Mutation Of NOD2/CARD15 Gene In Chinese Crohn's Disease

Background and Objiective:Crohn's disease(CD) is a main type of inflammatory bowel disease(IBD), and isa segmental transmural inflammatory disease of gastrointestinal tract. Themobidity of CD in asia is much lower than western countries, the incidenceincreased continuously in these years, including china. So far the cause and thepathogenesis remains unclear, the clinical manifestations are diverse, which isdifficult to diagnosis in early stage and easily misdiagnosed. It can't cure and easily torelapse.Recent studies indicating, participated under the environment factors, theimmuniting function disord of the hose taking along aesy infecting gene, leadingto CD in the end. Studies have revealed coding region polymorphisms inNOD2/CARD15 which are significantly associated with susceptibility to CD. Theyverified the three major polymorphisms in NOD2/CARD15—R702W, G908R and3020insC which is genetically associated with CD in European and Americanpopulation. They also testify it is associated with the clinical features and the positionof illness. The outcome was testify in German, Australian, and England population,and so on, but not in Japanese, Korea, Hongkong and Chinese Zhejiang population.In early days we carried out DNA sequencing of 4th,8th and 11th exons of NOD2/CARD15, none of them had R702W, G908R and 3020insC variants, but wefound an other variant P268S associated with CD of Chinese possibly, we found 4patients with CD had P268S variant, the incidence is 13.3％.Activates NF-κB is the main function of NOD2/CARD15 protein. TheNOD2/CARD15 protein is composed of two NH2 terminal caspase recruitmentdomains, a centrally located mucleotide binding domain, and multiple COOHterminal leucine rich repeats. The CARD-CARD is an effector whenNOD2/CARD15 protein Activates NF-κB, NOD2/CARD15 protein ActivatesNF-κB via the isophil interaction between the serine—threonine kinase(RICK) andthe CARD-CARD. LRRs acts as pattern recognition acceptor which can discriminatebacterial Lipopolysaccharide(L PS),peptid,hydrocarbon et al, so makeNOD2/CARD 15 protein oligomerizate and interact between NOD2/CARD 15 proteinand RICK, activates NF-κB in the end. NOD2/CARD15 variants associated withCD are not only deficient in their ability to activate NF-κB in response to LPS, butdebase innate immunity, which triggers an aberrabt inflammatory response in theintestinal tissue of CD patients.For further research on pathogenesis of CD and offering an instructor for CDtherapy, we enlarge the samples comparing the former research and carriy out DNAsequencing of 12 exons of NOD2/CARD15, the aims are to find coding regionpolymorphisms in NOD2/CARD15 which are significantly associated with CD inChinese population and find it's relationship to clinical features of CD patients.Methods:1. Forty eight consecutive Chinese patients with CD(including thirty formersamples and eighteen new samples),fifty ulcerative colitis(UC) and fifty healthycontrols were prospectively recruited from the NanFang Hospital. Peripheral bloodwas collected from patients and white blood cell was separated. Genomic DNA extraction was performed, then fifteen pairs of primers were designed to amplify thetwelve exons of NOD2/CARD15 gene.2. To amplify the twelve exons of NOD2/CARD15 gene of CD patients. Aftersucceeded amplify, purify the PCR production and directed sequence the targetregion of NOD2/CARD15 in ABI377 or ABI3730 sequencer made in American.Lastly contrasted with the genebank data to analyze the mutations ofNOD2/CARD15 gene in CD patients. If we find mutation that we amplify thecorresponding target region of NOD2/CARD15 of the fifty UC and fifty HC withuniform primer, purify the PCR production and directed sequence the target regionand contrasted with the genebank data with uniform means.3. According to the mutation of P268S found by DNA sequence, we design apair of primer, then to amplify the target region and use the method of restrictionfragment length polymorphism(RFLP) to testify the P268S mutation in CD patientsand find it's frequency in UC and health control.Results:1. The SNP of NOD2/CARD15 that is associated with CD inChinese population:From choosen by DNA sequence and testified by PCR-RFLP, five patients inCD have P268S variants, including three heterocyclic and two homogenesis, fourpatients were found in former research and one patients is found in our new samples,but not mutation in UC and HC. The C to T mutation at 802bp and the coding aminoacid changes from proline to cypress. The difference has statistic significance(Fisher's exact test P=0.003), which illuminate that the P268S variants inNOD2/CARD15 are possibly associated with susceptibility to CD. We don't find newmutation except for P268S in the other exons of NOD2/CARD15 gene. R702W, G908R and 3020insC variants were not found in any cases.2. The P268S is associated with CD clinical features:Of all the 48 CD patients, four P268S variants were found in twelve patientswho were younger than 20 years old, but only one in thirty six who were elder than20 years old. The difference has statistic significance (Fisher's exact testP=0.011), which illuminate that the P268S variants in NOD2/CARD15 are possiblyassociated with the age of patients, thirty six males have three P268S variants andtwelve females have two. The difference has no statistic significance(Fisher's exact test P=0.587), which illuminate that the P268S variants inNOD2/CARD15 are possibly not associated with the gender of patients. The locationof five P268S variants are all in ileum, but not in colon and the others. Thedifference has statistic significance (Fisher's exact test P=0.001), whichilluminate that the P268S variants in NOD2/CARD 15 are possibly associated with thelocation of lesion. Five P268S variants were found in sixteen patients who haveintestinal stricture, but none of them in thirty two patients who have no intestinalstricture. The difference has statistic significance (Fisher's exact testP=0.003), which illuminate that the P268S variants in NOD2/CARD15 are possiblyassociated with intestinal stricture, the patients who have P268S variants are easy tooccur intestinal stricture and ileum which need surgery. Four P268S variants werefound in twenty six patients whose state of illness were midrange, and only one ineleven patients whose state of illness were severe. The difference has nostatistic significance (Fisher's exact test P=0.289), which illuminate that theP268S variants in NOD2/CARD15 are possibly not associated with the state ofillness.Conclusion:1. Our study find the P268S variants in NOD2/CARD15 are possibly associated with susceptibility to CD in the Chinese population, but not associated withsusceptibility to UC, none of new mutations was found except for P268S in twelveexons ofNOD2/CARD 15.2. We don't find the three common of R702W, G908R and 3020insC ofNOD2/CARD 15 in European and American population in our study.