IL23R、PTPN2、10q21基因多态性与炎症性肠病相关性的初步研究
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摘要
背景、目的
炎症性肠病(Inflammatory bowel disease,IBD)包括克罗恩病(Crohn's disease,CD)和溃疡性结肠炎(Ulcerative colitis,UC)。近年来IBD的发病率呈上升趋势,特别是CD的发病率,随着双气囊小肠镜的应用,CD的发现率逐年上升。以往IBD一直被认为是一种自身免疫性疾病,而近年来多个研究证明IBD特别是CD具有明显的遗传易感性。目前较为一致的观点认为,炎症性肠病是携带遗传易感基因的宿主在环境因素作用下,自身免疫功能紊乱导致的一种非特异性炎症性疾病。因此遗传因素在炎症性肠病的发病过程中起着非常重要的作用。随着人类基因组计划的完成,对基因研究的重点已由全基因组序列测定转移到了对基因组中个体基因多态性和功能的研究。个体基因多态性的主要形式就是单链核苷酸多态性(Single nucleotide polymorphism,SNP),SNP被公认为继“限制性片段长度多态性”和“微卫星多态性”这两种遗传标记之后,出现的“第三代DNA遗传标记”,是目前全世界基因研究领域的热点之一。
2001年欧洲Hugot研究小组及美国Ogura研究小组几乎同时发现了人类第一个CD易感基因NOD2基因,后改名为CARD15,并证实该基因的3个单核苷酸多态性(SNP)位点Arg702Trp、Gly908Arg和Leu1007fsinsC是白种人CD的易感基因,但后来的研究证明此三个SNP位点与日本人及我国香港人、浙江人CD患者无关。我们也对广东地区CD患者NOD2基因的三个SNP位点进行研究发现其与CD无显著相关,并通过基因测序的方法发现了可能与中国人CD密切相关的SNP位点P268S,并成功构建了该位点的突变型真核表达载体,为后续功能的研究打下了基础。最近西方多个研究小组通过全基因扫描的方法分别对欧洲高加索人、犹太人、英国、法国、巴尔干、德国、加拿大(魁北克)等地区的IBD患者进行研究,发现了一些与IBD,特别是CD患者密切相关的SNP位点,主要位于IL23R、ATG16L1、PTPN2、10q21、NKX2、IRGM、MST1、DLG5、OCTN1、5p31等基因中。其中位于IL23R基因中的rs11209026、rs11805303位点、PTPN2基因中的rs2542151和位于染色体10q21中的rs10761659 SNP位点统计学意义较为明显;rs11209026位于IL23R基因的第10外显子中,正常为G,突变为A,导致381位的氨基酸由精氨酸(Arg)变为谷氨酰胺(Gln),Duerr等人研究的结果认为该SNP位点与欧洲白种人CD患者显著相关(P=1.56×10~(-3)),其OR值为0.26(犹太人为0.47);rs11805303位于IL23R基因第7和第8外显子之间的内含子之中,正常为C,突变为T,该碱基的改变增加了西方人CD发病的风险,杂合突变的OR值为1.39,纯合突变的OR值为1.86;rs2542151位于PTPN2基因上游约5.5kb的区域中,可能为PTPN2基因的启动子区或者上游控制元件,PTPN2基因编码一种关键的炎症负性调节因子—T细胞蛋白酪氨酸磷酸酶(T cell protein tyrosine phosphatase,TCPTP),在西方IBD患者当中,其危险等位基因为G,杂合子的OR值为1.3(1.14-1.48),纯合子的OR值为2.01(1.46-2.76),rs10761659位于10q21的基因间区,其危险等位基因为G,其杂合子的OR值为1.23(1.05-1.45),纯合子的OR值为1.55(1.30-1.84)。
本研究的目的通过基因测序和限制性片段长度多态性的方法探索与西方白种人及犹太人CD患者密切相关的上述四个多态性位点与IBD患者发病易感性的相关性,及其与临床特征的关系,为进一步研究IBD的发病机制及探索IBD易感基因打下基础,并有可能为临床治疗IBD提供一种新的基因治疗靶点。
材料和方法:
1、收集南方医院消化科及普外科确诊,临床资料完整,彼此无血源关系的广东及外省籍CD患者40例(包括前期收集病例24例和新收病例16例),UC患者40例(所选患者均为手术或者内镜下确诊的病例,并除外合并其它自身免疫性疾病),所选患者中男52例,女28例;平均年龄34岁;健康对照组(Healthy control,HC)取门诊健康体检者50例,其中男32例,女18例,平均年龄35岁;所有研究对象均抽取外周血5 ml。
2、应用特异性结合DNA的硅基质材料离心吸附柱法和独特的洗脱缓冲液系统抽提血液基因组DNA(按试剂盒说明书操作),抽提成功后DNA产物-70℃保存。
3、针对上述四个SNP位点用Primer5.0软件设计PCR引物,PCR方法扩增包含这四个SNP位点在内的DNA片段,扩增产物以1.5%琼脂糖凝胶电泳观察以确保目的片段扩增成功,然后采用切胶纯化试剂盒(TIANGEN公司提供)将目的片段纯化回收,分光光度计检测纯化产物浓度。
4、将纯化产物分别采用DNA直接测序法和聚合酶链反应—限制性片段长度多态性分析(restriction fragment length polymorphism polymerase chainreaction products,PCR-RFLP)进行研究;测序结果用chromas231判读SNP位点的基因型。酶切产物以2.5%琼脂糖凝胶电泳,根据电泳结果判读SNP位点的基因型。采用基因计数法计算四个SNP位点基因型频率和等位基因频率,最后进行统计分析。
结果:
1、IL23R(rs11209026和rs11805303)基因多态性与炎症性肠病发病的相关性:
