Endoscopic Feature And Etiology Of Crohn's Disease

Background and ObjectiveCrohn's disease(CD)is a main type of inflammatory bowel disease(IBD),and is a segmental transmural inflammatory disease of gastrointestinal tract.The mobidity of CD is about 0.01‰～0.1‰in west country.CD is seldom reported in the past, but in these years,the incidence increased continuously.So far the cause and the pathogenesis remain unclear,the clinical manifestations are diverse,which is difficult to diagnosis in early stage and easily misdiagnosed.It can't cure and easily to recur,which damaged people's health deeply.Recent studies indicating,participated under the environment factors,the immuniting function disord of the hose taking along aesy infecting gene,leading to CD in the end.Studies have revealed coding region polymorphisms in NOD2/CARD15 which are significantly associated with susceptibility to CD.They verified the three major polymorphisms in NOD2/CARD15-R702W,G908R and 3020insC which is genetically associated with CD in European and American population.They also testify it is associated with the clinical features and the position of illness.The outcome was testify in German,Australian,and England population,and so on,but not in Japanese,Korea,Hongkong and Chinese Zhejiang population.Hower,for the visible racial diversify between westerner and Chinese people,thepredisposing genes of CD probably not the same,furthermore,as other complex disease,modify of single gene can not lead to disease,may be interaction of genes and environment probably better explanat the etiopathogenesis of CD.therefore, it is necessary to find out the predisposing genes of CD in Chinese people by genome-wide association scan and transmission disquilibrium test. Methods1.155 patients with clinically suspicious of small intestinal diseases were studied.2.Forty eight consecutive Chinese patients with CD(including thirty former samples and eighteen new samples),fifty ulcerative colitis(UC)and fifty healthy controls were prospectively recruited from the NanFang Hospital.Peripheral blood was collected from patients and white blood cell was separated.Genomic DNA extraction was performed,then fifteen pairs of primers were designed to amplify the twelve exons of NOD2/CARD15 gene.3.To amplify the twelve exons of NOD2/CARD15 gene of CD patients.After succeeded amplify,purify the PCR production and directed sequence the target region of NOD2/CARD15 in ABI377 or ABI3730 sequencer made in American. Lastly contrasted with the genebank data to analyze the mutations of NOD2/CARD15 gene in CD patients.If we find mutation that we amplify the corresponding target region of NOD2/CARD15 of the fifty UC and fifty HC with uniform primer,purify the PCR production and directed sequence the target region and contrasted with the genebank data with uniform means.4.According to the mutation of P268S found by DNA sequence,we design a pair of primer,then to amplify the target region and use the method of restriction fragment length polymorphism(RFLP)to testify the P268S mutation in CD patients and find it's frequency in UC and health control.5.The site mutantion was induced by in vitro site-directed mutagenesis,identified by gene sequence.6.Protein expression was identified by western blot after transfection of mutant plasmid into HEK293T cells.7.The level of NF-κB was detected by western blot and immunofluorescence technique after LPS stimulated 30 minute.Results1.Among the 155 cases,lesions were found in 125 cases,with positive results accounting for 80.6%.These lesions mainly consisted of small intestinal ulcer(including Crohn's desease),chronic inflammation,Meckel's diverticulum, interstitialoma,vascular deformity and carcinoma of small intestine,etc.In 84 of the 92 patients suspicous of intestinal hemorrhage,the lesions were~- confirmed with a positive rate of 91.3%.In 24 of the 39 patients with abdomen pain,the etiologies were confirmed with a positive rate of 61.5%.In 16 of the 23 patients with diarrhoea, abdominal distention and malnutrition,the positive rate was 69.6%.The cause of the only one case with refractory hypoalbuminemia was confirmed.Among the 155 cases, 9 had lesions located in stomach and duodenum,115 in small intestine and one in large bowel,no lesion was found in 30 cases.Among the patients,43 were found to have small intestinal ulcer(18 in jejunum,20 in ileum,5 in both jejunum and ileum). In the 43 patients,27 complained of