Correlation Between Gene Polymorphism Of OCTN1,OCTN2,TNFα And Inflammatory Bowel Disease

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.Methods1.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.