We genotyped six PCSK9
polymorphisms, reconstructed haplotypes, and determined association of the biochemical, angiographic, and clinical phenotypes of coronary atherosclerosis with the haplotypes and genotypes in the LCAS population. The results are remarkable for the presence of significant copy-number-dependent association between haplotype 3 and plasma levels of LDL-C and TC and to a lesser extent MLD. Haplotype 3, which comprises the information content of Ln
STR polymorphism and five SNPs, is the only haplotype with amino acid glycine at position 670 in the protein. Consequently, the E670G cSNP was identified as the risk variant and others had no discernible effect. The results in a second independent population (TexGen), showing lower frequency of the risk allele in those with normal LDL-C levels, provided indirect evidence of support for the findings in the LCAS population. The findings are also in accord with the results of recent linkage mapping and identification of two mutations in PCSK9
in patients with ADH (2
). Nonetheless, because of the differences in the characteristics of the LCAS and TexGen populations, the results require confirmation in additional replicates and through experimentation. Studies in a larger sample size could establish the clinical significance of the observed associations of plasma levels of lipids and the haplotypes.
The strengths of the study are the prospective and placebo-controlled randomized design of LCAS and comprehensive phenotypic characterization, an essential component of LD studies. The study includes detection and analysis of novel polymorphisms including the Ln
polymorphism, and reconstruction of haplotypes comprising information on the content of six polymorphisms that collectively span the PCSK9
locus. Statistical analyses used a permutation test, which is considered robust and less prone to spurious association. Cognizant of the relatively high rate of spurious results in genetic association studies, we also calculated the FPRP, which was less than 8%, even under the most relaxed conditions. Finally, the findings are in accord with recent genetic linkage mapping and detection of mutations in PCSK9
in families with ADH (2
). Collectively, these findings suggest the presence of a significant association between PCSK9
E670G cSNP (haplotype 3) and plasma LDL-C and TC levels in non-Mendelian dyslipidemia.
Genetic linkage studies have established PCSK9
as a causal gene for familial ADH (2
), however, its function and the mechanisms by which PCSK9
mutations affect plasma LDL-C levels are largely unknown (18
encodes neural apoptosis-regulated convertase (NARC-1 aka PCSK9), a novel 691-amino-acid proprotein convertase expressed predominantly in the liver and intestine (19
). The protein is a member of subtilase subfamily with multiple domains () (20
) including signaling peptide (aa 1–30), pro-segment (aa 31–147), catalytic (aa 148–425), and cysteine-rich C-terminal (aa 526 to 691) domains. Human PCSK9 is synthesized as a zymogen and undergoes autocatalytic intramolecular processing in the endoplasmic reticulum, a step necessary for exiting the endoplasmic reticulum. The E670G cSNP is located in the cysteine-rich C-terminal domain, which is involved in regulation of autoprocessing, because deletion of this domain leads to accumulation of the processed PCSK9 (19
). The biological role of PCSK9 in regulating plasma LDL-C levels also remains elusive. Recent studies suggest that PCSK9 negatively regulates expression of LDL-C receptors in the liver through a post-translational mechanism before the internalization and recycling of the receptor (21
). Adenoviral-mediated overexpression of murine Pcsk9 results in near-complete depletion of the LDL-C receptor, whereas inactivation of the catalytic activity of Pcsk9 has no effect (22
). Accordingly, PCSK9 mutations are expected to increase the activity of the enzyme (gain of function). Other effects of PCSK9 mutations comprise decreased zymogen processing of PCSK9, reduced LDL-C receptor density (23
), and an increased production rate of apoB100 (24
). Finally, PCSK9 could interfere with the ability of the LDL-C receptor to bind to LDL-C (25
). Whether E670G cSNP impairs the effects of PCSK9 on LDL-C receptor abundance and/or activity or the production rate of apoB100 remains unknown.
Despite a strong association of the plasma LDL-C levels with haplotype 3 (E670G cSNP), MLD showed only a modest association. This is not surprising because the effect of gene variants is expected to be stronger on the immediate (gene products) and weaker on the remote phenotypes (such as atherosclerosis or death) because of the contribution of the competing factors to the distant phenotypes. A recent study in a Japanese population implicated an intronic SNP (C-161T) and a cSNP (I474V) in influencing plasma LDL-C levels but reported no association with MI (26
). We genotyped the I474V SNP and detected an MAF of 0.15, which is considerably higher than that in the Japanese population (MAF = 0.03). The I474V cSNP was in partial LD with the E670G cSNP and was not an independent predictor of the phenotypes in the LCAS population. The MAF of the C(−161)T SNP, determined in 50 subjects, was 0.059. Because of the relatively low frequency and its location in a noncoding region, we did not genotype the C(−161)T SNP in the LCAS population.
Our approach for genetic studies of complex trait, pending the completion of the HapMap project and resolution of the superiority of the SNP-centric or haplotype-centric approach, is based on analysis of common (common disease-common variant hypothesis) putatively functional SNPs (pfSNPs) in the gene/locus of interest, complemented by haplotype reconstruction. Accordingly, we analyzed six common SNPs, including two cSNPs, one amino acid repeat polymorphism, and one regulatory (3′ untranslated region) SNP. There are at least six additional SNPs in PCSK9, as shown in , that were genotyped only in 50 subjects. The MAFs of these SNPs are shown in . We did not genotype the entire LCAS population for these SNPs because they were in near-complete LD with those analyzed and/or because they were located in introns. Thus, we cannot exclude the possible presence of additional common haplotypes in the LCAS population. Finally, because we analyzed only the common SNPs, based on common disease-common variant hypothesis, we cannot exclude the possibility of uncommon alleles contributing to plasma LDL-C levels in the LCAS population.
The sample size of LCAS provided 80% power, at an α
value of 0.05, to detect a 20% difference in the mean baseline plasma LDL-C levels for haplotypes that were present in at least 25 heterozygous subjects. Considering the relatively low frequency of the approximately half of the haplotypes, a larger sample size would be necessary to detect effects of the rare haplotypes or smaller effects of the common haplotypes. In addition, mean MLD values in the placebo and fluvastatin groups changed only by −0.11 and −0.04 mm, respectively, which are consistent with the results of other angiographic regression/progression studies (27
), but less than 10% of the baseline MLD. Thus, a much larger sample size and/or a longer duration of follow up may be necessary to detect the potential effects of genetic variants on MLD. Similarly, the number of clinical events could be too small to detect the potential impact of genotypes or haplotypes on the clinical events.
In conclusion, we have identified and analyzed novel and known polymorphisms in PCSK9 locus, reconstructed haplotypes encompassing the information content of six polymorphisms, and shown that haplotype 3, representative of the E670G cSNP, is an important determinant of plasma LDL-C and TC levels and is associated with the severity of coronary atherosclerosis in the LCAS population. Identification of molecular mechanism(s) by which PCSK9 variants affect plasma LDL-C levels could provide new insight into the pathogenesis of atherosclerosis and development of new drug targets.