PTPN22-1123G>C与广东地区人群RA易感性及血清学指标的关联性研究
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摘要
研究背景和目的：
类风湿性关节炎(Rheumatoid arthritis, RA)是一种以关节滑膜炎为特征的慢性全身性自身免疫性疾病。以慢性、对称性、多滑膜关节炎和关节外病变为主要临床表现,滑膜炎持久反复发作,可导致关节内软骨和骨的破坏,关节功能障碍,甚至残废。该病好发于手、腕、足等小关节,反复发作,呈对称分布。血管炎病变累及全身各个器官,故本病又称为类风湿病。RA呈全球性分布,国外本病的患病率约为1%～2%,我国初步调查发现患病率为0.32%～0.34%,虽低于国外,但由于我国人口众多,估计患者总数约有360万之多,如此庞大的RA患病人群每年都给我国社会、经济造成巨大的损失,也是造成我国人群丧失劳动力和致残的主要病因之一。RA病因未明,目前多认为遗传、感染、环境和激素四大因素的交互作用是参与。RA发病的重要因素,文献报道遗传因素可以解释的易感性占50-60%。
主要组织相容性抗原复合物(MHC)基因族是目前已知的与RA发病最为紧密的遗传因素。随着HapMap工程的结束,人们认识到人类基因组中最为复杂的变异是单个核苷酸多态性(Single Nucleotide Polymorphisms, SNPs),与RA相关的易感基因的SNP的筛查成为类风湿病学的研究热点。SNP是指在基因组中变异频率大于1%的单核苷酸变异,包括碱基的置换、颠换、缺失和插入。SNP在基因组中分布相当广泛,近来的研究表明在人类基因组中每300碱基对就会出现一次,随着技术进步,高通量基因筛选和分析技术的应用,大量与自身免疫性疾病相关的低到中等危险度的SNP被确认。
最近研究发现,蛋白酪氨酸磷酸酶非受体型22(protein tyrosine phosphatase nonreceptor 22, PTPN22)基因的一个单核苷酸多态性,即1858C转换为1858T(rs2476601),使密码子620编码的P1基序中一个高度保守的关键氨基酸由精氨酸转变为色氨酸(R620W),导致Lyp不能正常发挥作用,增加了个体对自身免疫性疾病的易感性。PTPN22基因定位于1号染色体短臂1区3带(1p13.1-1p13.3)上,编码淋巴细胞特异性的蛋白酪氨酸磷酸酶(lymphoid protein tyrosine phosphatase, Lyp)。LYP从属于蛋白酪氨酸磷酸酶(PTPs)家族,是造血组织细胞特异性的PTPs,LYP主要通过C端命名为P1的富含脯氨酸基序与CSK蛋白酪氨酸激酶的SH3结构域连接,使已磷酸化的Src家族Lck、Fyn和ZAP-70激酶脱磷酸化,同时协同CSK抑制T细胞活化,在T细胞活化的信号传导中起到重要的负调节作用,一旦PTPN22基因发生变异,Lyp的功能或表达量发生改变,可引起T细胞信号传导障碍,导致自身免疫性疾病的发生。
2004年Nature genetic杂志首次报道了PTPN221858T变异和1型糖尿病(typeⅠdiabetes, TID)发生有关,随后在多种自身免疫性疾病中检测到这种基因变异。Begovich等报道了PTPN22变异与类风湿关节炎之间具有重要相关性,他们进一步应用HLA-DRB1基因分型分组研究并没有改变这种联系,说明PTPN22变异是RA的独立发病危险因素。Lee等发现只有RF阳性的RA患者与PTPN22 C1858T相关,并且强烈提示为1858T／T纯合子,但其他学者的研究发现在RF阳性和RF阴性的RA患者中都存在此种相关性。Steer等对英国人群的研究更是发现PTPN22 C1858T不仅与RA的发生有关,并与RA病情的严重程度相关。
