Novel PCSK9 (Proprotein Convertase Subtilisin Kexin Type 9) Variants in Patients With Familial Hypercholesterolemia From Cape Town

Abstract

Objective: Familial hypercholesterolemia (FH) is characterized by elevated low-density lipoprotein-cholesterol and markedly increased cardiovascular risk. In patients with a genetic diagnosis, low-density lipoprotein receptor ( LDLR ) mutations account for >90% of cases, apolipoprotein B ( APOB ) mutations for ≈5% of cases, while proprotein convertase subtilisin kexin type 9 ( PCSK9 ) gain of function mutations are rare (<1% of cases). We aimed to evaluate the functional impact of several novel PCSK9 variants in a cohort of patients with FH by genetic cascade screening and in vitro functionality assays. Approach and Results: Patients with clinically diagnosed FH underwent genetic analysis of LDLR , and if negative, sequential testing of APOB and PCSK9 . We analyzed cosegregation of hypercholesterolemia with novel PCSK9 variants. Gain of function status was determined by in silico analyses and validated by in vitro functionality assays. Among 1055 persons with clinical FH, we identified nonsynonymous PCSK9 variants in 27 (2.6%) patients and 7 of these carried one of the 4 previously reported gain of function variants. In the remaining 20 patients with FH, we identified 7 novel PCSK9 variants. The G516V variant (c.1547G>T) was found in 5 index patients and cascade screening identified 15 additional carriers. Low-density lipoprotein-cholesterol levels were higher in these 15 carriers compared with the 27 noncarriers (236±73 versus 124±35 mg/dL; P< 0.001). In vitro studies demonstrated the pathogenicity of the G516V variant. Conclusions: In our study, 1.14% of cases with clinical FH were clearly attributable to pathogenic variants in PCSK9 . Pathogenicity is established beyond doubt for the G516V variant.

Lipoprotein metabolism in familial hypercholesterolemia

“…PCSK9 has also been shown to promote LDLR decay via an intracellular route. Similar to LDLR gene defects, PCSK9 GOF mutations lower the abundance of the LDLR at the cell surface in many different ways ( 46 , 47 ). For instance, genetic variants on PCSK9 can display either higher transcriptional activity, or resistance to cleavage by furin, or increased affinity for the LDLR.…”

“…Given that PCSK9 acts primarily as a secreted protein, the extracellular activity of wild-type and PCSK9 variants can be comparatively assessed by adding the recombinant PCSK9 proteins in the culture medium of hepatoma cell lines (e.g., HepG2) prior to measurement of cell surface LDLR expression and fluorescent LDL cellular uptake by flow cytometry ( 47 , 50 , 51 , 52 ). Another valuable parameter that provides important information about PCSK9 variants is to measure their affinity for the LDLR at the cell surface and in endosomes.…”

“…Another valuable parameter that provides important information about PCSK9 variants is to measure their affinity for the LDLR at the cell surface and in endosomes. This can be evaluated by solid-phase immunoassay at pH 7.4 and 5.2, respectively, using the recombinant LDLR ectodomain to capture PCSK9 ( 47 , 51 ). Given that some PCSK9 variants have been shown to inhibit LDLR prior to secretion, this intracellular activity can be assessed in human embryonic kidney 293 cells coexpressing the LDLR ectodomain and either wild-type or PCSK9 variants.…”

“…Given that some PCSK9 variants have been shown to inhibit LDLR prior to secretion, this intracellular activity can be assessed in human embryonic kidney 293 cells coexpressing the LDLR ectodomain and either wild-type or PCSK9 variants. The amount of LDLR ectodomain secreted in the culture medium can be assessed by Western blot and recapitulates the ability of PCSK9 variants to impact the translocation of the LDLR from intracellular compartments to the cell surface ( 47 , 52 ).…”

A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants

“…Our results demonstrate the usefulness of these complementary approaches that all together may seem tedious but that can easily be restricted to transient transfections followed by flow cytometry analysis for assessment of GOF and LOF status [21,22,31].…”