| Backcground & ObjectiveRheumatoid arthritis (RA) is an autoimmune disease characterized by arthrosynovitis, with chronic, symmetrical, multi-arthritis and extra-articular synovial lesions as the major clinical manifestations. Persistent recurrent synovitis may lead to intra-articular cartilage and bone destruction, joint dysfunction, or even disability. Arthritis often occurs in the hand, wrist, feet and other small joints, breaking out repeatedly and symmetrically. Vasculitis lesions involve systemic organs. So the disease is also known as rheumatoid arthritis. RA distribute wordwidely, afflicting up to 1%-2% of the population abroad. Initial investigations revealed that the incidence rate of RA ranges from 0.32%-0.34% in our country, lower than abroad. Due to the large population, RA was estimated to afflict a total of 3.6 million chinese people, cause enormous social and economic losses, and it is the main contribution to labour loss and disability. Although the etiology of RA is still obscure, heredity, infection, environmental, hormone and their interactions contribute a lot to RA pathogenesis. It was reported that genetic factors accounted for 50%-60% of the total susceptibility.Major histocompatibility complex(MHC) has been regarded as the most relevant genetic factors. With the end of the HapMap project, it was recognized that the single nucleotide polymorphism (SNPs) is the most frequent variation of human genome, and it became increasingly appealing to screening the SNP associated with RA susceptibility. Single nucleotide polymorphism refers to the single nucleotide variations of variation frequency greater than 1% in the genome, including base substitution, transversion, deletion and insertion. SNP distributed fairly widely in the genome, recent studies have shown that in the human genome, an SNP occurred to each of 300 base pairs. As technology advances and the application of high-throughput genetic screening and analysis technology, a large number of low-to-moderate risk of the SNP was confirmed to be associated with autoimmune diseases.Recently, a non-synonymous single nucleotide transition of 1858C-T, makes a highly conserved key amino acid of P1 motif at codon 620 changes from arginine to tryptophan, leading to malfunction of Lyp and increasing individual's susceptibility to autoimmune diseases. The protein tyrosine phosphatase, nonreceptor 22 gene (PTPN22) located on chromosome 1p13.3-p13.1 encodes a lymphoid-specific phosphatase(Lyp) that is important in negative control of T-cell activation and in T-cell development. Lyp is an intracellular protein tyrosine phosphatase (PTP) with a molecular weight of 110 kDa that contains an N-terminal catalytic domain and a noncatalytic C-terminus with four proline-rich domains. Lyp is a hematopoietic cell specific PTPS, a member of protein tyrosine phosphatase family. Lyp connects with SH3 domain of CSK protein tyrosine kinase through the C terminal named P1 proline-rich motif, dephosphorizes phosphorized LCK, FYN AND ZAP-70 of SRC family, and meanwhile cooperates with CSK to inhibit activation of T cell, playing an important negative regulatory role in T cell activation signal transduction. Once a variation occurs on the PTPN22 gene and the function of Lyp changes, T cell signal transduction will be blocked, resulting in the autoimmune diseases.2004 Nature genetic journal initially reported the PTPN22 C1858T variant related to the occurrence of type 1 diabetes mellitus (TID). Later, the gene mutation were detected in a variety of autoimmune diseases.2004 Begovich et al [19] reported the association between PTPN22 polymorphism and rheumatoid arthritis (RA). They studied the relationship between the risk gene and RA patients or RA-prone family respectively, and found a statistically significant difference of the risk alleles frequencies in RA patients(28%) and the general population (17%). Further study of HLA-DRB1 grouping did not change the association, demonstrating that PTPN22 polymorphism were independent risk factors for RA. Lee et al [20] found that only RF-positive RA patients were associated with PTPN22 R620W, especially for the 1858T/T homozygotes, but other scholars [21,22]found that such relation existed in both RF-positive and RF-negative RA patients. Smyth et al[23] also reported the link between PTPN22 1858T and Graves' disease and confirmed the association with RA again. Steer et al [24] further found in a study of the UK population that PTPN22 SNP not only related to RA