然而,PTPN22 C1858T与RA、1型糖尿病及Graves'病等的发病相关性仅仅局限于欧美等高加索人群中,该位点在亚洲人群中并不存在多态性。胡必成等人利用PCR-RFLP基因分型方法对132例SLE患者和156例健康对照PTPN22的1858位的基因型进行分析,发现1858位点均为C等位基因。Begovich等人对100名中国汉族人和21名非洲人的研究发现,PTPN22 1858T的频率为零。Zhang等人2007年对中国15个人群1085位个体的研究共检出31例1858C／T杂合子个体,T等位基因的比例为1.43%,而且种族分布差异很大,汉族人群中T碱基的频率为零。本实验室的赵丽等的研究也未发现中国人群中T碱基的存在。对日本人和朝鲜半岛的人群研究同样未发现T碱基。总体来说,亚洲人群1858位点虽有突变,但未达到多态性水平。有人根据PTPN22 C1858T在不同人群中分布的差异推测：1.C1858T是PTPN22基因中唯一的与自身免疫性疾病相关联的多态性位点,该基因与亚洲人群自身免疫性疾病无相关性。2.PTPN22基因中存在尚未发现的与亚洲人群自身免疫性疾病相关联的多态性位点,这些位点与C1858T连锁。3.PTPN22基因中存在独立于C1858T以外的新的功能性位点。后续的研究逐渐印证了前人的推测,表明C1858T以外的新的功能性位点可能存在。Carlton等对48个北美RA患者PTPN22基因重新测序发现了15个新的SNP,随后在两个独立的样本对照试验中检测了包括新发现的SNP在内的38个位点多态性,发现多个位点的SNP与RA的发病相关。在单倍体分析中,Carlton将携带PTPN22 1858T等位基因的病例去除,发现不包含PTPN22 1858T等位基因的单倍体与RA依然具有相关性,说明PTPN22 C1858T并不能完全解释PTPN22与RA之间的关系。日本学者Kawasaki等通过对TID患者PTPN22全基因扫描的研究发现日本人群有5个新的SNP位点,其中-1123 G>C突变可能是另外一个与自身免疫病发病甚为相关的、出现频率较高的突变位点,在急性发作性TID病人中达54.9%,明显高于正常人群的46.1%,具有显著意义,这在韩国以及高加索人都得到了证实,并且似乎比1858T变异更容易传递到子代。Viken在挪威人群中也发现-1123G>C与RA发病具有显著相关性,而且,-1123G>C与+1858C>T紧密连锁。可见,-1123G>C在西方人群中普遍存在,极有可能是与RA发病相关的又一遗传位点。本课题的前期实验也发现,PTPN22-1123G>C在RA组和健康对照组间的分布有显著性差异。为此我们相信,PTPN22基因中存在独立于C1 858T的新的SNP。
为此,本课题拟增加RA患者病例数,同时我们还选取国外文献中发现的与自身免疫性疾病相关联的新的PTPN22基因SNP位点(rs33996649和rs1310182),采用PCR-RFLP技术结合测序技术来检测这三个位点在RA患者和健康人群中中的分布差异,分析它们与广东地区人群RA发病的相关性；采用荧光定量PCR技术对PTPN22基因的表达水平进行相对定量来了解rs2488457／-1123G>C与基因表达之间的关联；联合RF、anti-CCP、GPI、血沉(ESR)和C-反应蛋白(CRP)等的检测,研究其早期诊断价值及特异性,为临床RA的早期诊断、治疗提供依据。
方法：
1.研究对象
收集广州南方医院风湿科2008年1月～2010年1月门诊及住院RA患者494例,其中男性75例,女419例,年龄3-84岁,平均年龄44.99±0.65岁；所有RA患者均符合1987年美国类风湿病协会(ARA)修订的RA诊断分类标准及2009年ACR/EULAR联合颁布新的RA诊断标准。对照组选择496例健康查体者,男性99例,女性397例,年龄7y82岁,平均年龄：43.52±0.60。研究对象均为广东地区汉族人群,无亲缘关系,两组年龄、性别差异无统计学意义(p>0.05),健康对照组排除了除RA外的自身免疫性疾病患者。
2.研究方法：
2.1采用PCR-RFLP技术结合测序技术检测三种SNP基因型
选取国外文献中新发现与自身免疫性疾病相关联的PTPN22基因SNP位点(rs2488457,rs33996649和rs1310182),采用PCR-RFLP技术结合测序技术来检测这三个位点在两组中的分布。根据SNP位点上是否存在核酸内切酶的酶切位点,利用软件primer premier 5.0设计针对三种SNP靶片段上的引物,对研究对象的DNA进行扩增,用核酸内切酶消化PCR产物,消化产物琼脂糖凝胶电泳观察结果。随机抽取10%测序,与PCR-RFLP结果对比。
