Functional Characterization of p.(Arg160Gln) PCSK9 Variant Accidentally Found in a Hypercholesterolemic Subject

Abstract

Familial hypercholesterolaemia (FH) is an autosomal dominant dyslipidaemia, characterised by elevated LDL cholesterol (LDL-C) levels in the blood. Three main genes are involved in FH diagnosis: LDL receptor (LDLr), Apolipoprotein B (APOB) and Protein convertase subtilisin/kexin type 9 (PCSK9) with genetic mutations that led to reduced plasma LDL-C clearance. To date, several PCSK9 gain-of-function (GOF) variants causing FH have been described based on their increased ability to degrade LDLr. On the other hand, mutations that reduce the activity of PCSK9 on LDLr degradation have been described as loss-of-function (LOF) variants. It is therefore important to functionally characterise PCSK9 variants in order to support the genetic diagnosis of FH. The aim of this work is to functionally characterise the p.(Arg160Gln) PCSK9 variant found in a subject suspected to have FH. Different techniques have been combined to determine efficiency of the autocatalytic cleavage, protein expression, effect of the variant on LDLr activity and affinity of the PCSK9 variant for the LDLr. Expression and processing of the p.(Arg160Gln) variant had a result similar to that of WT PCSK9. The effect of p.(Arg160Gln) PCSK9 on LDLr activity is lower than WT PCSK9, with higher values of LDL internalisation (13%) and p.(Arg160Gln) PCSK9 affinity for the LDLr is lower than WT, EC50 8.6 ± 0.8 and 25.9 ± 0.7, respectively. The p.(Arg160Gln) PCSK9 variant is a LOF PCSK9 whose loss of activity is caused by a displacement of the PCSK9 P’ helix, which reduces the stability of the LDLr-PCSK9 complex.

Deep generative models of LDLR protein structure to predict variant pathogenicity

“…While AF2 was not designed to predict the effects of variants ( https://alphafold.ebi.ac.uk/faq ), the use of AF2 structures for variant prediction is an active area of research with the expectation that improved structural modeling of the human proteome may provide insight toward the mechanism of disease-associated variants ( 27 ). Recently, AF2 has been used to model pathogenic variants in LDLR ( 28 ) and PCSK9 ( 29 ) to determine effects on protein-protein interactions and the catalytic domain, respectively. One approach for variant prediction is to directly model a variant sequence with AF2 and observe any conformational changes in the predicted structure or model confidence.…”

Cardiovascular Disease, Atherosclerosis and Familial Hypercholesterolemia: From Molecular Mechanisms Causing Pathogenicity to New Therapeutic Approaches

“…Finally, Larrea-Sebal et al [12] conducted a functional characterization of a genetic variant in the Protein convertase subtilisin/kexin type 9 (PCSK9) gene, which was detected in a patient with suspected familial hypercholesterolemia (FH). Various techniques, including autocatalytic cleavage efficiency, protein expression and an LDLr activity assay, were employed to assess the functionality of the PCSK9 variant.…”