Association Analysis Of Single Nucleotide Polymorphism Of TNF-alpha Related Genes With Rheumatoid Arthritis And Its Molecular Mechanism

Objective The single nucleotide polymorphism of TNF-alpha,TNFR1,TNFR2,BAFF,BAFF-R and HLA were analyzed both in Chinese Han RA patients and normal people,and the relationship between gene polymorphism and the laboratory and clinical indicators of RF,CRP,ESR,PLT,HAQ,DAS28,anti CCP antibody and VAS score in RA patients were estimated.In this study,we also explored the mechanism of TNF-alpha activate the B cell through MKK3-p38 MAPK and NF-?B signaling pathway.Methods One hundred and forty RA patients and 149 healthy controls were recruited from the First Affiliated Hospital of Anhui Medical University between Jul 2014 and Mar 2017 in Chinese Han population.Each patient collected 5ml EDTA anticoagulant peripheral vein blood,extracted DNA,using PCR-LDR method to detect TNF-?,TNFR1,TNFR2,BAFF,BAFF-R and HLA gene SNP loci,and applied SHEsis online software for analysis.RF,CRP,ESR,PLT,anti CCP antibody,HAQ,DAS28,VAS scores and other laboratory and clinical indexes were collected,and the correlation between SNP and these indexes were analyzed.Forty RA patients and 40 healthy controls were randomly selected,PBMCs separated from peripheral venous blood were cultured in vitro or direct detected by Western blot and flow cytometry.Serum was isolated and used for the detection of TNF-? level by use ELISA.PBMCs were treated with 100ng/ml TNF-? for 48 h.The supernatant was discarded and the cells were re-suspended with PBS.The percentages of CD19+B cells,CD19+CD27+B cells and CD19+CD20+CD27+ B cells were detected by flow cytometry.To detect the percentage of B cells expressing TNFR2,TNFR2 monoclonal antibody was added and the second antibodies(Anti human CD19-APC antibody,FITC labeled goat anti mouse antibody)were added after they were washed.CD19+TNFR2+B cells were detected by flow cytometry.The MKK3,Phospho-p38 MAPK and Phospho-NF-?B p65 proteins were detected with western blotting method.?-actin was used as normalization.Results(1)In this study we analyzed the genotypes of TNF-alpha,TNFR1,TNFR2,BAFF,BAFF-R and HLA genes SNPs in RA patients and normal controls.We found that the TNFR2 rs1061622 gene G allele frequency was significantly different between the normal population and RA patients(22.8% vs 15.7%,P=0.031,OR=0.631,95%CI:0.414~0.960),however,the other SNPs frequencies between RA patients and normal people were not significantly different.The frequency of TNFR2 gene rs1061622 GG genotype in normal controls was significantly higher than that of RA patients,while the TT genotype was significantly lower than that in RA patients,suggesting that the TNFR2 gene rs1061622 is significantly associated with RA.The frequency distribution of HLA gene in normal control has significant difference(P<0.05),but this SNP was eliminated because it is not consistent with HWE.(2)The relationship between the clinical score and laboratory indexes and the SNPs were analyzed respectively.The MAF distribution of TNFR2 gene rs1061622 locus was significantly associated with the DAS28 score.The G allele frequency of rs1061622 in high and medium were 16.7% and 18.5%,respectively,while the G allele frequency of the low DAS28 group was significantly decreased(4.2%,P=0.012,Bonferroni Test).There is no significant relationship between the other clinical score and laboratory indexes(RF,CRP,ESR,PLT,anti CCP antibody,HAQ,VAS score)and the four SNPs genotypes.(3)The level of TNF-? in RA patients were significantly higher than that in healthy control.The percentages of B cells(CD19+),activated B cells(CD19+CD27+),memory B cells(CD19+CD20+CD27+)and CD19+TNFR2+B cells in RA patients were higher than that in healthy controls.The expression levels of MKK3,Phospho-p38 MAPK and Phospho-NF-?B p65 in B cells of RA patients were significantly higher than those of healthy controls.In TNF-alpha stimulated B cell,the percentages of B cells(CD19+),activated B cells(CD19+CD27+),memory B cell(CD19+CD20+CD27+)and CD19+TNFR2+B cells were significantly increased.And the expression levels of MKK3,Phospho-p38 MAPK and Phospho-NF-?B p65 also raised in the peripheral blood B cells of healthy people.Conclusion The results showed that the frequency of G alleles at the rs1061622 locus of the TNFR2 gene was significantly different between the normal population and the RA patients Chinese Han population.The minor allele frequency distribution of rs1061622 was significantly related to high DAS28 value.Abnormal proliferation and activation of B cells occurred in RA patients.By binding to TNFR2,TNF-alpha promotes the proliferation and differentiation of B cells through the activation of MKK3-p38 MAPK and NF-?B signaling pathway.