2.2采用SYBR Green荧光染料法检测PTPN22基因相对表达量
根据PTPN22 mRNA序列设计扩增引物,以管家基因GAPDH为内参,采用2-△△CT分析方法对PTPN22基因的表达量进行相对定量。比较PTPN22 mRNA的相对表达量在-1123G>C三种基因型之间的差别。
2.3采用ELISA法检测患者血浆GPI水平
收集患者血清,以PTPN22-1123G>C多态性位点将RA分为三组,另外设置健康对照组和非RA的自身免疫性疾病对照组,共5组,用ELISA法检测各组血浆样本中GPI的相对表达量。
2.4临床特征分析
以PTPN22 G-1123C多态性位点将.RA分为三组,同时我们以RF、anti-CCP、血沉(ESR)、C-反应蛋白(CRP)、X线损伤等指标阳性时和阴性时,比较PTPN22 G-1123C多态性位点的基因型分布在病例组和对照组之间的差异。
3.统计方法
应用SPSS 13.0统计软件包处理数据。计量资料用均数±标准差(x±s)表示,计数资料用率表示。RA组及健康对照组三个SNP位点基因型频率经Hardy-Weinberg遗传平衡定律检验样本是否具有群体代表性；三个SNP位点基因型分布以及等位基因频率在两组之间的比较采用R×C列联表的x2检验；对于样本含量<40和理论数<1的计数资料采用列联表的精确概率法；各组间均数比较采用单因素方差分析(one-way ANOVA),方差分析显著时进行多重比较,方差齐采用Bonferroni法,方差不齐采用Games-howell法。以P<0.05为差异有统计学意义；构成比的比较采用卡方分析(Chi-square test)。
结果：
1.PTPN22-1123G>C/rs2488457多态性位点存在三种基因型,分别为CC型、CG型和GG型,不同基因型在RA患者和健康对照组中的分布频率分别为17.0%、45.7%和37.3%以及15.9%、37.5%和46.6%,总体分布有显著性差异(x2=9.964,P=0.01)。与野生型纯合子GG相比,CG型增加发病风险(1.517,95%CI：1.154-1.995,P=0.003),突变型纯合子CC并不增加RA的发病风险(1.328,95%CI：0.924-1.909,P=0.138)。
rs1310182多态性位点的三种基因型,分别为CC型、CT型和TT型,不同基因型在RA患者和健康对照组中的分布频率分别为2.2%、22.0%和75.8%及1.6%、25.2%和73.2%。总体分布无显著性差异(Z=1.762,P=0.414)。与野生型纯合子TT相比,CT和CC型均未增加RA的发病风险。rs33996649多态性位点的检测并未发现突变碱基的存在,该位点不存在多态性。
2.荧光定量PCR结果显示,RA患者组PTPN22 mRNA显著高于健康对照组(P=0.04)：PTPN22 mRNA的表达在三种基因型的RA患者中无显著性差异,但我们观察到PTPN22 mRNA的表达由CC到GG有逐渐上升的趋势。
3.RA组GPI值显著高于非RA对照组(P=0.000)和健康对照组(P=0.000)。三种不同基因型RA比较时,GG组GPI值显著高于CC组(P=0.032),GG组和CG组之间(P=0.380)、CC组和CG组之间(0.833)比较无显著性差别。
4.RF+和RF-患者的PTPN22-1123G>C基因分型与对照组均有差别,表明PTPN22-1123G>C与RA发病易感性具有相关性。我们尚未观察到-1123G>C基因型分布在anti-CCP、ESR、CRP以及X线损伤为阴性和阳性时的差别。
结论：
1. PTPN22 -1123G>C与广东地区汉族人群RA的发病有关联,并且具有明显的杂合子优势,由于CG基因型在RA和对照组中的分布分别为45.7%和37.5%,说明PTPN22-1123G>C可能只是一种低危险度的SNP。PTPN22-1123G>C与中国人群RA易感性的关联可能具有普遍性。rs1310182与中国广东人群RA发病不具有关联性。-33996649与中国广东人群RA发病无关联性。
