Patient-Specific Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cells as a Model to Study Autosomal Recessive Hypercholesterolemia.

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.