Human population based genetic studies to elucidate the biology of NAFLD

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 DESCRIPTION (provided by applicant): Nonalcoholic fatty liver disease (NAFLD) is caused by hepatic steatosis (lipid accumulation), affects up to 29 million adults in the U.S. and will become the leading cause of liver disease worldwide by 2020. There are few effective ways to prevent or treat NAFLD. A better understanding of NAFLD etiology is needed to improve its diagnosis and treatment. We previously determined that NAFLD is heritable (genetically influenced) and identified common (minor allele frequency (MAF) >5%) single nucleotide polymorphisms (SNPs) at 5 loci that associate with hepatic steatosis, first in ~7,000 individuals of European ancestry and then across ancestries. Variants at the 5 loci explain ~20% of heritability, suggesting the existence of more influential causal variants. We now aim to identify 1. causal genetic variants 2. gene(s) they work through to exert their effects, and 3. mechanism(s) by which they lead to disease. We tripled the sample size and ancestral diversity of our discovery sample, thus improving our ability to identify and fine-map causal variants. We will impute genome- wide genotypes to new denser and ancestrally diverse imputation panels, increasing our ability to identify causal common and now low-frequency (MAF 1-5%) variants across ancestries. We will use new analytical methods to perform not only single variant but also conditional and gene-based analyses across ancestries allowing us to efficiently identify causal single variants and genes. We will use new variant and gene annotations algorithms to prioritize variants and genes for functional follow up. Using this approach we identify new putative causal NAFLD variants and genes missed by previous analyses. Finally, we developed a human cell line model of NAFLD that we use to show that altering the function of three genes prioritized by the above approaches results in increased liver triglyceride accumulation, suggesting this as a common mechanism by which these genes cause NAFLD. We hypothesize that causal NAFLD-promoting human genetic variants have effects across ancestries and exert their effects in a cell-autonomous manner in hepatocytes to increase triglyceride accumulation. To test this hypothesis, we will harmonize the computed tomography hepatic steatosis phenotype and genotypes from 10 discovery cohorts (n>21K), impute genotypes to the 1000 Genomes cosmopolitan reference panel, and perform single variant, gene-based, and conditional meta- analyses across groups to identify, fine-map, and annotate putative causal variants and genes. We will follow- up top associating variants in >4,000 histologically confirmed NAFLD cases and ~3,000 controls to replicate our findings. We will knockdown and overexpress putative causal genes (wild-type and variant) in human liver cell lines and determine their effect on triglyceride accumulation and genetic mechanism of action. We will begin with three genes prioritized from our previous NAFLD meta-analysis and proceed to new genes identified from the 1000 Genomes meta-analysis. This work will help define the genetic and metabolic mechanisms that cause NAFLD and inform development of new biomarkers as well as potential therapeutics for this condition.