Genetic analyses of hepatic steatosis GWAS associated loci. E. K. Speliotes, Y. Chen, A. W. Tai University of Michigan, Ann Arbor, MI.

   Nonalcoholic Fatty Liver Disease (NAFLD) is a heritable and prevalent disease, affecting about 30% of the population. A characteristic feature of NAFLD is hepatic steatosis, the presence of excess fat (mostly triglycerides (TG)) in the liver. Using genome wide association analysis (GWAS) we identified genetic variants in PNPLA3 and GCKR, and near LYPLAL1 that associate with population based hepatic steatosis. How these variants result in increased liver steatosis is not known. Here we aim to characterize the genetic mechanism by which genetic variants at these loci may affect nearby genes to result in hepatic triglyceride accumulation. HuH-7 and HepG2 liver cell lines were infected with lentiviruses expressing wildtype PNPLA3, GCKR, and LYPLAL1 as well as the mutants PPP1R3B(I148M) and GCKR(P446L) or with shRNAs to PNPLA3, GCKR, and LYPLAL1 and stably expressing cell lines were selected. Overexpression/knockdown was quantified using Western/Northern blotting analysis. Stable cell lines were loaded with oleic acid, hepatic steatosis was measured using LipidTOXTM (Life Technology), and total cellular triglyceride was quantified using a Triglyceride Determination Kit (Sigma-Aldrich). Overexpression of wildtype and to a larger extent mutant PNPLA3 but not knockdown of PNPLA3 resulted in increased steatosis/TG accumulation. Overexpression of wildtype GCKR but not mutant GCKR resulted in increased steatosis/TG accumulation whereas knockdown of GCKR also resulted in increased steatosis/TG accumulation. Overexpression of LYPLAL1 resulted in decreased steatosis/TG accumulation and knockdown in increased steatosis/TG accumulation. These results suggest that variants in PNPLA3 exert their effect through an increase/gain-of-function mechanisms, those in GCKR and near LYPLAL1 likely through a loss-of-function mechanism.

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