2. PTPN22-1123G>C可能使PTPN22 mRNA的表达异常,而且,PTPN22 mRNA和血清GPI的变化趋势一致,表明Lyp的表达量与GPI的产生具有相关性。
3.我们尚未发现PTPN22-1123G>C与RA患者anti-CCP、ESR、CRP以及X线损伤之间的关联。
本研究创新点：
1.增加了大量的病例数进一步证实PTPN22-1123G>C多态性与中国广东人群RA发病相关。
2.发现PTPN22-1123G>C多态性可能改变PTPN22启动子的转录调节功能。野生型G等位基因转变为C等位基因后,表达下调。
3.发现PTPN22-1123G>C与RA患者血清中GPI的浓度相关,PTPN22 mRNA水平的变化趋势与血清GPI的水平一致,为探讨Lyp与GPI之间的关系提供依据。
Backcground & Objective
Rheumatoid 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. Subject
For 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 method
2.1 Genotyping of the three SNPs in case-control groups
Three 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 method
A 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 serum
Serum 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 index
RA 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 statics
Quantitative 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.
类风湿性关节炎(Rheumatoid arthritis, RA)是一种以关节滑膜炎为特征的慢性全身性自身免疫性疾病。以慢性、对称性、多滑膜关节炎和关节外病变为主要临床表现,滑膜炎持久反复发作,可导致关节内软骨和骨的破坏,关节功能障碍,甚至残废。该病好发于手、腕、足等小关节,反复发作,呈对称分布。血管炎病变累及全身各个器官,故本病又称为类风湿病。RA呈全球性分布,国外本病的患病率约为1%～2%,我国初步调查发现患病率为0.32%～0.34%,虽低于国外,但由于我国人口众多,估计患者总数约有360万之多,如此庞大的RA患病人群每年都给我国社会、经济造成巨大的损失,也是造成我国人群丧失劳动力和致残的主要病因之一。RA病因未明,目前多认为遗传、感染、环境和激素四大因素的交互作用是参与。RA发病的重要因素,文献报道遗传因素可以解释的易感性占50-60%。
主要组织相容性抗原复合物(MHC)基因族是目前已知的与RA发病最为紧密的遗传因素。随着HapMap工程的结束,人们认识到人类基因组中最为复杂的变异是单个核苷酸多态性(Single Nucleotide Polymorphisms, SNPs),与RA相关的易感基因的SNP的筛查成为类风湿病学的研究热点。SNP是指在基因组中变异频率大于1%的单核苷酸变异,包括碱基的置换、颠换、缺失和插入。SNP在基因组中分布相当广泛,近来的研究表明在人类基因组中每300碱基对就会出现一次,随着技术进步,高通量基因筛选和分析技术的应用,大量与自身免疫性疾病相关的低到中等危险度的SNP被确认。
