PTPN22基因多态性与中国广东汉族人群RA发病的相关性研究
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摘要
研究背景
类风湿性关节炎(RA)是一种常见的结缔组织病,呈全球性分布,在我国的患病率约为0.32%～0.36%,专家估计目前我国RA患病人数约500万,如此庞大的RA患病人群,给我国社会、经济造成巨大的损失,也是导致劳动者丧失劳动力和致残的主要病因之一。目前RA的病因和发病机制仍不甚清楚,相关因素较多,研究表明基因遗传因素的作用约占60%。以往有关RA基因遗传因素的研究主要集中在主要组织相容性抗原复合物(major histocompatibilitycomplex,MHC)的研究方面,约占其发病遗传风险的三分之一。由于全基因组扫描、基因芯片等技术的不断发展,有关在MHC以外区域与不同研究背景人群RA发病有关的易感基因不断被发现,如肽基精氨酸脱亚氨酶4(peptidylarginine deiminase type 4,PADI4)、溶质转运蛋白SLC22A4基因(solute carrier family 22 members 4,SLC22A4),可结晶片段受体样因子3(FCRL3)、转录激活因子4(STAT4)基因及PTPN22等,而PTPN22是目前公认的除MHC之外与自身免疫性疾病发病相关的最重要的危险因子,其基因多态性研究已成为RA发病及其易感性研究中的热门焦点之一。PTPN22基因位于染色体1p13.3-13.1,包含16个外显子和15个内含子,其编码的淋巴酪氨酸磷酸酶(Lyp)属于酪氨酸磷酸酶(Ptps)家族。目前研究较多的位点是PTPN221858 C→T基因的突变,其主要致病机制文献报道认为是精氨酸突变为色氨酸后破坏了Lyp与Src酪氨酸激酶C端(Csk)的紧密结合,潜在改变了Lyp作为T细胞活性负调节剂的正常功能,导致T细胞活性增强,免疫稳态遭到破坏,从而诱发自身免疫性疾病的发生。虽然有大量文献报道PTPN22 1858 C→T的单核苷酸多态性(SNP)与多种自身免疫性疾病的发病有关,但是都主要集中在欧美白种人群,在亚洲黄种人及非洲黑人发生的频数几乎为0,研究认为这一SNP位点的发生存在明显的遗传异质性。2006年日本学者Kawasaki等通过基因测序发现PTPN22基因存在五个新的SNP位点,其中启动子区域的-1123G→C SNP与亚洲人群(日本及韩国人)急性发作型Ⅰ型糖尿病(TID)发生密切相关,认为此位点是除C1858T SNP之外又一个与自身免疫性疾病发病相关、出现频率较高的突变位点。鉴于PTPN22-1123G→C SNP与亚洲人群(日本及韩国人)急性发作型Ⅰ型糖尿病(TID)的发生密切相关以及中国人群同属亚洲黄种人,与日本及韩国人具有相似的遗传背景,我们推测PTPN22-1123 G→C SNP与自身免疫性疾病RA的发病具有相关性。为此,本论文以中国广东汉族RA人群为主要研究对象,采用高效液相色谱分析(denaturing high performance liquidchromatography,DHPLC)与DNA测序相结合及聚合酶链式反应—限制性片段长度多态性(polymerase chain reaction restriction fragment length polymorphism,PCR-RFLP)分析法与DNA测序相结合的检测技术,研究中国广东汉族RA患者PTPN22-1123 G→C基因多态性与RA发病的相关性及PTPN22 1858 C→T SNP在中国广东人群中的发生频率,同时采用荧光定量PCR技术对PTPN22基因的表达进行相对定量,了解G-1123C SNP对RA患者PTPN22基因表达的影响,为探讨RA的发病机制及临床早期诊断提供实验室的理论依据。
研究方法
1研究对象
RA组为2007年3月～2008年12月在南方医科大学附属南方医院就诊的门诊无血缘关系RA病人,其诊断依据为美国大学1987年修订的类风湿学诊断标准。RA患者148人,男性27人,女性121人,其中湖南籍10人,江西4人,福建省3人,贵州1人、河南省1人,广东129人。对照组为无血缘关系的同期医院门诊健康体检者。男性41人,女性111人,广东省152人。两组均为汉族,排除其它自身免疫性疾病和严重心、肝、肾疾病。
2研究方法
2.1酚—氯仿法抽提DNA,设计特异性引物,PCR扩增目的片段。
2.2采用PCR-RFLP,结合DNA测序技术检测PTPN22基因C1858T多态性。
2.3采用DHPLC法,结合DNA测序技术检测PTPN22基因G-1123C多态性。
2.3运用SPSS13.0进行二分类的Logistic回归分析,以所有个体是否发病为因变量Y,年龄、性别为自变量X进行分析,观察去除性别、年龄因素后各基因型与RA患者发病的相关性。根据RA病人X线检查是否出现关节损伤、RF的阴阳性、发病起始年龄的临床特征,将RA患者分为六组,并与健康对照组分别进行比较,分析G-1123C SNP位点的发生与各临床特征之间的相关性。
2.4收集148例RA患者,均为临床确诊病人,其中52名患者临床特征资料完整齐全,按照RA患者-1123 SNP位点基因型的不同,将52名RA患者分为突变杂合子(GC型)RA组、突变纯合子(CC型)RA组、以及野生型(GG型)RA组三个不同组别,分别对三组间RA患者的各个临床特征进行统计分析,观察三组不同基因型RA患者的临床特征间有无统计学差异。
2.5采用SYBR Green荧光染料定量PCR检测所有突变病人组及健康对照组PTPN22基因的表达量,以GAPDH管家基因为内参基因,采用△CT法计算,运用独立样本t检验进行统计学分析,观察两组样本PTPN22基因的表达量是否存在统计学差异。以2~(-△△CT)法计算两组表达量的差别。
2.6统计方法
采用基因计数法计算PTPN22两个SNP位点基因型频率和等位基因频率:各组间基因型和等位基因分布比较采用SPSS13.0统计软件进行Pearson x~2检验;运用二分类logistic回归分析去除性别和年龄因素的影响,以了解各基因型与RA发病相关的OR值。携带C基因与RA临床特征的相关性分析,以Pearsonx~2检验逐一分析。以X±s表示样本的均数,两样本均数的比较采用t检验,两个独立样本率的比较采用x~2,多个样本均数的比较采用one-way ANOVA,多个样本率的比较采用多个独立样本非参数检验,定义双侧P＜0.05为有统计学差异。
结果
1、PTPN22 G-1123C位点与中国广东汉族人群RA的发病有关,RA病人组与对照组PTPN22 G-1123C SNP基因型的发生频数存在差异(P=0.016);两组间携带C突变基因的频数有显著性差异(P=0.000;OR,1.837;95%CI,1.328～2.541)。logistic回归分析进一步提示:去除性别及年龄的影响因素后,两组间基因型频率仍存在差异(P=0.021),其中突变杂合子GC型与RA的发病明显相关(P=0.006;OR,2.051;95%CI,1.234～3.410)。
