Discovery of novel therapeutic targets for NASH through deep phenotyping of human knockouts and mechanistic studies

The progression of nonalcoholic fatty liver disease (NAFLD) to fibrotic non-alcoholic steatohepatitis (NASH) is emerging as the leading cause of liver disease. Until mid-2021, the genetic etiology of NAFLD/NASH in humans has been limited to a handful of loci. To address this gap, we and others have recently identified 657 novel loci associated with several quantitative traits linked with NAFLD/NASH. While these new and prior discovery studies have established secure links between genetic loci and NAFLD/NASH risk, a number of uncertainties continue to exist about such findings, including: (a) the causal gene(s) at these loci; (b) the directional impact of these loci on disease risk; and (c) the biological mechanisms leading to disease risk. Moreover, all prior discovery efforts have been limited to: (d) common variant discovery platforms; (e) populations of European descent; and (f) use of proxy definitions of NAFLD/NASH based on AST and ALT levels. We aim to resolve these uncertainties and find loci of high therapeutic relevance by capitalizing on the Pakistan Genomic Resource (PGR), that has leveraged high levels of consanguinity in Pakistan and assembled the largest cohort of human knockouts (KOs) in the world and a cohort of 4,000 ultrasonography (USG)-confirmed NAFLD cases. Specifically, in Aim 1, we will: (i) perform multi-ethnic association analyses for rare and common variants in 700,000 participants (PGR + UK Biobank) for serum AST and ALT levels and conduct validation studies in 4,000 USG-confirmed NAFLD cases and 4,000 controls; (ii) conduct fine-mapping studies and colocalization analyses; (iii) perform rare variant gene burden tests, focusing on predicted loss of function (pLoF) and predicted damaging variants (pDM) variants; (iv) conduct pathway analyses and (v) for 10 highly prioritized loci, conduct call-back studies of pLoF carriers, along with their family members, to drive causal gene identification and translation. In Aim 2, we will investigate two high priority genes already identified from the analyses proposed in Aim 1, WWP2 and CASP8, to illustrate how such genes can be studied mechanistically and in experimental NASH. For WWP2, we hypothesized based on its E3 ligase activity that it is protective by down- regulating a key protein in fibrotic NASH, TAZ (WWTR1), which is now supported by our exciting preliminary in vitro and in vivo data. CASP8 is an example of a NASH-promoting gene based on data from Aim 1 and prior mouse studies, but the mechanisms are uncertain and likely not related to its canonical cell-death function. Here we will test the hypothesis that PGR pLoF/pDM variants at WWP2 and CASP8, when introduced into hepatocytes of mice lacking the endogenous genes, affect NASH as predicted from the human data and our hypotheses. We will also probe the 2 genes' mechanisms in vitro and in experimental NASH and how the mechanism is affected by pLoF/pDM variants. In Aim 3, we will investigate impact of genetic loci on disease progression by leveraging available serial VCTE measurements available in 4,000 USG-confirmed NAFLD cases enrolled in PGR. Second, to investigate pleiotropy and safety implications, we will conduct association tests with multiple traits in > 700,000 participants and conduct in-depth deep phenotyping clinical studies on human KOs and consanguineous family members.