Arsenic contamination of drinking water is a major public health issue in many countries, increasing risk for a wide array of diseases, including cancer. There is inter-individual variation in arsenic metabolism efficiency and susceptibility to arsenic toxicity; however, the basis of this variation is not well understood. Here, we have performed the first genome-wide association study (GWAS) of arsenic-related metabolism and toxicity phenotypes to improve our understanding of the mechanisms by which arsenic affects health. Using data on urinary arsenic metabolite concentrations and approximately 300,000 genome-wide single nucleotide polymorphisms (SNPs) for 1,313 arsenic-exposed Bangladeshi individuals, we identified genome-wide significant association signals (P<5×10−8) for percentages of both monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) near the AS3MT gene (arsenite methyltransferase; 10q24.32), with five genetic variants showing independent associations. In a follow-up analysis of 1,085 individuals with arsenic-induced premalignant skin lesions (the classical sign of arsenic toxicity) and 1,794 controls, we show that one of these five variants (rs9527) is also associated with skin lesion risk (P = 0.0005). Using a subset of individuals with prospectively measured arsenic (n = 769), we show that rs9527 interacts with arsenic to influence incident skin lesion risk (P = 0.01). Expression quantitative trait locus (eQTL) analyses of genome-wide expression data from 950 individual's lymphocyte RNA suggest that several of our lead SNPs represent cis-eQTLs for AS3MT (P = 10−12) and neighboring gene C10orf32 (P = 10−44), which are involved in C10orf32-AS3MT read-through transcription. This is the largest and most comprehensive genomic investigation of arsenic metabolism and toxicity to date, the only GWAS of any arsenic-related trait, and the first study to implicate 10q24.32 variants in both arsenic metabolism and arsenical skin lesion risk. The observed patterns of associations suggest that MMA% and DMA% have distinct genetic determinants and support the hypothesis that DMA is the less toxic of these two methylated arsenic species. These results have potential translational implications for the prevention and treatment of arsenic-associated toxicities worldwide.

A large fraction of human genes are regulated by genetic variation near the transcribed sequence (cis-eQTL, expression quantitative trait locus), and many cis-eQTLs have implications for human disease. Less is known regarding the effects of genetic variation on expression of distant genes (trans-eQTLs) and their biological mechanisms. In this work, we use genome-wide data on SNPs and array-based expression measures from mononuclear cells obtained from a population-based cohort of 1,799 Bangladeshi individuals to characterize cis- and trans-eQTLs and determine if observed trans-eQTL associations are mediated by expression of transcripts in cis with the SNPs showing trans-association, using Sobel tests of mediation. We observed 434 independent trans-eQTL associations at a false-discovery rate of 0.05, and 189 of these trans-eQTLs were also cis-eQTLs (enrichment P<0.0001). Among these 189 trans-eQTL associations, 39 were significantly attenuated after adjusting for a cis-mediator based on Sobel P<10-5. We attempted to replicate 21 of these mediation signals in two European cohorts, and while only 7 trans-eQTL associations were present in one or both cohorts, 6 showed evidence of cis-mediation. Analyses of simulated data show that complete mediation will be observed as partial mediation in the presence of mediator measurement error or imperfect LD between measured and causal variants. Our data demonstrates that trans-associations can become significantly stronger or switch directions after adjusting for a potential mediator. Using simulated data, we demonstrate that this phenomenon is expected in the presence of strong cis-trans confounding and when the measured cis-transcript is correlated with the true (unmeasured) mediator. In conclusion, by applying mediation analysis to eQTL data, we show that a substantial fraction of observed trans-eQTL associations can be explained by cis-mediation. Future studies should focus on understanding the mechanisms underlying widespread cis-mediation and their relevance to disease biology, as well as using mediation analysis to improve eQTL discovery.

General: All reagents were bought from commercial sources and used as received unless otherwise noted. All reactions were performed under an inert N 2 atmosphere and all solvents were distilled before use unless otherwise noted. CH 2 Cl 2 was distilled over CaH 2 . Chromatographic purification of products was accomplished by using forced-flow chromatography on EM Science Geduran silica gel 60 (35-75 µm). Thin layer chromatography was performed on EM Science silica gel 60 F254 plates (250 µm). Visualization of the developed chromatogram was accomplished by UV lamp and/or by staining with aqueous potassium permanganate/K 2 CO 3 solutions, or aqueous ceric ammonium molybdate (CAM) solutions. Nuclear magnetic resonance (NMR) spectra were acquired on Bruker DRX-500/400 operating at 125/100 MHz for 1 H and 13 C, respectively; residual protio solvent signals were used as internal standards for calibration purposes. Data for 1 H NMR are reported as follows: chemical shift (ppm), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), integration, coupling constant (Hz). Freshly prepared samples (ca. 1 mg/mL in CH 2 Cl 2 ) were subject to electrospray mass spectra (ESI-MS) analysis. A 5 μL sample was injected by flow injection with the use of an autosampler and the data were recorded on with an m/z range of 0-3000. Both the nebulizing and drying gas are purified nitrogen. Typical instrumental parameters were: drying gas temperature 350 °C, nebulizer pressure 35 psi, drying gas flow 12.0 L/min, and fragmentor voltage 0 or 70 V.

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