| Research backgroundRheumatoid Arthritis(RA) is one of the most common systemic autoimmune disorders,showing the global distribution,afflicting up 0.32-0.36%of the population in our country,estimating five million people in china,causing enormous economy losses,and it is the main reason leads the workers lossing their labor ability and causes disability.The etiology of RA is complex and multifactorial,but genetic factors indicate a significant causal role.The etiology of RA is complex and multifactorial,genetic factors indicate a significant causal role,with heritability being estimated at 60%.Much of the research involving RA susceptibility factors has focused on the major histocompatibility complex(MHC) region on chromosome 6p21,which has been shown to account for approximately one-third of the genetic risk for RA.More recently,linkage data from genome-wide screens or mapping of multicase RA families identified a number of non-MHC chromosomal regions as possible RA susceptibility loci,and analyses of selected candidate genes within such regions revealed several gene variants as being associated with RA in some populations.Recently,a non-MHC gene has repeatedly and convincingly been found to be associated with RA and several other autoimmune diseases,namely the protein tyrosine phosphatase nonreceptor 22(PTPN22) gene,which be identifed as the key susceptibility gene of RA.The PTPN22(protein tyrosine phosphatase nonreceptor22) gene,located on chromosome 1p13,encodes a lymphoid-specific phosphatase(Lyp), including sixteen exons and fifteen introns,the PTPN22 variant(1858C→T) engenders a substitution in Arg620Trp that disrupts Lyp binding to Csk and would thereby predictably reduce or abrogate the inhibitory effect of the Lyp-Csk interaction on T cell activation.The PTPN22 1858T variant may be the etiologic relevance in multiple autoimmune diseases.Although the recent evidence showing that the 1858C→T SNP is susceptibility to multiple autoimmune diseases in European,the frequency of the 1858C→T SNP is almost absence in Asian and African people,showing a obviously genetic heterogeneity.By sequencing 21 exons, 16 introns,and the promoter region of PTPN22 on chromosome 1p13,Kawasaki etal found five novel SNPs,the snp of 1123G＞C(rs2488457) in the promoter region is associated with type 1 diabetes in Asian populations.The PTPN22-1123C variant may be another mutation associates with multiple autoimmune diseases and highly appears.Given the 1858C→T SNP relevants to the susceptibilities of multiple autoimmune diseases in European Caucasian populations,furthermore,both chinese people and japanese people have a similar genetic background,belong to Asia people of yellow race,we assume that the snp of 1123G＞C might also be etiologically relevant to the RA patients of china.Recently,there is no association data about the PTPN22 1123G＞C relevants to RA predisposition in yellow Asia people.To determine whether association of the PTPN22 1123G＞C variant with RA occurs in RA cohorts of the han population in the Guangdong province of china,the frequency of this variant was investigated by denaturing high performance liquid chromatography and DNA sequencing.The mechanism of the PTPN22 1123G＞C influences the transcription activity of PTPN22 is poorly understood.To investigate wheather this variant impacts the transcription activities of PTPN22,fluorescence quantitative PCR was used to assay wheather the gene expression of PTPN22 was difference between those carrying the-1123C risk variant and those not carrying the risk variant.The purpose of this study is to provide a theoretical basis to the deeply research of the exact mechanism how the PTPN22 1123G＞C SNP influences the transcription activities of PTPN22,and provides new ideas to the early diagnosis of RA.Finally,use the polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP) analysis and DNA sequencing technology to detecte the frequency of C1858T polymorphism in RA patients of the han population in the Guangdong provinc of china.Research Methods1 subjectsFor the RA patient cohorts,patients were recruited from South Medical University hospital from March 2007 to December 2008,all patients with RA met the American College of Rheumatology(formerly,the American Rheumatism Association) 1987 revised criteria for RA,includeing 27 males and 121 females,all the patients are from Guangdong province,except 10 people from Hunan Province;4 people from Jiangxi Province;and 3 people from Fujian Province.Control subjects for this study included 41 males and 111 females,none of the healthy anonymous volunteers with other inflammatory arthritis or severe heart,kidney and liver diseases.2 Research Methods2.1 Phenol-chloroform extracts the genomic DNA,designing specific primers,and PCR amplifies the fragment.2.2 Use the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP)analysis and DNA sequencing technology to detecte the C1858T polymorphism of PTPN22 gene. 2.3 Using the denaturing high performance liquid chromatography(DHPLC) method, combined with DNA sequencing technology to detect the G-1123C polymorphism of PTPN22 gene.2.4 Relative risk/odds ratios(ORs) excluded the impact of age and gender were calculated by using standard logisticregression methods and were processed with the SPSS 13.0 