| Objective To establish the routine diagnostic methods of TNF-alpha and its receptors gene polymorphisms using PCR-HRM assay for performing a case-control study between the TNF-alpha and its receptors gene loci (-238,-308,-857,-863, R1-383and R2-exon) with the susceptibility of Rheumatoid Arthritis(RA) and the association of serological indicators.Methods The research subjects were Han population in northwest China (493patients of RA and275healthy controls), the genomic DNA of which was extracted from peripheral venous blood. The polymorphisms of TNF-alpha and its receptors gene loci (-238,-308,-857,-863, R1-383and R2-exon) were genotyped using PCR-HRM assay. The distributions of six SNPs were analyzed using SPSS17.0and the online analysis software SHEsis between RA with control groups, and RA groups stratified based on gender, and serological indicators.Results (1)There were obvious differences in TNFA-308GA genotype and A allele frequencies between RA patients with healthy controls(x2=4.764, P=0.029, OR=0.578; x2=4.540, P=0.033, OR=0.594). The differences were found mainly in female (χ2=4.334, P=0.031, OR=0.508;χ2=4.143, P=0.042, OR=0.526), rather than in male (x2=0.559, P=0.454, OR=0.729; χ2=0.529, P=0.467, OR=0.741) after having stratified based on gender. The frequencies of GA genotype and A allele in the ACPA-positive RA group were significantly lower than in the healthy controls(χ2=5.244,P=0.022, OR=0.517; χ2=4.993, P=0.025, OR=0.533), no difference was found in the ACPA-negative RA group(x2=2.264, P=0.132, OR=0.543; χ2=2.139, P=1.144, OR=0.560). The frequencies of GA genotype and A allele in the RF-positive RA patients were significantly lower than in the healthy controls(χ2=4.067,P=0.044, OR=0.565; x2=3.867, P=0.049, OR=0.582), no difference was found in the RF-negative RA group(χ2=2.033, P=0.154, OR=0.607; χ:=1.921, P=0.166, OR=0.623).(2)The frequency of TNFA-857CT+TT in the RA patients was higher than in the healthy controls(χ2=6.231, P=0.013, OR=1.461), and no difference was found of T allele(χ2=2.623, P=0.105, OR=1.219). No differences of genotypes and alleles were found between female RA patients with female controls (χ2=3.576, P=0.059, OR=1.471; χ2=1.263, P=0.261, OR=1.203), neither in male (χ2=2.182,P=0.140, OR=1.448; χ2=1.090. P=0.296, OR=1.233). The frequencies of CT+TT in the ACPA-positive RA group and ACPA-negative RA group were both higher than in the healthy group(χ2=6.776,P=0.009. OR=1.540;χ2=4.651, P=0.031, OR=1.614), no differences were found of T allele(χ2=2.862, P=0.091, OR=1.540; χ2=3.047,P=0.081, OR=1.355). No differences were found of CT+TT and T between the RF-positive RA and healthy groups(χ2=2.634. P=0.105, OR=1.309; χ2==0.619, P=0.431, OR=1.111), the frequencies of CT+TT and T in the RF-negative RA group were higher than in the healthy group (χ2=9.439,P=0.002,OR=1.856; χ2=6.452, P=0.011, OR=1.487).(3)The frequencies of TNFA-863CA genotype and A allele were higher in female controls than in female RA patients (χ2=5.903, P=0.015, OR=0.519; χ2=5.479, P=0.019, OR=0.548), and no difference was found in male (x2=0.382, P=0.536, OR=1.242; χ2=0.352,P=0.553, OR=1.221). No differences were found between RA patients, ACPA-positive/negative RA, RF-positive/negative RA with healthy group (P>0.05).(4)No differences in TNFR2-exon genotypes and alleles were found between RA patients with healthy controls (P>0.05), either when stratified by gender or RF (P>0.05). The frequencies of TNFR2-exon TG+GG genotype and G allele were lower in ACPA-negative RA than in healthy group (x2=4.541,P=0.033OR=0.584; x2=3-990, P=0.046, OR=0.633), no difference was found in ACPA-positive RA (P>0.05).(5)There were no statistical differences in the frequencies of TNFR1-383and TNFA-238gene polymorphisms between RA patients with healthy controls (P>0.05), either when stratified by gender, ACPA, or RF (P>0.05).(6)The loci of TNFA-238,-308,-857and-863formed5haplotypes, and only the frequency of G-G-C-A haplotype was obviously different between RA patients and healthy controls (χ2=14.561,P<0.001,OR=0.403).Conclusion TNFA-308and TNFA-857are associated with RA susceptibility, while TNFA-863are only associated with RA susceptibility in female; The G-G-C-A haplotype formed by TNFA-308,-238,-238and-863SNPs is against RA occurrence; TNFA-308, TNFA-857and TNFR2-exon are associated with the status of ACPA and RF in RA patients; TNFA-238and TNFR1-383gene polymorphisms are not associated with RA susceptibility and the status of serological indicators. |
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