2、PTPN22基因C1858T多态性在中国广东地区汉族人群中发生的频率为0,C1858T SNP位点与中国广东汉族人群RA的发病无关,所检测的148例RA患者及151例健康对照组基因表型为CC野生型。
3、PTPN22-1123G＞C SNP与RA患者类风湿因子(RF)的产生有关,PTPN22-1123G＞C SNP基因分型与RF因子阳性RA患者相关P=0.041＜0.05,而与RF因子阴性RA患者无关P=0.862＞0.05。
4、比较三组不同基因型RA患者的临床特征,无统计学差异,P值均＞0.05。
5、实时荧光定量PCR检测结果显示,突变病人组(包括突变杂合子及突变纯合子型RA病人)PTPN22mRNA表达量是对照组(野生型健康对照组人员)的3.742倍,P=0.025＜0.05,突变病人组外周血白细胞PTPN22mRNA表达量升高。
结论
1、PTPN22 G-1123C位点与中国广东汉族人群RA的发病具有相关性,而PTPN22 C1858T SNP位点与中国广东汉族人群RA的发病无关;提示PTPN22 G-1123C位点的基因多态性可能是类风湿关节炎发病的一个危险因素。突变杂合子GC型发病危险度是对照组GG型的2.051倍,而突变纯合子型与RA的发病无关,PTPN22 G-1123C SNP存在杂种优势现象。
2、PTPN22 G-1123C SNP与RA患者RF因子的产生有关,与RA患者的发病起始年龄、X线检查是否存在骨关节损伤无关;三组不同基因型RA患者的临床特征,无统计学差异,可能与本次研究中收集的样本量较少有关。
3、突变病人组PTPN22mRNA表达量高于野生型健康对照组,可能是由于启动子区域G-1123C位点基因突变后影响了LYP蛋白的转录表达,机体为代偿这一功能缺陷而作出的保护性反应。PTPN22 G-1123C SNP致病机制可能是通过-1123C影响与转录因子激活蛋白4(AP-4)的中心基序区域(-1130gcaaGCTGaa-1121)的结合,使得PTPN22基因mRNA的转录激活受阻,LYP蛋白合成减少,最终影响T细胞信号中已磷酸化的重要酪氨酸激酶Src家族脱磷酸化,破坏了Lyp协同Csk抑制T细胞信号转导的功能,T细胞活性上调,免疫平衡遭到破坏,显著增加RA患者的发病危险性。
4、本次实验为研究RA患者的发病机制提供了新的思路,并且为进一步研究PTPN22 G-1123C SNP影响LYP表达量的确切作用机制提供了理论依据。
Research background
Rheumatoid 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 Methods
1 subjects
For 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 Methods
2.1 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 Methods
The 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. Results
1,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).
Conclusion
1,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.
类风湿性关节炎(RA)是一种常见的结缔组织病,呈全球性分布,在我国的患病率约为0.32%～0.36%,专家估计目前我国RA患病人数约500万,如此庞大的RA患病人群,给我国社会、经济造成巨大的损失,也是导致劳动者丧失劳动力和致残的主要病因之一。目前RA的病因和发病机制仍不甚清楚,相关因素较多,研究表明基因遗传因素的作用约占60%。以往有关RA基因遗传因素的研究主要集中在主要组织相容性抗原复合物(major histocompatibilitycomplex,MHC)的研究方面,约占其发病遗传风险的三分之一。由于全基因组扫描、基因芯片等技术的不断发展,有关在MHC以外区域与不同研究背景人群RA发病有关的易感基因不断被发现,如肽基精氨酸脱亚氨酶4(peptidylarginine deiminase type 4,PADI4)、溶质转运蛋白SLC22A4基因(solute carrier family 22 members 4,SLC22A4),可结晶片段受体样因子3(FCRL3)、转录激活因子4(STAT4)基因及PTPN22等,而PTPN22是目前公认的除MHC之外与自身免疫性疾病发病相关的最重要的危险因子,其基因多态性研究已成为RA发病及其易感性研究中的热门焦点之一。PTPN22基因位于染色体1p13.3-13.1,包含16个外显子和15个内含子,其编码的淋巴酪氨酸磷酸酶(Lyp)属于酪氨酸磷酸酶(Ptps)家族。目前研究较多的位点是PTPN221858 C→T基因的突变,其主要致病机制文献报道认为是精氨酸突变为色氨酸后破坏了Lyp与Src酪氨酸激酶C端(Csk)的紧密结合,潜在改变了Lyp作为T细胞活性负调节剂的正常功能,导致T细胞活性增强,免疫稳态遭到破坏,从而诱发自身免疫性疾病的发生。虽然有大量文献报道PTPN22 1858 C→T的单核苷酸多态性(SNP)与多种自身免疫性疾病的发病有关,但是都主要集中在欧美白种人群,在亚洲黄种人及非洲黑人发生的频数几乎为0,研究认为这一SNP位点的发生存在明显的遗传异质性。2006年日本学者Kawasaki等通过基因测序发现PTPN22基因存在五个新的SNP位点,其中启动子区域的-1123G→C SNP与亚洲人群(日本及韩国人)急性发作型Ⅰ型糖尿病(TID)发生密切相关,认为此位点是除C1858T SNP之外又一个与自身免疫性疾病发病相关、出现频率较高的突变位点。鉴于PTPN22-1123G→C SNP与亚洲人群(日本及韩国人)急性发作型Ⅰ型糖尿病(TID)的发生密切相关以及中国人群同属亚洲黄种人,与日本及韩国人具有相似的遗传背景,我们推测PTPN22-1123 G→C SNP与自身免疫性疾病RA的发病具有相关性。为此,本论文以中国广东汉族RA人群为主要研究对象,采用高效液相色谱分析(denaturing high performance liquidchromatography,DHPLC)与DNA测序相结合及聚合酶链式反应—限制性片段长度多态性(polymerase chain reaction restriction fragment length polymorphism,PCR-RFLP)分析法与DNA测序相结合的检测技术,研究中国广东汉族RA患者PTPN22-1123 G→C基因多态性与RA发病的相关性及PTPN22 1858 C→T SNP在中国广东人群中的发生频率,同时采用荧光定量PCR技术对PTPN22基因的表达进行相对定量,了解G-1123C SNP对RA患者PTPN22基因表达的影响,为探讨RA的发病机制及临床早期诊断提供实验室的理论依据。