通过DNA测序,我们发现中国人部分CD患者位于IL23R中的这两个SNP位点基因型频率及等位基因频率与健康对照组比较差异均无统计学意义(P＞0.05);rs11805303位点基因型频率UC组与HC组比较,P＞0.05,差异无显著性;而等位基因频率比较,P＜0.05(Fisher精确概率检验P=0.024),差异具有统计学意义;但rs11805303位点基因多态性与中国部分UC患者的性别、疾病的活动性及发病部位比较P值均大于0.05;提示rs11805303位点的多态性可能与中国部分UC患者有相关性。
2、PTPN2(rs2542151)和10q21(rs10761659)基因多态性与炎症性肠病发病的相关性:
通过聚合酶链反应—限制性片段长度多态性分析(PCR-RFLP),结果显示:①PTPN2(rs2542151)位点:CD组基因型频率、等位基因频率和基因携带者频率分别与HC组比较P值均大于0.05,而UC组基因型频率和等位基因频率与HC比较P＜0.05(分别为:0.002和0.006),且3例纯合子GG均出现于UC患者,提示该位点可能与中国部分UC患者的发病易感性有关。②10q21(rs10761659)位点:UC组的基因型频率、等位基因频率和基因携带者频率分别与HC组比较P值均大于0.05,CD组等位基因频率与HC组比较P值也大于0.05,而基因型频率与HC组比较P=0.044,提示该位点可能与中国UC患者易感性无关,但该位点基因型可能与中国部分CD患者有相关性。但这两个位点与相应疾病的临床特征均无显著相关(P值均大于0.05)。
结论:
1、IL23R中两个与西方人CD密切相关的SNP位点(rs11209026和rs11805303)的基因多态性与中国部分CD患者的发病无显著相关性,而rs11805303位点的多态性可能与中国部分UC患者有相关性,且该碱基的改变降低了UC的发病风险,但这个SNP位点基因多态性与UC患者的病变特点并无显著相关;
2、位于PTPN2基因上游区域中的rs2542151位点可能是中国部分UC患者的一个易感基因,其纯合子GG增加了UC的发病风险,但该位点基因多态性与UC的临床特点无显著相关。
3、位于染色体10q21中的rs10761659位点可能是中国部分CD患者的一个易感基因,其纯合子AA增加了CD的发病风险,但这个SNP位点基因多态性与CD患者的病变特点并无显著相关。
Background and Objective:
Inflammatory bowel disease(IBD) includes Crohn's disease(CD) and ulcerative colitis(UC).Its etiology and pathogenesis are still unknown.Most of the views presume that a kind of non-specific inflammatory diseases can be induced under the role of environmental factors and autoimmune disorders for the host who carry the genetic susceptibility gene.In recent years,with the Human Genome Project completed,human have been transferred the focus about gene research from the whole genome sequence to individual gene polymorphism and functional studies. Single-chain nucleotide polymorphism(SNP) is the main form of individual gene polymorphism;the SNP is recognized as the "third generations of DNA genetic marker "following the restriction fragment length polymorphism and microsatellite polymorphism,which is one of the hottest fields in the world.
Hugot Research Group in Europe and Ogura team in the United States simultaneously found out the first predisposing gene of CD—NOD2,which was renamed CARD15 in 2001.They certified this gene includes three SNPs sites: Arg702Trp,Gly908Arg,Leu1007fsinsC,and further confirmed the SNPs site are belong to Caucasian's CD patients predisposing gene but don't related with the CD patients bom from Japan,Hong Kong and ZheJiang province.We have researched the three SNPs site about NOD2 gene of CD patients born from Guangdong,the results indicated there isn't correlating with them,but we found out the closely related SNPs site—P268S from Chinese by detecting gene sequence,and constructed eukaryotic expression vector,the further study are carrying out.
