Genetic Regulation of Nonalcoholic Fatty Liver Disease

摘要 30%-40%的美国成年人患有非酒精性脂肪性肝病(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 由于肝脏中过多的脂肪堆积(即肝脏脂肪变性)。公认的风险变量TM6SF2 Rs58542926(E167K)和PNPLA3 rs738409(I148M)均导致细胞内脂质积累增加。而当 基于患者的细胞模型是发现新的遗传因素的宝贵工具，大多数是NAFLD 模型建立在肝脏来源或肝细胞样细胞的基础上，这些细胞的产生既耗时又昂贵。自.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，使用患者来源的 诱导多能干细胞(IPSCs)在其未分化状态作为NAFLD遗传模型。在预赛中 研究发现油酸对IPSC诱导分化为肝细胞样细胞(IHEPS)前后均有作用。 携带TM6SF2 rs58542926-T的危险等位基因较非携带者有更大的脂质蓄积，差异 在基因编辑将rs58542926恢复为非危险等位基因后，这些未被完全挽救的基因。这一发现 强调了IPSCs和iHEPs询问NAFLD的遗传易感性的潜力，以及 提示rs58542926与NAFLD之间的关联不是由TM6SF2单独驱动的。Rs58542926为 与调节SUGP1蛋白水平的rs10401969处于近乎完美的连锁不平衡。在试点研究中， SUGP1基因敲除可增加人肝癌细胞系的甘油三酯水平，而在体内则是敲除 导致肝脏脂肪变性。虽然我们的发现暗示SUGP1 rs10401069是另一个原因变量 到目前为止，对这种单倍型的机制研究主要集中在TM6SF2。在目标1中，我们 建议测试SUGP1 rs10401969是否通过以下方式调节细胞和动物模型中的NAFLD：i)识别 与NAFLD相关的细胞表型在来自SUGP1和rs10401969的iHEPs之间存在差异。 非携带者；ii)在非携带者中引入rs10401969和rs58542926危险等位基因，并进行概括性检验 细胞表型；iii)证实SUGP1在体内下调和过度表达对NAFLD的调节；以及iv) 确定介导NAFLD表型的SUGP1靶基因。在目标2中，我们将建立未分化的IPSC 作为NAFLD遗传风险变异的细胞模型：i)比较NAFLD相关的细胞表型 IPSCs与iHEPs的比较；ii)确认IPSCs可以模拟单个SNP效应，并测试 选自NAFLD遗传风险分数分布极端的IPSCs的表型不同；和iii) 检测非酒精性脂肪肝患者和对照组IPSCs的细胞表型差异。把这些研究放在一起 通过将SUGP1定义为NAFLD的遗传贡献者，将显著推进NAFLD遗传学领域，以及 建立IPSCs作为一种新的细胞模型来识别NAFLD基因变异并使其发挥功能。成功者 这些目标的实现将揭示NAFLD潜在的新的生物学基因驱动因素，最终可能 用于制定NAFLD诊断、筛查和管理的精准医学标准。

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.