The lipoprotein lipase (LPL) enzyme plays a major role in lipid metabolism, primarily by regulating the catabolism of triglyceride (TG)-rich lipoprotein particles. The gene for LPL is an important candidate for affecting the risk of atherlosclerosis in the general population. Previously, we have shown that the HindIII polymorphism in intron 8 of the LPL gene is associated with plasma TG and HDL-cholesterol variation in Hispanics and non-Hispanic whites (NHWs). However, this polymorphism is located in an intron and hence may be in linkage disequilibrium with a functional mutation in the coding region or intron-exon junctions of the LPL gene. The aim of this study was to initially screen the LPL coding region and the intron-exon junctions by single-strand conformation polymorphism (SSCP) analysis for mutation detection in a group of 86 individuals expressing the phenotype of high TG/low HDL, followed by association studies in a population-based sample of 1,014 Hispanics and NHWs. Four sequence variations were identified by SSCP and DNA sequencing in the coding region of the gene, including two missense mutations (D9N in exon 2 and N291S in exon 6), one samesense mutation (V108V in exon 3), and one nonsense mutation (S447X in exon 9). Multiple regression analyses, including these four mutations and the HindIII polymorphic site, indicate that the association of the HindIII site with plasma TG (P=0.001 in NHWs and P=0.002 in Hispanics) and HDL-cholesterol (P=0.007 in NHWs and P=0.127 in Hispanics) is independent of all other LPL variable sites examined. These observations reinforce the concept that the intronic 8 HindIII site is functional by itself and provide a strong rationale for future comprehensive functional studies to delineate its biological significance.

Lipoprotein Lipase (LPL) plays a pivotal role in lipid metabolism by hydrolyzing triglyceride (TG) rich lipoprotein particles. Abnormalities in normal LPL function are associated with the risk of coronary artery disease (CAD). A number of genetic variants have been identified in the LPL gene that affects different functions of the LPL protein. A common HindIII polymorphism in intron 8 (T/ G) of the LPL gene has been found to be associated with altered plasma TG and HDL-cholesterol, and CAD risk in several studies, but its functional significance is unknown. It has been shown that certain intronic sequence contain regulatory elements that are important for transcription and translational regulation of a gene. In this study we tested the hypothesis that this polymorphism affects the binding site of a transcription factor that regulates the transcription of LPL gene. Electrophoretic mobility shift assays revealed that the HindIII site binds to a transcription factor and that the mutant allele has lower binding affinity than the wild type allele. Transcription assays containing the entire intron 8 sequence along with full-length human LPL promoter were carried out in COS-1 and human vascular smooth muscle cells. The mutant allele was associated with significantly decreased luciferase expression level compared to the wild type allele in both the muscle (3.394 ± 0.022 vs. 4.184 ± 0.028; P=4.7 × 10 −6 ) and COS-1 (11.603 ± 0.409 vs. 14.373 ± 1.096; P<0.0001) cells. In conclusion, this study demonstrates for the first time that the polymorphic HindIII site in the LPL gene is functional because it affects the binding of a transcription factor and it also has an impact on LPL expression.

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Endothelial lipase (EL) plays a pivotal role in HDL metabolism. We sought to characterize EL and its interaction with HDL as well as its natural variants genetically, functionally and structurally. We screened our biethnic population sample (n = 802) for selected missense mutations (n = 5) and identified T111I as the only common variant. Multiple linear regression analyses in Hispanic subjects revealed an unexpected association between T111I and elevated LDL-C (p-value = 0.012) and total cholesterol (p-value = 0.004). We examined lipase activity of selected missense mutants (n = 10) and found different impacts on EL function, ranging from normal to complete loss of activity. EL-HDL lipidomic analyses indicated that EL has a defined remodeling of HDL without exhaustion of the substrate and a distinct and preference for several fatty acids that are lipid mediators and known for their potent pro- and anti-inflammatory properties. Structural studies using homology modeling revealed a novel α/β motif in the C-domain, unique to EL. The EL dimer was found to have the flexibility to expand and to bind various sizes of HDL particles. The likely impact of the all known missense mutations (n = 18) on the structure of EL was examined using molecular modeling and the impact they may have on EL lipase activity using a novel structure-function slope based on their structural free energy differences. The results of this multidisciplinary approach delineated the impact of EL and its variants on HDL. Moreover, the results suggested EL to have the capacity to modulate vascular health through its role in fatty acid-based signaling pathways.

Endothelial lipase (LIPG) and zinc finger protein 202 (ZNF202) are two pivotal genes in high density lipoprotein (HDL metabolism). We sought to determine their genetic contribution to variation in HDL-cholesterol levels by comprehensive resequencing of both genes in 235 individuals with high or low HDL-C levels. The selected subjects were 141 Whites (High HDL Group: n = 68, xMathClass-op¯=76.90mg/dl; Low HDL Group: n = 73, xMathClass-op¯=32.55mg/dl) and 94 Hispanics (High HDL Group: n = 46, xMathClass-op¯=74.85mg/dl; Low HDL Group: n = 48, xMathClass-op¯=29.95mg/dl). We identified a total of 185 and 122 sequence variants in LIPG and ZNF202, respectively. We found only two missense variants in LIPG (T111I and N396S) and two in ZNF202 (A154V and K259E). In both genes, there were several variants unique to either the low or high HDL group. For LIPG, the proportion of unique variants differed between the high and low HDL groups in both Whites (p = 0.022) and Hispanics (p = 0.017), but for ZNF202 this difference was observed only in Hispanics (p = 0.021). We also identified a common haplotype in ZNF202 among Whites that was significantly associated with the high HDL group (p = 0.013). These findings provide insights into the genetics of LIPG and ZNF202, and suggest that sequence variants occurring with high frequency in non-exonic regions may play a prominent role in modulating HDL-C levels in the general population.