Wellcome Sanger Institute, Hinxton, United Kingdom.
Wellcome Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.
Hepatology. 2021 Dec;74(6):2998-3017. doi: 10.1002/hep.32063. Epub 2021 Sep 27.
NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human-induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology.
We used isogenic hiPSC lines with either a knockout (PNPLA3 ) of the PNPLA3 gene or with the I148M variant (PNPLA3 ) to model PNPLA3-associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD-like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3 hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid-induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3 cells to be more susceptible to ethanol-induced and methotrexate-induced toxicity. The PNPLA3 cells exhibited an intermediate phenotype between the wild-type and PNPLA3 cells.
Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins.
非酒精性脂肪性肝病（NAFLD）是一个日益严重的公共健康负担。然而，NAFLD 的发病机制尚未完全阐明，遗传因素的重要性直到最近才被认识到。基因组研究表明，NAFLD 的进展与 patatin-like phospholipase domain-containing protein 3（PNPLA3）中的 I148M 变体之间存在很强的关联。尽管如此，对于该基因及其变体如何影响疾病发展的机制知之甚少。为了研究这些机制，我们开发了一种体外模型，该模型利用了人诱导多能干细胞（hiPSC）的独特特性和 CRISPR/CAS9 基因编辑技术。
我们使用具有 PNPLA3 基因敲除（PNPLA3 ）或 I148M 变体（PNPLA3 ）的同基因 hiPSC 系来模拟与 PNPLA3 相关的 NAFLD。由此产生的 hiPSC 分化为肝细胞，用不饱和或饱和游离脂肪酸处理，以诱导 NAFLD 样表型，并通过各种功能、转录组和脂质组学测定进行表征。PNPLA3 肝细胞表现出更高的脂质积累以及对脂质诱导的应激反应的改变模式。有趣的是，PNPLA3 的缺失也导致了对异生素代谢的减少，并使 PNPLA3 细胞更容易受到乙醇和甲氨蝶呤诱导的毒性。PNPLA3 细胞表现出介于野生型和 PNPLA3 细胞之间的中间表型。
综上所述，这些结果表明 I148M 变体诱导功能丧失，导致脂肪变性，并增加对肝毒素的易感性。