Western research groups recently study the gene susceptibility of the IBD patients who came from the Caucasus of Europe,the United Kingdom,France,the Balkans,Germany,Canada,and other regions in Europe by gene scanning,they found some of SNPs sites about IBD and especially related with CD patients,which located the gene of IL23R、ATG16L1、PTPN2、10q21、NKX2、IRGM、MST1、DLG5、OCTN1、5p31 and so on.The site of rs11209026、rs11805303 in IL23R gene、rs2542151 in PTPN2 gene and rs10761659 in chromosome 10q21 had statistical significance comparing with control.The normal rs11209026 in the exon 10 of IL23R gene is G,when it is mutated to A,the 381 amino acid arginine acid(Arg) become glutamine(Gln).Duerr believed that the SNP mutation were closely correlated with the European CD patients(p=1.56×10-3),OR=0.26(Jews 0.47).this indicating that the base-point change is a protection change;possible mechanism is that the function of its encoded protein is changed by mutations affecting the amino acid, thereby blocking the receptor binding after IL23 and the downstream Signal transduction pathways;rs11805303 lies in exon 7 and exon 8 of IL23R gene,normal C and T mutations in the base of the changes increased the risk of morbidity Westerners CD,heterozygous mutations OR=1.39,homozygous mutation OR=1.86; its influence gene function mechanism is not clear,may be due to the impact of changes in base gene transcription or mRNA transcription after shearing,and even as a remote-control devices affect gene function.Rs2542151 lies in PTPN2 gene, encoding a T-cell protein tyrosine phosphatase(TCPTP) - a key negative regulator of inflammation and the risk for the G allele,heterozygous OR=1.3(1.14-1.48), homozygous OR=2.01(1.46-2.76).Rs10761659 lies in 10 q21 area between the gene and the risk allele is the G,heterozygous OR=1.23(1.05-1.45),homozygous OR= 1.55(1.3-1.84);their specific mechanism is still further studies.
The purpose of this study is to prove the four SNPs which related to western CD patients whether associate with Chinese IBD patients and clinical characteristics by gene sequencing and restriction fragment length polymorphism methods,Our purpose is to further study the pathogenesis of IBD and patterns IBD susceptibility gene mapping.maybe also provide a new means of gene therapy for the treatment of IBD
Methods:
1.Forty Chinese Crohn's disease(CD) patients(including twenty-four former samples and sixteen new samples)、forty ulcerative colitis(UC) and fifty healthy controls(HC) were prospectively recruited from the Nan Fang Hospital.Peripheral blood was collected from patients and controls.
2.Extract genomic DNA from the blood(in kit brochures operation),after the success of extraction,preserve them in - 70℃.
3.Design primers using Primer5.0 software.To amplify the fragments includes the four SNPs.After succeeded amplify,(amplified products by 1.5%agarose gel electrophoresis to ensure that the observation fragment was successfully amplified).Purify the PCR production and directed sequence the target region in SIB3730XL sequencer.Then,assessment genotype with chromas2.0 and Last contrasted the gene bank data to analyze the polymorphism of SNPs in IBD patients and controls.If we find mutation that we amplify the corresponding target region and purify the PCR production and directed sequence the target region and contrasted gene bank data with uniform means.Two SNPs use the method of restriction fragment length polymorphism(RFLP) to testify the polymorphism in CD patients and find it's frequency in UC and health control.
Results:
1.IL23R(rs11209026 and rs11805303) gene polymorphism associated with the pathogenesis of inflammatory bowel disease:
Through DNA sequencing,we found that there was no significant difference in genotype frequencies and allele frequency with these two SNPs which lies in IL23R between Chinese Crohn's disease(CD) patients and normal population(P＞0.05); rs11805303 sites genotype frequencies of ulcerative colitis(UC) group compared with the control group,P＞0.05,there was no significant difference too,but allele frequencies in UC(UC is 41.2%)compared with normal control(normal control is 59.0%),P＜0.05(P=0.018),the differences were statistically significant;but rs11805303 gene polymorphism was not associated with the incidence age,gender, disease activity and the location of UC patients(P＞0.05);these cue us:rs11805303 polymorphism loci may be related to the incidence of partial UC patients in Chinese people.
2.PTPN2(rs2542151) and 10q21(rs10761659) gene polymorphism associated with the pathogenesis of inflammatory bowel disease:
Through restriction fragment length polymorphism(RFLP),PTPN2(rs2542151) gene polymorphism compared with IBD:genotype frequency,allele frequency and the gene-phore frequency in CD Group compared with normal control group,P＞0.05, and UC group genotype frequencies and allele frequency compared with normal controls,P＜0.05(P=0.002,P=0.006 respectively),suggesting that the site(rs2542151) may be related to UC susceptibility but unrelated with CD patients.10q21 (rs10761659) gene polymorphism compared with IBD:the UC group carriers of genotype frequency,allele frequency and the gene-phore frequency compared with normal control group P＞0.05,but CD group genotype frequencies compared with normal control,P=0.044,suggesting that the site(rs10761659) may be unrelated with Chinese UC patients,but genotype of the sites may be associated with Chinese CD patients.
