The Analysis Of HLA-DR4 Phenotype And Polymorphism Of TNFα-308,TNFRII+196 In Rheumatoid Arthritis

Backgroud:Rheumatoid Arthritis(RA) is an autoimmune disease which etiology is unknown. Heredity and immunological regulation have played significant roles in pathogenesis. Special human leucocyte antigen(HLA) which was confronted with external stimuli or altered by itself had been changed by shared epitope and dosage effect of gene. Nucleated cells who carried HLA become the target of immunological activation and T cell was activated, then a series of immunogenic molecules were released such as cytokines which induced the occurrence of RA. So we want to keep up investigating furthurly if the interaction among HLA-DR₄ phenotype and Tumor Necrosis Factor(TNF) and its receptor Tumor Necrosis Factor Recepor(TNFR) by Single Nucleotide Polymorphism (SNP) could influence the occurrence of RA.Objective:By investigating the interactions among HLA-DR₄ phenotype and TNF and TNFR by SNP, we want to acquire the information about the effects of three genes on RA risk of onset and concentration of cytokines and clinical manifestation which could help us to apprehend furthurly the pathogenesis of RA.Methods:Our study objects were 112 RA patients and 129 Healthy Subjects. Genomic DNA of white blood cell were extracted, then PCR-SSP was used to determine phenotype of HLA-DR₄ and PCR-RFLP were used to genotype for the A/G polymorphism at -308 position of TNF-αgene and the T/G polymorphism at +196 position of TNFRâ…¡gene. Levels of TNF-α,sTNFRâ…¡were measured by using ELISA. Finally clinical features of RA patients including sex,age,disease duration,VAS of rest pain,tender joints of 28,swollen joints of 28,grip,time of morning stiffness,ESR,CRP,RF,function of joints,radiologiucal stages were measured.Results:(1) The difference in positive rate of HLA-DR₄ phenotype between RA patients (50%) and healthy subjects (26.36%) had statistical significance (P=0.000). Positive HLA-DR₄ increased risk of onset in RA patients 2.794 times than that in healthy subjects[95%Confidence Index(CI):1.630-4.790]. Stratified analysis for sex showed Positive HLA-DR₄ increased risk of onset in female RA patients 3.556 times than that in female healthy subjects [95%CI: 1.811 -6.979].(2) The difference of composition in allelotype AA/GA/GG (0/8/104 vs 2/23/104, P=0.010),rare allelotype (AA+GA)/GG (8/104 vs 25/104, P=0.006) and allele A/G (8/216 vs 27/231, P = 0.004) on TNFα-308 between RA patients and healthy subjects had statistical significance. Rare allelotype (AA+GA) and allele A on TNFα-308 increased risk of onset in RA patients 3.125 times (95%CI: 1.347-7.248) and 3.156 times (95%CI: 1.403-7.097) than that in healthy subjects. Stratified analysis for sex showed the difference of composition in allelotype AA/GA/GG (0/2/34 vs 1/12/32, P=0.015),rare allelotype (AA+GA)/GG (2/34 vs 13/32, P=0.007) and allele A/G (2/70 vs 14/76, P=0.007) on TNFα-308 between male RA patients and male healthy subjects still existed. Rare allelotype (AA+GA) and allele A/G on TNFα-308 increased risk of onset in male RA patients 6.906 times (95%CI: 1.444-33.031) and 6.447 times (95%CI: 1.415-29.383) than that in male healthy subjects .(3) The difference of composition in allelotype GG/TG/TT (5/62/45 vs 9/56/64),rare allelotype (GG+TG)/TT (67/45 vs 65/64) and allele G/T (72/152 vs 74/184) on TNFRâ…¡+196 between RA patients and healthy subjects had no statistical significance. Stratified analysis for sex showed the difference in allelotype GG/TG/TT (3/49/24 vs 8/28/48, P=0.000) on TNFRâ…¡+196 between female RA patients and female healthy subjects had statistical significance. The difference in rare allelotype (GG+TG) /TT on TNFRâ…¡+196 between female RA patients and female healthy subjects (52/24 vs 36/48, P=0.001) and between male RA patients and male healthy subjects (15/21 vs 29/16, P=0.041) had statistical significance,which increased risk of onset in female RA patients 2.889 times(95%CI: 1.510-5.526) than that in female healthy subjects and lowered risk of onset in male RA patients 0.394 times (95%CI: 0.160-0.970) than that in male healthy subjects. However, there was no statistical significancant difference in allele G/T on TNFRâ…¡+196 between RA patients and healthy subjects.(4) Among 5 kinds of allele genotypes combination with HLA-DR₄ and TNFα-308 ,HLA-DR₄(+)TNFα-308(GG) increased risk of onset in RA patients 2.931 times (95%CI:1.642-5.233) than that in HLADR₄(-) TNFα-308(GG) of healthy subjects.