Knowledge-driven multi-locus analysis reveals gene-gene interactions influencing HDL cholesterol level in two independent EMR-linked biobanks

Genome-wide association studies (GWAS) are routinely being used to examine the genetic contribution to complex human traits, such as high-density lipoprotein cholesterol (HDL-C). Although HDL-C levels are highly heritable (h²∼0.7), the genetic determinants identified through GWAS contribute to a small fraction of the variance in this trait. Reasons for this discrepancy may include rare variants, structural variants, gene-environment (GxE) interactions, and gene-gene (GxG) interactions. Clinical practice-based biobanks now allow investigators to address these challenges by conducting GWAS in the context of comprehensive electronic medical records (EMRs). Here we apply an EMR-based phenotyping approach, within the context of routine care, to replicate several known associations between HDL-C and previously characterized genetic variants: CETP (rs3764261, p = 1.22e-25), LIPC (rs11855284, p = 3.92e-14), LPL (rs12678919, p = 1.99e-7), and the APOA1/C3/A4/A5 locus (rs964184, p = 1.06e-5), all adjusted for age, gender, body mass index (BMI), and smoking status. By using a novel approach which censors data based on relevant co-morbidities and lipid modifying medications to construct a more rigorous HDL-C phenotype, we identified an association between HDL-C and TRIB1, a gene which previously resisted identification in studies with larger sample sizes. Through the application of additional analytical strategies incorporating biological knowledge, we further identified 11 significant GxG interaction models in our discovery cohort, 8 of which show evidence of replication in a second biobank cohort. The strongest predictive model included a pairwise interaction between LPL (which modulates the incorporation of triglyceride into HDL) and ABCA1 (which modulates the incorporation of free cholesterol into HDL). These results demonstrate that gene-gene interactions modulate complex human traits, including HDL cholesterol.     

HDL2 and HDL3 cholesterol in hepatic cirrhosis

In order to further investigate plasma lipoproteins abnormalities secondary to serious liver damage, we studied plasma lipids and lipoproteins, and in particular HDL subfractions (HDL2, HDL3), in 12 patients with cirrhosis of the liver and in 12 sex, age and weight matched healthy volunteers. Enzymatic methods were used to determine total cholesterol and triglycerides, while the extractive method of Abell et al. was used for the determination of HDL-cholesterol levels after LDL and VLDL precipitation with polyanions (MnCl2 and Na-heparin) and of HDL3-cholesterol values after HDL2 precipitation with dextran-sulphate 15,000 m.w. Total cholesterol and HDL-cholesterol levels were significantly lower in cirrhotic patients compared to normal subjects. We must emphasize that only HDL3-cholesterol was decreased in cirrhotics, whereas HDL2-cholesterol values were normal or high. We suggest that a diminished activity of hepatic triglyceride lipase might account for the decrease in HDL3-cholesterol in liver cirrhosis.     

HDL and reverse cholesterol transport. Role of cholesterol ester transfer protein]

As most of peripheral cells are not able to catabolize cholesterol, the transport of cholesterol excess from peripheral tissues back to the liver, namely "reverse cholesterol transport", is the only way by which cholesterol homeostasis is maintained in vivo. Reverse cholesterol transport pathway can be divided in three major steps: 1) uptake of cellular cholesterol by the high density lipoproteins (HDL), 2) esterification of HDL cholesterol by the lecithin: cholesterol acyltransferase and 3) captation of HDL cholesteryl esters by the liver where cholesterol can be metabolized and excreted in the bile. In several species, including man, cholesteryl esters in HDL can also follow an alternative pathway which consists in their transfer from HDL to very low density (VLDL) and low density (LDL) lipoproteins. The transfer of cholesteryl esters to LDL, catalyzed by the Cholesteryl Ester Transfer Protein (CETP), might affect either favorably or unfavorably the reverse cholesterol transport pathway, depending on whether LDL are finally taken up by the liver or by peripheral tissues, respectively. In order to understand precisely the implication of CETP in reverse cholesterol transport, it is essential to determine its role in HDL metabolism, to know the potential regulation of its activity and to identify the mechanism by which it interacts with lipoprotein substrates. Results from recent studies have demonstrated that CETP can promote the size redistribution of HDL particles. This may be an important process in the reverse cholesterol transport pathway as HDL particles with various sizes have been shown to differ in their ability to promote cholesterol efflux from peripheral cells and to interact with lecithin: cholesterol acyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of HDL unesterified and esterified cholesterol by human endothelial cells. Modulation by HDL phospholipolysis and cell cholesterol content