Conclusion:
1.The two SNPs polymorphism(lies in IL23R gene) which related to Western CD patients was not associated with Chinese CD patients,and rs11805303 polymorphism loci may be a genetic marker of Chinese UC patients,However,there was no significant correlation between the SNPs loci gene polymorphism and characteristics of UC patients;
2.It is not similar to western results,rs2542151 polymorphism which related to Western CD patients likely to be a susceptibility gene sites in Chinese UC patients, homozygous mutant GG have increased the risk of UC.But the SNP loci gene polymorphism is no significant correlation with UC clinical characteristics.
3.rs10761659 polymorphism which related to Western CD patients likely to be a susceptibility gene sites in Chinese CD patients,homozygous mutant AA have increased the risk of CD.But the SNP loci gene polymorphism is no significant correlation with CD clinical characteristics.
炎症性肠病(Inflammatory bowel disease,IBD)包括克罗恩病(Crohn's disease,CD)和溃疡性结肠炎(Ulcerative colitis,UC)。近年来IBD的发病率呈上升趋势,特别是CD的发病率,随着双气囊小肠镜的应用,CD的发现率逐年上升。以往IBD一直被认为是一种自身免疫性疾病,而近年来多个研究证明IBD特别是CD具有明显的遗传易感性。目前较为一致的观点认为,炎症性肠病是携带遗传易感基因的宿主在环境因素作用下,自身免疫功能紊乱导致的一种非特异性炎症性疾病。因此遗传因素在炎症性肠病的发病过程中起着非常重要的作用。随着人类基因组计划的完成,对基因研究的重点已由全基因组序列测定转移到了对基因组中个体基因多态性和功能的研究。个体基因多态性的主要形式就是单链核苷酸多态性(Single nucleotide polymorphism,SNP),SNP被公认为继“限制性片段长度多态性”和“微卫星多态性”这两种遗传标记之后,出现的“第三代DNA遗传标记”,是目前全世界基因研究领域的热点之一。
2001年欧洲Hugot研究小组及美国Ogura研究小组几乎同时发现了人类第一个CD易感基因NOD2基因,后改名为CARD15,并证实该基因的3个单核苷酸多态性(SNP)位点Arg702Trp、Gly908Arg和Leu1007fsinsC是白种人CD的易感基因,但后来的研究证明此三个SNP位点与日本人及我国香港人、浙江人CD患者无关。我们也对广东地区CD患者NOD2基因的三个SNP位点进行研究发现其与CD无显著相关,并通过基因测序的方法发现了可能与中国人CD密切相关的SNP位点P268S,并成功构建了该位点的突变型真核表达载体,为后续功能的研究打下了基础。最近西方多个研究小组通过全基因扫描的方法分别对欧洲高加索人、犹太人、英国、法国、巴尔干、德国、加拿大(魁北克)等地区的IBD患者进行研究,发现了一些与IBD,特别是CD患者密切相关的SNP位点,主要位于IL23R、ATG16L1、PTPN2、10q21、NKX2、IRGM、MST1、DLG5、OCTN1、5p31等基因中。其中位于IL23R基因中的rs11209026、rs11805303位点、PTPN2基因中的rs2542151和位于染色体10q21中的rs10761659 SNP位点统计学意义较为明显;rs11209026位于IL23R基因的第10外显子中,正常为G,突变为A,导致381位的氨基酸由精氨酸(Arg)变为谷氨酰胺(Gln),Duerr等人研究的结果认为该SNP位点与欧洲白种人CD患者显著相关(P=1.56×10~(-3)),其OR值为0.26(犹太人为0.47);rs11805303位于IL23R基因第7和第8外显子之间的内含子之中,正常为C,突变为T,该碱基的改变增加了西方人CD发病的风险,杂合突变的OR值为1.39,纯合突变的OR值为1.86;rs2542151位于PTPN2基因上游约5.5kb的区域中,可能为PTPN2基因的启动子区或者上游控制元件,PTPN2基因编码一种关键的炎症负性调节因子—T细胞蛋白酪氨酸磷酸酶(T cell protein tyrosine phosphatase,TCPTP),在西方IBD患者当中,其危险等位基因为G,杂合子的OR值为1.3(1.14-1.48),纯合子的OR值为2.01(1.46-2.76),rs10761659位于10q21的基因间区,其危险等位基因为G,其杂合子的OR值为1.23(1.05-1.45),纯合子的OR值为1.55(1.30-1.84)。
本研究的目的通过基因测序和限制性片段长度多态性的方法探索与西方白种人及犹太人CD患者密切相关的上述四个多态性位点与IBD患者发病易感性的相关性,及其与临床特征的关系,为进一步研究IBD的发病机制及探索IBD易感基因打下基础,并有可能为临床治疗IBD提供一种新的基因治疗靶点。
材料和方法:
1、收集南方医院消化科及普外科确诊,临床资料完整,彼此无血源关系的广东及外省籍CD患者40例(包括前期收集病例24例和新收病例16例),UC患者40例(所选患者均为手术或者内镜下确诊的病例,并除外合并其它自身免疫性疾病),所选患者中男52例,女28例;平均年龄34岁;健康对照组(Healthy control,HC)取门诊健康体检者50例,其中男32例,女18例,平均年龄35岁;所有研究对象均抽取外周血5 ml。
2、应用特异性结合DNA的硅基质材料离心吸附柱法和独特的洗脱缓冲液系统抽提血液基因组DNA(按试剂盒说明书操作),抽提成功后DNA产物-70℃保存。
3、针对上述四个SNP位点用Primer5.0软件设计PCR引物,PCR方法扩增包含这四个SNP位点在内的DNA片段,扩增产物以1.5%琼脂糖凝胶电泳观察以确保目的片段扩增成功,然后采用切胶纯化试剂盒(TIANGEN公司提供)将目的片段纯化回收,分光光度计检测纯化产物浓度。
4、将纯化产物分别采用DNA直接测序法和聚合酶链反应—限制性片段长度多态性分析(restriction fragment length polymorphism polymerase chainreaction products,PCR-RFLP)进行研究;测序结果用chromas231判读SNP位点的基因型。酶切产物以2.5%琼脂糖凝胶电泳,根据电泳结果判读SNP位点的基因型。采用基因计数法计算四个SNP位点基因型频率和等位基因频率,最后进行统计分析。
结果:
1、IL23R(rs11209026和rs11805303)基因多态性与炎症性肠病发病的相关性:
通过DNA测序,我们发现中国人部分CD患者位于IL23R中的这两个SNP位点基因型频率及等位基因频率与健康对照组比较差异均无统计学意义(P＞0.05);rs11805303位点基因型频率UC组与HC组比较,P＞0.05,差异无显著性;而等位基因频率比较,P＜0.05(Fisher精确概率检验P=0.024),差异具有统计学意义;但rs11805303位点基因多态性与中国部分UC患者的性别、疾病的活动性及发病部位比较P值均大于0.05;提示rs11805303位点的多态性可能与中国部分UC患者有相关性。
2、PTPN2(rs2542151)和10q21(rs10761659)基因多态性与炎症性肠病发病的相关性:
通过聚合酶链反应—限制性片段长度多态性分析(PCR-RFLP),结果显示:①PTPN2(rs2542151)位点:CD组基因型频率、等位基因频率和基因携带者频率分别与HC组比较P值均大于0.05,而UC组基因型频率和等位基因频率与HC比较P＜0.05(分别为:0.002和0.006),且3例纯合子GG均出现于UC患者,提示该位点可能与中国部分UC患者的发病易感性有关。②10q21(rs10761659)位点:UC组的基因型频率、等位基因频率和基因携带者频率分别与HC组比较P值均大于0.05,CD组等位基因频率与HC组比较P值也大于0.05,而基因型频率与HC组比较P=0.044,提示该位点可能与中国UC患者易感性无关,但该位点基因型可能与中国部分CD患者有相关性。但这两个位点与相应疾病的临床特征均无显著相关(P值均大于0.05)。
结论:
1、IL23R中两个与西方人CD密切相关的SNP位点(rs11209026和rs11805303)的基因多态性与中国部分CD患者的发病无显著相关性,而rs11805303位点的多态性可能与中国部分UC患者有相关性,且该碱基的改变降低了UC的发病风险,但这个SNP位点基因多态性与UC患者的病变特点并无显著相关;
2、位于PTPN2基因上游区域中的rs2542151位点可能是中国部分UC患者的一个易感基因,其纯合子GG增加了UC的发病风险,但该位点基因多态性与UC的临床特点无显著相关。
3、位于染色体10q21中的rs10761659位点可能是中国部分CD患者的一个易感基因,其纯合子AA增加了CD的发病风险,但这个SNP位点基因多态性与CD患者的病变特点并无显著相关。
Background and Objective:
Inflammatory bowel disease(IBD) includes Crohn's disease(CD) and ulcerative colitis(UC).Its etiology and pathogenesis are still unknown.Most of the views presume that a kind of non-specific inflammatory diseases can be induced under the role of environmental factors and autoimmune disorders for the host who carry the genetic susceptibility gene.In recent years,with the Human Genome Project completed,human have been transferred the focus about gene research from the whole genome sequence to individual gene polymorphism and functional studies. Single-chain nucleotide polymorphism(SNP) is the main form of individual gene polymorphism;the SNP is recognized as the "third generations of DNA genetic marker "following the restriction fragment length polymorphism and microsatellite polymorphism,which is one of the hottest fields in the world.
