the α = 0.02 level in one or more of the other cohorts. We found 5 factors to be validated (FDR of 2%).

Figure 2. “Manhattan plot” style graphic showing the environment-wide association with T2D.

Y-axis indicates −log10(p-value) of the adjusted logistic regression coefficient for each of the environmental factors. Colors represent different environmental classes as represented in Figure 1A. Within each environmental class, factors are arranged left to right in order from lowest to highest odds ratio (OR). Plot symbols represent different cohorts: 1999–2000 (diamonds), 2001–2002 (square), filled dot (2003–2004), circle (2005–2006). Red horizontal line is −log10(α) = 1.8 (α = 0.02). Validated factors significant in 2 or more NHANES cohorts are in bold face (α = 0.02 in two or more cohorts, FDR of 2%) with larger plot points. Other significant factors (α = 0.02) are annotated with numeric label corresponding to the environmental factor class color key on the right. Figure abbreviations: Validated factors: t-β-carotene: trans β-carotene; c-β-carotene: cis β-carotene; PCB170: 2,2′,3,3′,4,4′,5-Heptachlorobiphenyl. Group 1 (dioxins): 1-hxcdd: 1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin; 2-hxcdd: 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin. Group 2 (furans): OCDF: 1,2,3,4,6,7,8,9-Octachlorodibenzofuran. Group 3 (heavy metals): Ur: uranium; Sb: antimony; Pb: Lead. Group 4 (nutrients): tot-β-car: total β-carotene; α-car: alpha-carotene; retnl: retinol; Vita. D: vitamin D; δ-t: delta-tocopherol. Group 5 (organochlorine pestcides): DDE: dichlorodiphenyltrichloroethylene. Group 6 (PCB): PCB169: 3,3′,4,4′,5,5′-hexachlorobiphenyl; PCB138: 2,2′,3,4,4′,4′,5-Hexachlorobiphenyl; PCB195: 2,2′,3,3′,4,4′,5,6-Octachlorobiphenyl; PCB183: 2,2′,3,4,4′,5′,6-Heptachlorobiphenyl; PCB199: 2,2′,3,3′,4,5,5′,6′-Octachlorobiphenyl; PCB178: 2,2′,3,3′,5,5′,6-Heptachlorobiphenyl; PCB187: 2,2′,3,4′,5,5′,6-Heptachlorobiphenyl; PCB180: 2,2′,3,4,4′,5,5′-Heptachlorobiphenyl; PCB146: 2,2′,3,4′,5,5′-Hexachlorobiphenyl; PCB196: 2,2′,3,4,4′,5,5′,6-Octachlorobiphenyl. Group 7 (bacteria): H2: Herpes Simplex 2; HSBA: Hepatitis B Surface Antibody.