Correlation Between Gene Polymorphism Of ATG16L1, IRGM And Crohn's Disease

Background and Objiective:Crohn'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 in asia is much lower than western countries, the incidence increased continuously in these years, including china. So far the cause and the pathogenesis remains unclear, the clinical manifestations are diverse, which is difficult to diagnosis in early stage and easily misdiagnosed. It can't cure and easily to relapse.Human health can not be separated from the homeostasis of the internal environment, and the homeostasis of cells depend on the balance of macromolecule material's biosynthesis and biolysis. Autophagy is one of the most important mechanism to achieve this dynamic balance and the mechanism of autophagy is the same in most eukaryotic cells which include from cerevisiae fermentum to human. There are two main pathway of the eukaryotic's degradation- the degradation of protease and autophagy, but only autophagy has the efficacy of degradating the whole cell. Autophagy is the degradation pathway of the macromolecule material and celll organ which develop in cytolysosome. Its major contribution are degradating and clearing the damaged cellular structure, effete cell organ, biomacromolecule that haven't need and so on. Meanwhile, it provides feedstock for the construction of cell organ-recirculation of the cellular structure. Thus, autophagia is a very important regulation mechanisms of the refreshment, development, differentiation of the cells and the remodeling of texture. Autophag phenomenon can be a active effect on the prevention of diseases (such as nerves degenerative disease, tumors, cardiomyopathy, the infection of causative organisms ) and the prevention ageing and increasing in life span. Therefore, it has a very important applied value on the research of the autophagy and cell death by autophagy. The discovery of Autophagia gene give us cognitions on the molecular level .The relation between autophagia and infection of causative organism display in the killing of extracelluar pathogenic bacteria by phagocytes and lymphocyte. When Pathogens enter the cells by internalization, autophagia is the capital mode of defense. The related study found that the rise and fall of the autophagy attributes to many factors. The defect of nourishment, glucagons induce autophagy and insuline restrains it. The point of action of these effects is impacting the concentration of amino acids. When the concentration of amino acids depress, autophagy starts to generate amino acids which guarantee organs take, on the contrary, restrain autophagy.When the autophagy process disturbed, the worn-out cell parts and harmful bacteria will to persist when they would otherwise be destroyed. These cell components and bacteria may trigger an inappropriate immune system response, leading to chronic inflammation in the intestinal walls and the digestive problems characteristic of Crohn disease.Besides, according to the theory of abnormal immunoregulation of CD, infections, virus and drugs may destroy the intestinal epithelium barrier, and that will result in the intestinal permeability increasing, intestinal epithelial cells swelling, absorption function decreasing and nutrition deficiency. The swelling of intestinal epithelial cells also has the role of inhibiting the autophagy, just as what the insulin does (for example, in the hepatic tissue, the increase of the cell volume promotes the synthesis of glycogen, fat and protein and inhibits the degradation of autophagic protein). The mechanism is the increase of intracellular amino acid concentration and the change of intracellular ion concentration. In addition, the response of mucous membrane in susceptible host can be induced by ingested multiplicity antigen in the enteric cavity and then exposed intestines tissue to multiplicity antigen. And iterative exciting the intestinal tract makes immunologic system have excess response and fault discrimination, activate the macrophages and lymphocytes and then releasing the cell factor and mediators of inflammation. This process induces cell immunity reaction and humoral immune reaction. Immunologic process once primed that the immune inflammatory reaction will be amplified step by step. Finally, it will make immune inflammatory reaction and appearance the appearance and pathological change.Nowadays, it has been confirmed by many studies at home and abroad that CD is a kind of complicated polygene disease. People gradually accepted the idea that the interaction between gene susceptibility and intestinal bacteria antigen is an important factor of CD occurrence. That is to say, affected by environment, immune function of the host carrying predisposing genes becomes disordered and finally the disease occurs. And with the development of gene studies, it is found that polymorphism site of N0D2/CARD15,which was firstly confirmed related to CD susceptibility of occidental has no obvious relationship with that of Chinese. It's have been discovered by foreign studies that both the ATG16L1 gene which is associated with autophagocytosis and the