Human HDL (1.070-1.210), doubly labelled with ³H/¹⁴C-labelled unesterified cholesterol and ³H-labelled esterified cholesterol were incubated for 1–5 h with monolayer cultures of human endothelial cells. HDL were preincubated for 60–120 min the presence of albumin and with/without purified phospholipase A₂ (control HDL, phospholipase A₂ HDL) before dilution in the cell culture medium. Average phosphatidyl-choline (PC) degradation was 62.10% ± 2.57% (range 45–80%). A purified lipase /phospholipase A₁ from guinea pig pancreas was used in some experiments (range of PC hydrolysis: 16–70%). (1) ³H/¹⁴C-labelled unesterified cholesterol and ³H-labelled esterified cholesterol appeared in cells during 0–5 h incubations. Trypsin treatment allowed a simple adsorption of HDL onto the cell surface to be avoided, and most of the ³H-labelled esterified cholesterol transferred to cells was hydrolysed. Cell uptake of radioactive cholesterol increased as a function of HDL concentration but no saturation was achieved at the highest lipoprotein concentration used (200 μg cholesterol/ml). Flux of ³H/¹⁴C-labelled unesterified cholesterol was related to the cell cholesterol content, suggesting that it might partly represent an exchange process. The cell cholesterol content was slightly increased after 5 h incubation with HDL (+16%). (2) Pretreatment of HDL with purified phospholipase A₂ doubled on average the amount of cell recovered ³H-labelled esterified cholesterol, while the flux of ³H/¹⁴C-labelled unesterified cholesterol was enhanced by 15–25%. Both transfer and cell hydrolysis of ³H-labelled esterified cholesterol were increased. A stimulation was also observed using purified lipase/phospholipase A₁, provided that a threshold phospholipid degradation was achieved (between 27 and 45%). (3) Endothelial cells were conditioned in different media so as to modulate their charge in cholesterol. The uptake of ³H-labelled esterified cholesterol was found to be significantly higher in cholesterol-enriched cells compared to the sterol-depleted state. Finally, movements of ³H-labelled esterified cholesterol from HDL to endothelial cells were essentially unaffected by cell density or by the presence of partially purified cholesterol ester transfer protein. The possible roles of the transfer of HDL esterified cholesterol to endothelial cells and its modulation by phospholipases are discussed.     

Models for the longitudinal genetic analysis of same-age twins: application to HDL cholesterol.

Models are presented for the analysis of longitudinal data from same-age twins which permit the exploration of a remarkably diverse array of alternative explanations for continuity and change during development. Data of this type permit the detection of new sources of genetic or environmental covariation during development that are not expressed at earlier ages and, because they include the effects of age-specific genes, the resulting heritability estimates are more reliable than those obtained from relatives who differ in age. The proposed models were applied to measurements of HDL cholesterol obtained on 81 pairs of monozygotic (MZ) twins and 69 dizygotic (DZ) pairs at 11, 12.5 and 14 years of age. All three MZ co-twin correlations were substantially higher than the self correlations across occasions, suggesting that new sources of genetic or environmental covariation must be expressed during early adolescence. This interpretation was confirmed by analysis of the full covariance matrices which showed that only models which assumed the expression of new or age-specific genes could explain the observed pattern of covariation. Because they include the effects of age-specific genes, the resulting heritabilities (0.80-0.83) were substantially higher than many previous estimates.     

Density profile and cholesterol concentration of serum lipoproteins in experimental animals and human subjects on hypercholesterolaemic diets

The density profile of Sudan black stained serum lipoproteins was studied in human subjects and various animal species on diets supplemented with cholesterol. In the animals studied (rabbits, calves, mice, chickens, rats and guinea-pigs), the feeding of cholesterol resulted in an elevation of serum cholesterol levels together with marked changes in the density profile and the cholesterol concentration of the serum lipoproteins. Large differences between animal species in their response to dietary cholesterol were found. In a human subject, an increased concentration of serum cholesterol due to the consumption of a diet supplemented with six egg yolks per day was reflected in an elevated level of LDL cholesterol, while changes in the density profile of stained serum lipoproteins were not observed. In subjects with familial type III and type IV hyperlipoproteinaemia, marked differences in the density profile of lipoproteins were found when compared with that of normolipoproteinaemic subjects. The density profile of stained lipoproteins in the type III patients was remarkably similar to that in cholesterol-fed chickens and lean Zucker rats.     