研究方法
1研究对象
RA组为2007年3月～2008年12月在南方医科大学附属南方医院就诊的门诊无血缘关系RA病人,其诊断依据为美国大学1987年修订的类风湿学诊断标准。RA患者148人,男性27人,女性121人,其中湖南籍10人,江西4人,福建省3人,贵州1人、河南省1人,广东129人。对照组为无血缘关系的同期医院门诊健康体检者。男性41人,女性111人,广东省152人。两组均为汉族,排除其它自身免疫性疾病和严重心、肝、肾疾病。
2研究方法
2.1酚—氯仿法抽提DNA,设计特异性引物,PCR扩增目的片段。
2.2采用PCR-RFLP,结合DNA测序技术检测PTPN22基因C1858T多态性。
2.3采用DHPLC法,结合DNA测序技术检测PTPN22基因G-1123C多态性。
2.3运用SPSS13.0进行二分类的Logistic回归分析,以所有个体是否发病为因变量Y,年龄、性别为自变量X进行分析,观察去除性别、年龄因素后各基因型与RA患者发病的相关性。根据RA病人X线检查是否出现关节损伤、RF的阴阳性、发病起始年龄的临床特征,将RA患者分为六组,并与健康对照组分别进行比较,分析G-1123C SNP位点的发生与各临床特征之间的相关性。
2.4收集148例RA患者,均为临床确诊病人,其中52名患者临床特征资料完整齐全,按照RA患者-1123 SNP位点基因型的不同,将52名RA患者分为突变杂合子(GC型)RA组、突变纯合子(CC型)RA组、以及野生型(GG型)RA组三个不同组别,分别对三组间RA患者的各个临床特征进行统计分析,观察三组不同基因型RA患者的临床特征间有无统计学差异。
2.5采用SYBR Green荧光染料定量PCR检测所有突变病人组及健康对照组PTPN22基因的表达量,以GAPDH管家基因为内参基因,采用△CT法计算,运用独立样本t检验进行统计学分析,观察两组样本PTPN22基因的表达量是否存在统计学差异。以2~(-△△CT)法计算两组表达量的差别。
2.6统计方法
采用基因计数法计算PTPN22两个SNP位点基因型频率和等位基因频率:各组间基因型和等位基因分布比较采用SPSS13.0统计软件进行Pearson x~2检验;运用二分类logistic回归分析去除性别和年龄因素的影响,以了解各基因型与RA发病相关的OR值。携带C基因与RA临床特征的相关性分析,以Pearsonx~2检验逐一分析。以X±s表示样本的均数,两样本均数的比较采用t检验,两个独立样本率的比较采用x~2,多个样本均数的比较采用one-way ANOVA,多个样本率的比较采用多个独立样本非参数检验,定义双侧P＜0.05为有统计学差异。
结果
1、PTPN22 G-1123C位点与中国广东汉族人群RA的发病有关,RA病人组与对照组PTPN22 G-1123C SNP基因型的发生频数存在差异(P=0.016);两组间携带C突变基因的频数有显著性差异(P=0.000;OR,1.837;95%CI,1.328～2.541)。logistic回归分析进一步提示:去除性别及年龄的影响因素后,两组间基因型频率仍存在差异(P=0.021),其中突变杂合子GC型与RA的发病明显相关(P=0.006;OR,2.051;95%CI,1.234～3.410)。
2、PTPN22基因C1858T多态性在中国广东地区汉族人群中发生的频率为0,C1858T SNP位点与中国广东汉族人群RA的发病无关,所检测的148例RA患者及151例健康对照组基因表型为CC野生型。
3、PTPN22-1123G＞C SNP与RA患者类风湿因子(RF)的产生有关,PTPN22-1123G＞C SNP基因分型与RF因子阳性RA患者相关P=0.041＜0.05,而与RF因子阴性RA患者无关P=0.862＞0.05。
4、比较三组不同基因型RA患者的临床特征,无统计学差异,P值均＞0.05。
5、实时荧光定量PCR检测结果显示,突变病人组(包括突变杂合子及突变纯合子型RA病人)PTPN22mRNA表达量是对照组(野生型健康对照组人员)的3.742倍,P=0.025＜0.05,突变病人组外周血白细胞PTPN22mRNA表达量升高。
结论
1、PTPN22 G-1123C位点与中国广东汉族人群RA的发病具有相关性,而PTPN22 C1858T SNP位点与中国广东汉族人群RA的发病无关;提示PTPN22 G-1123C位点的基因多态性可能是类风湿关节炎发病的一个危险因素。突变杂合子GC型发病危险度是对照组GG型的2.051倍,而突变纯合子型与RA的发病无关,PTPN22 G-1123C SNP存在杂种优势现象。
2、PTPN22 G-1123C SNP与RA患者RF因子的产生有关,与RA患者的发病起始年龄、X线检查是否存在骨关节损伤无关;三组不同基因型RA患者的临床特征,无统计学差异,可能与本次研究中收集的样本量较少有关。
3、突变病人组PTPN22mRNA表达量高于野生型健康对照组,可能是由于启动子区域G-1123C位点基因突变后影响了LYP蛋白的转录表达,机体为代偿这一功能缺陷而作出的保护性反应。PTPN22 G-1123C SNP致病机制可能是通过-1123C影响与转录因子激活蛋白4(AP-4)的中心基序区域(-1130gcaaGCTGaa-1121)的结合,使得PTPN22基因mRNA的转录激活受阻,LYP蛋白合成减少,最终影响T细胞信号中已磷酸化的重要酪氨酸激酶Src家族脱磷酸化,破坏了Lyp协同Csk抑制T细胞信号转导的功能,T细胞活性上调,免疫平衡遭到破坏,显著增加RA患者的发病危险性。
4、本次实验为研究RA患者的发病机制提供了新的思路,并且为进一步研究PTPN22 G-1123C SNP影响LYP表达量的确切作用机制提供了理论依据。
Research background
Rheumatoid 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 Methods
1 subjects
For 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 Methods
2.1 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 Methods
