Disease Modeling of Autosomal Recessive Hypercholesterolemia using Human Induced pluripotent Stem Cells

ABSTRACT

Autosomal recessive hypercholesterolemia (ARH) is an inherited disease causing by mutations in the low-density lipoprotein receptor (LDLR) adaptor protein-1 (LDLRAP1) gene, which leads to elevate the serum levels of low-density lipoprotein cholesterol (LDL-C). ARH patients have varying responses to statin therapy, and the pathophysiological mechanism(s) behind this diversity is unknown. This can be related in part to the lack of appropriate cellular models to investigate the effect of LDLRAP1 mutations on the role of encoded protein LDLRAP1 in LDLR internalization. The purpose of this research was to confirm the use of patient-specific induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) as an applicable tool to model ARH disorder. Using Sendai viral vectors, fibroblasts of an ARH patient harboring a novel variant c.649G>T in LDLRAP1 gene were reprogrammed into hiPSCs. Furthermore, we employed clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) for knocking out the LDLRAP1 gene of two different human iPSC lines. Control iPSCs, ARH-knockout iPSC lines, and ARH patient-derived iPSCs differentiated into HLCs efficiently. The absence of adaptor protein LDLRAP1 in HLCs derived from knockout and patient iPSCs was discovered by western blot analysis, and this was linked to the reduction in uptake of LDL-C in ARH-knockout/mutant HLCs in compare with control HLCs. Consequently, we discovered that the identified point-mutation c.649G>T in LDLRAP1 gene results in the absence of the LDLRAP1 protein, which is identical to what occurs when LDLRAP1 is knocked out. Subsequently, LDL uptake in ARH-knockout/mutant HLCs decreased but not completely abolished. Since, the existence of LDLRAP1 point mutation c.649G>T or knockout of LDLRAP1 cause to absence of adaptor protein LDLRAP1, remaining LDL uptake could be influenced via LDLRAP1-independent endocytosis processes. Therefore, can be a valuable tool for understanding more about LDLRAP1 mutations and the encoded protein function.