LPL Gene Polymorphism Is Associated With Lipid Levels And Coronary Heart Disease Risk Analysis And Research Related Functions

Part1:Association of SNPs in lipoprotein lipase gene with lipid levels and coronary heart disease risk in Chinese Han populationBackground and objective:Several single nucleotide polymorphisms (SNP) in the lipoprotein lipase(LPL) gene have been reported to be associated with lipid levels and coronary heart disease (CHD) risk in recent genome wide association studies (GWAS). Some studies have also reported the effect of interaction between LPL SNPs and smoking on influencing lipid levels and CHD risk. However, those studies were rarely reported in Chinese populations. The aim of this study was to examine the associations of LPL SNPs with lipid levels and risk of CHD in Chinese Han population and evaluate the SNP-smoking interaction.Materials and Methods:Genome wide genotyping have been conducted in1,515CHD cases and5,019controls, and118SNPs in and around LPL were analyzed in this study. SAS, PLINK and R software packages were used in the analyses. To detect the potential interaction, an extra interaction term for SNP and smoking was included in the regression model.To give a further detection of the relationship between rs2197089and lipid levels, an independent sample consisting of4320individuals were genotyped for rs2197089using TaqMan assays. A total of9339samples were analyzed for the association between rs2197089and lipid levels.Results:The118SNPs analyzed in this study were all in Hardy-Weinberg equilibrium. After adjusting for age and sex, we found that rs263and rs316as well as the SNPs in strong linkage disequilibrium (LD) with them were associated with CHD risk under the additive model. SNPs in3’region and downstream were found to interact with smoking to influence CHD risk. In subgroup analyses, two independent SNPs, rs11570892and rs10503669were associated with CHD in smokers (P=0.022and0.044, respectively), but not in nonsmokers. Haplotype analyses found that rs263T-rs316A was marginally associated with CHD risk (P=0.0576). Haplotype rs11570892A-rs10503669A and rs11570892G-rs10503669C significantly interact with smoking on CHD risk (P=0.0306and0.0265,respectively).The previous reported lipid-related SNPs in GWAS,such as rs2083637, rs325, rs326, rs328, rs10096633, rs12678919, rs4406409, rs17482753and rs10503669were successfully replicated in the5019control samples (P values from1.8×10-2to7.5×10-9). In analysis of a larger sample consisting of9339individuals, we observed significant association between rs2197089and decreased TG (P=0.0006), but not HDL-C level (P=0.0881). Evidence of interaction between cigarette smoking and rs2197089was detected (P=0.0362). In smokers, significant association between rs2197089and increased HDL-C level was found (P=0.0068).Conclusions:The results of the present study suggested that LPL SNPs were associated with CHD risk and lipid levels. LPL SNPs may influence susceptibility to CHD through interacting with smoking. The association between rs2197089and HDL-C may be influenced by smoking in the Chinese Han population. Part2:Functional study of the lipoprotein lipase gene SNPsBackground and objective:The GWAS reported SNPs were mostly located in non-coding regions. For example, the lipid-associated SNPs of the LPL gene were mainly in introns and3’downstream region. Whether the reported SNPs are functional variants or just genetic markers in LD with the causal variants is unknown. SNPs in non-coding regions are more likely to affect gene regulation or pre-mRNA splicing or mRNA stability. SNPs in evolutionary conserved regions or transcription factor binding sites are more likely to be functional. Those located in microRN A binding sites have also been reported to affect disease susceptibility. In this study, we evaluated the functionality of LPL SNPs using bioinformatics and experimental methods.Materials and Methods:LPL gene sequence and SNP information were searched in NCBI, UCSC, HapMap and1000genome project databases. VISTA Genome Browser and UCSC Genome Browser were used to identify evolutionary conserved regions and transcription factor binding sites in LPL gene region. SNPs in these evolutionary conserved regions or transcription factor binding sites were searched. eQTL analysis was conducted for LPL SNPs. Patrocles, MicroSNiPer and miRanda were used to look for microRNA binding sites in the3’-UTR of LPL and identify SNPs in the binding sites. Finally, expression studies using real-time PCR and western blot analysis were carried out in peripheral blood mononuclear cells (PBMC), and luciferase reporter assays were conducted to test and verify the important predicted results.Results:Two genomic regions in3’downstream of LPL, from19832kb to19833kb and19845kb to19846kb (NCBI37/hg19), were found to be highly conserved. Two lipid-related SNPs, rs17482753and rs7841189, were located in these conserved regions respectively, which indicated the potential functionality of these two SNPs in transcription regulation. eQTL analysis found that14SNPs in the LPL region were associated with LPL expression, including some lipid-related SNPs such as rs1800590(T-93G) in the promoter and rs1059611in the3’-UTR. The expression study showed that rs1059611was associated with LPL mRNA and protein expression in PBMC (P=0.0334and0.0167, respectively). Several SNPs were found to be located in microRNA binding sites. The lipid-related and gene expression related SNP rs1059611was located in the binding site of miR-136, which was predicted by the three methods. The3’-UTR luciferase reporter assays showed that miR-136mimic transfected were functional, but no difference was observed for the effects of the rs1059611T and C alleles.Conclusions:Two lipid-related SNPs, rs17482753and rs7841189, may have potential functionality in transcription regulation. Several lipid-associated SNPs in the LPL region were associated with LPL expression and rs1059611was associated with LPL expression in PBMC. miR-136was shown to bind to the3’-UTR of LPL. Further studies are still needed to evaluate the functionality of SNPs in LPL.