Introduction

Proprotein convertase subtilisin/kexin 9 (PCSK9) in chromosome 1p34.1-p32 is a proprotein convertase that belongs to the subtilase subfamily (Seidah et al. 2003). A related protein is the subtilisin/kexin isoenzyme-1/site-1 protease, which plays a key role in cholesterol homeostasis by processing sterol regulatory element-binding protein (SREBP) (Brown and Goldstein 1999). The expression of PCSK9 mRNA has been reported to be down regulated by dietary cholesterol in C57BL/6 mice and to be up regulated in SREBP transgenic mice (Maxwell et al. 2003). Mutations in PCSK9 have been reported in affected members of two families with autosomal dominant hypercholesterolemia (OMIM 603776) (Abifadel et al. 2003). These observations indicate that PCSK9 plays an important role in cholesterol metabolism. Thus, it is possible that common genetic variations in PCSK9 might affect the cholesterol level in the general population.

To investigate the effects of common variants in PCSK9 on cholesterol level, we detected common variants in PCSK9 by sequencing and conducted an association study using a large cohort representing the general population in Japan. We found that two polymorphisms, intron 1/C(-161)T and exon 9/I474V, were associated with levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). We next investigated the association between these polymorphisms and the incidence of myocardial infarction (MI).

Subjects and Methods

Subjects

  1. 1.

    The Suita population: Selection criteria and design of the Suita Study have been described previously (Shioji et al. 2004, in press; Mannami et al. 1997). The sample consisted of 14,200 men and women aged 30–79 years, stratified by gender and 10-year age groups, who were selected randomly from the municipal population registry. They were all invited by letter to attend regular cycles of follow-up examinations (every 2 years). The basic population sampling started in 1989 with a cohort study base, and 51.7% (n=7,347) of the subjects responded to the invitation letter and had paid their initial visit to the National Cardiovascular Center by February 1997. The participants visited the center every 2 years for regular health checkups. DNA from leukocytes was initially collected from participants who visited the center between May 1996 and February 1998. In the present study, the genotypes were determined in 1,880 consecutive subjects who visited the center between April 2002 and February 2003 (n=1,880, Table 1). Subjects with ischemic heart disease were excluded.

    Table 1 Suita population characteristics. BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, PR pulse rate, % CVA percentage of subjects with cerebrovascular accident, % OMI percentage of subjects with old myocardial infarction, % HT percentage of subjects with hypertension, % DM percentage of subjects with diabetes mellitus, % HLP percentage of subjects with hyperlipidemia, % drinking percentage of subjects with a drinking habit, % smoking percentage of subjects with a smoking habit
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  2. 2.

    The MI group: Selection criteria and design of the MI group have been described previously (Takagi et al. 2002). This group consisted of 649 patients with MI (553 men and 96 women) who were enrolled in the Division of Cardiology at the National Cardiovascular Center between May 2001 and April 2003 (Table 2).

    Table 2 Miocardial infarction (MI) group characteristics. BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, PR pulse rate, % CVA percentage of subjects with cerebrovascular accident, % OMI percentage of subjects with old myocardial infarction, % HT percentage of subjects with hypertension, % DM percentage of subjects with diabetes mellitus, % HLP percentage of subjects with hyperlipidemia
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Written informed consent was obtained from each subject after a full explanation of the study, which was approved by the Ethics Committee and the Committee on Genetic Analysis and Genetic Therapy of the National Cardiovascular Center.

DNA studies

All 12 exonic regions were sequenced for polymorphisms in 48 healthy subjects. Selected polymorphisms were determined by the TaqMan method. Detailed information will be provided upon request.

Statistical analysis

Values are expressed as mean±standard error of the mean (SEM). Since the distribution of triglyceride (TG) values was skewed, a logarithmic transformation was used for the statistical test; however, untransformed means are shown in Tables 1, 2, 5, 6. LDL-C was calculated by Friedewald’s formula [(LDL-C)=(TC) - (HDL-cholesterol) - (TG/5). We excluded those whose HDL-cholesterol (HDL-C) or TG levels were ≥2.6 mM or 4.53 mM respectively]. All statistical analyses were performed with the JMP statistical package (SAS Institute Inc.). Values of P<0.05 were considered to indicate statistical significance. The residuals of lipid levels were calculated by adjusting for gender, age, body mass index (BMI), smoking (cigarettes/day), and consumption of alcohol (ethanol g/week). Data were analyzed using a contingency table analysis and Student’s t-test. Hardy-Weinberg equilibrium was calculated by a chi-square test. R-square values between polymorphisms were analyzed using the SNPAlyze statistical package (Dynacom Inc.).

Results

Direct sequencing identified 21 polymorphisms (Table 3). We regarded r-20.5 as tight linkage (Table 4). Polymorphisms with frequencies of ≤0.03 in the intronic region and 3’-untranslated region were neglected in further analyses. Polymorphisms that were not accompanied by an amino acid change in the exonic regions were also neglected. Accordingly, we selected and genotyped nine polymorphisms for the following association study.

