Autosomal recessive hypercholesterolemia (ARH) is a rare monogenic disorder caused by pathogenic variants in the low-density lipoprotein receptor (LDLR) adaptor protein 1 (LDLRAP1) gene, encoding for the LDLRAP1 protein, which impairs internalization of hepatic LDLR. There are variable responses of ARH patients to treatment and the pathophysiological mechanism(s) for this variability remains unclear. This is in part caused by absence of reliable cellular models to evaluate the effect of LDLRAP1 mutations on the LDLRAP1 protein function and its role in LDLR internalization. Here, we aimed to validate patient-specific induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) as an appropriate tool to model ARH disease. Fibroblasts from an ARH patient carrying the recently reported nonsense mutation, c.649G>T, were reprogrammed into hiPSCs using Sendai viral vectors. In addition, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create an LDLRAP1 gene (also known as ARH) knockout in two different human iPSC lines. ARH patient-derived iPSCs, ARH-knockout iPSC lines, and control iPSCs were efficiently differentiated into HLCs. Western blot analysis demonstrated the absence of LDLRAP1 in HLCs derived from patient and knockout iPSCs, and this was associated with a decreased low-density lipoprotein cholesterol (LDL-C) uptake in ARH-mutant/knockout HLCs compared to control HLCs. In conclusion, we determined that the recently described c.649G>T point mutation in LDLRAP1 induces absence of the LDLRAP1 protein, similar to what is seen following LDLRAP1 knockout. This causes a decreased, although not fully absent, LDL-uptake in ARH-mutant/knockout HLCs. As knockout of LDLRAP1 or presence of the c.649G>T point mutation results in absence of LDLRAP1 protein, residual LDL uptake might be regulated by LDLRAP1-independent internalization mechanisms. Patient-specific iPSC-derived HLCs can therefore be a powerful tool to further decipher LDLRAP1 mutations and function of the protein.

中文翻译：

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患者特异性诱导的多能干细胞衍生的肝细胞样细胞作为研究常染色体隐性高胆固醇血症的模型

常染色体隐性遗传高胆固醇血症 (ARH) 是一种罕见的单基因疾病，由低密度脂蛋白受体 (LDLR) 衔接蛋白 1 ( LDLRAP1 ) 基因的致病变异引起，该基因编码 LDLRAP1 蛋白，损害肝脏 LDLR 的内化。ARH 患者对治疗有不同的反应，这种变化的病理生理机制仍不清楚。这部分是由于缺乏可靠的细胞模型来评估LDLRAP1的影响LDLRAP1 蛋白功能的突变及其在 LDLR 内化中的作用。在这里，我们旨在验证患者特异性诱导多能干细胞 (iPSC) 衍生的肝细胞样细胞 (HLC) 作为模拟 ARH 疾病的合适工具。使用仙台病毒载体将携带最近报道的无义突变 c.649G>T 的 ARH 患者的成纤维细胞重新编程为 hiPSC。此外，我们使用成簇的规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 来创建LDLRAP1基因（也称为ARH) 在两个不同的人类 iPSC 系中敲除。ARH 患者衍生的 iPSC、ARH 敲除 iPSC 系和对照 iPSC 被有效分化为 HLC。蛋白质印迹分析表明，源自患者和敲除 iPSC 的 HLC 中不存在 LDLRAP1，与对照 HLC 相比，这与 ARH 突变/敲除 HLC 中低密度脂蛋白胆固醇 (LDL-C) 的摄取减少有关。总之，我们确定最近描述的LDLRAP1 中的c.649G>T点突变导致LDLRAP1蛋白的缺失，类似于LDLRAP1敲除后所见的情况。这导致 ARH 突变体/敲除 HLC 中的 LDL 摄取减少，尽管并非完全不存在。作为LDLRAP1的敲除或存在c.649G>T点突变导致 LDLRAP1 蛋白缺失，残留的 LDL 摄取可能受 LDLRAP1 非依赖性内化机制的调节。因此，患者特异性 iPSC 衍生的 HLC 可以成为进一步破译LDLRAP1突变和蛋白质功能的有力工具。