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

• 批准号: 10504666
• 负责人: Danish Saleheen
• 金额: $ 67.79万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 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/10504666
• 关键词: Address; Adverse effects; Affect; Alleles; Back; Biochemistry; Biological; Biological Markers; CASP8 gene; Callback; Cardiovascular system; Cell Death; Cells; Clinical; Clinical Research; Clinical assessments; 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 Predisposition to Disease; Genetic Variation; Genetic study; Genome; Genomics; Genotype; Hepatic; Hepatocyte; 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; Phenotype; Population; Population Genetics; Proteins; Proxy; Resources; Risk; Role; Safety; Secure; Serum; Supervision; Testing; Therapeutic; Therapeutic Studies; Translations; Ultrasonography; Uncertainty; Variant; base; biobank; body system; causal variant; clinical phenotype; cohort; consanguineous family; disorder risk; elastography; exome; genetic analysis; genetic variant; 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; non-drug; nonalcoholic steatohepatitis; novel; pleiotropism; pre-clinical; preclinical study; rare variant; therapeutic target; trait; translational scientist; ubiquitin-protein ligase; ultrasound; validation studies; vibration; whole genome

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.

非酒精性脂肪性肝病（NAFLD）进展为纤维化非酒精性脂肪性肝炎（NASH）， 成为肝脏疾病的主要原因。直到2021年中期，人类NAFLD/NASH的遗传病因已经确定 仅限于少数几个位点。为了解决这一差距，我们和其他人最近确定了657个新的基因座， 与NAFLD/NASH相关的几个数量性状。虽然这些新的和先前的发现研究 尽管基因位点与NAFLD/NASH风险之间建立了可靠的联系，但仍存在许多不确定性 关于这些发现，包括：（a）这些位点的致病基因;（B）这些位点对疾病的方向性影响 风险;（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， （d）共同的变异发现平台;（e）欧洲裔人口;（f）使用替代定义 NAFLD/NASH基于AST和ALT水平。我们的目标是解决这些不确定性，并找到高治疗性的位点， 通过利用巴基斯坦基因组资源（PGR），利用高水平的血缘关系， 在巴基斯坦，他们聚集了世界上最大的人类基因敲除（科斯）队列， 超声检查（USG）证实的NAFLD病例。具体而言，在目标1中，我们将：（i）执行多民族联合 分析700，000名参与者（PGR + UK Biobank）的血清AST和ALT水平的罕见和常见变异 并在4,000例美国政府确认的NAFLD病例和4,000例对照中进行验证研究;（ii）进行精细绘图 研究和共定位分析;（iii）进行罕见变异基因负荷测试，重点是预测的基因丢失， 功能（pLoF）和预测的破坏性变体（pDM）变体;（iv）进行途径分析和（v）10个高度 优先化位点，对pLoF携带者沿着家庭成员进行回调研究，以驱动致病基因 识别和翻译。在目标2中，我们将研究已经从分析中鉴定的两个高优先级基因 在目标1，WWP 2和CASP 8中提出，以说明如何从机制上研究这些基因，并在 实验性NASH对于WWP 2，我们假设基于其E3连接酶活性，它通过下调- 调节纤维化NASH中的关键蛋白TAZ（WWTR 1），这一点现在得到了我们令人兴奋的初步体外研究的支持。 和体内数据。基于Aim 1和之前小鼠的数据，CASP 8是NASH促进基因的一个例子 研究，但其机制是不确定的，可能与其典型的细胞死亡功能无关。在这里我们将测试 假设当引入小鼠肝细胞时，PCR pLoF/pDM在WWP 2和CASP 8处发生变异， 缺乏内源性基因，影响NASH，如从人类数据和我们的假设预测的。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对这一机制的影响 变体。在目标3中，我们将利用现有的系列研究， 入组PGR的4，000例USG确认的NAFLD病例中的VCTE测量值可用。第二，调查 多效性和安全性的影响，我们将进行关联测试与多个性状在> 700，000参与者， 对人类科斯和近亲家族成员进行深入的表型临床研究。