The 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. Results
1,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).
Conclusion
1,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.
引文
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18.Ikegami H,Kawabata Y,Noso S,et al.Genetics of type 1 diabetes in Asian and Caucasian populations.Diabetes Res Clin Pract,2007 Sep,77 Suppl 1:S116-21.
19.张志红,张学龙,陈峰,等.类风湿性关节炎相关基因PTPN22 C1858T在中国6个民族中的遗传多态性研究[J].国际遗传学杂志,2007,(02):81-84.
20.王曙,赵泽飞,姜晓华,等.LYP/PTPN22与中国上海汉族人群中Graves病的关系[J].上海交通大学学报(医学版),2007,(08):984-986.
21.胡必成,崔天盆,吴健民.PTPN22基因的变异(R620W)与湖北汉族人群SLE 的易感性关系[J].华中医学杂志,2008,32(01):12-4.
22.Begovich AB,Carlton VE,Honigberg LA,et al.A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase(PTPN22) is associated with rheumatoid arthritis[J].Am J Hum Genet.2004;75:330-7.
23.Kawasaki E,Awata T,Ikegami H,et al.Systematic search for single nucleotide polymorphisms in a lymphoid tyrosine phosphatase gene(PTPN22):association between a promoter polymorphism and type 1 diabetes in Asian populations.Am J Med Genet A.2006 Mar 15;140(6):586-93.
24.Ichimura M,Kaku H,Fukutani T,etal.Associations of protein tyrosine phosphatase nonreceptor 22(PTPN22) gene polymorphisms with susceptibility to Graves' disease in a Japanese population.Thyroid,2008,18(6):625-30.
25.葛金梅,张忠英,彭宣宪,等.SNP的研究现状及在MMPs研究中的应用[J].世界华人消化杂志,2005,(17):2128-2132.
26.叶建伟.变性高效液相色谱分析的技术特征及其应用[J].肾脏病与透析肾移植杂志,2003,(02):197-200.
27.Arnett FC,Edworthy SM,Bloch DA,et al.The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum 1988;31:315-24.
28.Viken MK,Olsson M,Fl(?)m ST,et al.The PTPN22 promoter polymorphism -1123G>C association cannot be distinguished from the 1858C>T association in a Norwegian rheumatoid arthritis material.Tissue Antigens,2007Sep,70(3):190-7.
29.Gregersen PK,Lee HS,Batliwalla F,et al.PTPN22:setting thresholds for autoimmunity.Semin Immunol 2006:18:214-23.
30.于志云,张进安,买尔哈巴,等.PTPN22基因多态性与自身免疫甲状腺病的相关性[J].细胞与分子免疫学杂志,2008,(08):804-807.
31.Comings DE,MacMurray JP.2000.Molecular heterosis:A review.Mol Genet Metab 71:19-31.
32.Comings DE,MacMurray JP,Gonzalez N,Ferry L,et al.Association of the serotonin transporter gene with serum cholesterol levels and heart disease.Mol Genet Metab 67:248-253.
33.Pooley EC,Fairburn CG;Cooper Z,et al.A 5-HT2C receptor promoter polymorphism(HTR2C-759C/T) is associated with obesity in women,and with resistance to weight loss in heterozygotes.Am J Med Genet Part B Neuropsychiatr Genet 126B:124-127.
34.ComingsDE,Gonzalez NS,ChengLiSC,et al.A"lineitem" approach to the identification of genes involved in polygenic behavioral disorders:The adrenergic a2A(ADRA2A)gene.AmJMed Genet Part B Neuropsychiatr Genet 118B:110-114.
35.Little KY,McLaughlin DP,Zhang L,et al.Cocaine,ethanol,and genotype effects on human midbrain serotonin transporter binding sites and mRNA levels.Am J Psychiatry 155:207-213.
