Correlation Between Gene Polymorphism Of IL23R,PTPN2,10q21 And Inflammatory Bowel Disease

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 IBDMethods: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.