Identification and in vitro characterization of two new PCSK9 Gain of Function variants found in patients with Familial Hypercholesterolemia

Ornella Guardamagna,Carola Giacobbe,Aitor Etxebarria,Maria Donata Di Taranto,Marco Gentile,Asier Benito-Vicente,Giuliana Fortunato,Kepa Belloso Uribe,Paolo Rubba,Cesar Martín

doi:10.1038/s41598-017-15543-x

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by pathogenic variants in genes encoding for LDL receptor (LDLR), Apolipoprotein B and Proprotein convertase subtilisin/kexin type 9 (PCSK9). Among PCSK9 variants, only Gain-of- Function (GOF) variants lead to FH. Greater attention should be paid to the classification of variants as pathogenic. Two hundred sixty nine patients with a clinical suspect of FH were screened for variants in LDLR and the patients without pathogenic variants were screened for variants in PCSK9 and APOB. Functional characterization of PCSK9 variants was performed by assessment of protein secretion, of LDLR activity in presence of PCSK9 variant proteins as well as of the LDLR affinity of the PCSK9 variants. Among 81 patients without pathogenic variants in LDLR, 7 PCSK9 heterozygotes were found, 4 of whom were carriers of variants whose role in FH pathogenesis is still unknown. Functional characterization revealed that two variants (p.(Ser636Arg) and p.(Arg357Cys)) were GOF variants. In Conclusions, we demonstrated a GOF effect of 2 PCSK9 variants that can be considered as FH-causative variants. The study highlights the important role played by functional characterization in integrating diagnostic procedures when the pathogenicity of new variants has not been previously demonstrated.

Highlights

Familial Hypercholesterolemia (FH) is a severe genetic hyperlipidaemia characterized by increased levels of LDL cholesterol accumulating in tissues and leading to premature atherosclerosis, tendon xanthomas and corneal arcus
Two different types of pathogenic variants have been identified in this gene: 1. loss of function (LOF) variants producing a less functioning protein, causing an increase of LDL receptor (LDLR) amounts on the cell membrane and, hypocholesterolemia; 2. gain of function (GOF) variants producing a more functioning protein that degrades LDLR more efficiently, decreasing its levels and causing FH8
In the remaining 81 patients the genetic screening of Proprotein convertase subtilisin/ kexin type 9 (PCSK9) revealed the presence of 7 rare missense variants at heterozygous status (2.6% of total patients): 3 variants previously identified in Familial hypercholesterolemia (FH) patients; 4 variants never associated to FH before

Summary

Introduction

Familial Hypercholesterolemia (FH) is a severe genetic hyperlipidaemia characterized by increased levels of LDL cholesterol accumulating in tissues and leading to premature atherosclerosis, tendon xanthomas and corneal arcus. Female Male to decreased LDLR levels on the cell membrane available for LDL uptake[3]. Another mechanism independent of endocytosis, indicating an intracellular action of PCSK9 in LDLR expression regulation, was observed[4]. Functional characterization by in vitro assays is the most effective and reliable method to evaluate the pathogenic role of gene variants and it is especially required for evaluating PCSK9 variants. Recent guidelines support this concept and suggest that, among different criteria, functional assays can provide strong evidence of pathogenicity[11]. We report the characterization of 4 rare variants in the PCSK9 gene following 3 different approaches

Methods

Results

Conclusion