Association Research Of Genetic Susceptibility With Serum Markers Of Inflammatory Bowel Disease In Han Population Of China

Inflammatory bowel disease(IBD) is a group of chronic intestinal diseases including of Crohn's disease(CD) and ulcerative colitis(UC).The incidence of IBD shows a significant increase tendency in recent years.The causes and pathogenesis of IBD are not very clear so far.It is reported that the interaction between genetic factors, environmental factors and immune factors are involved in pathogenesis and its clinical phenotype of IBD.Recent studies on genetic factors of IBD found that gene mutation is not only related to disease susceptibility,also associated with disease phenotype,such as NOD2 mutation is frequently observed in ileum type of CD.The discovery of NOD2,the first CD susceptible gene,is a groundbreaking event.It suggests that the onset of IBD is associated with enteric bacteria and natural immunity.DEFB1 and ATG16L1 gene are associated with immunity defense.The genetic variations in the DEFB1 was associated with CD in Hungary populations.But there is no study on the related with IBD in Han population of China.The relative large-sample research about the relationship between IBD and ATG16L1,as a CD susceptibility gene identified recently,was not found in China.Besides of genetic factors,some specific antibodies would arouse concerns since they are evidence that IBD is related to immune reaction.Antineutrophil cytoplasmic antibodies(p-ANCA),Anti-saccharomyces cerevisiae antibodies(ASCA),Pancreatic antibodies(PAB)and Goblet cell autoantibodies(GAB) were included.These four antibodies have been widely used for the diagnosis of IBD abroad.Lots of studies were focus on p-ANCA and ASCA in China.The results of these studies showed that both p-ANCA and ASCA have some diagnostic value with low sensitivity.There was a few of studies on PAB and GAB in China.The association of PAB and GAB with clinical phenotype is controversial.IBD is heterogeneity disease.Studies based on genetic heterogeneity and serum heterogeneity could help to precisely define IBD clinical type,to make detail therapy plan and determine prognosis.In this study,the authors investigate IBD genetic susceptibility and antibodies detection based on current experimental condition.It aims to:1.Investigating the relationship between single nucleotide polymorphism of natural immunity related genes DEFB1 & ATG16L1,IBD genetic susceptibility,and IBD clinical phenotype.2.Investigating application value of combined detection of p-ANCA,ASCA,PAB and GAB in diagnosis and differential diagnosis of IBD,and the relationship with IBD clinical phenotype.Part 1.The Study of DEFB1 And ATG16L1 Gene Polymorphism with Genetic Susceptibility of Inflammatory Bowel Disease in Han Population of ChinaMethods:247 IBD patients(including 97 with CD and 150 with UC) and 200 healthy individuals of Chinese Han population were collected.Totally four SNPs of DEFB1(rs11362,rs1800972)and ATG16L1(,rs2241880,rs1045095)gene were genotyped by Polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP).10%of objective was selected to determine the sequence to confirm the PCR-RFLP.Genotype-phenotype analysis was performed with respect to disease susceptibility stratified by clinical feature.Results:The frequency of allele G of SNP site rs11362 in patients with UC is much higher than in controls(70 vs 60%,P=0.012).The distribution of genotype of AA,AG,GG of SNP rs11362 in UC patients has the significant difference,comparing with controls(P =0.006).It was found that multivariate-adjusted odds ratio(OR;95%confidence interval)for GG genotype compared with AA and AG were 2.04(95%CI:1.32-3.06). Haplotype analysis found that the haplotype G-C for the SNP site rs11362-rs1800972 were at significantly increased risk for UC(OR:1.621,95%CI:1.010-2.610).In subgroup analysis,GG genotype frequencies between UC patients with early onset of disease(age at diagnosis≤25,lower quartile)and controls showed significant difference (P=0.001,OR=3.29,95%CI:1.56-7.09).A significantly higher frequency of the GG genotype of SNP site rs11362 was observed among UC patients with extensive colitis(P=0.003,OR=2.27,95%CI:1.31-3.91) and severe course of disease behaviors(P= 0.009,OR=2.55,95%CI:1.24-5.24) respectively,as compared with healthy controls.No significant differences were noted in the DEFB1 gene SNP site rs1800972 and in the ATGI6LI gene SNP site rs2241880,rs1045095 polymorphisms among the patients with CD,UC and the control subjects.No association of above 3 SNP genotypes with disease subgroups in UC and CD was observed.Summary:These results indicated that rs11362 polymorphism in the DEFB1 might be associated with the risk for UC,but not for CD.It may also determine disease phenotype such as:early onset,severe degree and extensive colitis.The association of rs2241880 and rs1045095 polymorphism of ATGI6LI with IBD was