title: The E32K variant of PCSK9 exacerbates the phenotype of familial hypercholesterolaemia by increasing PCSK9 function and concentration in the circulation.
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.
authors.affiliation: Department of Lipidology, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8640, Japan. nogutito@med.kanazawa-u.ac.jp
authors.forename: Tohru
authors.initials: T
authors.lastname: Noguchi
authors.forename: Shoji
authors.initials: S
authors.lastname: Katsuda
authors.forename: Masa-Aki
authors.initials: MA
authors.lastname: Kawashiri
authors.forename: Hayato
authors.initials: H
authors.lastname: Tada
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nohara
authors.forename: Akihiro
authors.initials: A
authors.lastname: Inazu
authors.forename: Masakazu
authors.initials: M
authors.lastname: Yamagishi
authors.forename: Junji
authors.initials: J
authors.lastname: Kobayashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mabuchi
mesh_terms: D000328:Adult; D044466:Asian People / Q000235:genetics; D008078:Cholesterol, LDL / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D005091:Exons; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics*; D007438:Introns; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases / Q000097:blood / Q000235:genetics* / Q000502:physiology
chemical_list: D008078:Cholesterol, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases