OCTN1、OCTN2及TNFα基因多态性与炎症性肠病相关性的研究
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摘要
背景和目的:
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的发病率尤其是CD的发病率呈上升趋势,随着双气囊小肠镜的应用,CD的检出率逐年上升。以往一直认为IBD是一种自身免疫性疾病,而近年来诸多研究证明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是单链核苷酸多态性(Singl enucleotide 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,但后续的研究证明此三个SNP位点与日本人及我国香港人、浙江人CD患者无关。我们课题组对广东地区CD患者NOD2基因的上述三个SNP位点进行研究发现其与CD无显著相关,但通过基因测序的方法发现了可能与中国人CD密切相关的P268S位点,并已成功构建了该位点的突变型真核表达载体。此后,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OCTN1、OCTN2、TNFα、IL23R、ATG16L1、PTPN2、10q21、NKX2、IRGM、MST1、DLG5、5p31等基因中。其中位于OCTN1基因中的rs1050152位点、OCTN2基因中的rs2631367位点和位于TNFα基因中的G-308A位点具有显著的统计学意义。rs1050152位于OCTN1基因第9外显子中,正常为C,突变为T,导致第503位的氨基酸由亮氨酸(Leu)变成苯丙氨酸(Phe),而影响对肉毒碱的转运,使细胞内肉毒碱含量下降,导致脂肪酸氧化受抑,能量产生不足,最终出现细胞代谢紊乱;rs2631367位于OCTN2基因启动子区,正常为G,突变为C,其发病机制可能是该突变破坏了OCTN2基因的热休克元件(heart shock element),从而影响肉毒碱的转运,Peltekova等首次报道了以上两个SNP位点与欧洲CD患者明显相关,存在其中一个突变可使CD的患病风险提高2.5倍,同时具有两个变异发病风险提高4倍;G-308A位于TNFα基因启动子区,Wilson等首先报道了TNFα基因启动子区域内-308位点的双等位基因多态性,各项研究表明TNFα在IBD的发病中起重要作用,G-308A位点多态性可影响TNFα的生物学作用从而可能参与IBD的发病。
本研究目的是通过基因测序和限制性片段长度多态性的方法初步探索与西方人CD患者相关的上述三个多态性位点与中国部分汉族人IBD患者的相关性,进一步为IBD的发病机制提供理论依据,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、收集南方医科大学南方医院消化科及普外科确诊、临床资料完整、彼此无血缘关系的广东及外省籍(湖南、湖北、江西、福建、广西等)CD和UC患者(诊断标准采用2007年制定的IBD诊断标准,所有患者均除外合并其他自身免疫性疾病)各45例。UC患者中病变位于直肠6例,左半结肠12例,右半结肠6例,全结肠21例,其中男26例、女19例,平均年龄(35.58±15.81)岁;CD患者中病变部位位于小肠19例,结肠14例,小肠合并结肠9例,直肠3例,其中男28例、女17例,平均年龄(32.91±10.54)岁。对照组取门诊健康体检者50例,其中男32例、女18例,平均年龄(37.62±12.62)岁。所有研究对象均抽取外周血5ml。
2、CD、UC及健康对照组均于清晨空腹抽取外周静脉血5ml置于加入枸橼酸钠的抗凝管中,-20℃保存。按TIANGEN公司提供的试剂盒说明书操作,应用特异性结合DNA的硅基质材料离心吸附柱法提取血液白细胞基因组DNA,提取成功后置-70℃保存。
3、针对上述三个SNP位点用Primer5.0软件设计PCR引物,PCR方法扩增分别包含这三个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,并用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 chain reactionproducts,PCR-RFLP)进行研究;测序结果用chromas2判读SNP位点的基因型。酶切产物以2.0%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三个SNP位点基因型频率和等位基因频率,最后进行统计学分析。
结果:
1、OCTN1基因多态性位点rs1050152与中国部分汉族炎症性肠病的相关性:
通过DNA测序发现,OCTN1基因第9外显子上的rs1050152位点只有少数发生杂合突变(CT),所有三组研究对象均未发现纯合突变(TT),其中CD组有3例,UC组有4例,健康对照组有6例发生杂合突变,以上发生突变的样本均通过反向测序验证。CD患者该位点基因型频率为6.7%,等位基因频率为3.3%,UC患者该位点基因型频率为8.9%,等位基因频率为4.4%。CD组、UC组与对照组该位点基因型频率和等位基因频率(12.0%和6.0%)比较三组间差异均无统计学意义(x~2值分别=0.812和0.770;P值分别=0.715和0.727)。提示rs1050152位点的多态性可能与中国汉族人群IBD无明显相关性。
2、OCTN2基因多态性位点rs2631367与中国部分汉族炎症性肠病的相关性:
DNA测序发现CD患者、UC患者及健康对照者该位点基因型均全部为野生型纯合子GG,即未发现G-207C突变型基因型。提示rs2631367位点的多态性可能与中国汉族人群IBD无明显相关性。
3、TNFα基因多态性位点G-308A与中国部分汉族炎症性肠病的相关性:
通过聚合酶链反应—限制性片段长度多态性分析(PCR-RFLP),CD患者、UC患者及健康对照者该位点基因型均全部为野生型纯合子GG,即未发现G-308A突变型基因型。提示G-308A位点的多态性可能与中国汉族人群IBD无明显相关性。
结论:
1、国外报道的与西方人CD易感性相关的OCTN1基因多态性位点rs1050152和OCTN2基因多态位点rs2631367在中国部分汉族人CD、UC和正常健康对照组之间并无显著性差异,考虑OCTN1/2基因中与西方人CD易感性相关的以上两个主要SNP位点与中国部分汉族IBD患者易感性可能无关。
2、位于TNFα基因启动子区域的G-308A位点未检测出突变型基因型,考虑TNFα基因G-308A位点多态性可能与中国部分汉族人群IBD无明显相关性。
Background and Objectives:
Inflammatory bowel disease(IBD) includes Crohn's disease(CD) and ulcerativecolitis(UC).The incidence of IBD especially CD is going up year by year, but itsetiology and pathogenesis are still unknown.Most of the views presume that it's akind of non-specific inflammatory disease that can be induced under the role ofenvironmental factors and autoimmune disorders for the host who carry the geneticsusceptibility genes.In recent years,with the Human Genome Project have beencompleted,human have transferred the focus on gene research from the wholegenome sequence to individual gene polymorphism and functional studies. Singlechain nucleotide polymorphism(SNP) is the main form of a individual gene polymorphism,the SNP is recognized as "the third generation of DNA genetic marker "following the restriction fragment length polymorphism and microsatellite polymorphism,which is one of the hottest fields in the world.
The Hugot Research Group in Europe and Ogura team in the United States simultaneously found out the first predisposing gene of CD,names NOD2,which was renamed CARD15 in 2001.They certified that this gene includes three SNP sites:Arg702Trp,Gly908Arg and Leu1007fsinsC,and further confirmed the SNP sites are belong to Caucasian's CD patients predisposing gene but don't related with the CDpatients born from Japan、Hong Kong and ZheJiang province of China.We haveresearched the three SNP sites about NOD2 gene of CD patients born fromGuangdong,and the results indicated there have no correlation with them,but wefound out the closely related SNP site(names P268S) from Chinese by detecting genesequence,and have constructed eukaryotic expression vector,and the further researchare carrying out.
Recently several research groups in Western studied on the gene susceptibility ofthe IBD patients who come from the Caucasus of Europe,the United Kingdom,France,the Balkans,Germany,Canada,and other regions in Europe by gene scanning,they found out some SNP sites about IBD,especially about CD patients,whichlocated in the gene of OCTN1、OCTN2,TNFα、IL23R、ATG16L1、PTPN2、10q21、NKX2、IRGM、MST1、DLG5、5p31 and so on.Among them,the sites of rs1050152 in OCTN1 gene,rs2631367 in OCTN2 gene and G-308A in TNFαgene haveapparente statistical significance compared with controls. Rs1050152 sites in the ninthexon of OCTN1gene,normally,it is C,when it is mutated to T,the 503 amino acidleucine(Leu) trans into phenylalanine(Phe).This mutation effects the transportationof camitine for cells,and this leads to degression of carnitine inside cells and cellularmetabolic disorder.Rs2631367 sites in the promotor of OCTN2 gene,normally,it is G,when it is mutated to C,the heart shock element of OCTN2 gene is destroyed,andthen effects the transportation of carnitine for cells.Peltekova and his teammate foundout that the above two SNPs are associated with CD in Europe population for the firsttime.The onset risk of CD will raise 2.5 times when one of them mutates,and it willraise 4 times when all of them mutate.G-308A sites in the promotor of TNFαgene,Wilson found out its diallele gene polmorphisms for the first time.Many studiesindicate that tumor necrosis factor alpha plays very important role in the onset of IBD,and the polmorphisms of G-308A site can effect its biological activity.
The purpose of this study is to prove the three SNPs which related to western CD patients whether associate with Chinese Han population IBD patients by genesequencing and restriction fragment length polymorphism methods,Our purpose is tofurther study the pathogenesis of IBD and patterns IBD susceptibility gene mapping.Maybe,we will also provide a new means of gene therapy for the treatment of IBD.
Methods
1.Forty-five Chinese Crohn's disease(CD) patients(including twenty-six male samples and ninteen female samples),forty-five ulcerative colitis(UC) patients(including twenty-eight male samples and seventeen female samples) and fifty healthycontrols(HC) were prospectively recruited from the Nan Fang Hospital. In ulcerative colitis cases,diseased region in rectum 6 sample,left hemicolon 12 sample,right hemicolon 6 sample,hole colon 21 sample,among the total male 26, female 19、mean age was 35.58±15.81;in Crohn's disease case,diseased region in small intestine 19 sample,colon 14 sample,small intestine complicating with colon 9 sample,rectum 3 sample,among the total male 28,female 17,mean age was 32.91±10.54,Peripheral blood was collected from patients and white blood cell was separated.
2.Extract genomic DNA from the blood(in kit brochures operation),after the success of extraction,preserve them under-70℃.
3.Design primers using Primer5.0 software.To amplify the fragments includes the three SNPs.After succeeded amplify,(amplified products by 1.5% agarosegelelectrophoresis to ensure that the observation fragment was successfullyamplified).Purify the PCR production and directed sequence the target region inSIB3730XL sequencer.Then,assessment genotype with chromas2.0 and lastcontrasted 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 contrastedgene bank data with uniform means.One SNPs use the method of restriction fragmentlength polymorphism(RFLP) to testify the polymorphism in CD patients and find it'sfrequency in UC and health control.
Results:
1.OCTN1(rs1050152) gene polymorphism associated with the pathogenesis ofinflammatory bowel disease:
By DNA sequencing,we found that There was no statistical significance of thegenotype frequency and the allele frequency of SNP site between case groups of CDpatients、UC patients and normal control group:P=0.715,P=0.727.As a result, we can conclude that there is no correlation between thepolymorphism site rs1050152 of gene OCTN1 and Chinese susceptibility of IBD.
2.OCTN2(rs2631367) gene polymorphism associated with the pathogenesis of inflammatory bowel disease:
By DNA sequencing,we can't find any mutational genetype,As a result,we can conclude that there is no correlation between the polymorphism site rs2631367 ofgene OCTN2 and Chinese susceptibility of IBD.
3.TNFα(G-308A) gene polymorphism associated with the pathogenesis of inflammatory bowel disease:
Through restriction fragment length polymorphism(RFLP),we can't find anymutational genetype,As a result,we can conclude that there is no correlation betweenthe polymorphism site G-308A of gene TNFαand Chinese susceptibility of IBD.
Conclusion:
1.Our research indicates that the SNP sites rs1050152 of OCTN1 gene andrs2631367 of OCTN2 which reported abroadly was no associated with susceptibilityto IBD in the Chinese Hart population.
2.we can't find any mutational genetype in all the three groups,there isn't association between the SNP site G-308A of TNFαgene and Chinese susceptibility of IBD.
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的发病率尤其是CD的发病率呈上升趋势,随着双气囊小肠镜的应用,CD的检出率逐年上升。以往一直认为IBD是一种自身免疫性疾病,而近年来诸多研究证明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是单链核苷酸多态性(Singl enucleotide 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,但后续的研究证明此三个SNP位点与日本人及我国香港人、浙江人CD患者无关。我们课题组对广东地区CD患者NOD2基因的上述三个SNP位点进行研究发现其与CD无显著相关,但通过基因测序的方法发现了可能与中国人CD密切相关的P268S位点,并已成功构建了该位点的突变型真核表达载体。此后,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OCTN1、OCTN2、TNFα、IL23R、ATG16L1、PTPN2、10q21、NKX2、IRGM、MST1、DLG5、5p31等基因中。其中位于OCTN1基因中的rs1050152位点、OCTN2基因中的rs2631367位点和位于TNFα基因中的G-308A位点具有显著的统计学意义。rs1050152位于OCTN1基因第9外显子中,正常为C,突变为T,导致第503位的氨基酸由亮氨酸(Leu)变成苯丙氨酸(Phe),而影响对肉毒碱的转运,使细胞内肉毒碱含量下降,导致脂肪酸氧化受抑,能量产生不足,最终出现细胞代谢紊乱;rs2631367位于OCTN2基因启动子区,正常为G,突变为C,其发病机制可能是该突变破坏了OCTN2基因的热休克元件(heart shock element),从而影响肉毒碱的转运,Peltekova等首次报道了以上两个SNP位点与欧洲CD患者明显相关,存在其中一个突变可使CD的患病风险提高2.5倍,同时具有两个变异发病风险提高4倍;G-308A位于TNFα基因启动子区,Wilson等首先报道了TNFα基因启动子区域内-308位点的双等位基因多态性,各项研究表明TNFα在IBD的发病中起重要作用,G-308A位点多态性可影响TNFα的生物学作用从而可能参与IBD的发病。
本研究目的是通过基因测序和限制性片段长度多态性的方法初步探索与西方人CD患者相关的上述三个多态性位点与中国部分汉族人IBD患者的相关性,进一步为IBD的发病机制提供理论依据,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、收集南方医科大学南方医院消化科及普外科确诊、临床资料完整、彼此无血缘关系的广东及外省籍(湖南、湖北、江西、福建、广西等)CD和UC患者(诊断标准采用2007年制定的IBD诊断标准,所有患者均除外合并其他自身免疫性疾病)各45例。UC患者中病变位于直肠6例,左半结肠12例,右半结肠6例,全结肠21例,其中男26例、女19例,平均年龄(35.58±15.81)岁;CD患者中病变部位位于小肠19例,结肠14例,小肠合并结肠9例,直肠3例,其中男28例、女17例,平均年龄(32.91±10.54)岁。对照组取门诊健康体检者50例,其中男32例、女18例,平均年龄(37.62±12.62)岁。所有研究对象均抽取外周血5ml。
2、CD、UC及健康对照组均于清晨空腹抽取外周静脉血5ml置于加入枸橼酸钠的抗凝管中,-20℃保存。按TIANGEN公司提供的试剂盒说明书操作,应用特异性结合DNA的硅基质材料离心吸附柱法提取血液白细胞基因组DNA,提取成功后置-70℃保存。
3、针对上述三个SNP位点用Primer5.0软件设计PCR引物,PCR方法扩增分别包含这三个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,并用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 chain reactionproducts,PCR-RFLP)进行研究;测序结果用chromas2判读SNP位点的基因型。酶切产物以2.0%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三个SNP位点基因型频率和等位基因频率,最后进行统计学分析。
结果:
1、OCTN1基因多态性位点rs1050152与中国部分汉族炎症性肠病的相关性:
通过DNA测序发现,OCTN1基因第9外显子上的rs1050152位点只有少数发生杂合突变(CT),所有三组研究对象均未发现纯合突变(TT),其中CD组有3例,UC组有4例,健康对照组有6例发生杂合突变,以上发生突变的样本均通过反向测序验证。CD患者该位点基因型频率为6.7%,等位基因频率为3.3%,UC患者该位点基因型频率为8.9%,等位基因频率为4.4%。CD组、UC组与对照组该位点基因型频率和等位基因频率(12.0%和6.0%)比较三组间差异均无统计学意义(x~2值分别=0.812和0.770;P值分别=0.715和0.727)。提示rs1050152位点的多态性可能与中国汉族人群IBD无明显相关性。
2、OCTN2基因多态性位点rs2631367与中国部分汉族炎症性肠病的相关性:
DNA测序发现CD患者、UC患者及健康对照者该位点基因型均全部为野生型纯合子GG,即未发现G-207C突变型基因型。提示rs2631367位点的多态性可能与中国汉族人群IBD无明显相关性。
3、TNFα基因多态性位点G-308A与中国部分汉族炎症性肠病的相关性:
通过聚合酶链反应—限制性片段长度多态性分析(PCR-RFLP),CD患者、UC患者及健康对照者该位点基因型均全部为野生型纯合子GG,即未发现G-308A突变型基因型。提示G-308A位点的多态性可能与中国汉族人群IBD无明显相关性。
结论:
1、国外报道的与西方人CD易感性相关的OCTN1基因多态性位点rs1050152和OCTN2基因多态位点rs2631367在中国部分汉族人CD、UC和正常健康对照组之间并无显著性差异,考虑OCTN1/2基因中与西方人CD易感性相关的以上两个主要SNP位点与中国部分汉族IBD患者易感性可能无关。
2、位于TNFα基因启动子区域的G-308A位点未检测出突变型基因型,考虑TNFα基因G-308A位点多态性可能与中国部分汉族人群IBD无明显相关性。
Background and Objectives:
Inflammatory bowel disease(IBD) includes Crohn's disease(CD) and ulcerativecolitis(UC).The incidence of IBD especially CD is going up year by year, but itsetiology and pathogenesis are still unknown.Most of the views presume that it's akind of non-specific inflammatory disease that can be induced under the role ofenvironmental factors and autoimmune disorders for the host who carry the geneticsusceptibility genes.In recent years,with the Human Genome Project have beencompleted,human have transferred the focus on gene research from the wholegenome sequence to individual gene polymorphism and functional studies. Singlechain nucleotide polymorphism(SNP) is the main form of a individual gene polymorphism,the SNP is recognized as "the third generation of DNA genetic marker "following the restriction fragment length polymorphism and microsatellite polymorphism,which is one of the hottest fields in the world.
The Hugot Research Group in Europe and Ogura team in the United States simultaneously found out the first predisposing gene of CD,names NOD2,which was renamed CARD15 in 2001.They certified that this gene includes three SNP sites:Arg702Trp,Gly908Arg and Leu1007fsinsC,and further confirmed the SNP sites are belong to Caucasian's CD patients predisposing gene but don't related with the CDpatients born from Japan、Hong Kong and ZheJiang province of China.We haveresearched the three SNP sites about NOD2 gene of CD patients born fromGuangdong,and the results indicated there have no correlation with them,but wefound out the closely related SNP site(names P268S) from Chinese by detecting genesequence,and have constructed eukaryotic expression vector,and the further researchare carrying out.
Recently several research groups in Western studied on the gene susceptibility ofthe IBD patients who come from the Caucasus of Europe,the United Kingdom,France,the Balkans,Germany,Canada,and other regions in Europe by gene scanning,they found out some SNP sites about IBD,especially about CD patients,whichlocated in the gene of OCTN1、OCTN2,TNFα、IL23R、ATG16L1、PTPN2、10q21、NKX2、IRGM、MST1、DLG5、5p31 and so on.Among them,the sites of rs1050152 in OCTN1 gene,rs2631367 in OCTN2 gene and G-308A in TNFαgene haveapparente statistical significance compared with controls. Rs1050152 sites in the ninthexon of OCTN1gene,normally,it is C,when it is mutated to T,the 503 amino acidleucine(Leu) trans into phenylalanine(Phe).This mutation effects the transportationof camitine for cells,and this leads to degression of carnitine inside cells and cellularmetabolic disorder.Rs2631367 sites in the promotor of OCTN2 gene,normally,it is G,when it is mutated to C,the heart shock element of OCTN2 gene is destroyed,andthen effects the transportation of carnitine for cells.Peltekova and his teammate foundout that the above two SNPs are associated with CD in Europe population for the firsttime.The onset risk of CD will raise 2.5 times when one of them mutates,and it willraise 4 times when all of them mutate.G-308A sites in the promotor of TNFαgene,Wilson found out its diallele gene polmorphisms for the first time.Many studiesindicate that tumor necrosis factor alpha plays very important role in the onset of IBD,and the polmorphisms of G-308A site can effect its biological activity.
The purpose of this study is to prove the three SNPs which related to western CD patients whether associate with Chinese Han population IBD patients by genesequencing and restriction fragment length polymorphism methods,Our purpose is tofurther study the pathogenesis of IBD and patterns IBD susceptibility gene mapping.Maybe,we will also provide a new means of gene therapy for the treatment of IBD.
Methods
1.Forty-five Chinese Crohn's disease(CD) patients(including twenty-six male samples and ninteen female samples),forty-five ulcerative colitis(UC) patients(including twenty-eight male samples and seventeen female samples) and fifty healthycontrols(HC) were prospectively recruited from the Nan Fang Hospital. In ulcerative colitis cases,diseased region in rectum 6 sample,left hemicolon 12 sample,right hemicolon 6 sample,hole colon 21 sample,among the total male 26, female 19、mean age was 35.58±15.81;in Crohn's disease case,diseased region in small intestine 19 sample,colon 14 sample,small intestine complicating with colon 9 sample,rectum 3 sample,among the total male 28,female 17,mean age was 32.91±10.54,Peripheral blood was collected from patients and white blood cell was separated.
2.Extract genomic DNA from the blood(in kit brochures operation),after the success of extraction,preserve them under-70℃.
3.Design primers using Primer5.0 software.To amplify the fragments includes the three SNPs.After succeeded amplify,(amplified products by 1.5% agarosegelelectrophoresis to ensure that the observation fragment was successfullyamplified).Purify the PCR production and directed sequence the target region inSIB3730XL sequencer.Then,assessment genotype with chromas2.0 and lastcontrasted 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 contrastedgene bank data with uniform means.One SNPs use the method of restriction fragmentlength polymorphism(RFLP) to testify the polymorphism in CD patients and find it'sfrequency in UC and health control.
Results:
1.OCTN1(rs1050152) gene polymorphism associated with the pathogenesis ofinflammatory bowel disease:
By DNA sequencing,we found that There was no statistical significance of thegenotype frequency and the allele frequency of SNP site between case groups of CDpatients、UC patients and normal control group:P=0.715,P=0.727.As a result, we can conclude that there is no correlation between thepolymorphism site rs1050152 of gene OCTN1 and Chinese susceptibility of IBD.
2.OCTN2(rs2631367) gene polymorphism associated with the pathogenesis of inflammatory bowel disease:
By DNA sequencing,we can't find any mutational genetype,As a result,we can conclude that there is no correlation between the polymorphism site rs2631367 ofgene OCTN2 and Chinese susceptibility of IBD.
3.TNFα(G-308A) gene polymorphism associated with the pathogenesis of inflammatory bowel disease:
Through restriction fragment length polymorphism(RFLP),we can't find anymutational genetype,As a result,we can conclude that there is no correlation betweenthe polymorphism site G-308A of gene TNFαand Chinese susceptibility of IBD.
Conclusion:
1.Our research indicates that the SNP sites rs1050152 of OCTN1 gene andrs2631367 of OCTN2 which reported abroadly was no associated with susceptibilityto IBD in the Chinese Hart population.
2.we can't find any mutational genetype in all the three groups,there isn't association between the SNP site G-308A of TNFαgene and Chinese susceptibility of IBD.
引文
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2.Zhi FC,Xiao B,Jiang B,Wan TM,Guo Y,Zhou D,Wang LH,Chen JF,Xie L,Pan DS,Zhou DY.Double-balloon enteroscopy detecting smallintestinal bleeding.Chin Med J(Engl),2005,118:1834-1837.
3.Shanahan F.Crohn's disease.Lancet,2002,359:62-69.
4.Ahmad T,Satsangi J,McGovern D,Bunce M,Jewell DP.Thegenetics of inflammatory boweldisease.AlimentParmarcol Ther,2001,15(6):731-748.
5.Hugot JP,Chamaillard M,Zouali H,Lesage S,C(?)zard JP,Belaiche J,Almer S,Tysk C,O'Morain CA,Gassull M,Binder V,Finkel Y,Cortot A,Modigliani R,Laurent-Puig P,Gower-Rousseau C,Macry J,Colombel JF,Sahbatou M,Thomas G.Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.Nature,2001,411:599-603.
6.Ogura Y,Bonen DK,Inohara N,Nicolae DL,Chen FF,Ramos R,Britton H,Moran T,Karaliuskas R,Duerr RH,Achkar JP,Brant SR,Bayless TM,Kirschner BS,Hanauer SB,Nunez G,Cho JH.A frame shift mutation in NOD2associated with susceptibility to Crohn's disease.Nature,2001,411:603-606.
7.Leong RW,Armuzzi A,Ahmad T,Wong ML,Tse P,Jewell DP,Sung JJ.NOD2/CARD15 gene polymorphisms and Crohn's disease in the Chinese population.Aliment Pharmacol Ther,2003,17:1465-1470.
8.高敏,曹倩,罗灵和,吴敏良,胡伟玲,姒健敏.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.
9.Inoue N,Tamura K,Kinouchi Y,Fukuda Y,Takahashi S,Ogura Y,Inohara N,Nunez G,Kishi Y,Koike Y,Shimosegawa T,Shimoyama T,Hibi T.Lack of common NOD2 variants in Japanese patients with Crohn's disease.Gastroenterology,2002,123:86-91.
10.张以洋,智发朝,周殿元,姜泊,赖卓胜,张迎春,钟长青,龙靖华,徐迪辉,张奕.克罗恩病CARD15/NOD2基因突变的研究.中华消化杂志,2006,26(7):456-459.
11.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.
12.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.
13.T(o|¨)r(o|¨)k HP,Glas J,Tonenchi L,Lohse P,M(u|¨)ller-Myhsok B,Limbersky O,Neugebauer C,Schnitzler F,Seiderer J,Tillack C,Brand S,Br(u|¨)nnler G,Jagiello P,Epplen JT,Griga T,Klein W,Schiemann U,Folwaczny M,Ochsenk(u|¨)hn T,Folwaczny C.Polymorphisms in the DLG5 and OCTN cation transporter genes in Crohn's disease.Gut,2005,54:1421-1427.
14.Waller S,Tremelling M,Bredin F,Godfrey L,Howson J,Parkes M.Evidence for association of OCTN genes and IBD5 with ulcerative colitis.Gut,2006,55:809-814.
15.Gazouli M,Mantzaris G,Archimandritis AJ,Nasioulas G,Anagnou NP.Single nucleotide polymorphisms of OCTN1,OCTN2,and DLG5 genes in Greek patients with Crohn's disease.World J Gastroenterol,2005,11(47):7525-7530.
16.Christopher G.Mathew.New links to the pathogenesis of Crohn disease provided by genome-wide association scans.Nature Reviews Genetics.2007,2203:1038-1048
17.Duerr RH,Taylor KD,Brant SR,Rioux JD,Silverberg MS,Daly MJ,Steinhart AH,Abraham C,Regueiro M,Griffiths A,Dassopoulos T,Bitton A,Yang H, Targan S, Datta LW, Kistner EO, Schumm LP, Lee AT, Gregersen PK, Barmada MM, Rotter JI, Nicolae DL,Cho JH. A genome-wide association study identifies IL23R as aninflammatory bowel disease gene. Science. 2006,314:1461-1463.
18. 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.
19. Tremelling M, Cummings F, Fisher SA, Mansfield J, Gwilliam R, Keniry A, Nimmo ER, Drummond H, Onnie CM, Prescott NJ, Sanderson J, Bredin F, Berzuini C, Forbes A, Lewis CM, Cardon L, Deloukas P, Jewell D, Mathew CG, Parkes M, Satsanqi J. IL23R variation determines susceptibility but not diseasephenotype in inflammatory bowel disease.Gastroenterology 2007, 132: 1657-1664.
20. Rioux JD, Xavier RJ, Taylor KD, Silverberg MS, Goyette P, Huett A, Green T, Kuballa P, Barmada MM, Datta LW, Shugart YY, Griffiths AM, Targan SR, Ippoliti AF, Bernard EJ, Mei L, Nicolae DL, Regueiro M, Schumm LP, Steinhart AH, Rotter JI, Duerr RH, Cho JH, Daly MJ, Brant SR, Taylor KD. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nature Genet, 2007, 39:596-603.
21. Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, Huse K, Albrecht M, Mayr G, De La Vega FM, Briggs J, G(?)nther S, Prescott NJ, Onnie CM, Hasler R, Sipos B, Folsch UR, Lengauer T, Platzer M, Mathew CG, Krawczak M, Schreiber S. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. NatureGenet.2007, 39:207-211.
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