software.Subdivision of the RA patients into six groups,such as the group of X-ray positive,X-ray positive,RF factor positive,RF factor negative,age at onset more than 42 years old,or age at on set less than 41 years old,respectively compared to the healthy control group.Stratification nalysis was performed on the RA case cohort to investigate the effect of sex,disease severity(as assessed by the presence of erosive disease),RF status,and age at disease onset.2.5 There are only 52 patients have complete data of clinical characteristics among the 148 cases of clinical diagnosis RA patients.According to the genotype of PTPN22-1123 SNP,RA patients were divided into heterozygous mutations(GC type) RA group,homozygous mutant(CC-type) RA group,as well as the wild-type (GG Type) RA group,respectively assessing clinical similarities among the three different patient cohorts.2.6 We detected the PTPN22 gene expression levels in the patients with mutation and healthy control group to GAPDH housekeeping genes as reference genes by fluorescent dye SYBR Green quantitative PCR,calculating by△CT,using an independent samples t test for statistical analysis,finally,using 2-△△CT to calculate the difference of gene expression between the two groups.2.7 Statistical MethodsThe frequencies of PTPN22 alleles were established by direct counting. Association was evaluated using the chi-square test.Relative risk/odds ratios(ORs) were calculated using standard logistic regression methods and were processed for the analyses using SPSS13.0 statistical software.A one-way ANOVA test was used to determine significance among multiple samples means,and a nonparametric Mann-Whitney test was used for assessing clinical similarities between the three patient cohorts.A P-value less than 0.05 was considered statistically significant. Results1,The PTPN22 gene G-1123C polymorphism is associated with RA in Chinese Guangdong Han population.RA patients were different from healthy control individuals in frequencies of PTPN22 G-1123C genotypes(P=0.016) and carded C allele(P=0.000;OR,1.837;95%CI,1.328～2.541).Excluding the impact of sex and age,logistic regression analysis suggested that there were still statistically significant difference in the frequencies of PTPN22 G-1123C genotype(P= 0.021).The presence of CT heterozygote was very strongly associated with RA((P= 0.006;OR,2.051;95%CI,1.234～3.410),but the CC homozygotes was not(P= 0.427).2,The frequency of PTPN22 gene C1858T polymorphism in Chinese Guangdong han population was 0,only one phenotype CC existed in the148 RA patients and 151 healthy control group members.Therefore,this SNP site was not associated with RA in Chinese Guangdong Han population.3,The PTPN22-1123 allele was association with the presence of RF.The allele frequency of PTPN22G-1123C was association with rheumatoid factor-positive (RF+) RA(P=0.041).4,None of the clinical characteristics was found significant difference among the three RA patient cohorts.All the P-value were more than 0.05.5,The result of real-time fluorescence quantitative PCR showed that the PTPN22 mRNA expression of mutation patient group(including the heterozygous and homozygous mutant-type RA patients) was 3.742 times to the control group (wild-type health control).Conclusion1,The polymorphism of PTPN22 gene C1858T SNP was not associated with RA in Chinese Guangdong Hart population,while the other SNP G-1123C may be related to RA susceptibility,suggesting that the polymorphism of PTPN22 G-1123C was possible another risk allele for RA,the OR of heterozygous mutation is 2.051 times to the control group,but CC homozygotes was no correlation,suggesting molecular heterosis.2,The allele frequency of PTPN22G-1123C was association with the presence of RF, and no association with the effect of sex,disease severity(as assessed by the presence of erosive disease),and age at disease onset.None of the clinical characteristics was found significant difference among the three RA patient cohorts. It is essential that the number of our inception cohort should be expanded in the future studies.3,The PTPN22 mRNA expression of mutant patients group was higher than the wild-type healthy control group,suggesting that it may be a compensatory mechanism to the function defect of mutant patients.Examining the DNA sequence around the -1123G＞C site,we could find it showed its perfect match with that of the binding site for transcription factor activatorprotein 4(AP-4),a member of the basic helix-loop-helix-zipper family of transcription factor on the antisense strand.The -1123G＞C is located at the critical region of the coremotif of the AP-4 binding consensus sequence(「1130-gcaaGCTGaa-1121;core motif is shown in uppercase, the position「-1123G is in bold) and the「-1123C allele did not correspond to binding elements of any known transcription factors.Thus,it is suggested that heterosis at the PTPN22 promoter may be related to the regulation of the expression of Lyp and confered the development of specific disease subphenotype. 4.Our research provides a new way of thinking for the further study of the patheogenesis of RA,and offers a theoretical basis to the further study of the exact mechanism of PTPN22 G-1123C SNP impact LYP expression. |
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