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.

非酒精性脂肪性肝病 (NAFLD) 进展为纤维化非酒精性脂肪性肝炎 (NASH) 已成为肝病的主要原因。到 2021 年中期，人类 NAFLD/NASH 的遗传病因已仅限于少数基因座。为了解决这一差距，我们和其他人最近确定了 657 个相关的新位点具有与 NAFLD/NASH 相关的多个数量性状。虽然这些新的和先前的发现研究已经尽管已在基因位点和 NAFLD/NASH 风险之间建立了安全联系，但仍存在一些不确定性关于这些发现，包括： (a) 这些基因座的致病基因； (b) 这些位点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，所有先前的发现努力都受到限制 (d) 通用变体发现平台； (e) 欧洲人后裔； (f) 使用代理定义 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效果的位点通过利用巴基斯坦基因组资源（PGR）来提高相关性，该资源利用了高水平的血缘关系在巴基斯坦组建了世界上最大规模的人类基因敲除 (KO) 队列，共 4,000 人超声检查 (USG) 确诊的 NAFLD 病例。具体来说，在目标 1 中，我们将： (i) 进行多民族交往对 700,000 名参与者（PGR + 英国生物银行）的血清 AST 和 ALT 水平的罕见和常见变异进行分析对 4,000 例经 USG 确诊的 NAFLD 病例和 4,000 例对照进行验证研究； (ii) 进行精细测绘研究和共定位分析； (iii) 进行罕见变异基因负荷测试，重点关注预测的基因缺失功能（pLoF）和预测破坏性变异（pDM）变异； (iv) 进行路径分析和 (v) 对 10 个高度优先位点，对 pLoF 携带者及其家庭成员进行回调研究，以驱动因果基因识别和翻译。在目标 2 中，我们将研究已从分析中识别出的两个高优先级基因目标 1、WWP2 和 CASP8 中提出，以说明如何从机制和方法上研究这些基因实验性 NASH。对于 WWP2，我们根据其 E3 连接酶活性假设它通过下调来保护调节纤维化 NASH 中的关键蛋白 TAZ (WWTR1)，现在我们令人兴奋的体外初步实验支持了这一点和体内数据。 CASP8 是基于 Aim 1 和先前小鼠数据的 NASH 促进基因的一个例子研究，但其机制尚不确定，并且可能与其典型的细胞死亡功能无关。这里我们将测试当将 PGR pLoF/pDM 引入小鼠肝细胞时，WWP2 和 CASP8 上的 PGR pLoF/pDM 变异的假设正如人类数据和我们的假设所预测的那样，缺乏内源基因会影响 NASH。我们也会探讨2个基因在体外和实验NASH中的机制以及pLoF/pDM如何影响该机制变种。在目标 3 中，我们将利用现有的序列研究遗传位点对疾病进展的影响 VCTE 测量可用于 PGR 中登记的 4,000 例 USG 确诊的 NAFLD 病例。二、调查多效性和安全性影响，我们将在超过 700,000 名参与者中进行多种特征的关联测试，对人类KO和近亲家庭成员进行深入的表型临床研究。