refractory hemorrhage,9 abdominal pain,4 abdominal distention,2 malnutrition and one diarrhoea.12 patients were with single intestinal ulcer,and 31 with multiple.For cases of Meckel's diverticulum, interstitialoma,carcinoma,vascular deformity and intestinal adhesion of small intestine in this series,diagnoses made by double balloon endoscopy combined with morphology were completely consistent with those found in operation.However,for ulcer lesions(mainly Crohn's disease),there was diversity in the diagnoses between the two methods,the coincidence was 57.1%.Two patients had complication,one with perforation of small intestine and the other acute intestinal stasis2.From choosen by DNA sequence and testified by PCR-RFLP,five patients in CD have P268S variants,including three heterocyclic and two homogenesis,four patients 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 amino acid changes from proline to cypress.The difference has statistic significance( Fisher's exact test P=0.003),which illuminate that the P268S variants in NOD2/CARD 15 are possibly associated with susceptibility to CD.We don't find new mutation except for P268S in the other exons of NOD2/CARD15 gene.R702W, G908R and 3020insC variants were not found in any cases.3.Of all the 48 CD patients,four P268S variants were found in twelve patients who were younger than 20 years old,but only one in thirty six who were elder than 20 years old.The difference has statistic significance(Fisher's exact test P=0.011),which illuminate that the P268S variants in NOD2/CARD15 are possibly associated with the age of patients.thirty six males have three P268S variants and twelve females have two.The difference has no statistic significance (Fisher's exact test P=0.587),which illuminate that the P268S variants in NOD2/CARD15 are possibly not associated with the gender of patients.The location of five P268S variants are all in ileum,but not in colon and the others.The difference has statistic significance(Fisher's exact test P=0.001),which illuminate that the P268S variants in NOD2/CARD15 are possibly associated with the location of lesion.Five P268S variants were found in sixteen patients who have intestinal stricture,but none of them in thirty two patients who have no intestinal stricture.The difference has statistic significance(Fisher's exact test P=0.003),which illuminate that the P268S variants in NOD2/CARD15 are possibly associated with intestinal stricture,the patients who have P268S variants are easy to occur intestinal stricture and ileum which need surgery.Four P268S variants were found in twenty six patients whose state of illness were midrange,and only one in eleven patients whose state of illness were severe.The difference has no statistic significance(Fisher's exact test P=0.289),which illuminate that the P268S variants in NOD2/CARD15 are possibly not associated with the state of illness.4.The mutant plasmids were correctly constructed.Both wildtype and mutant constructs were able to express NOD2/CARD15 protein.And Both wildtype and mutant protein could increase NF-κB activation in HEK293T cells stimulated by LPS.Conclusion1.For CD,Double-balloon endoscopy is efficient and safe for it's diagnosis, especially in detecting the lesion of small intestinal.However,for ulcer of small intestine,this method even combined with biopsy is sometimes unable to determine its nature.So surgery may be feneficial in this condition.2.The P268S variants in NOD2/CARD15 are possibly associated with susceptibility to CD in the Chinese population,but not associated with susceptibility to UC.3.None of new mutations was found except for P268S in twelve exons of NOD2/CARD15. 4.The three common of R702W,G908R and 3020insC of NOD2/CARD15 in European and American population are not associated with susceptibility to CD in the Chinese population.5.The P268S variants is associated with the age of patients,the location of lesion and it's complication,but not with the gender and the state of illness.the patients who have P268S variants are easy to occur intestinal stricture and ileum which need surgery,are usually younger than 20 years old and whose location of lesion are usually in ileum.All of which indicate that NOD2/CARD15 are possibly associated with susceptibility to CD in the Chinese population6.Both wildtype and mutant Nod2 could enhance activated level of NF-κB in HEK293T cells stimulated by LPS.But the difference between the two type was not obvious.