not observed.Part 2.The Clinical Significance of Combined Measurement of Serological Marker in Inflammatory Bowel Disease in Chinese PopulationsMethods:A total of 217 patients with IBD(UC 108,CD 109),59 patients with non-IBD intestinal disease controls(DC) and 112 healthy controls(HC) were recruited in our research.ASCA were detected by enzyme-linked immunosorbent(ELISA) assay. p-ANCA,PAB and GAB were measured by indirect immunofluorescence(IIF) assay.Results:1.The distribution of these antibodies in IBD:The prevalence of p-ANCA in UC,CD,DC and HC group was 60.2%,5.5%,3.4%and 3.4%,respectively.The prevalence of GAB in UC,CD,DC and HC group was 30.6%,3.7%,0%and 1.8%, respectively.The prevalence of p-ANCA and GAB in UC group was statistically different compared with that in CD or DC(P＜0.05).The prevalence of ASCA in CD,UC,DC and HC group was 34.9%,13%,10.2%and 16.1%,respectively.The prevalence of PAB in CD,UC,DC and HC group was 41.3%,6.5%,3.4%and 0%,respectively.The prevalence of ASCA and PAB in CD group was statistically different compared with that in CD or DC(P＜0.05).Among the UC patients with negative for p-ANCA,13.6%(6/44) are positive for GAB.Among the CD patients negative for ASCA,34.3%(24/70) are positive for PAB.2.The significance of combined marker in diagnosis of IBD:The sensitivity of p-ANCA or GAB alone to diagnose UC was 59.3%and 27.8%,the sensitivity was increased to 64.8%.The sensitivity of ASCA or PAB alone to diagnose CD was 35.8%.If combination them, the sensitivity was increased to 57.8%;3.The significance of combined marker in the differentiation of IBD:The sensitivity,specificity of combination of positive ASCA and negative p-ANCA to diagnose CD was 57.8%and 96.3%respectively.If combined with PAB and GAB,the specificity and PPV increased to 100%,but the sensitivity was 12.8%for CD.The sensitivity and specificity of combination of positive p-ANCA and negative ASCA to diagnose UC was 50%and 94.5%, respectively.If combined with PAB and GAB,the specificity and PPV increased to 100%,but the sensitivity was 19.4%for UC;4.The significance of combined marker in the differentiation between colonic CD and UC:The sensitivity,specificity and PPV of combined of p-ANCA and ASCA for differentiation between type of colonic CD and UC were 60%,96.3%and 28.6%,respectively.If combined with PAB and GAB, the specificity and PPV were 100%,but the sensitivity was 0.9%for type of colonic CD.The sensitivity,specificity and PPV of combined of p-ANCA and ASCA for differentiation of UC from type of colonic CD were 50%,94.5%and 28.9%,respectively.The specificity and PPV were 100%,and the sensitivity was 16.7%for UC,when combined with PAB and GAB.5.The significance of combined marker in the differentiation between IBD and DC:The specificity of these antibodies alone for differentiation between IBD and DC were above 95%.The specify of combined of PAB and GAB was higher,compared combination of p-ANCA and ASCA.6.An association of these markers with clinical feature:No statistically significant association of p-ANCA and GAB with clinical feature were seen in patients with UC,as well as ASCA in patients with CD.The percentage of PAB positivity was different between CD patients with≤40 and＞40 age at diagnosis(43.2%vs 20%,P=0.018).The percentage of PAB positivity was different between CD patients with EIM and without EIM(58.8%vs 31.5%,P=0.031).Summary:p-ANCA and GAB were specific for UC,while ASCA and PAB were specific for CD.Compared with these antibodies alone,it improved the sensitivity in diagnosis of UC if the combination of p-ANCA and GAB.Meanwhile the combination of ASCA and PAB,it improved the sensitivity in diagnosis of CD.If the combination of p-ANCA,ASCA,PAB and GAB,it improved the differentiation between UC and CD,expecially in isolated colonic disease,compared with the combination of p-ANCA and ASCA.GAB alone is useful for differentiation between IBD and non-IBD intestinal disorders.Antibody response to PAB was associated with early onset and extensive colitis in Chinese populations.Expression of p-ANCA and GAB was not associated with disease behavior of UC.ASCA was not associated with disease behavior of CD.Conclusion:1.These results suggested that polymorphisms of the DEFB1 gene were associated with an increased risk of UC,but not for CD.It may also determine disease phenotype such as:early onset,severe degree and extensive colitis.2.The association of rs2241880 and rs1045095 polymophism of ATG16L1 gene with IBD was not observed.3.The combination of p-ANCA,ASCA,PAB and GAB improved the differentiation between UC and CD,particularly in isolated colonic disease,compared with the combination of p-ANCA and ASCA testing4.Antibody response to PAB was associated with early onset and extensive colitis in Chinese populations,p-ANCA and GAB expression was not associated with disease behavior of UC.ASCA was not associated with disease behavior of CD.