Association Of Inflammatory Bowel Disease With FUT2 And FUT3 Polymorphisms And Their Expression In Chinese Patients

Backgrounds and aims:Inflmmatory bowel disease(IBD),mainly comprising ulcerative colitis(UC)and Crohn's disease(CD),has a rising global incidence and morbidity rate in recent years.Although the etiology and pathogenesis of IBD is not yet fully clarified,it has been mostly accepted that IBD is a condition involved in multiple factors such as heredity,environment,and immunity,triggering an inappropriate immune response to normal constitutions of the gut microbiota in an individual with genetic predisposition.which leads to the injury of intestinal tissue.Of these factors,the imbalance of micriobiota has been thought to be the trigger of the onset of IBD[1].Data from clinical and colitis animal model studies supported that the composition of intestinal microbiota is closely related to the inflammation of the intestinal tissue[2].Histo-blood group antigens(HBGA),including ABH and Lewis antigens,are mainly expressed in the intestine,saliva and other secretion gland.The expression of HBGA in the intestine has been shown to affect the constitution of intestinal microbiota[3-5]Fucosyltransferases(FUTs)-encoding genes FUT2 and FUT3 are in charge of the formation of ABH and Lewis antigens by adding a fucose to precursor substrate.Blood group precursor becomes H antigen after fucosylation by a-(1,2)-fucosyltransferase encoded by FUT2 In the presence of alpha-3-N-acetylgalactosamine transferase encoded by Gene A.or D-galactosyl transferase encoded by Gene B.H antigen would further become A antigen or B antigen,which together compose the ABH blood group antigen.FUT3.which encodes ?-(1,3/1,4)-fucosyltransferase,together with FUT2,determines the formation of Lewis antigens.If the loss-of-function mutation occurred in FUT2 gene,the precursor of blood groups will not be catalyzed to H antigen,but be directly transformed to Lewis a antigen by the help of ?-(1,3/1,4)-fucosyltransferase encoded by FUT3.From a study on the CD patients and healthy controls,Philipp Rausch and the colleagues revealed that FUT2(rs601338)polymorphism is strongly correlated with the constitution of the intestinal microbiota,while the expression of ABH and Lewis antigens also affect the composition of the intestinal microbiota[6].Therefore,FUT2 and FUT3 genes might influence the composition of intestinal microbiota through determining the intestinal expression of HBGA,thus potentially play a key role in the pathogenesis of IBD.Studies has showed that polymorphims of FUT2 are associated with a group of autoimmune disease such as CD[7,8],type ? diabetes[9]and primary sclerotic cholangitis[10].However,the conclusions drawn from these studies seem to be inconsistent[11-12].For example,McGovern DP and the colleages found that the single nucleotide polymorphism(SNP)in FUT2(rs601338)was not associated with the predisposition of UC in Caucasians[8],while another study from Finland suggested that the carrier with wildtype GG of this SNP in FUT2 had an increased risk of UC[31].Moreover,the conclusions of the association between FUT2 and CD drawn from different ethic background were also inconsistency.For example,one study showed that non-secretor status in Caucasian polulation increased the risk of CD[8]while in Japanese population the FUT2 non-secretor status played a protective role in patients with CD[14].As far as we know,the investigation of the association between FUT3 polymorphisms and IBD is limited.Therefore,it is necessary to investigate the relationship between IBD and the SNPs of FUT2 and FUT3 in a cohort of Han patients from Zhejiang province of China.Part I:Association of Ulcerative Colitis with FUT2 and FUT3 Polymorphisms and their expressions in colonic tissuesMethods During January 2005 and October 2015,a total of 485 UC patients were recruited from the Second Affiliated Hospital and the first Affiliated Hospital of Wenzhou Medical University and Wenzhou People's Hospital.Five hundred and eighty age-and sex-matched healthy controls,who underwent routine health check-up,were also collected from the medical examination center of the Second Affiliated Hospital of Wenzhou Medical University.Peripheral blood was collected from each study individual.Genomic DNA was extracted from the peripheral blood.Then the polymorphisms of FUT2(rs281377,rs1047781 and rs601338)and FUT3(rs28362459,rs3745635 and rs3894326)was genotyped using SNaPshot assays.By an immunohistochemistry method,seven of these UC patients presenting inflammatory lesions in the sigmoid colon were selected to evaluate the expression of Lewis a and b antigens in the sigmoid colon.In seven patients with confirmed benign colonic polyps,specimens of normal sigmoid colon mucosa were also obtained as controls.Then the expression of Lewis a and b antigens in the UC patients and the controls were compared.Results The frequencies of mutant allele(A)and genotype(GA+AA)in FUT3(rs3745635)were higher in UC patients than in controls(17.0%vs 13.3%,P=0.016,;30.7%vs 25.0%,P=0.038,respectively).There is no statistical significance of mutant allele and genotype frequencies of FUT2(rs281377,rs1047781 and rs601338)and FUT3(rs28362459,rs3745635 and rs3894326)between the two groups(all P>0.05).Stratified analyses revealed that mutant allele(G)and genotype(TG+GG)of