Identification And Evaluation Potential Biomarkers Of Rheumatoid Arthritis At Multiple Levels

Rheumatoid arthritis(RA)is a complex systemic autoimmune disease characterized by hyperplastic synovial tissue and destruction of articular cartilage and adjacent bone.Its high disability rate,poor prognosis and low cure rate are heavy burden on society and family.The pathogenesis of RA is complex and the specific factors remain largely unknown.It is believed that the disease is caused by environmental,infection,endocrine and genetic factors,among which genetic factors can explain about 50-60%of the susceptibility of RA.It is an urgent mission for rheumatology to study the pathogenesis and find new targets for early diagnosis and treatment of RA.Circulating cytokines are considered to be the best potential new biomarkers which play critical roles in the complex pathogenesis of RA,but the specific cytokines are still in need of being discovered.Human Cytokine Antibody Array 440 is a reliable method for screening potential biomarkers which is used to identify RA-related cytokines.In addition,GWASs have identified lots of functional genetic loci of RA.These loci can be used as potential biomarkers,which need further screening and evaluation.However,most of them are in non-coding regions,which cause difficulties in clarifying their effects on diseases pathogenesis.Also,till now,the majority of functional SNPs remain unrevealed in RA studies.Therefore,it is necessary to annotate the GWAS results of RA comprehensively.Although GWAS have identified thousands of genetic variants associated with human complex traits.However,the genes or functional DNA elements through which these variants exert their effects on the traits are often unknown.Therefore,a new method(SMR)is needed to synthesize the existing GWAS and eQTL data to identify new RA-related novel causal genes and explore their potential functional significance.Chapter 1Objective:The cytokines play critical roles in the complex pathogenesis of rheumatoid arthritis(RA),but the specific cytokines are still in need of being discovered.This study performed a multi-stage study to identify novel RA cytokines in plasma and further understand the pathological mechanisms of the identified cytokines.Materials and methods:The plasma cytokine protein array profile was evaluated by using Human Cytokine Antibody Array 440 in 18 subjects(RA:healthy control=9:9).ELISA assay was used at the validation stage in 80 subjects(RA:healthy control=40:40).Further functional experiments on RA fibroblast-like synoviocytes(RA-FLS)and T lymphocytes(Jurkat cell)were performed to identify the pathological mechanisms of the highlighted PDGF-BB for RA.Receiver operating characteristics(ROC)curve and Area under curve(AUC)was used to evaluation cytokines.Results:A total of 7 significant cytokines(IL-10,TNF-α,MMP-3,IL-18,IL-7,IFN-γand PDGF-BB)differentially expressed between RA patients and controls(fold change>2,P value<0.05)(all up-regulated in RA patients).The expression level of plasma PDGF-BB was higher in RA patients than in controls(P=0.005).Further,the PDGF-BB obviously prompts cell proliferation of MH7A cell,probably by inhibiting cell apoptosis and accelerating the cell cycle.The PDGF-BB can promote MH7A cell migration.The PDGF-BB has functional effects of on Jurkat cells,but probably weaker than on MH7A cells.PDGF-BB can promote Jurkat cell proliferation and the secretion of inflammatory factors,however,it has no obvious effect on cell apoptosis,cycle and activation of Jurkat cell.The AUC was only 0.587 which indicate PDGF-BB has lower validity.Conclusion:This study evaluated the plasma cytokine protein array profile associated with RA and highlighted the importance of PDGF-BB.PDGF-BB can affect T lymphocytes bioactivities by promoting Jurkat cell proliferation and the secretion of inflammatory factors.PDGF-BB can also influence the pathogenesis and progress of RA by promoting the proliferation and migration of synovial cells,inhibiting apoptosis and changing the cycle process.However,PDGF-BB was not a suitable biomarker of RA owing to the low diagnostic value.Chapter 2Objective:Obtain RA associated SNPs from GWASs and conduct comprehensive functional annotation for all susceptibility loci to clarify their biological functions.Materials and methods:RA associated GWAS data was download from a public database and SNPs with P<5×10-8 were selected,and then the