(5) Among 6 kinds of allele genotypes combination with HLA-DR₄ and TNFRâ…¡+196, HLA-DR₄(+)TNFRâ…¡+196(TT) and HLA-DR₄(+) TNFRâ…¡+196(TG) increased risk of onset in RA patients 2.476 times (95%CI:1.120-5.473) and 6.136 times (95%CI:2.548-14.775) than that in HLA-DR₄(-)TNFRâ…¡+196(TT) of healthy subjects,HLA- DR₄(+)TNFRâ…¡+196(TG) increased risk of onset in RA patients 4.313 times (95%CI: 1.851-10.050) than that in HLA-DR₄(-)TNFRâ…¡+196(TG) of healthy subjects.(6) Among 7 kinds of allele genotypes combination with TNFα-308 and TNFRâ…¡+196, TNFα-308(GA)TNFRâ…¡+196(TT) and TNFα-308(GA) TNFRâ…¡+196(TG) lowered risk of onset in RA patients 0.212 times (95%CI: 0.056-0.804) and 0.282 times (95%CI:0.084-0.945) than that in TNFα-308(GG) TNFRâ…¡+196 (TG) of healthy subjects.(7) Among 11 kinds of allele genotypes combination with HLA-DR₄,TNFα-308 and TNFRâ…¡+196, HLA-DR₄(+)TNFα-308(GG) TNFRâ…¡+196(TT) and HLA-DR₄(+)TNFα-308(GG)TNFRâ…¡+196(TG) increased risk of onset in RA patients 2.64 times (95%CI:1.124-6.202) and 5.8 times (95%CI:2.297- 14.646) than that in HLA-DR₄(-)TNFα-308(GG)TNFRâ…¡+196(TT) of healthy subjects. HLA-DR₄(+)TNFα-308 (GG)TNFRâ…¡+196 (TG) increased risk of onset in RA patients 4 times (95%CI: 1.637-9.775) than that in HLA-DR₄(-)TNFα-308(GG)TNFRâ…¡+196(TG) of healthy subjects.(8) The difference of concentration of serum TNF-α(pg/ml) between RA patients and healthy subjects (34.56±21.04 vs 20.63±15.46,P=0.000) had statistical significance. The difference of TNF-αconcentration between certain allele genotype combination of RA patients and healthy subjects had statistical significance. By analysis of variance of factorial design, interactions existing in mfgene^*tnfrgene(P=0.037),dr₄^*tnfgene ^*tnfrgene(P=0.009) and group(P=0.000),group^*dr₄^*tnfgene(P=0.001) had statistical significant influence to level of TNF-α.(9) The difference of concentration of serum sTNFRâ…¡(ng/ml) between RA patients and healthy subjects (8.54±6.29 vs 5.48±4.41, P=0.000) had statistical significance. The difference of sTNFRâ…¡concentration between certain allele genotype combination of RA patients and healthy subjects had statistical significance. By analysis of variance of factorial design, interactions existing in group(P=0.000),group^*sex ^*dr4(P=0.038),group^*tnfgene(P=0.025),group^*tnfgene^*tnfrgene (P=0.037) had statistical significant influence to level of sTNFRâ…¡.(10) A single allelotype had a certain influence to clinical manifestations of RA. Patients of HLA-DR₄(+) had more earlier onset age (42.59±14.07 vs 47.36±10.89, P=0.047) and higher level (IU/mL)of RF (471.32±435.22 vs 308.13±331.34, P=0.028 ) than those of HLA-DR₄(-).There were no statistical significance between patients of TNFα-308(GG) and TNFα-308(GA). Among patients of three kind of TNFRâ…¡+196 allelotype GG/TG/TT, there were statistical significance in sex composition male/female (2/3 vs 13/49 vs 21/24,P=0.018) and level(IU/mL)of RF(917.00±674.77 vs 353.58±362.85 vs 380.93±365.39, P=0.008).(11) Allele genotypes combination also had a certain influence to clinical manifestations of RA. The difference of onset of disease between HLA-DR₄(+)TNFα-308(GG) and HLA-DR₄(-)TNFα-308(GG) (42.35±14.27 vs 47.71±10.95, P=0.033) had statistical significance. Combinations of TNFRâ…¡+196 with one or two of other Allele only had statistical significance in sex composition.By analysis of variance of factorial design, interactions existing in dr4(P=0.013),tnfrgene (P=0.005),sex^*tnfrgene(P=0.043) had statistical significant influence to level of RF.Conclusions:(1) Positive HLA-DR₄ phenotype increased risk of onset in RA patients especially female RA patients.(2) Polymorphism of TNF-αat position -308 increased risk of onset in RA patients especially male RA patients. (3) The relationship between polymorphism of TNFRâ…¡at position +196 and RA was sophisticated.(4) Certain allele genotype combination of HLA-DR₄,TNFα-308 and TNFRâ…¡+196 altered the risk of onset, TNFα-308(GG) increased slightly,TNFRâ…¡+196(TT) decreased slightly and TNFRâ…¡+196(TG) increased obviously the risk of onset based on HLADR4 phenotype.(5) The difference in level of TNF-αwas related to allele gene,disease,interactions between disease and allele gene. However, the difference in level of sTNFRâ…¡was related to disease,interactions among disease,sex and allele.(6) The influence of Single allelotype and Allele genotypes combination mainly embodyied in some aspcets such as onset age,sex composition and level of RF. Interaction of multi-factor leaded to difference in level of RF.