FUT3(rs28362459)were less dominant in patients with extensive colitis than in those with distal colitis(17.8%vs 27.7%,P=0.001;31.6%vs 47.9%,P<0.001,respectively).Same conclusions could be applied for the mutant allele(A)and genotype(GA+AA)of FUT3(rs3745635)in patients with extensive colitis compared to those with distal colitis(12.9%vs 19.3%,P=0.011;23.0%vs 35.0%,P=0.006,respectively).Haploview 4.2 software was employed to analyze the linkage disequilibrium(LD)and haplotype reconstruction.Two LD blocks were presented,including block rs281377-rs 1047781 in FUT2(D' = 0.96,r2 = 0.10)and block rs3894326-rs3745635-rs28362459 in FUT3[rs3894326/rs3745635(D'= 1,r2=0.02),rs3894326/rs28362459(D'= 0.86,r2= 0.30),rs3745635/rs28362459(D'=0.92,r'=0.45)].Furthermore,no haplotype frequencies differed significantly between UC patients and the controls(all P>0.05).Although expression of Lewis b antigen in the sigmoid colon did not differ between UC patients and controls,Lewis a antigen expression was higher in the cryptic epithelium of both inflammatory and non-inflammatory sigmoid colon of UC patients than controls(P=0.028).Conclusions Our findings indicated that the polymorphism in FUT3 and its intestinal expression of Lewis a antigen might be associated with the susceptibility of UC in Zhejiang Han population.Part ? Associations of FUT2 and FUT3 gene polymorphisms with Crohn's diseaseMethods:During March 2005 and March 2014,a total of 275 CD patients were recruited from the Second Affiliated Hospital and the first Affiliated Hospital of Wenzhou Medical University,Wenzhou Central Hospital and Wenzhou People's Hospital.The diagnosis of CD were based on colonoscope,gastroscope,small intestinal endoscope,and capsule endoscope,in collaboration with clinical,histopathological,and radiologic findings according to "consensus of the diagnosis and management of inflammatory bowel disease" released by the Digestive Diseases Branch of Chinese Medical Association(2012,Guangzhou).Five hundred and two age-matched and sex-matched healthy controls were also collected from the medical examination center of the Second Affiliated Hospital of Wenzhou Medical University.Diseases including cardiovascular and cerebrovascular diseases,metabolic diseases,liver and kidney dysfunction,tumors,and autoimmune diseases and those having inflammatory bowel disease family history have been ruled out of these controls.All the subjects were unrelated to each other in terms of heredity.Around 2ml peripheral blood from each study individual was collected.Genomic DNA was extracted from the peripheral blood,followed by multiplex PCR reaction and.purification steps according to the manufacturer's instructions.Then the polymorphisms of FUT2(rs281377.rs 1047781 and rs601338)and FUT3(rs28362459,rs3745635 and rs3894326)was genotyped using SNaPshot assays.Hardy-Weinberg equilibrium for each of the studied polymorphisms and the distributions of genotypes in the compared groups were evaluated by chi-squared test.Haploview 4.2 was employed to analyze the linkage disequilibrium(LD)and haplotype reconstruction.Results:Compared with controls,mutant allele and homozygote TT frequencies of FUT2(rs1047781)was significantly increased in CD patients(49.27%vs 43.33%,P=0.024;27.64%vs 16.53%,P<0.001,respectively).Same conclusions were drawn from the mutant allele A of FUT2(rs601338)in CD patients as compared with the controls(1.64%vs 0.50%,P=0.023;3.27%vs 1%,P=0.044,respectively).However,after Bonferroni correction(P value threshold was adjusted to 0.05/6=0.0083),only mutant homozygote TT frequencies of FUT2(rs 1047781)was still significantly increased in CD patients.Nevertheless,the allelic and genotypic distributions of FUT3 were not statistically different between CD patients and controls.Stratified analysis revealed that the polymorphisms of FUT2(rs1047781),FUT3(rs28362459)and FUT3(rs3745635)were significantly associated with the disease location of CD.The mutant allele(T)and genotype(AT+TT)of FUT2(rs1047781)were less prevalent in patients with ileocolonic CD than in colonic CD(41.67%vs 59.41%,P=0.001;63.33%vs 83.17%,P=0.002,respectively).The frequencies of mutant allele and genotype in FUT2(rs1047781)were also lower in patients with ileal CD compared to colonic CD(45.24%vs 59.41%,P=0.007;64.29%vs 83.17%,P=0.004,respectively).The mutant allele(A)and genotype(AA+AG)of FUT3(rs3745635)were more prevalent in patients with ileocolonic CD than in colonic CD(19.44%vs 9.41%,P=0.006;34.44%vs 16.83%,P=0.006,respectively).The frequencies of mutant genotype in FUT3(rs28362459)were also higher in patients with ileal CD compared to colonic CD(53.33%vs 34.65%,P=0.010).Haploview 4.2 software was employed to analyze the linkage disequilibrium and haplotype reconstruction,showing that LD were presented in block rs281377-rs 1047781 in FUT2(D'=0.90,r2=0.10)and in block rs3894326-rs3745635-rs28362459 in FUT3[rs3894326/rs3745635(D'=1.0,r2=0.01),rs3894326/rs28362459(D'=0.93,r2=0.29),rs3745635/rs28362459(D'=0.96,r2= 0.45)].Furthermore,the haplotype TT formed with FUT2(rs281377 and rs1047781)was more prevalent in CD patients than in controls(49%vs 43%,P=0.020).Conclusions:The polymorphic locus of FUT2(rs 1047781)might be associated with the susceptibility of CD.Mutations of FUT3(rs28362459 and rs3745635)might influence the lesion locations in CD patients.