Hugot Research Group in Europe and Ogura team in the United States simultaneously found out the first predisposing gene of CD—NOD2,which was renamed CARD15 in 2001.They certified this gene includes three SNPs sites: Arg702Trp,Gly908Arg,Leu1007fsinsC,and further confirmed the SNPs site are belong to Caucasian's CD patients predisposing gene but don't related with the CD patients bom from Japan,Hong Kong and ZheJiang province.We have researched the three SNPs site about NOD2 gene of CD patients born from Guangdong,the results indicated there isn't correlating with them,but we found out the closely related SNPs site—P268S from Chinese by detecting gene sequence,and constructed eukaryotic expression vector,the further study are carrying out.
Western research groups recently study the gene susceptibility of the IBD patients who came from the Caucasus of Europe,the United Kingdom,France,the Balkans,Germany,Canada,and other regions in Europe by gene scanning,they found some of SNPs sites about IBD and especially related with CD patients,which located the gene of IL23R、ATG16L1、PTPN2、10q21、NKX2、IRGM、MST1、DLG5、OCTN1、5p31 and so on.The site of rs11209026、rs11805303 in IL23R gene、rs2542151 in PTPN2 gene and rs10761659 in chromosome 10q21 had statistical significance comparing with control.The normal rs11209026 in the exon 10 of IL23R gene is G,when it is mutated to A,the 381 amino acid arginine acid(Arg) become glutamine(Gln).Duerr believed that the SNP mutation were closely correlated with the European CD patients(p=1.56×10-3),OR=0.26(Jews 0.47).this indicating that the base-point change is a protection change;possible mechanism is that the function of its encoded protein is changed by mutations affecting the amino acid, thereby blocking the receptor binding after IL23 and the downstream Signal transduction pathways;rs11805303 lies in exon 7 and exon 8 of IL23R gene,normal C and T mutations in the base of the changes increased the risk of morbidity Westerners CD,heterozygous mutations OR=1.39,homozygous mutation OR=1.86; its influence gene function mechanism is not clear,may be due to the impact of changes in base gene transcription or mRNA transcription after shearing,and even as a remote-control devices affect gene function.Rs2542151 lies in PTPN2 gene, encoding a T-cell protein tyrosine phosphatase(TCPTP) - a key negative regulator of inflammation and the risk for the G allele,heterozygous OR=1.3(1.14-1.48), homozygous OR=2.01(1.46-2.76).Rs10761659 lies in 10 q21 area between the gene and the risk allele is the G,heterozygous OR=1.23(1.05-1.45),homozygous OR= 1.55(1.3-1.84);their specific mechanism is still further studies.
The purpose of this study is to prove the four SNPs which related to western CD patients whether associate with Chinese IBD patients and clinical characteristics by gene sequencing and restriction fragment length polymorphism methods,Our purpose is to further study the pathogenesis of IBD and patterns IBD susceptibility gene mapping.maybe also provide a new means of gene therapy for the treatment of IBD
Methods:
1.Forty Chinese Crohn's disease(CD) patients(including twenty-four former samples and sixteen new samples)、forty ulcerative colitis(UC) and fifty healthy controls(HC) were prospectively recruited from the Nan Fang Hospital.Peripheral blood was collected from patients and controls.
2.Extract genomic DNA from the blood(in kit brochures operation),after the success of extraction,preserve them in - 70℃.
3.Design primers using Primer5.0 software.To amplify the fragments includes the four SNPs.After succeeded amplify,(amplified products by 1.5%agarose gel electrophoresis to ensure that the observation fragment was successfully amplified).Purify the PCR production and directed sequence the target region in SIB3730XL sequencer.Then,assessment genotype with chromas2.0 and Last contrasted the gene bank data to analyze the polymorphism of SNPs in IBD patients and controls.If we find mutation that we amplify the corresponding target region and purify the PCR production and directed sequence the target region and contrasted gene bank data with uniform means.Two SNPs use the method of restriction fragment length polymorphism(RFLP) to testify the polymorphism in CD patients and find it's frequency in UC and health control.
Results:
1.IL23R(rs11209026 and rs11805303) gene polymorphism associated with the pathogenesis of inflammatory bowel disease:
Through DNA sequencing,we found that there was no significant difference in genotype frequencies and allele frequency with these two SNPs which lies in IL23R between Chinese Crohn's disease(CD) patients and normal population(P＞0.05); rs11805303 sites genotype frequencies of ulcerative colitis(UC) group compared with the control group,P＞0.05,there was no significant difference too,but allele frequencies in UC(UC is 41.2%)compared with normal control(normal control is 59.0%),P＜0.05(P=0.018),the differences were statistically significant;but rs11805303 gene polymorphism was not associated with the incidence age,gender, disease activity and the location of UC patients(P＞0.05);these cue us:rs11805303 polymorphism loci may be related to the incidence of partial UC patients in Chinese people.
