SUMMARY Between 30-40% of American adults have nonalcoholic fatty liver disease (NAFLD), which currently has no therapeutic options and is predicted to become the leading cause of the liver failure in the US. NAFLD is initiated by excess lipid accumulation in the liver (i.e. hepatic steatosis). The well-established risk variants TM6SF2 rs58542926 (E167K) and PNPLA3 rs738409 (I148M) both lead to increased cellular lipid accumulation. While patient-derived cell-based models are a valuable tool for the discovery of novel genetic factors, most NAFLD models are based on liver-derived or hepatocyte-like cells, which are time-intensive and costly to generate. Since steatosis is a cell autonomous phenotype, we propose an entirely novel approach of using patient-derived induced pluripotent stem cells (iPSCs) in their undifferentiated state as a NAFLD genetic model. In preliminary studies we found that oleate-treated iPSC both before and after differentiation into hepatocyte-like cells (iHeps) with the TM6SF2 rs58542926-T risk allele have greater lipid accumulation compared to non-carriers, differences that were not completely rescued after gene editing to revert rs58542926 to the non-risk allele. This finding highlights the potential for iPSCs and iHeps to interrogate the genetic predisposition to NAFLD, as well as suggests that the association between rs58542926 and NAFLD is not driven by TM6SF2 alone. rs58542926 is in near perfect linkage disequilibrium with rs10401969, which regulates SUGP1 protein levels. In pilot studies, SUGP1 knock-down increased triglyceride levels in human hepatoma cell lines, while knock-down in vivo resulted in hepatic steatosis. While our findings implicate SUGP1 rs10401069 as an additional causal variant for NAFLD, to date, mechanistic studies of this haplotype have been focused on TM6SF2 alone. In Aim 1 we propose to test whether SUGP1 rs10401969 modulates NAFLD in cellular and animal models by: i) Identifying NAFLD relevant cellular phenotypes that differ between iHeps from SUGP1 and rs10401969 double-carriers vs. non-carriers; ii) Introduce the rs10401969 and rs58542926 risk-alleles in non-carriers, and test for recapitulation of cellular phenotypes; iii) Verify that SUGP1 knock-down and overexpression modulates NAFLD in vivo; and iv) Identify SUGP1 target genes that mediate NAFLD- phenotypes. In Aim 2, we will establish undifferentiated iPSCs as a cellular model for NAFLD genetic risk variants by: i) Comparing NAFLD-relevant cellular phenotypes in iPSCs compared to iHeps; ii) Confirming that iPSCs can model single SNP effects, and testing whether phenotypes differ in iPSCs selected from the extremes of the NAFLD genetic risk score distribution; and iii) testing for differences in cellular phenotypes in iPSCs from NAFLD cases vs. controls. Together these studies will significantly advance the field of NAFLD genetics by defining SUGP1 as a genetic contributor to NAFLD, and establishing iPSCs as a novel cellular model to ide...