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

通过人类基因敲除的深入表型分析和机制研究发现 NASH 的新治疗靶点

基本信息

批准号：
10698154
负责人：
Danish Saleheen
金额：
$ 66.18万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10698154
关键词：
Address Adverse effects Affect Alleles Biochemistry Biological Biological Markers CASP8 gene Callback Cardiovascular system Cell Death Cells Chromosome Mapping Clinical Clinical Research Consanguinity Data Data Analyses Disease Progression Enrollment Epidemiologist Ethnic Origin Etiology European FDA approved Family member Fibrosis Future Gene Dosage Gene Expression Genes Genetic Genetic Determinism Genetic Population Study Genetic Predisposition to Disease Genetic Variation Genotype Hepatic Hepatocyte Heterozygote Human Human Genetics In Vitro Infrastructure Kidney Knock-out Link Lipids Liver Liver diseases Maps Measurement Metabolic Molecular and Cellular Biology Mus Nature Obesity Epidemic Pakistan Participant Pathway Analysis Pathway interactions Pharmaceutical Preparations Phenotype Population Proteins Proxy Resources Risk Role Safety Secure Serum Testing Therapeutic Therapeutic Studies Translations Ultrasonography Uncertainty Variant biobank body system causal variant clinical phenotype cohort consanguineous family disorder risk elastography exome genetic analysis genetic variant genome resource genome sequencing genome-wide genome-wide analysis genomic locus human data in vivo interest loss of function multi-ethnic new therapeutic target non-alcoholic fatty liver disease nonalcoholic steatohepatitis novel participant enrollment pleiotropism pre-clinical preclinical study rare variant therapeutic target trait translational scientist ubiquitin-protein ligase ultrasound validation studies vibration whole genome

项目摘要

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.

非酒精性脂肪肝疾病（NAFLD）的进展到纤维化的非酒精性脂肪性肝炎（NASH）的进展为成为肝病的主要原因。直到2021年中，人类NAFLD/NASH的遗传病因学仅限于少数基因座。为了解决这一差距，我们和其他人最近确定了657个新颖的位点相关具有与NAFLD/NASH相关的几个定量特征。尽管这些新的和先前的发现研究已经遗传基因座与NAFLD/NASH风险之间建立的安全联系，继续存在许多不确定性关于此类发现，包括：（a）这些基因座的因果基因；（b）这些基因座对疾病的定向影响风险; （c）导致疾病风险的生物学机制。此外，所有先前的发现工作都受到限制至：（d）常见的变体发现平台；（e）欧洲血统的种群；（f）使用代理定义基于AST和ALT级别的NAFLD/NASH。我们旨在解决这些不确定性并找到高治疗的基因座利用高水平血缘的巴基斯坦基因组资源（PGR）来实现相关性在巴基斯坦，聚集了世界上最大的人类淘汰赛（KOS）和4,000个队列超声检查（USG）确认的NAFLD病例。具体来说，在AIM 1中，我们将：（i）执行多种族协会分析70万参与者（PGR + UK Biobank）的稀有和常见变体的血清AST和ALT级别并在4,000个USG确认的NAFLD病例和4,000个对照中进行验证研究；（ii）进行精细映射研究和共定位分析；（iii）执行稀有变体基因负担测试，重点是预测的丢失函数（plof）和预测损坏变体（PDM）变体；（iv）进行途径分析和（v）10高度优先考虑基因座，对plof携带者以及其家人进行呼叫研究，以驱动因果基因识别和翻译。在AIM 2中，我们将研究两个已经从分析中鉴定出的高优先基因在AIM 1，WWP2和CASP8中提出的，以说明如何机械和中如何研究此类基因实验纳什。对于WWP2，我们基于其E3连接酶的活性假设，它可以通过下降来保护调节TAZ纤维化NASH（WWTR1）中的关键蛋白和体内数据。 CASP8是基于AIM 1和先前鼠标的数据的启动NASH基因的示例研究，但是机制是不确定的，可能与其规范细胞死亡功能无关。在这里我们将测试当引入小鼠的肝细胞时，pgr plof/pdm变体在WWP2和CASP8处的假设缺乏内源基因，会影响人类数据和我们的假设所预测的NASH。我们也会在体外和实验性NASH中探测2种基因的机制，以及该机理如何受Plof/PDM的影响变体。在AIM 3中，我们将通过利用可用的系列来研究遗传基因座对疾病进展的影响在PGR中注册的4,000个USG确认的NAFLD案件中可用的VCTE测量值。第二，调查多效和安全性影响，我们将与> 700,000参与者的多个特征进行关联测试，对人类KO和血统家庭成员进行深入深入的深层表型临床研究。