最近研究发现,蛋白酪氨酸磷酸酶非受体型22(protein tyrosine phosphatase nonreceptor 22, PTPN22)基因的一个单核苷酸多态性,即1858C转换为1858T(rs2476601),使密码子620编码的P1基序中一个高度保守的关键氨基酸由精氨酸转变为色氨酸(R620W),导致Lyp不能正常发挥作用,增加了个体对自身免疫性疾病的易感性。PTPN22基因定位于1号染色体短臂1区3带(1p13.1-1p13.3)上,编码淋巴细胞特异性的蛋白酪氨酸磷酸酶(lymphoid protein tyrosine phosphatase, Lyp)。LYP从属于蛋白酪氨酸磷酸酶(PTPs)家族,是造血组织细胞特异性的PTPs,LYP主要通过C端命名为P1的富含脯氨酸基序与CSK蛋白酪氨酸激酶的SH3结构域连接,使已磷酸化的Src家族Lck、Fyn和ZAP-70激酶脱磷酸化,同时协同CSK抑制T细胞活化,在T细胞活化的信号传导中起到重要的负调节作用,一旦PTPN22基因发生变异,Lyp的功能或表达量发生改变,可引起T细胞信号传导障碍,导致自身免疫性疾病的发生。
2004年Nature genetic杂志首次报道了PTPN221858T变异和1型糖尿病(typeⅠdiabetes, TID)发生有关,随后在多种自身免疫性疾病中检测到这种基因变异。Begovich等报道了PTPN22变异与类风湿关节炎之间具有重要相关性,他们进一步应用HLA-DRB1基因分型分组研究并没有改变这种联系,说明PTPN22变异是RA的独立发病危险因素。Lee等发现只有RF阳性的RA患者与PTPN22 C1858T相关,并且强烈提示为1858T／T纯合子,但其他学者的研究发现在RF阳性和RF阴性的RA患者中都存在此种相关性。Steer等对英国人群的研究更是发现PTPN22 C1858T不仅与RA的发生有关,并与RA病情的严重程度相关。
然而,PTPN22 C1858T与RA、1型糖尿病及Graves'病等的发病相关性仅仅局限于欧美等高加索人群中,该位点在亚洲人群中并不存在多态性。胡必成等人利用PCR-RFLP基因分型方法对132例SLE患者和156例健康对照PTPN22的1858位的基因型进行分析,发现1858位点均为C等位基因。Begovich等人对100名中国汉族人和21名非洲人的研究发现,PTPN22 1858T的频率为零。Zhang等人2007年对中国15个人群1085位个体的研究共检出31例1858C／T杂合子个体,T等位基因的比例为1.43%,而且种族分布差异很大,汉族人群中T碱基的频率为零。本实验室的赵丽等的研究也未发现中国人群中T碱基的存在。对日本人和朝鲜半岛的人群研究同样未发现T碱基。总体来说,亚洲人群1858位点虽有突变,但未达到多态性水平。有人根据PTPN22 C1858T在不同人群中分布的差异推测：1.C1858T是PTPN22基因中唯一的与自身免疫性疾病相关联的多态性位点,该基因与亚洲人群自身免疫性疾病无相关性。2.PTPN22基因中存在尚未发现的与亚洲人群自身免疫性疾病相关联的多态性位点,这些位点与C1858T连锁。3.PTPN22基因中存在独立于C1858T以外的新的功能性位点。后续的研究逐渐印证了前人的推测,表明C1858T以外的新的功能性位点可能存在。Carlton等对48个北美RA患者PTPN22基因重新测序发现了15个新的SNP,随后在两个独立的样本对照试验中检测了包括新发现的SNP在内的38个位点多态性,发现多个位点的SNP与RA的发病相关。在单倍体分析中,Carlton将携带PTPN22 1858T等位基因的病例去除,发现不包含PTPN22 1858T等位基因的单倍体与RA依然具有相关性,说明PTPN22 C1858T并不能完全解释PTPN22与RA之间的关系。日本学者Kawasaki等通过对TID患者PTPN22全基因扫描的研究发现日本人群有5个新的SNP位点,其中-1123 G>C突变可能是另外一个与自身免疫病发病甚为相关的、出现频率较高的突变位点,在急性发作性TID病人中达54.9%,明显高于正常人群的46.1%,具有显著意义,这在韩国以及高加索人都得到了证实,并且似乎比1858T变异更容易传递到子代。Viken在挪威人群中也发现-1123G>C与RA发病具有显著相关性,而且,-1123G>C与+1858C>T紧密连锁。可见,-1123G>C在西方人群中普遍存在,极有可能是与RA发病相关的又一遗传位点。本课题的前期实验也发现,PTPN22-1123G>C在RA组和健康对照组间的分布有显著性差异。为此我们相信,PTPN22基因中存在独立于C1 858T的新的SNP。