SNP site of IRGM gene are significant correlated with CD, and also in the case of One SNP (rs2241880) of ATG16L1 and two SNP site(rsl3361189,rs4958847)of IRGM .Basing on previous researches, this study made a further research of polymorphism loci of autophagic genes ATG16L1 and IRGM which are related to the occidental CD susceptibility. We chose 3 SNP site of two genes associated with occidental CD susceptibility which has been reported by foreign studies and make a preliminary study on the relationship of SNP site of CD gene susceptibility between occidental and Chinese, and also on the clinical feature relativity between this SNP site and CD. The purpose of the research was to establish the foundation for further studying the pathogenesis of CD and to provide a theoretical basis.Methods:1. Forty consecutive Chinese patients with CD(including thirty former samples and eighteen new samples),forty ulcerative colitis(UC) and fifty healthy controls were prospectively recruited from the NanFang Hospital. In ulcerative colitis case, diseased region in rectum 6 sample, left hemicolon 11 sample, right hemicolon 4 sample, hole colon 19 sample, among the total male 27, female 13, mean age was 36.38±12.00; in Crohn's disease case, diseased region in small intestine 16 sample, colon 13 sample, small intestine complicating with colon 8 sample, rectum 3 sample; classify by activity of the Crohn's disease, be in paracmasis 6 sample, active phase 34 sample, among the total male 26, female 14, mean age was 32.7±9.46; there are fifty healthy control, 32 male, 18 female, the mean age is 34.58±9.37. Peripheral blood was collected from patients and white blood cell was separated. Genomic DNA extraction was performed, then three pairs of primers were designed to amplify the three single nucleotide Polymorphisms (SNP) site in ATG16L1 and IRGM gene.2. To amplify the 9th exons of ATG16L1 gene of forty CD patients. After succeeded amplify, purify the PCR production and directed sequence the target region of ATG16L1 gene in SIB3730XL sequencer made in American. Lastly contrasted with the genebank data to analyze the mutations of ATG16L1 gene in CD patients. If we find mutation that we amplify the corresponding target region of ATG16L1 of the forty 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.3. To succeed amplify the two target region which have the two SNP site in IRGM gene. According to the mutation of two target region found by DNA sequence, we design a pair of primer, then to amplify the target region, pure the production by polymerase chain reaction and use the method of restriction fragment length polymorphism(RFLP) to testify the two mutational site rsl3361189 and rs4958847 by restriction enzyme TasⅠ(TspEⅠ) and AluⅠin CD patients and find it's frequency in UC and health control.Results:1. The SNP of ATG16L1 that is associated with CD in Chinese population: The polymorphism site rs2241880 was measured by DNA sequencing and backward sequencing samples of heterozygous mutation and homozygous mutation. And it shows that base pairs A were replaced by G, codon ACT by GCT and threonine by alanine. There was no statistical significance of the genotype frequency and the allele frequency of SNP site between case group of CD patients and normal control group: P=0.669, P=0.973. And there was no statistical significance between case group of UC patients and normal control group: P=0.098, P=0.639. Comparing the CD group and UC group, P values of the genotype frequency and the allele frequency were 0. 416 and 0.633, respectively, which suggested that there was no statistical significance between the two groups. As a result, we can conclude that there is no correlation between the polymorphism site rs2241880 of gene ATG16L1 and Chinese susceptibility of IBD. 2. The SNP of IRGM that is associated with CD in Chinese population:In the patients, 36 cases have generated mutational, of which 12 cases have developed homozygosis mutational and 24 cases have developed heterozygosis, and in the 40 cases Crohn diseases, 24 cases mutational, 10 cases homozygosis, 14 cases heterozygosis, meanwhile the 50 healthy people have 46 mutational, 18 cases homozygosis, 28 cases heterozygosis. There is not statistically significant between the genotypic frequency(x~2=1.669 , P=0.796) and so is the allele frequency (x~2 = 0.416 , P=0.812). To check polymorphism sites of the IRGM gene rs4958847 by the enzyme cutting of PCR-RFLP, 40 cases patients were found positive and this illustrates that P268S mutational have obviously related with the age of onset of Corhn diaseases.Conclusion:1,Our research indicate that the SNP site rs2241880 of ATG16L1 gene which reported by abroad was no associated with susceptibility to CD in the Chinese population. There was no significant difference among CD, UC and health control. To consider that the SNP site rs2241880 have no related with susceptibility to CD in the Chinese population, didn't like in white people.2. We don't find the two SNP site in IRGM gene have correlation with susceptibility to CD in the Chinese population. It was difference with white people that reported by abroad.