PROJECT SUMMARY Fatty liver disease (FLD) is a major public health issue that affects millions of people worldwide. The two major subtypes of FLD include alcoholic FLD (AFLD) and nonalcoholic FLD (NAFLD) although the threshold of alcohol intake for subdivision is controversial. FLD is defined by excess fat in the liver and can lead to a more profound disease state of steatohepatitis (SH) in which there is liver inflammation/damage that may be reflected in hepatic fibrosis. SH can lead to liver failure or hepatocellular carcinoma. Major risk factors predisposing individuals to the development of FLD include biological factors (obesity, insulin resistance, type 2 diabetes) demographic characteristics (e.g., sex, age and ethnicity), behavioral and lifestyle-related variables (e.g., alcohol intake, dietary behavior and physical activity), and other environmental factors (e.g., infectious agents such as hepatitis viruses, microbiome variability, exposure to pollutants/contaminants/toxins). Hispanics are disparately impacted by NAFLD with the highest observed prevalence of NAFLD in the world. Risk for NAFLD is due to a complex mixture of genetic and environmental factors and their interactions that are still largely unidentified. Quantitative endophenotypes (i.e., biomarkers that are genetically correlated with disease risk) have important properties that can speed the discovery of disease-related genetic causal factors and aid in individual-level risk estimation. Here we propose a novel deep cellular phenotyping assessment of induced pluripotent stem cell (iPSC)-derived hepatocyte response to experimental lipid overload challenge, which mimics dietary lipid overfeeding. This experimental epidemiological study will allow us to rigorously test for genotype-by-environment interactions (GEI) to better understand the etiology/mechanisms of NAFLD risk and to enhance early detection and halt progression. Specifically, we will identify novel iPSC-derived hepatocellular endophenotypes involved in NAFLD risk in Mexican Americans, a health disparity population, using a high-throughput multiomic approach. The study will use existing data and cellular biosamples from 900 participants in our longitudinal Mexican American Family Study (MAFS), who have been extensively phenotyped (including liver MRI) and genetically characterized. Our specific aims include: 1) discovery of novel hepatocellular cellular endophenotypes for NAFLD risk by quantitative genome-wide RNA sequencing and functional evaluation of hepatocytes derived from existing iPSCs in 400 subjects; (2) discovery of multivariate hepatocellular transcriptional coherence endophenotypes using single-cell sequencing; and (3) confirmation of the highest ranking NAFLD endophenotypes using MRI-derived liver fat measures from an additional 500 MAFS subjects from the same families. Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease and it disparately affects minority p...