为此,本课题拟增加RA患者病例数,同时我们还选取国外文献中发现的与自身免疫性疾病相关联的新的PTPN22基因SNP位点(rs33996649和rs1310182),采用PCR-RFLP技术结合测序技术来检测这三个位点在RA患者和健康人群中中的分布差异,分析它们与广东地区人群RA发病的相关性；采用荧光定量PCR技术对PTPN22基因的表达水平进行相对定量来了解rs2488457／-1123G>C与基因表达之间的关联；联合RF、anti-CCP、GPI、血沉(ESR)和C-反应蛋白(CRP)等的检测,研究其早期诊断价值及特异性,为临床RA的早期诊断、治疗提供依据。
方法：
1.研究对象
收集广州南方医院风湿科2008年1月～2010年1月门诊及住院RA患者494例,其中男性75例,女419例,年龄3-84岁,平均年龄44.99±0.65岁；所有RA患者均符合1987年美国类风湿病协会(ARA)修订的RA诊断分类标准及2009年ACR/EULAR联合颁布新的RA诊断标准。对照组选择496例健康查体者,男性99例,女性397例,年龄7y82岁,平均年龄：43.52±0.60。研究对象均为广东地区汉族人群,无亲缘关系,两组年龄、性别差异无统计学意义(p>0.05),健康对照组排除了除RA外的自身免疫性疾病患者。
2.研究方法：
2.1采用PCR-RFLP技术结合测序技术检测三种SNP基因型
选取国外文献中新发现与自身免疫性疾病相关联的PTPN22基因SNP位点(rs2488457,rs33996649和rs1310182),采用PCR-RFLP技术结合测序技术来检测这三个位点在两组中的分布。根据SNP位点上是否存在核酸内切酶的酶切位点,利用软件primer premier 5.0设计针对三种SNP靶片段上的引物,对研究对象的DNA进行扩增,用核酸内切酶消化PCR产物,消化产物琼脂糖凝胶电泳观察结果。随机抽取10%测序,与PCR-RFLP结果对比。
2.2采用SYBR Green荧光染料法检测PTPN22基因相对表达量
根据PTPN22 mRNA序列设计扩增引物,以管家基因GAPDH为内参,采用2-△△CT分析方法对PTPN22基因的表达量进行相对定量。比较PTPN22 mRNA的相对表达量在-1123G>C三种基因型之间的差别。
2.3采用ELISA法检测患者血浆GPI水平
收集患者血清,以PTPN22-1123G>C多态性位点将RA分为三组,另外设置健康对照组和非RA的自身免疫性疾病对照组,共5组,用ELISA法检测各组血浆样本中GPI的相对表达量。
2.4临床特征分析
以PTPN22 G-1123C多态性位点将.RA分为三组,同时我们以RF、anti-CCP、血沉(ESR)、C-反应蛋白(CRP)、X线损伤等指标阳性时和阴性时,比较PTPN22 G-1123C多态性位点的基因型分布在病例组和对照组之间的差异。
3.统计方法
应用SPSS 13.0统计软件包处理数据。计量资料用均数±标准差(x±s)表示,计数资料用率表示。RA组及健康对照组三个SNP位点基因型频率经Hardy-Weinberg遗传平衡定律检验样本是否具有群体代表性；三个SNP位点基因型分布以及等位基因频率在两组之间的比较采用R×C列联表的x2检验；对于样本含量<40和理论数<1的计数资料采用列联表的精确概率法；各组间均数比较采用单因素方差分析(one-way ANOVA),方差分析显著时进行多重比较,方差齐采用Bonferroni法,方差不齐采用Games-howell法。以P<0.05为差异有统计学意义；构成比的比较采用卡方分析(Chi-square test)。
结果：
1.PTPN22-1123G>C/rs2488457多态性位点存在三种基因型,分别为CC型、CG型和GG型,不同基因型在RA患者和健康对照组中的分布频率分别为17.0%、45.7%和37.3%以及15.9%、37.5%和46.6%,总体分布有显著性差异(x2=9.964,P=0.01)。与野生型纯合子GG相比,CG型增加发病风险(1.517,95%CI：1.154-1.995,P=0.003),突变型纯合子CC并不增加RA的发病风险(1.328,95%CI：0.924-1.909,P=0.138)。