2.PTPN2(rs2542151) and 10q21(rs10761659) gene polymorphism associated with the pathogenesis of inflammatory bowel disease:
Through restriction fragment length polymorphism(RFLP),PTPN2(rs2542151) gene polymorphism compared with IBD:genotype frequency,allele frequency and the gene-phore frequency in CD Group compared with normal control group,P＞0.05, and UC group genotype frequencies and allele frequency compared with normal controls,P＜0.05(P=0.002,P=0.006 respectively),suggesting that the site(rs2542151) may be related to UC susceptibility but unrelated with CD patients.10q21 (rs10761659) gene polymorphism compared with IBD:the UC group carriers of genotype frequency,allele frequency and the gene-phore frequency compared with normal control group P＞0.05,but CD group genotype frequencies compared with normal control,P=0.044,suggesting that the site(rs10761659) may be unrelated with Chinese UC patients,but genotype of the sites may be associated with Chinese CD patients.
Conclusion:
1.The two SNPs polymorphism(lies in IL23R gene) which related to Western CD patients was not associated with Chinese CD patients,and rs11805303 polymorphism loci may be a genetic marker of Chinese UC patients,However,there was no significant correlation between the SNPs loci gene polymorphism and characteristics of UC patients;
2.It is not similar to western results,rs2542151 polymorphism which related to Western CD patients likely to be a susceptibility gene sites in Chinese UC patients, homozygous mutant GG have increased the risk of UC.But the SNP loci gene polymorphism is no significant correlation with UC clinical characteristics.
3.rs10761659 polymorphism which related to Western CD patients likely to be a susceptibility gene sites in Chinese CD patients,homozygous mutant AA have increased the risk of CD.But the SNP loci gene polymorphism is no significant correlation with CD clinical characteristics.
引文
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29. Wee C, Muise ES, Ennis M, et al. Promoter analysis of the murine T- cell protein tyrosine phosphatase gene [J] .Gene, 1999, 237:351-360.
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33. Lam MH, Michell BJ, Fodero - Tavoletti, et al. Cellular stress regulates the nucleocytoplasmic distribution of the protein tyrosine phosphatase TCPTP [J].J Biol Chem, 2001, 276:37700 - 37707.
34. Iversen LF, Moller KB. Structure determination of T cell protein tyrosine phosphatase [J]. J Biol Chem, 2002,20:20.
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36. Simoncic PD, Lee - Loy A, Barber DL, et al. The T cell protein tyrosine phosphatase is a negative regulator of Janus family kinases 1 and 3[J]. Curr Biol, 2002,12(6):446-453.
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2.Zhi FC,Xiao B,Jiang B,et al.Double-balloon enteroscopy detecting small intestinal bleeding.Chin Med J(Engl),2005,118:1834-1837.
3.Ahmad T,Satsangi J,Mcgovern D,et al.Thegenetics of inflammatory bowil diseas.AlimentParmarcol Ther,2001,15(6):731-748.
4.HugotJP,Chamaillard M,ZoualiH,etal.Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.Nature,2001,411:599-603.
5.Ogura Y,Bonen DK,Inohara N,etal.A frame shift mutation in NOD2associated with susceptibility to Crohn's disease.Nature,2001,411:603-606.
6.Leong RW,Armuzzi A,Ahmad T,etal.NOD2/CARD15 gene polymorphisms and Crohn's disease in the Chinese population.Aliment Pharmacol Ther,2003,17:1465-1470.
7.高敏,曹倩,罗灵和,等.NOD2/CARD15基因多态性与克罗恩病患者相关性研究.中华内科杂志,2005,44(3):210-212.
8.Inoue N,Tamura K,Kinouchi Y,etal.Lack of common NOD2 variants in Japanese patients with Crohn's disease.Gastroenterology,2002,123:86-91.
9.张以洋,智发朝,周殿元,等。克罗恩病CARD15/NOD2基因突变的研究.中华消化杂志,2006,26(7):456-459.
10.龙靖华,智发朝,张迎春,张以洋,钟长青,姚国鹏,陈正彦,林勇,智佳,关婧.NOD2/CARD15基因突变与中国人克罗恩病相关性的研究.胃肠病学,2007,12(6):327-330.
11.钟长青,智发朝,王继德,龙靖华,张迎春.P268S突变型NOD2/CARD15真核表达载体的构建及其体外表达.胃肠病学,2007,12(6):331-334.
12. Christopher G. Mathew. New links to the pathogenesis of Crohn disease provided by genome-wide association scans. Nature Reviews Genetics. 2007,2203:1038-1048
13. Duerr, R. H. et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science. 2006,314:1461-1463
14. The Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls.Nature. 2007, 447:661-678.
15. Tremelling, M. et al. IL23R variation determines susceptibility but not disease phenotype in inflammatory bowel disease. Gastroenterology 2007, 132:1657-1664.
