Genetic variants in PCSK9 in the Japanese population: Rare genetic variants in PCSK9 might collectively contribute to plasma LDL cholesterol levels in the general population
Introduction
Elevated plasma concentration of low-density lipoprotein cholesterol (LDL-C) is a major risk factor for the development and progression of atherosclerosis. Plasma concentrations of LDL-C are determined primarily by the activity of the LDL receptor (LDLR) in the liver. Recently, the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene has been found to be involved in the post-transcriptional regulation of the LDLR. PCSK9 encodes a protein of 692 amino acids which is a member of the subtilisin-like protein convertase family [1], [2] and is expressed most abundantly in the liver, kidneys and small intestine [2]. PCSK9 consists of several domains: a signal peptide, a prosegment, a subtilase-like catalytic domain and a C-terminal domain [3]. It is synthesized as a soluble zymogen which undergoes autocatalytic intramolecular cleavage in the endoplasmic reticulum (ER) between the prosegment and the catalytic domain [1], [2]. After cleavage, the mature PCSK9 exits the ER and is efficiently secreted [2]. The only known substrate of PCSK9 is itself; no other substrate(s) for PCSK9 have yet been identified. Although even the physiological substrate remains unknown, PCSK9 has been shown to play a role in cholesterol metabolism by regulating the number of cell-surface LDLRs [3], [4], [5].
Overexpression of the wild-type Pcsk9 gene in mice results in hypercholesterolemia because of a reduced number of LDLRs [3], [4], [5]. The reduced number of LDLRs due to PCSK9 is not accompanied by changes in LDLR mRNA levels; therefore, it is likely that PCSK9 is involved in the post-transcriptional regulation of the LDLR [4], [5]. Degradation of the LDLR by PCSK9 is dependent on the catalytic activity of PCSK9 [5], [6]. In contrast, mice expressing no PCSK9 have markedly increased hepatic LDLR levels, resulting in accelerated LDL clearance [7]. These findings indicate that PCSK9 normally acts to limit the number of LDLRs at the cell surface. Thus, PCSK9 mutations which disrupt normal function, i.e., loss-of-function mutations, are presumed to increase the number of LDLRs, resulting in hypocholesterolemia. In fact, the nonsense mutations identified by Cohen et al. are associated with a 40% reduction in mean plasma levels of LDL-C [8]. On the other hand, some mutations in the PCSK9 gene cause hypercholesterolemia [9], [10], [11], which are probably due to gain-of-function mechanisms. These mutations in PCSK9 might promote the degradation of LDLRs in hepatocytes [3], [4], [5]. Recently, Cameron et al. demonstrated that loss-of-function mutations in PCSK9 increase the number of cell-surface LDLRs, while gain-of-function mutations decrease the number of LDLRs, based on studies on HepG2 cells transfected with mutant PCSK9 constructs [12].
Since mutations in PCSK9 can cause severe hypercholesterolemia [9], [10], [11] as well as hypocholesterolemia [8], [13], sequence variants of PCSK9 might contribute to variations in the plasma levels of LDL-C. Shioji et al. [14] have identified the two common single nucleotide polymorphisms (SNPs), and Chen et al. [15] identified a haplotype associated with differences in plasma LDL-C levels. Kotowski et al. performed a systematic examination of the relationship between sequence variations in PCSK9 and plasma levels of LDL-C in the general population [16]. They analyzed sequence variations in PCSK9 in individuals of the examined population who had lower and higher LDL-C levels and found that three missense mutations and two noncoding sequence variants were significantly associated with lower levels of LDL-C, while a single noncoding variant was associated with a modest increase in LDL-C levels. They concluded that sequence variants in PCSK9 contribute significantly to interindividual variations in plasma LDL-C levels, and report that the spectrum of PCSK9 alleles associated with LDL-C levels spanned a wide range of allele frequencies and magnitude of phenotypic effects.
In order to verify whether sequence variants in PCSK9 could be a determinant of LDL-C plasma levels in the Japanese general population, we performed sequence analyses in the proximal promoter and all exons of PCSK9 in individuals from the general population with the lowest and highest LDL-C levels and also in individuals taking antihypercholesterolemia medication since these individuals are presumed to have originally high levels of plasma LDL-C. Finally, we performed statistical analyses and compared the numbers of individuals with certain genetic variants between groups.
Section snippets
General population and the three investigated groups of individuals
DNA analysis was performed in individuals selected from the participants of the Suita cohort study, whose total sample included 3655 subjects. The study design of the Suita study has been described previously [17], [18], [19]. Briefly, the individuals were randomly selected from the municipal population registry, taking into consideration group stratification by gender and 10-year age divisions. The subjects visited the National Cardiovascular Center every 2 years for general health checkups.
Participant characteristics
The characteristics of the individuals in the low LDL-C, high LDL-C and treatment groups, and those of the treated and untreated individuals in the total population, are shown in Table 1. The LDL-C levels of the individuals in the low LDL-C group ranged from 29.2 to 88.0 mg/dl (mean ± S.D., 70.3 ± 13.2 mg/dl), and those in the high LDL-C group ranged from 169.8 to 300.8 mg/dl (mean ± S.D., 196.7 ± 19.2 mg/dl). The LDL-C levels of the subjects in the treatment group ranged from 148.0 to 204.4 mg/dl (mean ±
Discussion
In order to identify the genetic variants in PCSK9 affecting plasma LDL-C levels in the Japanese population, we screened the proximal promoter and the entire coding region sequences in 78 individuals with low LDL-C levels, 96 individuals with high LDL-C levels, and 96 individuals currently taking antihypercholesterolemia medication. All subjects were selected from a large sample of the general population (n = 3655).
Among the 33 detected sequence variants, only one missense mutation, R93C, was
Acknowledgments
The authors would like to thank Drs. Otosaburo Hishikawa, Yasushi Kotani, Katsuyuki Kawanishi and Toshifumi Mannami, and Mr. Tadashi Fujikawa for their support of our population survey in Suita City, Osaka, Japan. We are also grateful to the members of the Satsuki-Junyukai. We also thank Ms. Junko Ishikawa for assistance with the genetic variant analyses. We are grateful to Ms. Mikiko Kasahara, Mikiko Kojima, Hiroko Ueda, Keiko Yamaguchi and Mayumi Yoshimura for their support in medical
References (28)
- et al.
Functional characterization of Narc 1, a novel proteinase related to proteinase K
Arch Biochem Biophys
(2003) - et al.
NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol
J Biol Chem
(2004) - et al.
Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver
J Biol Chem
(2004) - et al.
A common PCSK9 haplotype, encompassing the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis
J Am Coll Cardiol
(2005) - et al.
A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol
Am J Hum Genet
(2006) - et al.
Absence of familial defective apolipoprotein B-100 in Japanese patients with familial hypercholesterolaemia
Lancet
(1995) - et al.
Three novel missense mutations of WNK4, a kinase mutated in inherited hypertension, in Japanese hypertensives: implication of clinical phenotypes
Am J Hypertens
(2004) - et al.
The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation
Proc Natl Acad Sci USA
(2003) - et al.
Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype
Proc Natl Acad Sci USA
(2004) - et al.
Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment
Proc Natl Acad Sci USA
(2005)
Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9
Proc Natl Acad Sci USA
Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9
Nat Genet
Mutations in PCSK9 cause autosomal dominant hypercholesterolemia
Nat Genet
A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree
Hum Genet
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