| ChapterⅠAssociation study of genetic variations in RP1-276N6.2,TARID gene with coronary artery disease in the Chinese Han populationPurpose:To investigate the association of genetic vatiantions in RP1-276N6.2（rs611950、rs10499313、rs505000）and TARID（rs1966248、rs2327429、rs2327433、rs12190287、rs6569912）with risk of coronary artery disease（CAD）in the Chinese Han population and to explore the causal potentiality of RP1-276N6.2 and TARID in CAD.Methods:This study applied the case-control study method.We collected the cases’and control’s blood,clinical and epidemiological data in the hospitals being strictly accordant with the established inclusion and exclusion criteria.A total of 949 CAD patients and 892 controls were enrolled from the Han population in Chongqing,China.Eight SNPs（rs611950、rs10499313 and rs505000 in RP1-276N6.2,rs1966248、rs2327429、rs2327433、rs12190287 and rs6569912 in TARID）were genotyped based on the Taqman assay method.The Haploview program was employed to calculate the lingkage disequilibrium（LD）parameters（D’and r2）between paired SNPs.Haplotype analysis was performed with SHEsis online tool and Statistical analysis was conducted using SPSS 22.0software.GTEx database was applied to acquire the eQTL analysis results,and R 3.6.0 was used to complete the gene expression level correlation analysis and mapping.Results:1.In the case-control study,the CAD group had higher prevalence of smoking,higher fasting blood glucose and systolic pressure,but lower diastolic pressure,total cholesterol,high density lipoprotein and low density lipoprotein levels and had a lower prevalence of drinking,hypertension and diabetes,and all the above differences were statistically significant（P<0.05）.2.For the association analysis between variants and early-onset CAD,the differences in the frequency distribution of rs611950 genotype and allele in the case group and the control group were statistically significant（P<0.05）,and the T allele of the rs611950 can increase the risk of early-onset CAD than the C allele（OR=1.32,95%CI:1.05-1.66,P=0.02）,the rs10499313 AG genotype carriers’risk of early-onset CAD was higher than AA genotype carriers’（OR=2.11,95%CI:1.31-3.39,P<0.01）,the frequency distribution of rs505000 genotype and allele were lower in the case group compared with that in the control group（P<0.05）,and the allele G of rs505000 can reduce the risk of early-onset CAD than the C allele（OR=0.74,95%CI:0.59-0.93,P=0.01）,and the frequency of rs1966248 AA genotype in the case group was higher than that in the control group（P<0.05）;In the premature CAD and control group,the CG genotype of rs505000 can increase the risk of premature CAD than the CC genotype（OR=1.39,95%CI:1.03-1.89,P=0.03）.In the sex subgroup analysis,the difference in the frequency distribution of rs2327433 genotype bweteen the case and control group of male were statistically significant（P<0.05）,and the G allele of rs2327433can increase the CAD risk than the A allele in male（OR=1.38,95%CI:1.07-1.78,P=0.01）;In female subgroup,the frequency distribution of AG genotype at rs10499313 in CAD group was higher than that in control group（P<0.05）.In the MI group analysis,the G allele of rs505000 can reduce the risk of MI compared with the C allele（OR=0.78,95%CI:0.63-0.98,P=0.04）,and the CC genotype of rs2327429 can reduce the risk of MI than the TT genotype（OR=0.55,95%CI:0.31-0.97,P=0.04）.3.The results of eQTL analysis show that rs611950 genotype was significantly associated with the expression levels of RP1-276N6.2 and the well-known CAD-prone gene SLC22A3 in skeletal muscle and esophageal mucosa(P<1×10-5),and the genotype of three SNPs（rs2327429,rs656991and rs12190287）were correlated with the expression levels of TARID and atheroprotective gene TCF21(P<1×10-5)in coronary artery.4.For the correlation analysis of gene expression level,we found that there was a linear correlation between the expression levels of TARID and TCF21 in vascular tissues(R=0.62,P=6.28×10-67),and the separate analysis results were still statistically significant in carotid artery,tibial artery and coronary artery(R=0.45,P=6.97×10-12;R=0.42,P=2.27×10-13;R=0.50,P=7.52×10-9;respectively).Besides,the