Relationship of the parameters of body cholesterol metabolism with plasma levels of HDL cholesterol and the major HDL apoproteins

The inverse relationship between plasma levels of high density lipoprotein (HDL) and coronary heart disease rates has suggested that HDL might influence body stores of cholesterol. Therefore, we have investigated potential relationships between the parameters of body cholesterol metabolism and the plasma levels of HDL cholesterol and the major HDL apoproteins. The study involved 55 human subjects who underwent long-term cholesterol turnover studies, as well as plasma lipoprotein and apolipoprotein assays. In order to maximize the likelihood of detecting existing relationships, the subjects were selected to span a wide range of plasma levels of lipids, lipoproteins, and apolipoproteins. Single univariate correlation analyses suggested weak but statistically significant inverse relationships of HDL cholesterol and apoA-I levels with the following model parameters: production rate (PR), the mass of rapidly exchanging body cholesterol (M1), the minimum estimate of the mass of slowly exchanging body cholesterol (M3min), and of the mass of total exchangeable body cholesterol (Mtotmin). These correlations, however, were quantitatively quite small (/r/ = 0.28-0.42) in comparison to the strength of the univariate relationships between body weight and PR (r = 0.76), M1 (r = 0.61), M3min (r = 0.58), and Mtotmin (r = 0.78). Correlations for apoA-II and apoE levels were even smaller than those for apoA-I and HDL cholesterol. In additional analyses using multivariate approaches, HDL cholesterol, apoA-I, apoA-II, and apoE levels were all found not to be independent determinants of the parameters of body cholesterol metabolism (/partial r/ less than 0.17, P greater than 0.3 in all cases). Thus the weak univariate correlations reflect relationships of HDL cholesterol and apoA-I levels with physiological variables, such as body size, which are primarily related to the model parameters. We conclude that plasma levels of HDL cholesterol and apoproteins A-I, A-II, and E are not quantitatively important independent determinants of the mass of slowly exchanging body cholesterol or of other parameters of long-term cholesterol turnover in humans. These studies give no support to the hypothesis that the inverse relationship between HDL cholesterol levels and coronary heart disease rates is mediated via an influence of HDL on body stores of cholesterol.     

Dark chocolate consumption increases HDL cholesterol concentration and chocolate fatty acids may inhibit lipid peroxidation in healthy humans

Cocoa powder is rich in polyphenols and, thus, may contribute to the reduction of lipid peroxidation. Our aim was to study the effects of long-term ingestion of chocolate, with differing amounts of polyphenols, on serum lipids and lipid peroxidation ex vivo and in vivo. We conducted a 3 week clinical supplementation trial of 45 nonsmoking, healthy volunteers. Participants consumed 75 g daily of either white chocolate (white chocolate, WC group), dark chocolate (dark chocolate, DC group), or dark chocolate enriched with cocoa polyphenols (high-polyphenol chocolate, HPC group). In the DC and HPC groups, an increase in serum HDL cholesterol was observed (11.4% and 13.7%, respectively), whereas in the WC group there was a small decrease (-2.9%, p < 0.001). The concentration of serum LDL diene conjugates, a marker of lipid peroxidation in vivo, decreased 11.9% in all three study groups. No changes were seen in the total antioxidant capacity of plasma, in the oxidation susceptibility of serum lipids or VLDL + LDL, or in the concentration of plasma F2-isoprostanes or hydroxy fatty acids. Cocoa polyphenols may increase the concentration of HDL cholesterol, whereas chocolate fatty acids may modify the fatty acid composition of LDL and make it more resistant to oxidative damage.     

Measurement of normative HDL subfraction cholesterol levels by Gaussian summation analysis of gradient gels

This report describes development of a computerized method for analyzing polyacrylamide gradient gels of high density lipoproteins (HDL) by Gaussian summation, a simple technique to obtain standardized measurements of size and amount of HDL subfractions. Conditions for reproducibility and ranges of linearity were established. By Gaussian summation analysis, five or six HDL subfractions could be found in the plasma of most normolipidemic people. The relationship of staining intensity to cholesterol level was determined for Coomassie Blue R-250, permitting determination of the cholesterol levels in the individual subfractions, with standard errors of repeated measurements of 2% or less of the total HDL area, and accuracy, limited by the standard error of the chromogenicity, of 1-2 mg/dl for the least abundant fractions and 3-4 mg/dl for the most abundant subfractions. Levels of HDL2b measured by this method were statistically the same as levels of HDL2 measured by dextran sulfate-Mg2+ precipitation. Gaussian summation analysis of gradient gels was used to measure HDL subfraction cholesterol levels in subjects from the Baltimore Longitudinal Study on Aging to obtain normative levels for men and women for the major HDL subfractions. Comparisons of these levels with each other and with triglyceride and cholesterol levels showed that triglyceride levels were inversely correlated with levels of HDL2a and HDL2b, cholesterol levels were directly correlated with levels of HDL3b and HDL3a, and that HDL3b levels were inversely correlated with levels of both HDL2a and HDL2b.     

