In vitro functional characterization of PCSK9 variants identified in patients with Familial Hypercholesterolemia

Familial Hypercholesterolemia (FH) is a genetic disorder of lipoprotein metabolism, characterized by elevated plasma cholesterol levels, mostly carried by low-density lipoprotein (LDL). FH is mainly caused by mutations in three genes, LDLR, APOB, and PCSK9. Gain-of-function mutations in PCSK9 reduce LDL receptor levels, resulting in high levels of LDL cholesterol in the plasma. Loss-of-function mutations lead to higher levels of the LDL receptor, resulting in lower LDL cholesterol levels. The Human Genome Project led to a faster technological development related to sequencing methods, which allowed identifying many novel variants associated with FH. However, the mechanisms by which these variants influence cholesterol levels and their interference in therapeutic response are not fully understood. The aim of the present study was to perform an in vitro characterization of the effect of PCSK9 variants identified in FH patients using Next-Generation Sequencing. For the functional characterization of variants in the coding region of PCSK9, the impact of these variants on PCSK9-LDLR interaction was evaluated by molecular docking. HEK293FT cells were transiently transfected with different PCSK9 constructs, and the amount of cell surface LDLR and LDL internalization were determined by flow cytometry. For the variants in PCSK9 3UTR region, an in silico prediction of PCSK9 3UTR variants in miRNA seed regions and target sites was performed. To determine whether the predicted miRNAs directly interact with PCSK9 3UTR region, HEK293FT cells were co-transfected with a vector containing a PCSK9 3\'UTR region and a Gaussia luciferase reporter gene, together with an expression plasmid containing the miRNAs of interest. The effect of the predicted miRNAs on the expression of PCSK9 was evaluated using RT-qPCR and Western blot in HepG2 cells transiently transfected with miRNA mimics. Nine missense variants were identified in PCSK9 gene. E32K e R469W were chosen for further analysis. For R469W, a possible conformational change was observed that could increase the affinity of PCSK9 for LDLR, when compared to the wild-type. For E32K, a possible association with FH in a Brazilian family with Japanese ancestry was observed. E32K and R469W had a 5% and 11% decreased level of cell surface LDLR, respectively, as compared with WT-PCSK9. However, no significant reduction in the number of cell surface LDLR and LDL internalization was observed in transfected cells for both variants. Ten variants were found in PCSK9 3\'UTR region, of which three were selected for affecting the binding of four miRNAs. Our data demonstrated a significant downregulation of PCSK9 in cells transfected with miR-4721 and miR-564 miRNA mimics, compared to cells transfected with a scramble control (p=0,036 and p=0,010, respectively). However, no differences in luciferase expression were observed in cells transfected with these miRNAs, therefore, it was not possible to experimentally validate miRNA-mRNA interaction. PCSK9 variants found in our study may not fully explain FH phenotype but may contribute to the severity of the disease together with other variants in other genes. (AU)