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. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.
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, PCSK9E32K 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 PCSK9E32K in clinical-FH (6.42%) was significantly higher than that in controls (1.71%). Three cases representing homozygous FH phenotypes were double heterozygous for PCSK9E32K and LDLRC183S, C292X or K790X. Two cases were true homozygous for PCSK9E32K; to our knowledge, these are the first true homozygotes for gain-of-function PCSK9 mutations reported to date. The PCSK9E32K 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 PCSK9E32K 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 PCSK9E32K. CONCLUSIONS: We found 2 true homozygotes for PCSK9E32K and 3 double heterozygotes for PCSK9E32K and LDLR mutations associated with autosomal dominant hypercholesterolaemia. This study provided evidence that PCSK9E32K significantly affects LDL-C levels via increased mass and function of PCSK9, and could exacerbate the clinical phenotypes of patients carrying LDLR mutations. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.
Authors: David N Cooper; Michael Krawczak; Constantin Polychronakos; Chris Tyler-Smith; Hildegard Kehrer-Sawatzki Journal: Hum Genet Date: 2013-07-03 Impact factor: 4.132
Authors: Ariel Brautbar; Emili Leary; Kristen Rasmussen; Don P Wilson; Robert D Steiner; Salim Virani Journal: Curr Atheroscler Rep Date: 2015-04 Impact factor: 5.113
Authors: Paul N Hopkins; Joep Defesche; Sigrid W Fouchier; Eric Bruckert; Gérald Luc; Bertrand Cariou; Barbara Sjouke; Trond P Leren; Mariko Harada-Shiba; Hiroshi Mabuchi; Jean-Pierre Rabès; Alain Carrié; Charles van Heyningen; Valérie Carreau; Michel Farnier; Yee P Teoh; Mafalda Bourbon; Masa-Aki Kawashiri; Atsushi Nohara; Handrean Soran; A David Marais; Hayato Tada; Marianne Abifadel; Catherine Boileau; Bernard Chanu; Shoji Katsuda; Ichiro Kishimoto; Gilles Lambert; Hisashi Makino; Yoshihiro Miyamoto; Matthieu Pichelin; Kunimasa Yagi; Masakazu Yamagishi; Yassine Zair; Scott Mellis; George D Yancopoulos; Neil Stahl; Johanna Mendoza; Yunling Du; Sara Hamon; Michel Krempf; Gary D Swergold Journal: Circ Cardiovasc Genet Date: 2015-09-15