Statistical analysis of variations in total cholesterol, HDL, HDL2, HDL3, LDL-cholesterol, VLDL-triglycerides, apo-A and apo-B lipoproteins in dyslipidemias during antilipemic therapy

In our study, bezafibrate in short-acting formula and long-acting formula, administered to hyperlipidaemic patients, resulted in a significant lowering of atherogenic lipids and lipoproteins (chol.-T, LDL and VLDL-chol., apolipoprotein B and VDLD-TR), and a marked increase in the levels of HDL, HDL2, HDL3-chol. and apolipoprotein A with a protective action as regards vascular atherosclerotic damage. The long-acting formula showed a greater effectiveness.     

Testosterone replacement in hypogonadal men alters the HDL proteome but not HDL cholesterol efflux capacity

The effects of androgens on cardiovascular disease (CVD) risk in men remain unclear. To better characterize the relationship between androgens and HDL, we investigated the effects of testosterone replacement on HDL protein composition and serum HDL-mediated cholesterol efflux in hypogonadal men. Twenty-three older hypogonadal men (ages 51-83, baseline testosterone < 280 ng/dl) were administered replacement testosterone therapy (1% transdermal gel) with or without the 5α-reductase inhibitor dutasteride. At baseline and after three months of treatment, we determined fasting lipid concentrations, HDL protein composition, and the cholesterol efflux capacity of serum HDL. Testosterone replacement did not affect HDL cholesterol (HDL-C) concentrations but conferred significant increases in HDL-associated paraoxonase 1 (PON1) and fibrinogen α chain (FGA) (P = 0.022 and P = 0.023, respectively) and a decrease in apolipoprotein A-IV (apoA-IV) (P = 0.016). Exogenous testosterone did not affect the cholesterol efflux capacity of serum HDL. No differences were observed between men who received testosterone alone and those who also received dutasteride. Testosterone replacement in older hypogonadal men alters the protein composition of HDL but does not significantly change serum HDL-mediated cholesterol efflux. These effects appear independent of testosterone conversion to dihydrotestosterone. Further research is needed to determine how changes in HDL protein content affect CVD risk in men.     

Tweaking the cholesterol efflux capacity of reconstituted HDL

Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.     

Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL’s size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL’s content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL’s content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL’s ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL’s APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.     

Localization of genes for V+LDL plasma cholesterol levels on two diets in the opossum Monodelphis domestica

Plasma cholesterol levels among individuals vary considerably in response to diet. However, the genes that influence this response are largely unknown. Non-HDL (V+LDL) cholesterol levels vary dramatically among gray, short-tailed opossums fed an atherogenic diet, and we previously reported that two quantitative trait loci (QTLs) influenced V+LDL cholesterol on two diets. We used hypothesis-free, genome-wide linkage analyses on data from 325 pedigreed opossums and located one QTL for V+LDL cholesterol on the basal diet on opossum chromosome 1q [logarithm of the odds (LOD) = 3.11, genomic P = 0.019] and another QTL for V+LDL on the atherogenic diet (i.e., high levels of cholesterol and fat) on chromosome 8 (LOD = 9.88, genomic P = 5 × 10⁻⁹). We then employed a novel strategy involving combined analyses of genomic resources, expression analysis, sequencing, and genotyping to identify candidate genes for the chromosome 8 QTL. A polymorphism in ABCB4 was strongly associated (P = 9 × 10⁻¹⁴) with the plasma V+LDL cholesterol concentrations on the high-cholesterol, high-fat diet. The results of this study indicate that genetic variation in ABCB4, or closely linked genes, is responsible for the dramatic differences among opossums in their V+LDL cholesterol response to an atherogenic diet.     

Effect of exercise timing on postprandial lipemia and HDL cholesterol subfractions

The purpose ofthe study was to examine the effect of exercise timing on postprandiallipemia responses. Subjects were 21 recreationally trained men (ages 27 ± 1.7 yr). Each subject performed four trials:1) Control (fat meal only),2) Post (exercise 1 h after a fat meal), 3) 1 h-Pre(exercise 1 h before a fat meal), and4) 12 h-Pre (exercise 12 h before afat meal). In each trial, subjects had a standard fat meal to inducepostprandial hypertriglyceridemia. Blood samples were taken at 0 h(immediately before the fat meal) and at 2, 4, 6, 8, and 24 h after themeal. In the exercise trials, each subject exercised at 60% of maximalO₂ consumption for 1 h. Theresults indicated that triglyceride area under the curve scores inpremeal-exercise trials were lower (P < 0.05) than those in Post and Control. At 24 h, total high-densitylipoprotein (HDL)-cholesterol in the premeal-exercise trials was higher(P < 0.05) than that at 0 h, whereastotal HDL-cholesterol was not changed in Control and Post. At 24 h, HDLsubtype 2-cholesterol was higher (P < 0.05) in the premeal-exercise trials than in Control, which did not differ from Post. These results suggest that exercising before a fatmeal may have a beneficial effect on the triglyceride response and HDLmetabolism, which may blunt atherosclerotic process induced by the fatmeal.