Characterization of novel mutations in the catalytic domain of the PCSK9 gene.

OBJECTIVES: To expand our understanding of the structure and function of proprotein convertase subtilisin/kexin type 9 (PCSK9) by studying how naturally occurring mutations in PCSK9 disrupt the function of PCSK9. DESIGN: Mutations in PCSK9 were identified by sequencing of DNA from subjects with hypo- or hypercholesterolemia. The effect of the identified mutations on the autocatalytic cleavage and secretion of PCSK9, as well as the effect on PCSK9-mediated degradation of the low density lipoprotein receptors, were determined in HepG2 or HEK293 cells transiently transfected with mutant PCSK9-containing plasmids. The findings were collated to the clinical characteristics of the subjects possessing these mutations, and the phenotypic effects were analysed in terms of available structural data for PCSK9. RESULTS: Five novel mutations in PCSK9 were identified. Mutation R215H was a gain-of-function mutation which causes hypercholesterolemia. Mutation G236S and N354I were loss-of-function mutations due to failure to exit the endoplasmic reticulum or failure to undergo autocatalytic cleavage, respectively. Mutations A245T and R272Q were most likely normal genetic variants. By comparing the number of patients with gain-of-function mutations in PCSK9 with the number of familial hypercholesterolemia heterozygotes among subjects with hypercholesterolemia, the prevalence of subjects with gain-of-function mutations in PCSK9 in Norway can be estimated to one in 15,000. CONCLUSION: This study has provided novel information about the structural requirements for the normal function of PCSK9. However, more studies are needed to determine the mechanisms by which gain-of-function mutations in PCSK9 cause hypercholesterolemia.

Low-density lipoprotein receptor activity in Epstein-Barr virus-transformed lymphocytes from heterozygotes for the D374Y mutation in the PCSK9 gene.

OBJECTIVE: Missense mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have been found to cause autosomal dominant hypercholesterolemia. The objective of this study was to investigate possible mechanisms by which mutation D374Y in the PCSK9 gene causes hypercholesterolemia. MATERIAL AND METHODS: Binding and internalization of low-density lipoprotein LDL in Epstein-Barr virus (EBV)-transformed lymphocytes from D374Y heterozygotes were examined. The autocatalytic activity of the D374Y mutant was studied in transiently transfected HEK293 cells. RESULTS: As determined by Western blot analysis of transiently transfected HEK293 cells, the autocatalytic activity of the D374Y mutant was approximately 95% of the wild-type. Levels of PCSK9 mRNA in EBV-transformed lymphocytes from D374Y heterozygotes and normal controls were similar and less than 1/1000 of the level in HepG2 cells. The amount of cell surface LDL receptors (LDLRs) in EBV-transformed lymphocytes from five D374Y heterozygotes was non-significantly increased by 17% compared with the amount in normal controls. LDLR-dependent binding and internalization of LDL in EBV-transformed lymphocytes from D374Y heterozygotes were non-significantly reduced by 11% and 12%, respectively, compared to the corresponding values in normal controls. CONCLUSIONS: LDLR-mediated endocytosis of LDL is not reduced in EBV-transformed lymphocytes from D374Y heterozygotes. Because of the extremely low levels of PCSK9 mRNA in EBV-transformed lymphocytes, it is possible that the LDLR-dependent endocytosis of LDL could be more severely affected in hepatocytes from D374Y heterozygotes than in EBV-transformed lymphocytes.