Background

Plasma levels of high density lipoprotein cholesterol (HDL-C) are known to be heritable, but only a fraction of the heritability is explained. We used a high density genotyping array containing SNPs from HDL-C candidate genes selected on known biology of HDL-C metabolism, mouse genetic studies, and human genetic association studies. SNP selection was based on tagging-SNPs but also included low-frequency nonsynonymous SNPs.

Methods and Results

Association analysis in a cohort containing extremes of HDL-C (case-control, n=1733) provided a discovery phase, with replication in three additional populations for a total meta-analysis in 7,857 individuals. We replicated the majority of loci identified through genome wide association studies and present on the array (including ABCA1, APOA1/C3/A4/A5, APOB, APOE/C1/C2, CETP, CTCF-PRMT8, FADS1/2/3, GALNT2, LCAT, LILRA3, LIPC, LIPG, LPL, LRP4, SCARB1, TRIB1, ZNF664), and provide evidence suggestive of association in several previously unreported candidate gene loci (including ABCG1, GPR109A/B/81, NFKB1, PON1/2/3/4). There was evidence for multiple, independent association signals in five loci, including association with low frequency nonsynonymous variants.

Conclusions

Genetic loci associated with HDL-C are likely to harbor multiple, independent causative variants, frequently with opposite effects on the HDL-C phenotype. Cohorts composed of extreme individuals may be efficiently used in a case-control discovery of quantitative traits.

Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5′ UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5′ UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci.

Author Summary

Genetic association studies have identified genomic regions that affect quantifiable traits such as lipid levels. When a gene and a trait are found to be associated with one another, the gene is often further studied to determine its role in affecting the trait. One approach is to sequence the gene in individuals at the extremes of the trait's distribution with the hope of finding rare mutations that directly contribute to the trait. Until now studies using this approach have focused on genetic variation in the protein coding sequence of these genes and have been largely successful in identifying functionally important mutations. However, other studies have found an abundance of noncoding variation in the genome that may also contribute to the heritability of these traits. Here we seek to determine the contribution of such noncoding mutations to high density lipoprotein cholesterol (HDL-C) levels in humans using the HDL-C candidate gene LIPG as an example. Through a sequencing study in individuals with high and low HDL-C levels, we demonstrate that both rare and common noncoding mutations are influential contributors to the allelic spectrum of such traits and should be further characterized after initial association with the trait.

Objective

Pharmacological inhibition of the cholesteryl ester transfer protein (CETP) in humans increases high-density lipoprotein (HDL) cholesterol (HDL-C) levels; however, its effects on apolipoprotein A-I (apoA-I) containing HDL subspecies, apoA-I turnover, and markers of reverse cholesterol transport are unknown. The present study was designed to address these issues.

Methods and Results

Nineteen subjects, 9 of whom were taking 20 mg of atorvastatin for hypercholesterolemia, received placebo for 4 weeks, followed by the CETP inhibitor torcetrapib (120 mg QD) for 4 weeks. In 6 subjects from the nonatorvastatin cohort, the everyday regimen was followed by a 4-week period of torcetrapib (120 mg BID). At the end of each phase, subjects underwent a primed-constant infusion of (5,5,5-2H3)-L-leucine to determine the kinetics of HDL apoA-I. The lipid data in this study have been reported previously. Relative to placebo, 120 mg daily torcetrapib increased the amount of apoA-I in α1-migrating HDL in the atorvastatin (136%; P<0.001) and nonatorvastatin (153%; P<0.01) cohorts, whereas an increase of 382% (P<0.01) was observed in the 120 mg twice daily group. HDL apoA-I pool size increased by 8±15% in the atorvastatin cohort (P=0.16) and by 16±7% (P<0.0001) and 34±8% (P<0.0001) in the nonatorvastatin 120 mg QD and BID cohorts, respectively. These changes were attributable to reductions in HDL apoA-I fractional catabolic rate (FCR), with torcetrapib reducing HDL apoA-I FCR by 7% (P<0.10) in the atorvastatin cohort, by 8% (P<0.001) in the nonatorvastatin 120 mg QD cohort, and by 21% (P=0.01) in the nonatorvastatin 120 mg BID cohort. Torcetrapib did not affect HDL apoA-I production rate. In addition, torcetrapib did not significantly change serum markers of cholesterol or bile acid synthesis or fecal sterol excretion.

