Genetic Regulation of Nonalcoholic Fatty Liver Disease

非酒精性脂肪肝的基因调控

• 批准号: 10598158
• 负责人: Aras Nikodemas Mattis
• 金额: $ 56.02万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-08 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10598158
• 关键词: Adult; Age; Alleles; Alternative Splicing; American; Animal Model; Biology; Cell Differentiation process; Cell Line; Cell model; Cells; Development; Diagnostic tests; Disease model; Electronic Health Record; Fatty Liver; Fatty acid glycerol esters; Genes; Genetic; Genetic Diseases; Genetic Models; Genetic Predisposition to Disease; Genetic Risk; Genetic Variation; Haplotypes; Hepatic; Hepatocyte; Human; Hypertriglyceridemia; Impairment; Incubated; Individual; Linkage Disequilibrium; Lipids; Liver; Liver Failure; Liver diseases; Measures; Mediating; Modeling; Mus; Non obese; Oleates; Patients; Phenotype; Pilot Projects; Plasma; Proteins; Race; Regulation; Research; Testing; Therapeutic; Time; Triglycerides; Undifferentiated; Variant; causal variant; clinical diagnosis; cost; cytokine; disease diagnosis; disease phenotype; endoplasmic reticulum stress; genetic variant; hepatoma cell; in vivo; induced pluripotent stem cell; knock-down; lipid biosynthesis; lipid metabolism; mouse model; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; novel strategies; overexpression; precision medicine; risk variant; screening; sex; tool; transcriptome sequencing

SUMMARY Between 30-40% of American adults have nonalcoholic fatty liver disease (NAFLD), which currently has no therapeutic options and is predicted to become the leading cause of the liver failure in the US. NAFLD is initiated by excess lipid accumulation in the liver (i.e. hepatic steatosis). The well-established risk variants TM6SF2 rs58542926 (E167K) and PNPLA3 rs738409 (I148M) both lead to increased cellular lipid accumulation. While patient-derived cell-based models are a valuable tool for the discovery of novel genetic factors, most NAFLD models are based on liver-derived or hepatocyte-like cells, which are time-intensive and costly to generate. Since steatosis is a cell autonomous phenotype, we propose an entirely novel approach of using patient-derived induced pluripotent stem cells (iPSCs) in their undifferentiated state as a NAFLD genetic model. In preliminary studies we found that oleate-treated iPSC both before and after differentiation into hepatocyte-like cells (iHeps) with the TM6SF2 rs58542926-T risk allele have greater lipid accumulation compared to non-carriers, differences that were not completely rescued after gene editing to revert rs58542926 to the non-risk allele. This finding highlights the potential for iPSCs and iHeps to interrogate the genetic predisposition to NAFLD, as well as suggests that the association between rs58542926 and NAFLD is not driven by TM6SF2 alone. rs58542926 is in near perfect linkage disequilibrium with rs10401969, which regulates SUGP1 protein levels. In pilot studies, SUGP1 knock-down increased triglyceride levels in human hepatoma cell lines, while knock-down in vivo resulted in hepatic steatosis. While our findings implicate SUGP1 rs10401069 as an additional causal variant for NAFLD, to date, mechanistic studies of this haplotype have been focused on TM6SF2 alone. In Aim 1 we propose to test whether SUGP1 rs10401969 modulates NAFLD in cellular and animal models by: i) Identifying NAFLD relevant cellular phenotypes that differ between iHeps from SUGP1 and rs10401969 double-carriers vs. non-carriers; ii) Introduce the rs10401969 and rs58542926 risk-alleles in non-carriers, and test for recapitulation of cellular phenotypes; iii) Verify that SUGP1 knock-down and overexpression modulates NAFLD in vivo; and iv) Identify SUGP1 target genes that mediate NAFLD- phenotypes. In Aim 2, we will establish undifferentiated iPSCs as a cellular model for NAFLD genetic risk variants by: i) Comparing NAFLD-relevant cellular phenotypes in iPSCs compared to iHeps; ii) Confirming that iPSCs can model single SNP effects, and testing whether phenotypes differ in iPSCs selected from the extremes of the NAFLD genetic risk score distribution; and iii) testing for differences in cellular phenotypes in iPSCs from NAFLD cases vs. controls. Together these studies will significantly advance the field of NAFLD genetics by defining SUGP1 as a genetic contributor to NAFLD, and establishing iPSCs as a novel cellular model to identify and functionalize NAFLD genetic variants. The successful implementation of these aims will reveal new biology underlying genetic drivers of NAFLD that may ultimately be used to develop precision medicine standards for NAFLD diagnosis, screening and management.

总结 30-40%的美国成年人患有非酒精性脂肪肝（NAFLD），目前没有 治疗选择，预计将成为美国肝功能衰竭的主要原因。NAFLD启动 通过肝脏中的过量脂质积累（即肝脂肪变性）。已确立的风险变体TM 6SF 2 rs 58542926（E167 K）和PNPLA 3 rs738409（I148 M）均导致细胞脂质积累增加。而 患者来源的基于细胞的模型是发现新的遗传因素的有价值的工具， 模型是基于肝源性或肝细胞样细胞，这是时间密集型和昂贵的生产。以来 脂肪变性是一种细胞自主表型，我们提出了一种全新的方法， 诱导多能干细胞（iPSC）在其未分化状态作为NAFLD遗传模型。初步 我们的研究发现，油酸处理的iPSC在分化成肝细胞样细胞（iHeps）之前和之后， 与非携带者相比，携带TM 6SF 2 rs 58542926-T风险等位基因的患者有更大的脂质蓄积，差异 在基因编辑将rs 58542926恢复为非风险等位基因后没有完全获救。这一发现 强调了iPSC和iHep询问NAFLD遗传易感性的潜力，以及 这表明rs 58542926和NAFLD之间的关联不是由TM 6SF 2单独驱动的。rs 58542926是 与调节SUGP 1蛋白水平的rs 10401969接近完美连锁不平衡。在试点研究中， SUGP 1敲低可增加人肝癌细胞系中的甘油三酯水平，而在体内敲低SUGP 1可增加人肝癌细胞系中的甘油三酯水平。 导致肝脂肪变性。虽然我们的研究结果暗示SUGP 1 rs 10401069是一个额外的因果变异， NAFLD，迄今为止，该单倍型的机制研究仅集中在TM 6SF 2上。在目标1中， 提出通过以下方式测试SUGP 1 rs 10401969是否在细胞和动物模型中调节NAFLD：i）鉴定 NAFLD相关细胞表型在来自SUGP 1和rs 10401969双携带者的iHep与来自 非携带者; ii）在非携带者中引入rs 10401969和rs 58542926风险等位基因，并测试重演 iii）验证SUGP 1敲低和过表达在体内调节NAFLD;和iv） 鉴定介导NAFLD表型的SUGP 1靶基因。在目标2中，我们将建立未分化的iPSC 作为NAFLD遗传风险变体的细胞模型，通过：i）比较NAFLD相关细胞表型， i i）证实iPSC可以模拟单一SNP效应，并测试iPSC是否 表型在选自NAFLD遗传风险评分分布的极端的iPSC中不同;和iii） 测试来自NAFLD病例与对照的iPSC中细胞表型的差异。这些研究 通过将SUGP 1定义为NAFLD的遗传贡献者，将显著推进NAFLD遗传学领域， 建立iPSC作为新的细胞模型以鉴定和功能化NAFLD遗传变体。成功 这些目标的实现将揭示NAFLD遗传驱动因素的新生物学基础， 用于制定NAFLD诊断、筛查和管理的精准医学标准。