36.Juliger S,Bongartz M,Luty AJ,et al.Functional analysis of a promoter variant of the gene encodingthe interferon-gamma receptor chain I.Immunogenetics 54:675-680.
37.Plenge RM,Padyukov L,Remmers EF,et al.Replication of putative candidate-gene associations with rheumatoid arthritis in >4,000 samples from North America and Sweden:association of susceptibility with PTPN22,CTLA4,and PADI4.Am J Hum Genet,2005 Dec,77(6):1044-60.
38.Harrison P,Pointon JJ,Farrar C,et al.Effects of PTPN22 C1858T polymorphism on susceptibility and clinical characteristics of British Caucasian rheumatoid arthritis patients.Rheumatology(Oxford),2006 Aug,45(8):1009-11.
39.Steer S,Lad B,Grumley JA,et al.Association of 11602W in a protein tyrosine phosphatase gene with a high risk of rheumatoid arthritis in a British population:evidence for an early onset/disease severity effect.Arthritis Rheum,2005,52:358-60.
40.Karlson EW,Chibnik LB,Cui J,Plenge RM,et al.Associations between human leukocyte antigen,PTPN22,CTLA4 genotypes and rheumatoid arthritis phenotypes of autoantibody status,age at diagnosis and erosions in a large cohort study.Ann Rheum Dis,2008 Mar,67(3):358-63.
41.Orozco G,Pascual-Salcedo D,L(?)pez-Nevot MA,et al.Auto-antibodies,HLA and PTPN22:susceptibility markers for rheumatoid arthritis.Rheumatology (Oxford),2008 Feb,47(2):138-41.
42.Freeman WM,Walker SJ,Vrana KE.Quantitative RT-PCR:pitfalls and potential[J].Biotechniques,1999,26(1):112-22,124-5.
1.Lee YH,Rho YH,Choi SJ,et al The PTPN22 C1858T functional polymorphism and autoimmune diseases--a meta-analysis[J].Rheumatology(Oxford).2007Jan;46(1):49-56.
2.Gregersen PK,Silver J,Winchester RJ,et al The shared epitope hypothesis.An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis[J].Arthritis Rheum 1987;30:1205-13.
3.MacGregor AJ,Snieder H,Rigby AS,et al Characterizing the quantitative genetic contribution to rheumatoid arthritis using data from twins[J].Arthritis Rheum 2000;43:30-7.
4.Begovich AB,Carlton VE,Honigberg LA,et al A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase(PTPN22) is associated with rheumatoid arthritis[J].Am J Hum Genet 2004;75:330-7.
5.Karlson EW,Chibnik LB,Cui J,et al Associations between human leukocyte antigen,PTPN22,CTLA4 genotypes and rheumatoid arthritis phenotypes of autoantibody status,age at diagnosis and erosions in a large cohort study.Ann Rheum Dis.2008 Mar;67(3):358-63.
6.Go(?)bV,Dieud(?) P,Daveau R,et al Contribution of PTPN22 1858T,TNFRⅡ 196R and HLA-shared epitope alleles with rheumatoid factor and anti-citrullinated protein antibodies to very early rheumatoid arthritis diagnosis.Rheumatology(Oxford).2008 Aug;47(8):1208-12.
7.Kallberg H,Padyukov L,Plenge RM,et al Gene-gene and gene-environment interactions involving HLA-DRB1,PTPN22,and smoking in two subsets of rheumatoid arthritis.Am J Hum Genet.2007 May;80(5):867-75.
8.Hasegawa K,Martin F,Huang G,et al PEST domain-enriched tyrosine phosphatase(PEP) regulation of effector/memory T cells.Science.2004 Jan 30;303(5658):685-9
9.Gregersen PK,Lee HS,Batliwalla F,et al PTPN22:setting thresholds for autoimmunity.Semin Immunol.2006 Aug;18(4):214-23.
10.Zheng W,She JX.Genetic association between a lymphoid tyrosine phosphatase (PTPN22) and type 1 diabetes.Diabetes.2005 Mar;54(3):906-8.
11.王曙,赵泽飞,姜晓华,等.LYP/PTPN22与中国上海汉族人群中Graves病的关系[J].上海交通大学学报(医学版),2007,27(8):984-6.
12.张志红,张学龙,陈峰,等.类风湿性关节炎相关基因PTPN22 C1858T在中国6个民族中的遗传多态性研究[J].国际遗传学杂志,2007,30(02):81-4.
13.Kawasaki E,Awata T,Ikegami H,et al.Systematic search for single nucleotide polymorphisms in a lymphoid tyrosine phosphatase gene(PTPN22):association between a promoter polymorphism and type 1 diabetes in Asian populations.Am J Med Genet A.2006 Mar 15;140(6):586-93.
14.Viken MK,Olsson M,Fl(?)m ST,et al.The PTPN22 promoter polymorphism -1123G>C association cannot be distinguished from the 1858C>T association in a Norwegian rheumatoid arthritis material.Tissue Antigens.2007 Sep;70(3):190-7.
15.胡必成,崔天盆,吴健民.PTPN22基因的变异(R620W)与湖北汉族人群SLE 的易感性关系[J].华中医学杂志,2008,32(01):12-4.
16.Moil M,Yamada R,Kobayashi K,et al.Ethnic differences in allele frequency of autoimmune-disease-associated SNPs.1:J Hum Genet.2005;50(5):264-6.
17.Hinks A,Worthington J,Thomson W,et al The association of PTPN22 with rheumatoid arthritis and juvenile idiopathic arthritis.Rheumatology(Oxford) 2006:45:365-8.
18.Viken MK,Olsson M,Fl(?)n ST,et al.The PTPN22 promoter polymorphism -1123G>C association cannot be distinguished from the 1858C>T association in a Norwegian rheumatoid arthritis material.Tissue Antigens.2007 Sep;70(3):190-7.
