The E32K variant of PCSK9 exacerbates the phenotype of familial hypercholesterolaemia by increasing PCSK9 function and concentration in the circulation.

Department of Lipidology, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8640, Japan.
Atherosclerosis (Impact Factor: 3.71). 11/2009; 210(1):166-72. DOI: 10.1016/j.atherosclerosis.2009.11.018
Source: PubMed

ABSTRACT Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol trafficking by mediating degradation of cell-surface LDL receptors (LDLR). Gain-of-function PCSK9 mutations are known to increase plasma LDL-C levels. We attempted to find gain-of-function PCSK9 mutations in Japanese subjects and determine the frequency and impacts of these mutations, especially on circulating PCSK9 and LDL-C levels.
PCR-SSCP followed by direct sequence analysis was performed for all 12 exons and intronic junctions of the PCSK9 in 55 subjects with clinically diagnosed familial hypercholesterolaemia (clinical-FH), who were confirmed to have no LDLR mutations. Among the mutations detected, PCSK9 E32K was likely to be a gain-of-function mutation, and screening was performed by PCR-RFLP in clinical-FH and general Japanese controls. The levels of PCSK9 in plasma from subjects and in media of HepG2 cells transfected with PCSK9 constructs were measured by ELISA.
We detected 7 PCSK9 variants, including E32K. The frequency of PCSK9 E32K in clinical-FH (6.42%) was significantly higher than that in controls (1.71%). Three cases representing homozygous FH phenotypes were double heterozygous for PCSK9 E32K and LDLR C183S, C292X or K790X. Two cases were true homozygous for PCSK9 E32K; to our knowledge, these are the first true homozygotes for gain-of-function PCSK9 mutations reported to date. The PCSK9 E32K mutant had over 30% increased levels of PCSK9 in plasma from the subjects and in media of transiently transfected HepG2 cells as compared with those in controls. Furthermore, LDL-C levels in the PCSK9 E32K true homozygotes and heterozygotes were 2.10- and 1.47-fold higher than those in controls with comparable circulating PCSK9 levels, respectively, suggesting enhanced function of PCSK9 E32K.
We found 2 true homozygotes for PCSK9 E32K and 3 double heterozygotes for PCSK9 E32K and LDLR mutations associated with autosomal dominant hypercholesterolaemia. This study provided evidence that PCSK9 E32K significantly affects LDL-C levels via increased mass and function of PCSK9, and could exacerbate the clinical phenotypes of patients carrying LDLR mutations.

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    ABSTRACT: Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a newly-identified circulating protein in cholesterol metabolism in mammals, including humans, which has emerged as a new pharmacological target for hypocholesterolemia. It has been demonstrated that PCSK9 gene mutations are associated with hyper- or hypocholesterolemia. In the latter case, the incidence of coronary heart disease (CHD) is remarked reduced, suggesting that low level of low-density lipoprotein cholesterol (LDL-C) at birth is highly beneficial. Loss-of-function PCSK9 mutations will result in lower LDL-C levels and protect against CHD. Conversely, patients harboring gain-of-function PCSK9 mutations will suffer from familial autosomal dominant hypercholesterolemia (ADH), a disease characterized by elevated LDL-C plasma concentration. Although compelling evidence has suggested that PCSK9 can impair the LDL receptor (LDLR) pathway, its biological role in cholesterol metabolism remains to be defined. According to data from previous studies, PCSK9 appears a promising therapeutic target due to its role as a major LDLR regulator. Specific pharmacological inhibitors of PCSK9 have demonstrated a significant impact on plasma LDL-C concentrations. Therefore, understanding the relationship between PCSK9 and its genetic variants, on one hand, and the level of plasma LDL-C, on the other hand, may be clinically useful due to the fact that this protein has become a key target of lipid-lowering therapy. In this manuscript we mainly review recent data with regard to the association between PCSK9 genetic variants and plasma LDL-C concentrations, and outline the clinical implications.
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    ABSTRACT: Backgrounds Familial hypercholesterolemia (FH) is an autosomal dominant disease characterized by hypercholesterolemia, tendon xanthomas, and premature coronary heart disease. FH is caused by mutations of “FH genes,” which include the LDL-receptor (LDLR), apolipoprotein B-100 (APOB) or proprotein convertase subtilisin/kexin type 9 (PCSK9). We evaluated the usefulness of FH gene analysis for diagnosing homozygous FH (homo-FH), particularly in cases caused by gain-of-function (g-o-f) mutations in PCSK9 (PCSK9 E32K). Objectives To evaluate the frequency of homo-FH caused by PCSK9 E32K compared with FH due to other genetic causes and to report the phenotypic features of homo-FH caused by PCSK9 E32K. Methods Genomic DNA was prepared from white blood cells, and LDLR and PCSK9 mutations were identified using the Invader assay method. Results Of the 1055 hetero-FH patients, 62 patients (5.9%) carried the PCSK9 E32K mutation, while in the 82 alleles of 41 homo-FH patients, 13 (15.9%) had double mutations of LDLR allele and PCSK9 E32K mutation. Mean plasma total cholesterol (TC) (9.93 ± 2.95 mmol/L, mean ± SD) in true homo-FH cases with PCSK9 E32K or double hetero-FH cases with PCSK9 E32K and LDLR mutations were significantly lower than those in true homo-FH (18.06 ± 4.96 mmol/L) and compound heterozygous cases with LDLR mutations (14.84 ± 1.62 mmol/L). Mean plasma TC concentrations in the 59 hetero-FH cases with PCSK9 E32K (7.21 ± 1.55 mmol/L) were significantly lower than those (8.94 ± 1.53 mmol/L) in the hetero-FH by LDLR mutations. Conclusions FH caused by PCSK9 g-o-f mutations is relatively common in Japan and causes a mild type of homo- and hetero-FH compared with FH caused by LDLR mutations.
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