expression level of TARID gene in atherosclerotic plaques was significantly lower than that in normal tissues,and the difference was statistically significant(P=5.52×10-4).Conclusions:1.Stratified analysis shows that rs611950,rs10499313,rs505000 and rs1966248 are associated with the risk of early-onset CAD,and the CG genotype of rs505000 significantly increases the risk of late-onset CAD;rs2327433 and rs10499313 are associated with the risk of male CAD and female CAD,respectively;this study also finds that the allele of rs505000is associated with MI,and the CC genotype of rs2327429 can significantly reduce the risk of MI.2.eQTL analysis showes that genotypes of rs611950 in RP1-276N6.2and genotypes of rs2327429,rs6569912 and rs12190287 in TARID are not only correlated with host gene expression levels,but also significantly correlated with nearby CAD susceptibility gene expression levels in different tissues.3.There is a significant correlation between TARID and TCF21expression levels in coronary artery,and TARID is significantly down-regulated in atheromatous plaques.ChapterⅡConstruction of key LncRNA networks related to the pathogenesis of coronary heart disease by weighted gene co-expression network analysis of（WGCNA）Purpose:Applying the bioinformatics analysis mehtod based on weighted gene coexpression network analysis（WGCNA）to screen the key lncRNA genes that may be related to the occurrence and development of coronary heart disease,and analyzed candidate lncRNAs’potential biological functions,so as to provide scientific basis for the screening,diagnosis and treatment of coronary heart disease.Methods:The WGCNA approach was employed to analyze the gene expression profiles in peripheral blood monocytes of 141 CAD patients and controls and divide the differential genes into different modules which were used to estimate the association with clinical data,and the module highly related to CAD were selected to construct the network.The R package clusterprofiler was used to conduct gene ontology（GO）enrichment and KEGG biological pathway enrichment analysis of the selected module.The ceRNA regulatory network was constructed based on open data from four online databases.According to the gene significance（GS）and module membership（MM）,the hub lncRNA genes involved in the ceRNA network were screened,and the receiver operating characteristic（ROC）curve analysis was performed to judge the biomarker characteristics of the candidate lncRNAs.Results:The gene expression data of 48 normal samples and 93 coronary heart disease samples were analyzed,and volcano and heat maps were apllied to visualize the results of gene difference analysis.The expression matrices of5000 differentially expressed genes（including the top 2500 lncRNA and the top 2500 mRNA）and clinical data were used for WGCNA analysis.The results showed that the turquoise module was most related to clinical trait of CAD.In the turquoise module,the biological process of GO analysis indicated that genes were mainly involved in transcriptional regulation and immune responce,and KEGG pathway enrichment analysis showed that genes were mainly enriched in signal pathways related to inflammatory response and immune response.The ceRNA regulatory network was constructed for genes in the turquoise module,and the interaction networks of lncRNA-miRNA-mRNA were visualized using Cytoscape software.Combined with the parameters both gene significance（GS）and module membership（MM）,10 hub lncRNAs which also participating in the ceRNA network were screened.At the same time,ROC analysis indicated that the 10 lncRNAs had potential for biomarkers（AUC>0.9,P<0.05）and might be served as indicators for screening or diagnosis of coronary artery disease.Conclusions:Through WGCNA analysis,a total of 10 key lncRNAs were identified,including DGCR5,LINC00161,LINC00222,LINC00315,LINC00461,LINC00504,PWRN1,DSCR10,LINC00092 and TTTY14,which are likely to regulate the occurrence and development of coronary artery disease by competing endogenouse RNA network.In addition,the abovementioned lncRNAs has good potential for diagnostic markers. |