19.Plenge RM,Padyukov L,Remmers EF,et al.Replication of putative candidate-gene associations with rheumatoid arthritis in >4,000 samples from North America and Sweden: association of susceptibility with PTPN22, CTLA4, and PADI4.Am J Hum Genet.2005 Dec;77(6): 1044-60.
20.Harrison P, Pointon JJ, Farrar C, et al.Effects of PTPN22 C1858T polymorphism on susceptibility and clinical characteristics of British Caucasian rheumatoid arthritis patients.Rheumatology (Oxford).2006 Aug;45(8): 1009-11.
21.Michou L, Lasbleiz S, Rat AC, et al.Linkage proof for PTPN22, a rheumatoid arthritis susceptibility gene and a human autoimmunity gene.Proc Natl Acad Sci U S A.2007 Jan 30; 104(5): 1649-54.
22.Johansson M, Arlestig L, Hallmans G, et al.PTPN22 polymorphism and anti-cyclic citrullinated peptide antibodies in combination strongly predicts future onset of rheumatoid arthritis and has a specificity of 100% for the disease.Arthritis ResTher.2006;8(1):R19.
23.Orozco G, Pascual-Salcedo D, Lopez-Nevot MA, et al.Auto-antibodies, HLA and PTPN22: susceptibility markers for rheumatoid arthritis.Rheumatology (Oxford).2008 Feb;47(2): 138-41.
24.Karlson EW, Chibnik LB, Cui J, Plenge RM, et al.Associations between human leukocyte antigen, PTPN22, CTLA4 genotypes and rheumatoid arthritis phenotypes of autoantibody status, age at diagnosis and erosions in a large cohort study.Ann Rheum Dis.2008 Mar;67(3):358-63.
25.Goeb V, Dieude P, Daveau R, et al.Contribution of PTPN22 1858T, TNFRⅡ 196R and HLA-shared epitope alleles with rheumatoid factor and anti-citrullinated protein antibodies to very early rheumatoid arthritis diagnosis.Rheumatology (Oxford).2008 Aug;47(8):1208-12.
2.MacGregor AJ,Snieder H,Rigby AS,et al.Characterizing the quantitative genetic contributionto rheumatoid arthritis using data from twins.Arthritis Rheum,2000,43:30-7.
3.Hinks A,Barton A,John S,et al.Association between the PTPN22 gene and rheumatoid arthritis and juvenile idiopathic arthritis in a UK population:further support that PTPN22 is an autoimmunity gene.Arthritis Rheum,2005Jun,52(6):1694-9.
4.Carlton VE,Hu X,Chokkalingam AP,et al.PTPN22 genetic variation:evidence for multiple variants associated with rheumatoid arthritis.Am J Hum Genet,2005Oct,77(4):567-81.
5.Kilding R,Ilcs MM,Timms JM,et al.Additional genetic susceptibility for rheumatoid arthritis telomeric of the DRB1 locus.Arthritis Rheum 2004;50:763-9.
6.van Oene M,Wintle RF,Liu X,et al.Association of the lymphoid tyrosine phosphatase R620W variant with rheumatoid arthritis,but not Crohn's disease,in Canadian populations.Arthritis Rheum,2005 Jul,52(7):1993-8.
7.Lee HS,Remmers EF,Le JM,et al.Association of STAT4 with rheumatoid arthritis in the Korean population.Mol Med.2007 Sep-Oct;13(9-10):455-60.
8.Orozco G,S(?)nchez E,Gonz(?)lez-Gay MA,et al.Association of a functional single-nucleotide polymorphism of PTPN22,encoding lymphoid protein phosphatase,with rheumatoid arthritis and systemic lupus erythematosus.Arthritis Rheum,2005 Jan,52(1):219-24.
9.Hasegawa K, Martin F, Huang G, et al.PEST domain-enriched tyrosine phosphatase (PEP) regula-tion of effector/memory T cells.Science 2004,303:685-689.
10.Bottini N, Musumeci L, Alonso A, et al.A functional variant of lymphoid tyrosine phosphatase is asso-ciated with type I diabetes.Nat Genet 2004,36:337-338.
11.Majorczyk E, Jasek M, Pioski R, et al.Association of PTPN22 single nucleotide polymorphism with rheumatoid arthritis but not with allergic asthma .Eur J Hum Genet, 2007 Oct,15(10):1043-8
12.Martin Johanssonl, Lisbeth Arlestigl, Goran Hallmans2 ,et al.PTPN22 polymorphism and anti-cyclic citrullinated peptide antibodies in combination strongly predicts future onset of rheumatoid arthritis and has a specificity of 100% for the disease .Arthritis Res Ther, 2006,8(1):R19:1-6.
13.Lie BA, Viken MK, Odegard S, et al.Associations between the PTPN22 1858C->T polymorphism and radiographic joint destruction in patients with rheumatoid arthritis: results from a 10-year longitudinal study.Ann Rheum Dis,2007 Dec,66(12):1604-9.
14.Chelala C, Duchatelet S, Joffret ML, et al.PTPN22 R620W functional variant in type 1 diabetes and autoimmunity related traits.Diabetes,2007 Feb,56(2):522-6.
15.Kaufman KM, Kelly JA, Herring BJ,et al.Evaluation of the genetic association of the PTPN22 R620W polymorphism in familial and sporadic systemic lupus erythematosus.Arthritis Rheum, 2006 Aug,54(8):2533-40.
16.Y.H.Lee, Y.H.Rhol, S.J.Choil, et al.The PTPN22 C1858T functional polymorphism and autoimmune diseases-a meta-analysis.Rheumatology,2007,46:49-56.
17.Ray D,Tomar N,Gupta N,Goswami R,et al.Protein tyrosine phosphatase non-receptor type 22(PTPN22) gene R620W variant and sporadic idiopathic hypoparathyroidism in Asian Indians.Int J Immunogenet,2006 Aug,33(4):237-40.