16. 2006, 26(7):456-459.Rioux, J. D. et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nature Genet, 2007, 39:596-603.
17. Hampe, J. et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nature Genet.2007,39:207-211.
18. Peltekova, V. D. et al. Functional variants of OCTN cation transporter genes are associated with Crohn disease. Nature Genet. 2004, 36:471-475.
19. Rioux, J. D. et al. Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease. Nature Genet. 2001, 29:223-228.
20. Todd, J. A. et al. Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes. Nature Genet. Advance online publication, 2007,10:1038-1068.
21. Rioux, J. D. et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nature Genet. 2007, 39:596-604.
22. Oppmann B, Lesley R, Blom B, et al. Novel pi9 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12[J]. Immunity, 2000,13(5):715-725.
23. Amir F Sheibanie, Iman Tadmori, Huie Jing, et al. Prostaglandin E2 induces IL-23 production in bone marrow derived dendritic cells [J]. FASEB Journal, 2004, 18(11):1318-1320.
24. Parham C, Chirica M, Timans J, et al. A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbetal and a novel cytokine receptor subunit, IL-23R [J]. JImmunol ,2002 ,168(11):5699-5708
25. Sudeepta Aggarwal, Nico Ghilardi, Ming-Hong Xie, Frederic J. de Sauvage.Interleukin-23 Promotes a Distinct CD4 T Cell Activation State Characterized by the Production of Interleukin-17, 2003,278(3):1910 - 1914.
26. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B,Sedgwick JD,et al. IL-23 drives a pathogenic Tcell population that induces autoimmune inflammation. J Exp Med .2005, 201-233.
27. Yoichiro Iwakura and Harumichi Ishigame. The IL-23/IL-17 axis in inflammation. The Journal of Clinical Investigation. 2006, 116:1218-1222.
28. Zili Zhang, David J. Hinrichs, Huiying Lu, Hong Chen. After interleukin-12p40, are interleukin-23 and interleukin-17 the next therapeutic targets for inflammatory bowel disease? International Immunopharmacology.2007, 7:409-416.
29. Wee C, Muise ES, Ennis M, et al. Promoter analysis of the murine T- cell protein tyrosine phosphatase gene [J] .Gene, 1999, 237:351-360.
30. Simoncic PD, Lee - Loy A, Barber DL, et al. The T cell protein tyrosine phosphatase is a negative regulator of Janus family kinases 1 and 3[J]. Curr Biol,2002, 12(6):446-453.
31. Aoki N, Matsuda T. A nuclear protein tyrosine phosphatase TC - PTP is a potential negative regulator of the PRL - mediated signaling pathway:dephosphorylation and deactivation of signal transducer and activator of transcription 5a and 5b by TC - PTP in nucleus [J]. MolEndocrinol, 2002, 16(1):58-69.
32. Persson C , Savenhed C , Bourdeau A , et al. Site - selective regulation of platelet - derived growth factor beta receptor tyrosine phosphorylation by T -cell protein tyrosine phosphates[J] .Mol Cell Biol ,2004 ,24(5):2190- 2201.
33. Lam MH, Michell BJ, Fodero - Tavoletti, et al. Cellular stress regulates the nucleocytoplasmic distribution of the protein tyrosine phosphatase TCPTP [J].J Biol Chem, 2001, 276:37700 - 37707.
34. Iversen LF, Moller KB. Structure determination of T cell protein tyrosine phosphatase [J]. J Biol Chem, 2002,20:20.
35. Galic S , Hauser C , Kahn BB , et al. Coordinated regulation of insulin signaling by the protein tyrosine phosphatases PTP1B and TCPTP[J] .Mol Cell Biol, 2005,25(2):819 - 829.
36. Simoncic PD, Lee - Loy A, Barber DL, et al. The T cell protein tyrosine phosphatase is a negative regulator of Janus family kinases 1 and 3[J]. Curr Biol, 2002,12(6):446-453.
37. Yamamoto T, Sekine Y, Kashima K, et al. The nuclear isoform of protein -tyrosine phosphatase TC - PTP regulates interleukin - 6 - mediated signaling pathway through STAT3 dephosphorylation [J]. Biochem Biophys Res Commun, 2002, 297(4):811 - 817.
38. Tiganis T, Kemp BE, Tonks NK. The protein - tyrosine phosphatase TCPTP regulates epidermal growth factor receptor - mediated and phosphatidylinositol 3 - kinase -dependent signaling [J]. J Biol Chem, 1999,274(39):27768 - 27775.
39. Mattila E , Pellinen T, Nevo J , et al. Negative regulation of EGFR signalling through integrin-alphalbetal-mediated activation of protein tyrosine phosphatase TCPTP[J].Nat Cell Biol,2005,7(1):78-85.
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