occurrence, but also with RA severity.However, the association of PTPN22 C1858T with RA,1 diabetes and Graves' disease confined to Caucasian populations from Europe and America and the polymorphism dose not exist in the Asian. Hu, et al. genotyped PTPN22 1858C>T polymorphism of 132 cases of SLE patients and 156 healthy controls utilizing PCR-RFLP method and found that there were only C allele in1858 site. Begovich et al. studied 100 Han Chinese and 21 African and found that PTPN22 1858T frequency was close to zero. Zhang et al detected 31 individuals with 1858C/T heterozygous out of 1085 people from 15 Chinese population in 2007 and the frequency of T allele was 1.43%. The distribution of T allele varied remarkablely among different ethnics and the frequency in Han population was zero. Li Zhao, et al. have not detected the presence of T allele Chinese population too. The frequencies of T allele in population of Japan and the Korean Peninsula were analogue to that in Chinese. The distribution difference raised several possibilities on the association between the PTPN22 locus and RA susceptibility in Asian population. Firstly, it is possible that+1858C> T SNP is the only disease susceptibility site in PTPN22 and this SNP is not associated with type 1 diabetes in Asian population. Secondly, other potentially functional variants in PTPN22 may be responsible for the susceptibility to type 1 diabetes in these populations. Lastly, there is a possibility that the PTPN22 locus contains another(unidentified) functional variant in LD with the+1858C>T polymorphism. In order to characterize the extent of linkage disequilibrium(LD) across the gene and determine whether variants other than 1858C>T are susceptible to RA, Carlton et al sequenced the PTPN22 gene in 48 white North Americans with RA and genotyped two large RA case-control sample sets. They identified 15 new SNPs(including rs33996649/788G>A) and provided evidence for two additional SNPs (rs1310182 and rs3811021) associated with RA, whereas not related to the 1858C>T SNP. They concluded that 1858C>T does not completely explain the association between PTPN22 and RA, since significant differences between cases and controls persisted after the haplotype data were stratified by R620W. Kawasaki et al carried out a systemic search for PTPN22 and identified five novel SNPs, but not the+1858C>T SNP. Of these two frequent SNPs,-1123G>C, and +2740C> T were in strong linkage disequilibrium (LD), and the -1123G>C promoter SNP was associated with acute-onset but not slow-onset type 1 diabetes in the Japanese population. This association had been confirmed in population from Korea and Caucasus and it seemed that the -1123G>C SNP transmit to offspring more readily than +1858C>T SNP. So we believe that there is new SNP associated with RA susceptibility independent of +1858C>T. Due to the association of PTPN22+1858C>T with RA and the yellow-skin genetic background of Chinese, four PTPN22 SNPs previously proposed to be associated with autoimmune diseases were selected for genotyping:rs2488457 (promoter,-1123G>C), rs2476601 (exon 14,1858C>T), rs33996649(788G>A) and rs1310181 (intron 16). All the SNPs were tested using PCR-RFLP analysis, followed by agarose gel electrophoresis to study the genotype distribution of the four SNPs and analyze the association of these SNPs with RA pathogenesis in Chinese Hans of Guangdong province. Meanwhile, the promoter SNP-1123G>C was investigated to see if its genotypes are correlated to the expression level of PTPN22 in PBMC. Serum RF, Anti-ccp and GPI are detected jointly to study its early diagnostic value and specificity for clinical RA, early diagnosis, therapy.Method:1. SubjectFor a case-control design, we studied 494 patients with RA (75 males and 420 females, mean age:44.99±0.65) and 496 unrelated healthy controls (99 males and 397 females, mean age:43.52±0.60), of similar sex, age(±5 years) and ethnicity. All samples were collected from Laboratory department of Nanfang Hospital. Guangzhou. Patients were diagnosed according to the American Rheumatism Association 1987 revised criteria for the classification of RA. The subjects are all from Guangdong Han population, with no genetic relationship. Age and gender difference between groups was not statistically significant(p>0.05). healthy control with autoimmune diseases were excluded.2.Research method2.1 Genotyping of the three SNPs in case-control groupsThree PTPN22 SNPs previously proposed to be associated with autoimmune diseases were