Identifying multiple causative genes at a single GWAS locus. M. Flister1,2, S. Tsaih1, B. Endres1,2, A. Geurts1,2, J. Lazar1,3, M. Dwinell1,2, C. Moreno1, H. Jacob1,4 1) Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI; 2) Department of Physiology, Medical College of Wisconsin, Milwaukee, WI; 3) Department of Dermatology, Medical College of Wisconsin, Milwaukee, WI; 4) Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI.
Background: Genome-wide association studies (GWAS) can nominate candidate genes, but are largely unable to establish disease causality or differentiate between variants in linkage disequilibrium (LD). Moreover, some GWAS loci might contain multiple causative variants that contribute to the overall disease susceptibility at a single locus. However, most GWAS lack the statistical power to detect multigenic interactions, prompting us to adopt an alternative approach to testing multiple GWAS genes empirically. Here we present a new strategy using inbred rat strains to rapidly dissect the (+), (-), or (=) phenotypic effects of multiple genes at a single hypertension GWAS locus. Methods: Agtrap, Mthfr, Clcn6, Nppa, Nppb, and Plod1 were individually mutated in the Dahl SS rat by zinc finger nuclease injections (ZFN). ZFN-mutated and wild type (WT) control SS rats (n=8-25 per group) were assessed for BP and renal damage after 10 days on a 4% NaCl diet. Human haplotypes were functionally annotated using HapMap, 1000 Genomes, and ENCODE data. Results: Compared with WT, Nppa (+27mmHg, P<0.001) and Plod1 (+17mmHg, P<0.01) mutations increased BP in response to high salt diet (4% NaCl), whereas Clcn6 mutation decreased BP (-22mmHg, P<0.001). Compared with WT (915 mg/day), Plod1 (14712 mg/day, P<0.001) and Mthfr (13223 mg/day, P<0.05) mutations increased proteinuria, whereas Clcn6 (565 mg/day, P<0.05) and Agtrap (549 mg/day, P<0.05) mutation decreased protein excretion. Using HapMap and 1000 Genomes, we identified 226 SNPs in LD (r2<0.6) with disease associated haplotype blocks. Out of 9 disease-associated haplotypes, 6 had multiple SNPs in LD that caused nonsynonymous changes or were associated with differential expression of multiple genes, demonstrating that genetic interactions within the AGTRAP-PLOD1 locus were not only possible, but rather were highly plausible. Conclusions: Using rat and human data, we were able to draw previously unforeseen conclusions. Foremost, that multiple genes at a single GWAS locus can influence clinically relevant phenotypes. We present the first evidence that Nppa, Clcn6, Mthfr, Plod1, and Agtrap mutations cause divergent CVD phenotypes and some have the ability to modify renal phenotypes independently of BP. Combined, these data suggest that the current best-fit candidate gene interpretations of GWAS are in some cases only a simplified view of the complex genetic architecture underlying individually associated loci.
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