Elevated HDL-C is a significant negative risk factor for coronary artery disease.(27
) In the current study, the PLTP gene was chosen as a potential determinant of HDL-C levels. The effect of PLTP on HDL-C levels is attributed to its facilitated transfer of phospholipids from triglyceride-rich lipoproteins (TRL) to HDL, and the generation of pre-β-HDL in reverse cholesterol transport.
It is clear from the current findings that the rs2294213 variant of the PLTP gene is associated with the lipoprotein profile with an increased level of HDL-cholesterol in healthy subjects. Interrogation of the International HapMap database (http://www.hapmap.org
) yields only one SNP (rs2294212) in strong linkage disequilibrium (LD, r2
= 1.0) with rs2294213, located 128bp downstream (c.69+154G>C) in intron 1. Previous examinations of the human PLTP promoter have failed to include these two SNPs.(30
) Analysis of the two alleles represented by rs2294213 using AliBaba2.1 (http://www.gene-regulation.com/pub/programs/alibaba2
) showed the creation of the overlapping putative Simian-virus-40-protein-1 (Sp1) and Yin Yang Protein 1 (YY1) binding sites in minor allele carriers (c.69+26C). Promoter analysis of both the LDL-receptor(35
) and the HDL-receptor(36
) demonstrated that co-stimulation of the Sp1/YY1 sites modulates gene expression. A recent survey of SNPs and haplotypes in the PLTP gene provided evidence of an association between another intron 1 polymorphism (rs553359) and obesity-related traits.(37
) Unfortunately, rs553359 (and rs2294212) was not included in the amplicons designed to analyze exon 1 and exons 2 and 3. Moreover, rs553359 was not in LD (r2
<0.8) with rs2294213. Functional studies are required to formally demonstrate the functional impact of rs2294213.
PLTP’s role in phospholipid transfer from TRL, phospholipid and cholesterol acquisition from peripheral cells, and participation in hepatic synthesis of VLDL, indicates that PLTP is a central effector of HDL metabolism. In the present study, we explored further the impact of rs2294213, mapping to intron 1 of PLTP, that we previously demonstrated as being associated with anti-atherogenic changes in lipoprotein profiles.
Previously, we reported decreased PLTP activity in one (p.R235W) of four (p.E72G, p.S119A, p.S124Y, and p.R235W) missense mutations in the HypoA group using an in vitro
) Whereas the L106F missense mutation was only identified in one HyperA subject, it has the potential to further our understanding of PLTP structure and function. Predictions regarding the potential impact of this missense mutation on PLTP structure can be made within the context of the structural model proposed by Desrumaux and colleagues.(38
) This mutant is predicted to be “benign” when analyzed online by PolyPhen (at http://genetics.bwh.harvard.edu/cgi-bin/pph/polyphen.cgi
). The L106F mutation does not induce any gross structural abnormalities affecting secretion of the mutant protein. Though it appears to be benign, it is unclear if the L106F mutation results in more subtle functional differences that the current in vitro assay is not sufficiently sensitive to discern. Though the PLTP activity assay is used to assay the impact of these missense mutations, additional analysis of these sequence variations with respect to PLTP-mediated remodeling of HDL(7
), ABCA1-mediated efflux of cholesterol from peripheral tissues to HDL(9
), and its participation in hepatic VLDL synthesis(39
) is warranted.
In our previous report, we detailed potential study limitations that are also relevant to the current report.(12
) These include the fact that the participants recruited into the GRA were enrolled primarily from tertiary lipid clinics, which are most commonly referred for elevated LDL-C and could explain their elevated LDL-C levels in this study. Lastly, though measurement of sequence anomalies by DGGE and dHPLC has detection sensitivity approaching 100%(42
), some sequence anomalies may have gone undetected.
This is the first study to screen the entire PLTP gene for sequence anomalies in individuals with HyperA. Our findings show that the rs2294213 minor allele was enriched in individuals with HyperA. The inverse relationship between HDL-C and risk of coronary heart disease is firmly established. This and our previous report(12
) are the first in-depth sequence analyses of the PLTP gene in humans and identify a novel polymorphism associated with altered lipoprotein profiles. The strikingly progressive increase in representation of this minor allele with ascending HDL-C levels presents strong inference that PLTP is an important determinant of HDL metabolism and makes PLTP a potential target for pharmacological intervention.