A Novel Platform for Genetic Screening of NAFLD-Associated Genes Identifies UBE2G2 and Other Genes That Alter Hepatic Lipid Accumulation

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver failure that is prevalent in 70% of people with diabetes. Human genetic studies (i.e. GWAS) have identified dozens of potentially causal genes/loci increasing NAFLD susceptibility but translation into actionable drug targets has been impeded by a lack of cellular and molecular mechanistic studies. To address this knowledge gap, we developed a platform for high-throughput genetic perturbation and assessment of fatty acid-triggered lipid accumulation (i.e. hepatic steatosis) in human hepatocytes by combining mechanized cell culture in multi-well plates with high-content microscopy and automated image analysis. We then deployed this platform to screen 106 genes nominated by NAFLD biomarker GWAS, knocking out each gene via CRISPR and scoring the phenotypic changes of the resulting engineered cells in response to oleate treatment. Analysis of over 24,000 images across all gene knockouts revealed 32 genes whose deletion caused significant alterations in cellular lipid droplet (LD) numbers and/or droplet size distribution. Here, we highlight ubiquitin-conjugating enzyme E2 G2 (UBE2G2), whose knockout robustly increases the size of cellular LDs that form in response to oleate stimulation in both the primary screen and subsequent validation. UBE2G2 localizes to the LD surface and is essential for the sterol-induced degradation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Thus we propose that deletion of UBE2G2 may increase cholesterol derivative content in LDs, thereby increasing LD size. In summary, we demonstrate the successful development and use of a platform for genetic screening of hepatocyte steatosis in vitro and identify multiple NAFLD-associated genes of previously unknown function that likely operate during this early stage of NAFLD pathogenesis. We acknowledge input: Seunghan Lee, Lifeng Wang, David Frederick, Alessandro Pocai Disclosure S. Hu: None. S. Khandavilli: None. R.Y. Diao: None. J. Raum: Employee; Janssen Research & Development, LLC. N. Azhar: Employee; Janssen Research & Development, LLC. I. Bakaj: Employee; Janssen Research & Development, LLC. G. Cowley: Employee; Janssen Research & Development, LLC. B. Rady: Employee; Janssen Research & Development, LLC. D.F. Reilly: Employee; Janssen Research & Development, LLC. A.R. Majithia: None. Funding National Institutes of Health (RDK01DK123422)