In vitro and in silico functional characterization of variants in the PCSK9 gene identified in patients with Familial hypercholesterolemia

Abstract

Familial Hypercholesterolemia (FH) is a genetic disorder of lipoprotein metabolism, characterized by high plasma cholesterol, 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 increase its proteolytic activity, reducing LDL receptor concentration, therefore resulting in high levels of LDL cholesterol in the plasma. Loss-of-function mutations lead to a higher concentration 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 us to identify many novel variants associated with FH. However, the mechanisms by which these variants influence cholesterol levels and their interference in the therapeutic response are not fully understood. The aim of the present project is an in silico and in vitro characterization of the effect of variants on the PCSK9 gene identified in FH patients using New Generation Sequencing. Methods: (i): in silico analysis; (ii) molecular docking; (iii) HepG2 and HUVEC cell culture; (iv) site-directed mutagenesis of the variants using the Phusion Site-Directed Mutagenesis Kit; (v) transfection of the variants constructs using Lipofectamine 2000; (vi) evaluation of LDL uptake into transfected, HepG2 and HUVEC, cells by flow cytometry. This study has been in progress since July 2016, and in silico analyzes and molecular docking of the following variants have been concluded: R237W, A443T, R469W and Q619P. For variants R237W and R469W a possible conformational change was observed that could increase the affinity of PCSK9 for LDLR. Also, E32K was sequenced by Sanger, and it is possibly related to FH in a Brazilian family with Japanese ancestry. We expect that the results of this study may contribute to the knowledge of the possible functional impacts of these variants in HF patients. (AU)