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

• Published in: Atherosclerosis 2010-05, Vol.210 (1), p.166-172
• Main Authors: Noguchi, Tohru, Katsuda, Shoji, Kawashiri, Masa-aki, Tada, Hayato, Nohara, Atsushi, Inazu, Akihiro, Yamagishi, Masakazu, Kobayashi, Junji, Mabuchi, Hiroshi
• Format: Article
• Language: English
• Subjects: Adult; Asian Continental Ancestry Group - genetics; Atherosclerosis (general aspects, experimental research); Biological and medical sciences; Blood and lymphatic vessels; Cardiology. Vascular system; Cardiovascular; Cardiovascular system; Cholesterol, LDL - blood; Enzyme-Linked Immunosorbent Assay; Exons; Familial hypercholesterolaemia; Heterozygote; Homozygote; Humans; Hyperlipoproteinemia Type II - genetics; Introns; Investigative techniques of hemodynamics; Investigative techniques, diagnostic techniques (general aspects); LDLR; Medical sciences; Middle Aged; Mutation; PCSK9; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Proprotein Convertase 9; Proprotein Convertases; Serine Endopeptidases - blood; Serine Endopeptidases - genetics; Serine Endopeptidases - physiology
• ISSN: 0021-9150; 1879-1484
• DOI: 10.1016/j.atherosclerosis.2009.11.018

Abstract Objective 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. Methods 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. Results 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. Conclusions 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.