selected for genotyping:rs2488457 (promoter,-1123G>C), rs33996649(788G>A) and rs1310181(intron 16). All the SNPs were tested using PCR-RFLP analysis, followed by agarose gel electrophoresis. primer premier 5.0 was used to design primers for these SNPs according to the sequence around the site.2.2 To quantify PTPN22 mRNA expression relatively by flourescent dye SYBR Green methodA pair of primers, which covered the exonl/exon2 boundary, were designed to detect most of the variants of mRNA. The assay for the PTPN22 gene was analyzed relative to the endogenous control assay using the 2-ΔΔCt method.2.3 Assay of GPI in RA patient serumSerum was collected and divided into three groups according to genotype of patients, plus healthy control group and non-RA autoimmune disease control group. The relative expression level of plasma GPI was detected using ELISA method.2.4 Analysis of clinical indexRA patients were assigned to three groups according to genotypes of PTPN22-1123G>C, comparing distribution of PTPN22-1123G>C genotypes between case and control group, stratified by RF, CRP, anti-CCP and X ray injury.2.5 staticsQuantitative data was expressed as x±s and qualitative data was expressed as rate. Hardy-Weinberg equilibrium were calculated by HAPLOVIEW v3.32. The frequency distribution between cases and controls were compared by Pearson's chi-squared test or Fisher's exact test when suitable, utilizing the SPSS 13.0. Analyses of the expression data were performed by one-way ANOVA using SPSS 13.0. Multiple comparison were made using Bonferroni method or Games-howell method. P<0.05 was considered statistically significant.Result: 1. there wer three genotypes for PTPN22-1123G>C/rs2488457 polymorphic locus, namely CC, CG and GG genotype, distribution frequencies of different genotypes in RA patients and healthy controls were 17.0%,45.7%,37.3%and 15.9%,37.5%, 46.6% respectively, with a significant difference of the overall distribution (x2= 9.964, P= 0.07). Comparing with the wild genotype of GG homozygotes, CG genotype increased the morbidity of RA(1.517,95% CI:1.154-1.995), homozygous CC did not increase the morbidity of RA (1.328,95% CI:0.924-1.909).The three genotypes of rs1310182 polymorphic locus were CC, CT and TT genotype. Distribution frequency of the different genotypes in RA patients and health control group were 2.2%,22.0%,75.8% and 1.6%,25.2%,73.2% respectively. There was no significant difference in the overall distribution (x2= 1.762, P= 0.414). Compared with wild genotype of TT homozygous, CT and CC genotype did not increase the incidence rate of RA.rs33996649 was not detected in our subjects.2. Although no correlation was found between the -1123G>C genotypes and the expression level of the total transcripts when contrasting the three groups according to the genotypes, we observed a gradient growth in means through the groups.3. Concentration of GPI in RA group was significantly higher than that in non-RA control group (P=0.000) and healthy control group (P= 0:000). Concentration of GPI was significantly higher in GG group than that in CC group (P= 0.032). There was on difference between GG group and CG group (P= 0.380) or CC group and CG group (0.833).4. Both RF+ and RF- patients showed difference of PTPN22-1123G>C genotype distribution compared with the control group, indicating PTPN22-1123G> C is relevant to susceptibility of RA, but no direct correlation with RF. Confined to small sample size, we have not yet observed correlation of -1123G>C genotype distribution with status of anti-CCP, ESR, CRP, as well as X-injury.Conclusion:1. PTPN22-1123G> C is associated with the pathogenesis of RA in Han population Guangdong, shows a clear sign of Molecular heterosis and the association of PTPN22 promoter-1123G>C polymorphism with RA may be ubiquitous in Asian. It was reported in the literature that rs1310182 and rs33996649 are associated with RA in Caucasus population but not in Chinese population.2. PTPN22-1123G> C showed correlation with both PTPN22 mRNA expression and serum levels of GPI, moreover, PTPN22 mRNA and serum GPI changed consistently, indicating that the expression of Lyp is associated with GPI concentration.3. We have not found any correlation of PTPN22-1123G>C with other index of RA. With more and more RA patients observed, we will find new evidence for the relation between PTPN22-1123G>C and RA, provide new ideas for elaboration of the pathogenesis, prevention and treatment of RA.
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