Conclusions

These data indicate that partial inhibition of CETP via torcetrapib in patients with low HDL-C: (1) normalizes apoA-I levels within α1-migrating HDL, (2) increases plasma concentrations of HDL apoA-I by delaying apoA-I catabolism, and (3) does not significantly influence fecal sterol excretion.

Background

Lp(a), implicated in both atherogenesis and thrombosis pathways, varies significantly by demographic and metabolic factors, providing challenges for its use in Coronary Heart Disease (CHD) risk. The purpose of this study was to investigate whether type-2 diabetic subjects, relative to non-diabetics, might benefit more from Lp(a) measurement in the prediction of CHD risk, as measured by coronary artery calcium (CAC).

Methods

We performed cross sectional analyses in two community-based studies: the Penn Diabetes Heart Study [N=1299 with type-2 diabetes] and the Study of Inherited Risk of Coronary Atherosclerosis [N=860 without diabetes].

Results

Blacks had 2–3 fold higher Lp(a) levels than whites in diabetic and non-diabetic samples. There was significant difference by gender (interaction p<0.001), but not race, in the association of Lp(a) with CAC in type-2 diabetic subjects. In age and race adjusted analysis of diabetic women, Lp(a) was associated with CAC [Tobit regression ratio 2.76 (95% CI 1.73–4.40), p<0.001]. Adjustment for exercise, medications, Framingham risk score, metabolic syndrome, BMI, CRP and hemoglobin A1c attenuated this effect, but the association of Lp(a) with CAC remained significant [2.25, (1.34–3.79), p=0.002]. This relationship was further maintained in women stratified by race, or by the use of HRT or lipid lowering drugs. In contrast, Lp(a) was not associated with CAC in diabetic men, nor in non-diabetic men and women.

Conclusions

Lp(a) is a strong independent predictor of CAC in type-2 diabetic women, regardless of race, but not in men. Lp(a) does not relate to CAC in men or women without type-2 diabetes.

The benefit in reducing cardiovascular risk with statins has been attributed both to cholesterol lowering and pleiotropic effects. These pleiotropic effects are thought to include attenuation of the inflammatory response due to reduced prenylation of proteins in the inflammatory cascade. We conducted studies in normolipidemic subjects to determine if treatment with high-dose (80 mg) atorvastatin could reduce circulating levels of inflammatory markers. We also determined whether high-dose atorvastatin affected the inflammatory response of monocytes stimulated with lipopolysaccharide (LPS) ex vivo. We found that treatment with atorvastatin rapidly and significantly reduced plasma low-density lipoprotein (LDL) cholesterol levels in subjects treated for 2 weeks. However, statin treatment had no discernible effect on plasma levels of the inflammatory markers high-sensitivity C-reactive protein (hsCRP), tumor necrosis factor (TNF)-α, or interleukin (IL-6) and no effect on the cytokine response of monocytes following ex vivo stimulation with LPS. High-dose atorvastatin treatment of normolipidemic subjects with normal C-reactive protein levels has no effect on the inflammatory response assessed by monocyte stimulation with LPS ex vivo despite significant reductions in LDL cholesterol levels.

Summary

Objective

Resistin causes insulin resistance and diabetes in mice whereas in humans it is linked to inflammation and atherosclerosis. Few human genetic studies of resistin in inflammation and atherosclerosis have been performed. We hypothesized that the −420C>G putative gain-of-function resistin variant would be associated with inflammatory markers and atherosclerosis but not with metabolic syndrome or adipokines in humans.

Design and methods

We examined the association of three resistin polymorphisms, −852A>G, −420C>G and +157C>T, and related haplotypes with plasma resistin, cytokines, C-reactive protein (CRP), adipokines, plasma lipoproteins, metabolic syndrome and coronary artery calcification (CAC) in nondiabetic Caucasians (n = 851).

