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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., Competing Interests: The authors have declared that no competing interests exist.

Human endothelial lipase (EL) is a member of a family of lipases and phospholipases that are involved in the metabolism of plasma lipoproteins. EL displays a preference to hydrolyze lipids in HDL. We report here that a naturally occurring low frequency coding variant in the EL gene (LIPG), glycine-26 to serine (G26S), is significantly more common in African-American individuals with elevated HDL cholesterol (HDL-C) levels. To test the hypothesis that this variant results in reduced EL function, we extensively characterized and compared the catalytic and noncatalytic functions of the G26S variant and wild-type (WT) EL. While the catalytic-specific activity of G26S EL is similar to WT EL, its secretion is markedly reduced. Consistent with this observation, we found that carriers of the G26S variant had significantly reduced plasma levels of EL protein. Thus, this N-terminal variant results in reduced secretion of EL protein, plausibly leading to increased HDL-C levels.

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: Endothelial lipase (EL) is a plasma lipase that we previously reported to be significantly correlated with all features of the metabolic syndrome in humans, including directly with measures of adiposity and inversely with high-density lipoprotein cholesterol levels. We hypothesized that inflammation associated with obesity results in upregulation of EL. We determined the relationship between inflammatory markers and EL levels in a cohort of healthy persons recruited on the basis of family history of coronary disease. Furthermore, we directly tested the hypothesis that plasma EL concentrations would increase with induction of an inflammatory state by low-dose endotoxin in humans., Methods and Results: High-sensitivity C-reactive protein, interleukin 6, soluble tumor necrosis factor receptor II, soluble intercellular adhesion molecule 1, leptin, and adiponectin were measured in plasma of 858 subjects. Significant direct correlations (P<0.001 for all) were found between EL concentrations and high-sensitivity C-reactive protein (r=0.28), interleukin-6 (r=0.22), soluble tumor necrosis factor receptor II (r=0.22), soluble intercellular adhesion molecule 1 (r=0.24), and leptin (r=0.20). An inverse correlation was present with adiponectin (r=-0.15, P<0.001). Adiponectin inhibited the tumor necrosis factor-alpha-stimulated EL secretion from cultured human coronary endothelial cells in a dose-dependent manner. Experimental low-dose endotoxemia in 20 subjects resulted in a 2.5-fold increase in EL concentrations 12 to 16 hours after injection, which correlated temporally with decreases in both total and high-density lipoprotein phospholipid., Conclusions: In humans, plasma inflammatory markers are directly correlated with plasma EL concentrations, and experimental endotoxemia significantly increases plasma EL concentrations, proving that EL is upregulated by inflammation in humans. This mechanism may partially explain the low high-density lipoprotein cholesterol levels seen in obesity and metabolic syndrome.

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.

Lipoprotein lipase, HL, and endothelial lipase (EL) are proteoglycan-bound enzymes that regulate plasma lipoprotein levels through coordinated triglyceride (TG) lipase and phospholipase activity. We hypothesized that single nucleotide polymorphisms (SNPs) in lipase genes would have higher order impact on plasma lipoproteins beyond the influence of individual SNPs. In a sample of asymptomatic Caucasian subjects (n = 738), we used a two-stage approach, first identifying groups of subjects with similar multilocus lipase genotypes and then characterizing the relationships between genotype groups and plasma lipids. Using complementary methods, including a permutation test procedure and a mixed-effects modeling approach, we found a higher order interaction between four SNPs in three lipase genes (EL 2,237 3' untranslated region, EL Thr111Ile, HL -514C/T, and LPL HindIII) and plasma TG levels. Subjects who were heterozygous for all four lipase SNPs had significantly higher plasma TG levels beyond the effect of individual lipase SNPs and environmental factors, even after correcting for multiple comparisons. In conclusion, lipase genes had synergistic association with plasma TG beyond individual gene effects. Higher order multilocus genotype contributions to dyslipidemia and atherosclerotic cardiovascular disease need to be considered a priori because they may have an important effect even in the absence of significant main effects of the individual genes.

Background: Elevated high-density lipoprotein cholesterol (HDL-C) is associated with reduced risk of cardiovascular disease, and variation in HDL-C levels has been shown to be approximately 50% heritable. Overexpression of endothelial lipase (EL), a member of the lipoprotein lipase gene family, markedly reduces HDL-C levels in mouse models. We hypothesized that genetic variation in EL might be associated with elevated HDL-C., Methods and Results: All exons and 1.2 kilobase of promoter of the EL gene were sequenced in 20 unrelated human subjects with high HDL-C levels. A total of 17 variants were identified. Six of these were potentially functional and were confirmed by restriction enzyme analysis. Four variants result in amino acid changes (Gly26Ser, Thr111Ile, Thr298Ser, and Asn396Ser,) and 2 variants were in the promoter (-303A/C and -410C/G). The genotype frequencies of each variant were determined in 176 black controls, 165 white controls, and 123 whites with high HDL-C. The Thr111Ile variant was the most common, with an allele frequency of 10.3% in blacks, 31.2% in white controls, and 32.6% in the high HDL-C group. The remaining variants all had allele frequencies <5.0% but differed in frequency among the 3 groups. Interestingly, Gly26Ser, Thr298Ser, and -303A/C were found in the black and high HDL-C white cohorts but were absent in the control white group., Conclusions: Six new potentially functional variants in EL were discovered through sequencing of the EL gene in subjects with high HDL-C levels. Differences in allele frequencies exist between blacks and whites and between control subjects and those with high HDL-C levels.