The Association Between Genetic Variants And Expression Of SLC22A3-LPAL2-LPA And Coronary Artery Disease In The Hans Of Northeast China

PartⅠ The association between genetic variants of the SLC22A3-LPAL2-LPA genecluster and coronary artery disease risk and serum lipid levelPurpose:The case-control study investigated the association between SLC22A3-LPAL2-LPA genecluster polymorphisms (rs9346816, rs2221750, rs3127596, rs9364559, rs1367211, rs6415085,rs9347438and rs9355296) and coronary artery disease (CAD) risk in the Hans of NortheastChina.Methods:551well-characterized CAD cases and544healthy controls were included in this study.They were all recruited from the department of cardiology, second division of First Hospital,Jilin University between2009and2012. All the subjects including the case group and thecontrol group used for this study were Chinese of Han descent, and without bood relationship.Eight SNPs (rs9346816; rs2221750; rs3127596; rs9364559; rs1367211; rs6415085;rs9347438and rs9355296) were genotyped by real-time using the MassARRAY system(Sequenom; USA) in the SLC22A3-LPAL2-LPA gene cluster of Chinese Han sample. And theepidemiological data of all subjects were collected. Characteristics of subjects, alleles,genotypes and serum lipid levels analysis were performed with SPSS16.0software.Haplotype analysis was performed with SHEsis software. The Haploview program (version4.1) was applied to estimate the linkage disequilibrium (LD) measures (D’ and r2) betweenpaired SNPs. We also applied logistic regression analysis to correct the final P values.Results: 1. Compared with healthy controls, CAD patients were more likely to be smokers,hypertension and diabetes mellitus (P<0.05). Additionally, the CAD group had higher levelsof serum total cholesterol, LDL-C, HDL-C and Lp(a)(P<0.05).2. Rs9364559was associated with CAD and remained significant even after adjustmentof the conventional CAD risk factors by forward logistic regression analysis (χ2=4.436,P=0.039). The frequency of minor allele G in rs9364559was significantly higher in CADpatients than that in healthy controls. The genotypic association between rs9364559and CADand remained significant even after adjustment of the conventional CAD risk factors byforward logistic regression analysis (χ2=7.126, P=0.028).3. The haplotype analysis results showed that subjects with different blocks mayassociated with CAD (P<0.05). The haplotype GAAAGTG formed by7SNPs loci wasassociated with significantly reduced risk of CAD, while those with GGAAGTG wereassociated with significantly increased risk of CAD.8haplotypes formed by6SNPs loci wereassociated with increased risk of CAD in this population;6haplotypes formed by6SNPs lociwere associated with increased risk of CAD in this population.21haplotypes formed by5SNPs loci were associated with increased risk of CAD in this population;18haplotypesformed by5SNPs loci were associated with increased risk of CAD in this population.22haplotypes formed by4SNPs loci were associated with increased risk of CAD in thispopulation;27haplotypes formed by4SNPs loci were associated with increased risk of CADin this population.19haplotypes formed by3SNPs loci were associated with increased riskof CAD in this population;20haplotypes formed by3SNPs loci were associated withincreased risk of CAD in this population.3haplotypes formed by2SNPs loci were associatedwith increased risk of CAD in this population;6haplotypes formed by2SNPs loci wereassociated with increased risk of CAD in this population.4. There were significant differences among different genotypes of rs9346816sites onSLC22A3gene for LDL-C, TC level (P<0.05) and of rs6415085sites on LPA gene for Lp(a),LDL-C level (P<0.05). Conclusions:1. Smoking, diabetes mellitus and hypertension may be risk factors of CAD.2. The SLC22A3-LPAL2-LPA gene cluster is strongly associated with CAD risk andserum lipid level in the Hans of Northeast China.3. Rs9364559in the LPA gene may affect risk of CAD in the Hans of Northeast China.4. Many SNPs loci exist at the same time may have different effects on the risk ofcoronary heart disease.Part ⅡThe effect of SLC22A3-LPAL2-LPA gene cluster expression on CAD risk in theHans of Northeast ChinaPurpose:The purpose of this study was to analyze the effect of SLC22A3, LPAL2, LPA geneexpression on CAD risk in the Hans of Northeast China.Methods:First, seven SNPs (rs9346816; rs2221750; rs3127596; rs9364559; rs1367211; rs6415085and rs9347438) in the LPA gene were genotyped using Sequenom MassARRAY time of flightmass spectrometer (TOF) in92CAD patients as case group and32non-CAD subjects ascontrol group. Second, using real-time fluorescent quantitative PCR analyze the mRNAexpression levels of LPA gene in different genotypes of seven loci. All subjects were fromChinese Han ancestry and recruited from the department of cardiology, second division ofFirst Hospital, Jilin University in2012. SPSS16.0was used for above analyses.Results:1. Compared with control group, CAD group had more smokers and more individualswith hypertension (P<0.05). Additionally, compared with control group, CAD group hadhigher level of serum LDL-C, HDL-C and Lp (a)(P<0.05).2. Results showed that compared with control group, the case group has a higher mRNAexpression level of LPA and SLC22A3gene in the Hans of Northeast China (P<0.05).However, there was no difference for the mRNA expression level of LPAL2gene in case andcontrol groups. Additionally, there was no significant difference for the mRNA expression level of SLC22A3, LPAL2and LPA gene among different genotypes of seven loci (rs9346816,rs2221750, rs3127596, rs9364559, rs1367211, rs6415085and rs9347438) in the Hans ofNortheast China.Conclusions:1. Increased mRNA expression level of LPA and SLC22A3gene may be associatedwith CAD risk.2. The possible mechanisms that rs9346816, rs2221750, rs3127596, rs9364559,rs1367211, rs6415085and rs9347438at the SLC22A3-LPAL2-LPA gene cluser affect theoccurrence of CAD are not by influencing SLC22A3, LPAL2and LPA gene mRNA expressionlevels of the patients in the peripheral blood.Part Ⅲ The association between protein expression of SLC22A3-LPAL2-LPA gene cluserand CAD riskPurpose:To indentify the relationship between protein expression level of SLC22A3-LPAL2-LPAgene cluser and CAD; to clarify the effect of genetic variations in SLC22A3-LPAL2-LPA genecluser to SLC22A3protein expression levels.Methods:55unrelated patients with CAD and15controls were recruited from the department ofcardiology, second division of First Hospital, Jilin University in2012. Western blot was usedto test SLC22A3protein levels. Semi-quantitative analysis was carried with Quantity Onesoftware. SPSS16.0software was used to analyze the data.Results:1. SLC22A3protein levels in case group were significant higer than that in control group(P<0.05).2. There are no differences of SLC22A3protein levels between different genotypes ofseven loci (rs9346816, rs2221750, rs3127596, rs9364559, rs1367211, rs6415085andrs9347438) in the Hans of Northeast China (P>0.05).Conclusions: 1. Increased SLC22A3protein levels may be associated with CAD risk.2. Increased LPA and SLC22A3gene mRNA expression levels and SLC22A3proteinlevels of CAD patients in the peripheral blood may be the possible mechanisms thatSLC22A3-LPAL2-LPA gene cluser affect the occurrence of CAD.