Results

Resistin levels were higher, dose-dependently, with the −420G allele (CC 5·9 ± 2·7 ng/ml, GC 6·5 ± 4·0 ng/ml and GG 7·2 ± 4·8 ng/ml, trend P = 0·04) after age and gender adjustment [fold higher for GC + GG vs. CC; 1·07 (1·00–1·15), P < 0·05)]. The −852A>G single nucleotide polymorphism (SNP) was associated with higher soluble tumour necrosis factor-receptor 2 (sol-TNFR2) levels in fully adjusted models [1·06 (95% CI 1·01–1·11), P = 0·01)]. The estimated resistin haplotype (GGT) was associated with sol-TNFR2 (P = 0·04) and the AGT haplotype was related to CRP (P = 0·04) in the fully adjusted models. Resistin SNPs and haplotypes were not associated with body mass index (BMI), fasting glucose, insulin resistance, metabolic syndrome, adipokines or CAC scores.

Conclusions

Despite modest associations with plasma resistin and inflammatory biomarkers, resistin 5′ variants were not associated with metabolic parameters or coronary calcification. This suggests that resistin is an inflammatory cytokine in humans but has little influence on adiposity, metabolic syndrome or atherosclerosis.

Background

Niacin has multiple lipoprotein effects that may provide cardiovascular benefit when added to statin monotherapy.

Methods

In this randomized, placebo-controlled trial (n = 75) of magnetic resonance imaging of carotid atherosclerosis, we performed a secondary comparison of combination niacin-statin (simvastatin 20 mg/Niacin-ER 2G [S20/N]) to monotherapy with moderate (20 mg [S20]) and high-dose (80 mg [S80]) simvastatin on lipids, apolipoproteins (apo), low density lipoprotein (LDL) and high density lipoprotein (HDL) particle subclasses, and inflammatory markers.

Results

At baseline, average age was 71, 72% were male, 62.5% used statins, and average LDL-cholesterol was 111 mg/dL. At 12 months, S20/N, compared to S80, significantly reduced apoB (−36.6% vs −11.9%; P = .05) and lipoprotein(a) (−18% vs +3.5%; P = .001) and had at least an equivalent effect on LDL-cholesterol (−39.3% vs −24.3%; P = .24). The combination reduced the proportion of subjects with atherogenic LDL pattern-B (50% to 11.5%) compared to S80 (56% to 56%) (P = .01). Despite increases in plasma free fatty acids (+62.4%; F = 5.65, P = .005 vs S20 and S80), plasma triglycerides (−29.4%; F = 6.88, P = .002 vs S20 and S80), and very-low-density lipoprotein (−44.2%; F = 7.94, P < .001 vs S20 and S80), levels were reduced by S20/N. S20/N increased HDL-cholesterol levels (+18.1%) as compared to S20 (0%) and S80 (+5.9%) (P < .001 vs both statin arms), largely due to an increase in HDL particle size (+4.6%; P = .01 vs both statin arms).

Conclusions

We demonstrate that full-dose niacin/moderate-dose simvastatin combination has sustained benefits on atherogenic apoB lipoproteins, at least comparable to high-dose simvastatin, while also raising HDL-cholesterol. Results of large clinical trials will inform whether niacin-statin combinations reduce cardiovascular disease events.

Objectives

We evaluated the hypothesis that plasma levels of adiponectin and leptin are independently but oppositely associated with coronary calcification (CAC), a measure of subclinical atherosclerosis. In addition, we assessed which biomarkers of adiposity and insulin resistance are the strongest predictors of CAC beyond traditional risk factors, the metabolic syndrome and plasma C-reactive protein (CRP).

Background

Adipokines are fat-secreted biomolecules with pleiotropic actions that converge in diabetes and cardiovascular disease.

Methods

We examined the association of plasma adipocytokines with CAC in 860 asymptomatic, non-diabetic participants in the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA).

Results

Plasma adiponectin and leptin levels had opposite and distinct associations with adiposity, insulin resistance and inflammation. Plasma leptin was positively (top vs. bottom quartile) associated with higher CAC after adjusting for age, gender, traditional risk factors and Framingham Risk Scores (FRS) [tobit regression ratio 2.42 (95% CI 1.48–3.95, p=0.002)] and further adjusting for metabolic syndrome and CRP [ratio 2.31 (95% CI 1.36–3.94, p=0.002)]. In contrast, adiponectin levels were not associated with CAC. Comparative analyses suggested that levels of leptin, IL-6 and sol-TNFR2 as well as HOMA-IR predicted CAC scores but only leptin and HOMA-IR provided value beyond risk factors, the metabolic syndrome and CRP.

