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

非酒精性脂肪性肝病(NAFLD)向纤维性非酒精性脂肪性肝炎(NASH)的进展 成为导致肝病的主要原因。直到2021年年中，人类NAFLD/NASH的遗传病因学 仅限于少数几个基因座。为了解决这一差距，我们和其他人最近确定了657个相关的新基因座 有几个数量性状与NAFLD/NASH连锁。虽然这些新的和先前的发现研究已经 在遗传基因座和NAFLD/NASH风险之间建立了可靠的联系，一些不确定因素继续存在 关于这些发现，包括：(A)这些基因座上的致病基因(S)；(B)这些基因座对疾病的方向性影响 风险；以及(C)导致疾病风险的生物机制。此外，所有先前的发现努力都是有限的。 到：(D)共同变异发现平台；(E)欧洲后裔人口；和(F)使用 NAFLD/NASH基于AST和ALT水平。我们的目标是解决这些不确定性，并找到高效治疗的基因座。 通过利用巴基斯坦基因组资源(PGR)的相关性，该资源利用了高度的血缘关系 在巴基斯坦，并聚集了世界上最大的人类基因敲除(KO)队列和4000人 超声检查(USG)确诊的NAFLD病例。具体地说，在目标1中，我们将：(1)进行多族裔联合 PGR UK生物库70万名受试者血清AST和ALT罕见和常见变异分析 并对4,000例经美国政府确认的非酒精性脂肪肝病例和4,000名对照进行验证研究；(2)进行精细绘图 研究和共定位分析；(Iii)执行罕见的变异基因负荷测试，重点是预测的丢失 功能(PLoF)和预测的破坏性变异(Pdm)变体；(Iv)进行途径分析；和(V)高度 优先定位基因，对pLoF携带者及其家庭成员进行回拨研究，以驱动因果基因 识别和翻译。在目标2中，我们将研究已经从分析中确定的两个高优先级基因 在目标1，WWP2和CASP8中提出，以说明这些基因如何能够被机械地和在 实验性的纳什。对于WWP2，我们根据其E3连接酶活性推测，它是通过下调- 调控纤维化NASH中的关键蛋白TAZ(WWTR1)，这是我们在体外兴奋的初步研究支持的 以及活体数据。Casp8是一个基于Aim 1和之前的小鼠的数据的Nash促进基因的例子 研究，但机制是不确定的，很可能与其典型的细胞死亡功能无关。在这里，我们将测试 PGR pLoF/Pdm基因导入小鼠肝细胞后WWP2和CASP8突变的假说 缺乏内源性基因，会影响从人类数据和我们的假设预测的NASH。我们还将 探讨这两个基因在体外和实验性NASH中的作用机制以及pLoF/PDM对其作用机制的影响 变种。在目标3中，我们将通过利用可用的序列来研究遗传位点对疾病进展的影响 在登记在PGR中的4,000例USG确认的NAFLD病例中进行了VCTE测量。第二，调查 多效性和安全性影响，我们将在70万名参与者中进行多个特征的关联测试，并 对人类KO和血缘家庭成员进行深入的表型临床研究。

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.