Table 3 Polymorphisms and nucleotide sequence in PCSK9
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Table 4 Linkage disequilibrium among polymorphisms in PCSK9
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As shown in Table 5, intron 1/C(-161)T and exon 9/I474 V polymorphisms were associated with levels of TC and LDL-C in the Suita population. Since we only found one subject each who was homozygous for minor alleles, these subjects were categorized as heterozygotes. A gender-based subanalysis indicated that the exon 9/I474 V polymorphism significantly influenced the LDL-C level in both male and female subjects (Table 6). TC level in the IV(+VV) genotype of exon 9/I474 V was also lower than that in the II genotype in both male (P=0.1656) and female subjects (P=0.0133). Although P-values were not statistically significant, partially due to low statistical power, TC and LDL-C levels in the CT(+TT) genotype of intron 1/C(-161)T were lower than those in the CC genotype in both male and female subjects. No significant deviation from Hardy-Weinberg equilibrium was observed in these polymorphisms [C(-161)T: P=0.8290, I474 V: P=0.9971].

Table 5 Lipid levels among the PCSK9 polymorphisms (Suita population). BMI body mass index, TC total cholesterol, HDL-C high-density lipoprotein cholesterol. TG triglycerides, LDL-C low-density lipoprotein cholesterol, % drinking percentage of subjects with a drinking habit, % smoking percentage of subjects with a smoking habit
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Table 6 Lipid levels among the PCSK9 polymorphisms (gender-based subanalysis). TC total cholesterol, HDL-C high-density lipoprotein cholesterol, TG triglycerides, LDL-C low-density lipoprotein cholesterol
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We next evaluated whether intron 1/C(-161)T and exon 9/I474 V polymorphisms were associated with the incidence of MI. Distribution of these polymorphisms in subjects with MI were no different from those in the Suita population (Table 7). A gender-based subanalysis indicated that these polymorphisms did not influence the incidence of MI in either male or female subjects (data not shown), nor were they associated with lipid levels in patients with MI. One possible reason for this lack of association may be that a substantial proportion of the MI group had dyslipidemia and had been treated with hypolipidemic drugs.

Table 7 Association between PCSK9 polymorphisms and the incidence of myocardial infarction (MI)
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Discussion

While C(-161)T and I474 V polymorphisms have been reported previously (Abifadel et al. 2003), association studies have not been reported. The present study clarified that the C(-161)T and I474V polymorphisms were significantly associated with TC and LDL-C levels in the total population. Even in a gender-based subanalysis, the I474V polymorphism significantly influenced the LDL-C level in both male and female subjects. It is unclear whether these polymorphisms are functional variations or just in linkage disequilibrium with other important variants, and this question requires further investigation. Since Ile at amino acid number 474 was not conserved in either rats or mice, another polymorphism in tight linkage with I474 V may be influential. In fact, a polymorphism in the polypyrimidine-rich tract in intron 8/T(-57)C was almost completely concordant with I474V (r 2=1.00, Tables 3 and 4).

The minor allele frequencies of intron 1/C(-161)T and exon 9/I474 V polymorphisms were low. However, variances between residuals of TC in genotypes [C(-161)T: CC versus CT+TT, I474 V: II versus IV+VV] were similar [C(-161)T: F-ratio=0.2368, P=0.6266; I474 V: F-ratio=2.418, P=0.1201 (Levene’s test)]. Variances between residuals of LDL-C in the genotypes were also similar [C(-161)T: F ratio=0.1060, P=0.7448; I474 V: F ratio=0.4436, P=0.5055]. The sample power was 0.9234 (α-value: 0.05, sigma: 27.70, delta: 2.35, adjusted power: 0.8990, confidence limit: 0.2978–0.9996). Thus, these associations were thought to have adequate statistical power. It has been recommended that a single, nominally significant association should be viewed as tentative until it has been independently replicated at least once and preferably twice (Ioannidis et al. 2001). Accordingly, it will be necessary to verify the association between these PCSK9 polymorphisms and the levels of TC and LDL-C using a larger number of subjects from the Suita cohort or another population.

We found two polymorphisms that were associated with TC and LDL-C levels among nine polymorphisms of PCSK9 in the Suita population. However, if we apply Bonferroni’s correction for multiple tests, only exon 9/I474 V polymorphism can be considered significantly associated with the HDL level [intron 1/C(-161)T, TC: P=0.2565, LDL-C: P=0.2313; exon 9/I474 V, TC: P=0.0621, LDL-C: P=0.0063, P-values are corrected by multiplying by 9 (nine polymorphisms)]. Again, it will be necessary to verify the association between these PCSK9 polymorphisms and the levels of TC and LDL-C using a larger number of subjects from the Suita cohort or another population.

A high LDL-C level is a well-known coronary risk factor (Kannel et al. 1979). Although PCSK9 polymorphisms affected the LDL cholesterol level, they did not affect the incidence of MI. The intron 1/C(-161)T polymorphism was inversely associated with LDL-C level and incidence of MI, although these associations were not significant. This was thought to be due, at least in part, to the low statistical power. A much larger group of MI subjects might be necessary to detect the influence of these variants on the incidence of MI.

In conclusion, the present study provides the first evidence that common variants intron 1/C(-161)T and exon 9/I474 V in PCSK9 significantly affect TC and LDL-C levels in the general Japanese population.