Conclusion

In SIRCA, while both leptin and adiponectin levels were associated with metabolic and inflammatory markers, only leptin was a significant independent predictor of CAC. Of several metabolic markers, leptin and the HOMA-IR index had the most robust, independent associations with CAC.

Condensed Abstract

Adipokines are fat-secreted biomolecules with pleiotropic actions and represent novel markers for cardiovascular risk. We examined the association of plasma adipocytokines with CAC in 860 asymptomatic, non-diabetic Caucasians. Leptin was positively (top vs. bottom quartile) associated with higher CAC even after adjustment for age, gender, traditional risk factors, Framingham Risk Score, metabolic syndrome, and CRP [ratio 2.31 (95% CI 1.36–3.94, p=0.002)]. Adiponectin levels were not associated with CAC. Comparative analyses suggested that levels of leptin, IL-6 and sol-TNFR2 as well as HOMA-IR predicted CAC scores, but only leptin and HOMA-IR provided value beyond risk factors, the metabolic syndrome and CRP.

Objective

The study of PPAR-α activation on apoA-I production in humans has been limited to fibrates, relatively weak PPAR-α agonists that may have other molecular effects. We sought to determine the effect of a potent and highly specific PPAR-α agonist, LY518674, on apoA-I, apoA-II and apoB-100 kinetics in humans with metabolic syndrome and low levels of HDL cholesterol (C).

Methods and Results

Subjects were randomized to receive LY518674 (100 μg) once daily (n=13) or placebo (n=15) for 8 weeks. Subjects underwent a kinetic study using a deuterated leucine tracer to measure apolipoprotein production and fractional catabolic rates (FCR) at baseline and following treatment. LY518674 significantly reduced VLDL-C (−38%, p=0.002) and triglyceride (−23%, p=0.002) levels while LDL-C and HDL-C levels were unchanged. LY518674 significantly reduced VLDL apoB-100 (−12%, p=0.01) levels, due to an increased VLDL apoB-100 FCR with no change in VLDL apoB-100 production. IDL and LDL apoB-100 kinetics were unchanged. LY518674 significantly increased of the apoA-I production rate by 31% (p< 0.0001) but this was accompanied by a 33% increase in the apoA-I FCR (p=0.002), resulting in no change in plasma apoA-I. There was a 71% increase in the apoA-II production rate (p< 0.0001) accompanied by a 25% increase in the FCR (p< 0.0001), resulting in a significant increase in plasma apoA-II.

Conclusions

Activation of PPAR-α with of LY518674 (100 μg) in subjects with metabolic syndrome and low HDL-C increased the VLDL apoB-100 FCR consistent with enhanced lipolysis of plasma triglyceride. Significant increases in the apoA-I and apoA-II production rates were accompanied by increased FCRs resulting in no change in HDL-C levels. These data indicate a major effect of LY518674 on the production and clearance of apoA-I and HDL despite no change in the plasma concentration. The effect of these changes on reverse cholesterol transport remains to be determined.

Elevated plasma concentrations of HDL cholesterol (HDL-C) are associated with protection from atherosclerotic cardiovascular disease. Animal models indicate that decreased expression of endothelial lipase (LIPG) is inversely associated with HDL-C levels, and genome-wide association studies have identified LIPG variants as being associated with HDL-C levels in humans. We hypothesized that loss-of-function mutations in LIPG may result in elevated HDL-C and therefore performed deep resequencing of LIPG exons in cases with elevated HDL-C levels and controls with decreased HDL-C levels. We identified a significant excess of nonsynonymous LIPG variants unique to cases with elevated HDL-C. In vitro lipase activity assays demonstrated that these variants significantly decreased endothelial lipase activity. In addition, a meta-analysis across 5 cohorts demonstrated that the low-frequency Asn396Ser variant is significantly associated with increased HDL-C, while the common Thr111Ile variant is not. Functional analysis confirmed that the Asn396Ser variant has significantly decreased lipase activity both in vitro and in vivo, while the Thr111Ile variant has normal lipase activity. Our results establish that loss-of-function mutations in LIPG lead to increased HDL-C levels and support the idea that inhibition of endothelial lipase may be an effective mechanism to raise HDL-C.