rs1310182多态性位点的三种基因型,分别为CC型、CT型和TT型,不同基因型在RA患者和健康对照组中的分布频率分别为2.2%、22.0%和75.8%及1.6%、25.2%和73.2%。总体分布无显著性差异(Z=1.762,P=0.414)。与野生型纯合子TT相比,CT和CC型均未增加RA的发病风险。rs33996649多态性位点的检测并未发现突变碱基的存在,该位点不存在多态性。
2.荧光定量PCR结果显示,RA患者组PTPN22 mRNA显著高于健康对照组(P=0.04)：PTPN22 mRNA的表达在三种基因型的RA患者中无显著性差异,但我们观察到PTPN22 mRNA的表达由CC到GG有逐渐上升的趋势。
3.RA组GPI值显著高于非RA对照组(P=0.000)和健康对照组(P=0.000)。三种不同基因型RA比较时,GG组GPI值显著高于CC组(P=0.032),GG组和CG组之间(P=0.380)、CC组和CG组之间(0.833)比较无显著性差别。
4.RF+和RF-患者的PTPN22-1123G>C基因分型与对照组均有差别,表明PTPN22-1123G>C与RA发病易感性具有相关性。我们尚未观察到-1123G>C基因型分布在anti-CCP、ESR、CRP以及X线损伤为阴性和阳性时的差别。
结论：
1. PTPN22 -1123G>C与广东地区汉族人群RA的发病有关联,并且具有明显的杂合子优势,由于CG基因型在RA和对照组中的分布分别为45.7%和37.5%,说明PTPN22-1123G>C可能只是一种低危险度的SNP。PTPN22-1123G>C与中国人群RA易感性的关联可能具有普遍性。rs1310182与中国广东人群RA发病不具有关联性。-33996649与中国广东人群RA发病无关联性。
2. PTPN22-1123G>C可能使PTPN22 mRNA的表达异常,而且,PTPN22 mRNA和血清GPI的变化趋势一致,表明Lyp的表达量与GPI的产生具有相关性。
3.我们尚未发现PTPN22-1123G>C与RA患者anti-CCP、ESR、CRP以及X线损伤之间的关联。
本研究创新点：
1.增加了大量的病例数进一步证实PTPN22-1123G>C多态性与中国广东人群RA发病相关。
2.发现PTPN22-1123G>C多态性可能改变PTPN22启动子的转录调节功能。野生型G等位基因转变为C等位基因后,表达下调。
3.发现PTPN22-1123G>C与RA患者血清中GPI的浓度相关,PTPN22 mRNA水平的变化趋势与血清GPI的水平一致,为探讨Lyp与GPI之间的关系提供依据。
Backcground & Objective
Rheumatoid 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. Subject
For 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 method
2.1 Genotyping of the three SNPs in case-control groups
Three 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 method
A 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 serum
Serum 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 index
RA 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 statics
Quantitative 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.
引文
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