| High-density lipoproteins (HDL) encompass a highly heterogeneous group of particles which differ in size, charge, lipid and protein composition, and physiological function. It is well-established that plasma HDL-cholesterol (HDL-C) levels are inversely correlated with the risk of developing atherosclerotic cardiovascular disease in humans. Obese individuals frequently present with a dyslipidemia characterized not only by decreased HDL-C levels, but also a preponderance of smaller, denser HDL particles. In normolipidemic individuals, these particles are associated with potent anti-oxidative, anti-atherogenic properties. However, under dyslipidemic conditions, such as those observed in obesity and the metabolic syndrome, studies have shown that these particles are dysfunctional and exhibit both qualitative and quantitative alterations in their protein constituents. Evidence strongly suggests that these functionally defective HDL particles may facilitate rather than impede the progression of atherosclerosis, thereby placing obese individuals at an elevated risk for the development of cardiovascular co-morbidities, even with normal HDL cholesterol levels.;In a previous Metabolic Risk Complications of Obesity Genes (MRC-OB) study, the lipoprotein particle profile in 532 individuals from an obese, family-based cohort was analyzed, and a quantitative trait locus affecting HDL median particle diameter (HDLm) on human chromosome 12p13 (LOD=3.15) was identified. The interval contains 144 genes, none of which have a known role in lipid or lipoprotein metabolism.;We pursued a proteomic approach to prioritize potential candidate genes in this genomic region that lead to an altered composition of HDL particles in obese individuals, and may mediate the observed shift in median particle diameter. We isolated HDL fractions from human serum samples of thirteen obese and non-obese sibling pairs from our cohort using non-denaturing fast protein liquid chromatography to identify quantitative differences in the HDL proteome that could potentially mediate this effect. Proteins were isolated from HDL particles by chloroform extraction and quantified using isotopic labeling and tandem mass spectrometry.;From our proteomic analyses of obese and non-obese sibling pairs, we quantified an average of 58% of the previously reported HDL proteome. From these, we identified proteins that were significantly altered in obese individuals. However, none of the altered proteins are encoded by genes located on human chromosome 12p13. Therefore, we employed multiple bioinformatics approaches to identify interactions between the altered proteins in HDL and the genes in the QTL interval. We identified three genes (C1RL, CD163 and VWF) that could be linked to the altered proteins. Two of these genes, CD163 and VWF, were highlighted by all three bioinformatics approaches.;Based on the predominance of associations identified between altered HDL proteins and the VWF gene, coupled with the fact that this gene is relatively large, we selected VWF for detailed genotyping. In a candidate gene association study, we examined 85 single nucleotide polymorphisms (SNPs) across the VWF gene in 625 individuals from our obese cohort. These SNPs were analyzed for association with HDLm. In our cohort, six SNPs were marginally associated with HDLm (p<0.05). Four of these SNPs (rs216867, rs216873, rs216902, rs216800) are located within the same linkage disequilibrium block. On average, these SNPs account for 6.2% of the observed linkage in our cohort, suggesting that, in combination, these SNPs may potentially explain a greater percentage of the HDLm variance in our cohort. Overall, these results suggest that variation in the VWF gene contributes, at least in part, to the altered HDL particle size distribution characteristic of atherogenic dyslipidemias. Furthermore, this study illustrates the utility of proteomics analyses to identify genetic predispositions to quantitative traits such as HDL particle diameter. |
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