Background

Protease inhibitors (PIs) are associated with hypertriglyceridemia and atherogenic dyslipidemia. Identifying HIV-1-infected individuals who are at increased risk of PI-related dyslipidemia will facilitate therapeutic choices that maintain viral suppression while reducing risk of atherosclerotic diseases. Apolipoprotein C-III (apoC-III) gene variants, which vary by race/ethnicity, have been associated with a lipid profile that resembles PI-induced dyslipidemia. However, the association of race/ethnicity, or candidate gene effects across race/ethnicity, with plasma lipid levels in HIV-1-infected individuals, has not been reported.

Methods and Findings

A cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and PI exposure on plasma lipids was performed in AIDS Clinical Trial Group studies (n = 626). Race/ethnicity was a highly significant predictor of plasma lipids in fully adjusted models. Furthermore, in stratified analyses, the effect of PI exposure appeared to differ across race/ethnicity. Black/non-Hispanic, compared with White/non-Hispanics and Hispanics, had lower plasma triglyceride (TG) levels overall, but the greatest increase in TG levels when exposed to PIs. In Hispanics, current PI antiretroviral therapy (ART) exposure was associated with a significantly smaller increase in TGs among patients with variant alleles at apoC-III-482, −455, and Intron 1, or at a composite apoC-III genotype, compared with patients with the wild-type genotypes.

Conclusions

In the first pharmacogenetic study of its kind in HIV-1 disease, we found race/ethnic-specific differences in plasma lipid levels on ART, as well as differences in the influence of the apoC-III gene on the development of PI-related hypertriglyceridemia. Given the multi-ethnic distribution of HIV-1 infection, our findings underscore the need for future studies of metabolic and cardiovascular complications of ART that specifically account for racial/ethnic heterogeneity, particularly when assessing candidate gene effects.

This cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and protease inhibitor exposure on plasma lipids showed that race/ethnicity was a highly significant predictor of plasma lipids

Background

Endothelial lipase (EL), a new member of the lipase family, has been shown to modulate high-density lipoprotein (HDL-C) metabolism and atherosclerosis in mouse models. We hypothesized that EL concentrations would be associated with decreased HDL-C and increased atherosclerosis in humans.

Methods and Findings

Healthy individuals with a family history of premature coronary heart disease (n = 858) were recruited as part of the Study of the Inherited Risk of Atherosclerosis. Blood was drawn in the fasting state before and, in a subgroup (n = 510), after administration of a single dose of intravenous heparin. Plasma lipids were measured enzymatically, lipoprotein subclasses were assessed by nuclear magnetic resonance, and coronary artery calcification (CAC) was quantified by electron beam computed tomography. Plasma EL mass was measured using a newly developed enzyme-linked immunosorbent assay. Median EL mass in pre-heparin plasma was 442 (interquartile range = 324–617) ng/ml. Median post-heparin mass was approximately 3-fold higher, 1,313 (888–1,927) ng/ml. The correlation between pre-heparin EL mass and post-heparin EL mass was 0.46 (p < 0.001). EL mass concentrations in both pre- and post-heparin plasma significantly correlated with all NCEP ATPIII-defined metabolic syndrome factors: waist circumference (r = 0.28 and 0.22, respectively, p < 0.001 for each), blood pressure (r = 0.18 and 0.24, p < 0.001 for each), triglycerides (r = 0.22, p < 0.001; and 0.13, p = 0.004), HDL cholesterol (r = –0.11, p = 0.002; and –0.18, p < 0.001), and fasting glucose (r = 0.11 and 0.16, p = 0.001 for both). EL mass in both routine (odds ratio [OR] = 1.67, p = 0.01) and post-heparin (OR = 2.42, p = 0.003) plasma was associated with CAC as determined by ordinal regression after adjustment for age, gender, waist circumference, vasoactive medications, hormone replacement therapy (women), and established cardiovascular risk factors.

Conclusions

We report, to our knowledge for the first time, that human plasma EL concentrations, in both post-heparin and routine pre-heparin plasma, are significantly associated with metabolic syndrome features and with subclinical atherosclerosis. EL may be a pro-atherogenic factor in humans, especially in overweight individuals and those with metabolic syndrome.

Studies in rodents had suggested that endothelial lipase might play a role in the development of atherosclerosis. This study finds an association between EL levels and early-stage atherosclerosis in humans.