18.Ikegami H,Kawabata Y,Noso S,et al.Genetics of type 1 diabetes in Asian and Caucasian populations.Diabetes Res Clin Pract,2007 Sep,77 Suppl 1:S116-21.
19.张志红,张学龙,陈峰,等.类风湿性关节炎相关基因PTPN22 C1858T在中国6个民族中的遗传多态性研究[J].国际遗传学杂志,2007,(02):81-84.
20.王曙,赵泽飞,姜晓华,等.LYP/PTPN22与中国上海汉族人群中Graves病的关系[J].上海交通大学学报(医学版),2007,(08):984-986.
21.胡必成,崔天盆,吴健民.PTPN22基因的变异(R620W)与湖北汉族人群SLE 的易感性关系[J].华中医学杂志,2008,32(01):12-4.
22.Begovich AB,Carlton VE,Honigberg LA,et al.A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase(PTPN22) is associated with rheumatoid arthritis[J].Am J Hum Genet.2004;75:330-7.
23.Kawasaki E,Awata T,Ikegami H,et al.Systematic search for single nucleotide polymorphisms in a lymphoid tyrosine phosphatase gene(PTPN22):association between a promoter polymorphism and type 1 diabetes in Asian populations.Am J Med Genet A.2006 Mar 15;140(6):586-93.
24.Ichimura M,Kaku H,Fukutani T,etal.Associations of protein tyrosine phosphatase nonreceptor 22(PTPN22) gene polymorphisms with susceptibility to Graves' disease in a Japanese population.Thyroid,2008,18(6):625-30.
25.葛金梅,张忠英,彭宣宪,等.SNP的研究现状及在MMPs研究中的应用[J].世界华人消化杂志,2005,(17):2128-2132.
26.叶建伟.变性高效液相色谱分析的技术特征及其应用[J].肾脏病与透析肾移植杂志,2003,(02):197-200.
27.Arnett FC,Edworthy SM,Bloch DA,et al.The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum 1988;31:315-24.
28.Viken MK,Olsson M,Fl(?)m ST,et al.The PTPN22 promoter polymorphism -1123G>C association cannot be distinguished from the 1858C>T association in a Norwegian rheumatoid arthritis material.Tissue Antigens,2007Sep,70(3):190-7.
29.Gregersen PK,Lee HS,Batliwalla F,et al.PTPN22:setting thresholds for autoimmunity.Semin Immunol 2006:18:214-23.
30.于志云,张进安,买尔哈巴,等.PTPN22基因多态性与自身免疫甲状腺病的相关性[J].细胞与分子免疫学杂志,2008,(08):804-807.
31.Comings DE,MacMurray JP.2000.Molecular heterosis:A review.Mol Genet Metab 71:19-31.
32.Comings DE,MacMurray JP,Gonzalez N,Ferry L,et al.Association of the serotonin transporter gene with serum cholesterol levels and heart disease.Mol Genet Metab 67:248-253.
33.Pooley EC,Fairburn CG;Cooper Z,et al.A 5-HT2C receptor promoter polymorphism(HTR2C-759C/T) is associated with obesity in women,and with resistance to weight loss in heterozygotes.Am J Med Genet Part B Neuropsychiatr Genet 126B:124-127.
34.ComingsDE,Gonzalez NS,ChengLiSC,et al.A"lineitem" approach to the identification of genes involved in polygenic behavioral disorders:The adrenergic a2A(ADRA2A)gene.AmJMed Genet Part B Neuropsychiatr Genet 118B:110-114.
35.Little KY,McLaughlin DP,Zhang L,et al.Cocaine,ethanol,and genotype effects on human midbrain serotonin transporter binding sites and mRNA levels.Am J Psychiatry 155:207-213.
36.Juliger S,Bongartz M,Luty AJ,et al.Functional analysis of a promoter variant of the gene encodingthe interferon-gamma receptor chain I.Immunogenetics 54:675-680.
37.Plenge RM,Padyukov L,Remmers EF,et al.Replication of putative candidate-gene associations with rheumatoid arthritis in >4,000 samples from North America and Sweden:association of susceptibility with PTPN22,CTLA4,and PADI4.Am J Hum Genet,2005 Dec,77(6):1044-60.
38.Harrison P,Pointon JJ,Farrar C,et al.Effects of PTPN22 C1858T polymorphism on susceptibility and clinical characteristics of British Caucasian rheumatoid arthritis patients.Rheumatology(Oxford),2006 Aug,45(8):1009-11.
39.Steer S,Lad B,Grumley JA,et al.Association of 11602W in a protein tyrosine phosphatase gene with a high risk of rheumatoid arthritis in a British population:evidence for an early onset/disease severity effect.Arthritis Rheum,2005,52:358-60.
40.Karlson EW,Chibnik LB,Cui J,Plenge RM,et al.Associations between human leukocyte antigen,PTPN22,CTLA4 genotypes and rheumatoid arthritis phenotypes of autoantibody status,age at diagnosis and erosions in a large cohort study.Ann Rheum Dis,2008 Mar,67(3):358-63.
41.Orozco G,Pascual-Salcedo D,L(?)pez-Nevot MA,et al.Auto-antibodies,HLA and PTPN22:susceptibility markers for rheumatoid arthritis.Rheumatology (Oxford),2008 Feb,47(2):138-41.
42.Freeman WM,Walker SJ,Vrana KE.Quantitative RT-PCR:pitfalls and potential[J].Biotechniques,1999,26(1):112-22,124-5.
1.Lee YH,Rho YH,Choi SJ,et al The PTPN22 C1858T functional polymorphism and autoimmune diseases--a meta-analysis[J].Rheumatology(Oxford).2007Jan;46(1):49-56.