SNPs were annotated with UCSC to get their genomic region information.For SNPs in coding sequence,we predicted the potential effect of missense SNPs on protein functions using PROVEAN,SIFT and Polyphen2.For SNPs located in 3’-UTR,we predicted the miRNAs binding affinity with the UTR sequences using MirSNP database.For SNPs located in promoters and enhancers,we detected whether the promoter and enhancer SNPs would affect transcription factors(TFs)binding using the SNP2TFBS database.We also investigated which TFs they were enriched for disruption and our in-house samples were used to check whether these enriched TFs were differentially expressed.Results:a total of 11179 Asian-specific and 22959 Europe-specific SNPs were annotated.19 Asian-specific and 32 Europe-specific SNPs were predicted to be damaging by at least one algorithm,rs3819268,rs34536443and rs14398 were predicted to be damaging by all three algorithms.36 Asian-specific and 37 Europe-specific SNPs were predicted to be involved in microRNA targets.Asian-specific SNP rs9104 may regulate the target gene BTN3A2 by binding to hsa-microRNA 4474-5p.European-specific SNPs 1573298 may regulate TRIM10 gene by binding with hsa-mir-664a-3p.a total of 9 Asian-specific and 43 Europe-specific SNPs affect transcription factor binding in promoter regions.For SNPs in intronic region,we identified 1162 and 3261 SNPs were located in the enhancer regions from Asian and Europe population,respectively.Among these SNPs,281 Asian-specific and 839 Europe-specific SNPs might regulate enhancer activity through affecting TFs binding.These promoter SNPs were enriched for disruption of 7/7 TFs and enhancer SNPs were enriched of 8/21 TFs in Asian and Europe population,respectively.6 Asian-specific and 8 European-specific enriched TFs can be validated in our in-house samples(RA patients and controls).Conclusion:We identified 19 missense SNPs,36 3’UTR SNPs,9 promoter SNPs and 281 enhancer SNPs in Asian populations and six corresponding genes were differentially expressed in RA case-control groups.We also identified 32 missense SNPs,37 3’UTR SNPs,43 promoter SNPs and 839 enhancer SNPs in European populations and eight corresponding genes were differentially expressed in RA case-control groups.Chapter 3Objective:To identify RA associated causal genes using SMR and explore the biological function.Materials and methods:RA associated GWAS data was download from a public database,eQTL summary data include Westra eQTL summary data,CAGE eQTL summary data,GTEx-brain eQTL summary data and Geuvadis eQTL summary data.SMR was used to integrate GWAS and eQTLs to identify genes whose expression levels are associated with RA.GO,KEGG and protein-protein interactin(PPI)analysis were conducted to investigate the biological function of genes.Furthermore,the genes were validated in 3 GEO databases and our in-house RA patients and controls.Results:We prioritize 8,34 and 32 RA-related susceptibility genes from Asian,European and trans-ethnic populations using SMR method.Among these genes,2 Asian-specific,18 European-specific and 9 trans-racial genes are not the nearest annotated gene to the top associated GWAS SNP.A total of 42 genes are validated in the GEO database and our in-house samples and 11 genes are highest validated,including 2 Asian-specific(PADI4 and HLA-DQB1),6 European-specific(FCRL3,FADS1,SYNGR1,TRAF1,ABCF1 and CD40)and 9 trans-racial genes(IFNAR2,CD226,FADS1,FCRL3,ABCF1,SYNGR1,FLOT1,CD40 and PADI4)Six of them(FCRL3,FADS1,SYNGR1,TRAF1,IFNAR2 and CD226)are not top genes in GWAS studies(P>5×10-8).IFNAR2 is a new candidate.In addition,Three Asian-specific,eight European-specific and nine trans-racial genes were not validated in GEO databases and three Asian-specific,eight European-specific and nine trans-racial genes were not validated in GEO databases and internal samples.Conclusion:in this study,8,34 and 32 RA-related susceptibility genes were screened from Asian,European and trans-ethnic populations by SMR.A total of 42 genes were verified in 3 GEO databases and our in-house sample and 11 genes were highly validated,including 2 Asian-specific,6 European-specific and 9 trans-ethnic genes.among them,FCRL3、FADS 1、SYNGR1、TRAF1、IFNAR2 and CD226 were not the genes nearest the top GWAS SNPs.These results greatly improve our understanding of genetic susceptibility to RA;provide important insights into the ethnogenetic homogeneity and heterogeneity of RA in two races.