2.Gregersen PK,Silver J,Winchester RJ,et al The shared epitope hypothesis.An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis[J].Arthritis Rheum 1987;30:1205-13.
3.MacGregor AJ,Snieder H,Rigby AS,et al Characterizing the quantitative genetic contribution to rheumatoid arthritis using data from twins[J].Arthritis Rheum 2000;43:30-7.
4.Begovich AB,Carlton VE,Honigberg LA,et al A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase(PTPN22) is associated with rheumatoid arthritis[J].Am J Hum Genet 2004;75:330-7.
5.Karlson EW,Chibnik LB,Cui J,et al Associations between human leukocyte antigen,PTPN22,CTLA4 genotypes and rheumatoid arthritis phenotypes of autoantibody status,age at diagnosis and erosions in a large cohort study.Ann Rheum Dis.2008 Mar;67(3):358-63.
6.Go(?)bV,Dieud(?) P,Daveau R,et al Contribution of PTPN22 1858T,TNFRⅡ 196R and HLA-shared epitope alleles with rheumatoid factor and anti-citrullinated protein antibodies to very early rheumatoid arthritis diagnosis.Rheumatology(Oxford).2008 Aug;47(8):1208-12.
7.Kallberg H,Padyukov L,Plenge RM,et al Gene-gene and gene-environment interactions involving HLA-DRB1,PTPN22,and smoking in two subsets of rheumatoid arthritis.Am J Hum Genet.2007 May;80(5):867-75.
8.Hasegawa K,Martin F,Huang G,et al PEST domain-enriched tyrosine phosphatase(PEP) regulation of effector/memory T cells.Science.2004 Jan 30;303(5658):685-9
9.Gregersen PK,Lee HS,Batliwalla F,et al PTPN22:setting thresholds for autoimmunity.Semin Immunol.2006 Aug;18(4):214-23.
10.Zheng W,She JX.Genetic association between a lymphoid tyrosine phosphatase (PTPN22) and type 1 diabetes.Diabetes.2005 Mar;54(3):906-8.
11.王曙,赵泽飞,姜晓华,等.LYP/PTPN22与中国上海汉族人群中Graves病的关系[J].上海交通大学学报(医学版),2007,27(8):984-6.
12.张志红,张学龙,陈峰,等.类风湿性关节炎相关基因PTPN22 C1858T在中国6个民族中的遗传多态性研究[J].国际遗传学杂志,2007,30(02):81-4.
13.Kawasaki E,Awata T,Ikegami H,et al.Systematic search for single nucleotide polymorphisms in a lymphoid tyrosine phosphatase gene(PTPN22):association between a promoter polymorphism and type 1 diabetes in Asian populations.Am J Med Genet A.2006 Mar 15;140(6):586-93.
14.Viken MK,Olsson M,Fl(?)m ST,et al.The PTPN22 promoter polymorphism -1123G>C association cannot be distinguished from the 1858C>T association in a Norwegian rheumatoid arthritis material.Tissue Antigens.2007 Sep;70(3):190-7.
15.胡必成,崔天盆,吴健民.PTPN22基因的变异(R620W)与湖北汉族人群SLE 的易感性关系[J].华中医学杂志,2008,32(01):12-4.
16.Moil M,Yamada R,Kobayashi K,et al.Ethnic differences in allele frequency of autoimmune-disease-associated SNPs.1:J Hum Genet.2005;50(5):264-6.
17.Hinks A,Worthington J,Thomson W,et al The association of PTPN22 with rheumatoid arthritis and juvenile idiopathic arthritis.Rheumatology(Oxford) 2006:45:365-8.
18.Viken MK,Olsson M,Fl(?)n ST,et al.The PTPN22 promoter polymorphism -1123G>C association cannot be distinguished from the 1858C>T association in a Norwegian rheumatoid arthritis material.Tissue Antigens.2007 Sep;70(3):190-7.
19.Plenge RM,Padyukov L,Remmers EF,et al.Replication of putative candidate-gene associations with rheumatoid arthritis in >4,000 samples from North America and Sweden: association of susceptibility with PTPN22, CTLA4, and PADI4.Am J Hum Genet.2005 Dec;77(6): 1044-60.
20.Harrison P, Pointon JJ, Farrar C, et al.Effects of PTPN22 C1858T polymorphism on susceptibility and clinical characteristics of British Caucasian rheumatoid arthritis patients.Rheumatology (Oxford).2006 Aug;45(8): 1009-11.
21.Michou L, Lasbleiz S, Rat AC, et al.Linkage proof for PTPN22, a rheumatoid arthritis susceptibility gene and a human autoimmunity gene.Proc Natl Acad Sci U S A.2007 Jan 30; 104(5): 1649-54.
22.Johansson M, Arlestig L, Hallmans G, et al.PTPN22 polymorphism and anti-cyclic citrullinated peptide antibodies in combination strongly predicts future onset of rheumatoid arthritis and has a specificity of 100% for the disease.Arthritis ResTher.2006;8(1):R19.
23.Orozco G, Pascual-Salcedo D, Lopez-Nevot MA, et al.Auto-antibodies, HLA and PTPN22: susceptibility markers for rheumatoid arthritis.Rheumatology (Oxford).2008 Feb;47(2): 138-41.
24.Karlson EW, Chibnik LB, Cui J, Plenge RM, et al.Associations between human leukocyte antigen, PTPN22, CTLA4 genotypes and rheumatoid arthritis phenotypes of autoantibody status, age at diagnosis and erosions in a large cohort study.Ann Rheum Dis.2008 Mar;67(3):358-63.
25.Goeb V, Dieude P, Daveau R, et al.Contribution of PTPN22 1858T, TNFRⅡ 196R and HLA-shared epitope alleles with rheumatoid factor and anti-citrullinated protein antibodies to very early rheumatoid arthritis diagnosis.Rheumatology (Oxford).2008 Aug;47(8):1208-12.