Genetic Variations At Rs3129891 And Rs77005575 Are Associated With Reduced Expression Of Enteric ?-defensins In IBD Patients

Background and AimsInflammatory bowel disease(IBD)is a chronic idiopathic inflarmmation of the intestine,and its two major forms include ulcerative colitis(UC)and Crohn's disease(CD),based on clinical features and histopathology.Although its aetiology is still unclear,a strongly advocated view is that ineffective bacterial clearance resulted from the diminished expression of enteric antimicrobial peptides triggers and sustains the abnormal immune responses observed in IBD patients.Human enteric antimicrobial peptides are composed predominantly of human enteric a-defensins as well as lysozyme and secretory phospholipase A2(SPLA2),to a lesser extent.Genotype-phenotype correlation studies have revealed that NOD2 mutations(SNP8,rs2066844;SNP12,rs2066845;SNP13,rs2066847)are associated with ileal CD.Expression of enteric a-defensins has been shown to be diminished in terminal ileal biopsies from CD patients harboring these mutations,with diminished levels being more pronounced in the presence of a SNP13 mutation.However,it is still unclear which genetic variations are associated with mucosal expression of antimicrobial peptides in colonic CD and UC patientsThe purpose of this study was to determine which genetic variations can influence mucosal expression of antimicrobial peptides in colonic CD and UC.To address this issue,we compared mucosal levels of human enteric antimicrobial peptides in colonic CD and UC patients with respect to genotypes at 22 IBD-associated SNPs.These SNPs are associated with IBD,including rs2066844?rs2066845?rs2066847?rs35261698?rs2172252?rs3197999?rs4151651?rs6930777?rs77005575?rs9268832?rs3129891?rs3115674?rs2066842?rs10885394?rs10885395?rs3814570?rs11209026?rs11805303?rs12212067?rs2241880?rs2542151 and rs7234029?Material and MethodsPatientsSixteen patients undergoing a routine colonoscopy without significant pathology were randomly selected as controls.A cohort of 42 UC and 60 CD patients from Department of Gastroenterology at Nanfang Hospital were screened for mutant genotypes of 22 IBD-associated SNPs.Colonic biopsies were available from the 118 patients.In the case of UC and CD,samples were taken from macroscopically inflamed and non-inflamed mucosa.Samples were immediately snap frozen in liquid nitrogen.The diagnosis in all patients was based on standard criteria using clinical,endoscopic,and histopathological findings.All patients gave written informed consent before colonoscopy was performed.The study was approved by the clinical ethics committee at Nanfang Hospital.Mutation assayFrozen biopsies were disrupted mechanically and genomic DNA was extracted using MiniBEST Universal Genomic DNA Extraction kit Ver.5.0(TaKaRa)according to the manufacturer's protocol,Genotyping of genomic DNA for mutations of 22 IBD-associated SNPs was performed using MassARRAY MALDI-TOF System(SEQUENOM)according to the manufacturer's protocol.Real-time quantitative RT-PCRFrozen biopsies were disrupted mechanically and total RNA was extracted using the RNAiso Plus Kit(TaKaRa),then cDNA synthesis was performed using the PrimeScript RT reagent Kit(TaKaRa),and finally Real-time PCR was performed in triplicate using the LightCycler 480 System(Roche)according to the manufacturer's protocol.18SrRNA were used as internal controls.The 2-?CT method was used to analyze real-time PCR data.Statistical analysisThe results are shown as the mean ±SEM.Statistical significance was determined by one-way analysis of variance with Tukey's multiple comparisons under equal variances or with Dunnett T3's multiple comparisons under unequal variances;a value of P<0.05 was considered statistically significant.Results1.SNP mutation analysesAnalyses of 118 specimens from 16 controls,42 UC and 60 CD patients failed to show mutant genotypes at rs2066844?rs2066845?rs2066847?rs11209026?rs4151651?rs6930777,but showed mutant genotypes at rs11805303?rs12212067?rs2241880?rs2542151?rs7234029?rs35261698?rs2172252?rs3197999?rs77005575?rs9268832?rs3129891?rs10885394?rs10885395?rs3814570.However,homozygous mutant rs77005575 genotype CC and rs3129891 genotype AA were not found in controls.In addition,mutant genotypes at rs2066842 and rs3115674 were only found in CD patients and controls,respectively.2.Colonic gene expression of HD5 and HD6 was decreased in UC and CD patients carrying rs3129891 mutant genotypes,while the mRNA expression of lysozyme and SPLA2 was not significantly influenced3.Colonic gene expression of HD5 and HD6 was decreased in UC patients carrying rs77005575 mutant genotypes,while the mRNA expression of lysozyme and SPLA2 was not significantly influenced4.Colonic gene expression of HD5,HD6,lysozyme and SPLA2 in CD patients was not affected by NOD2 rs77005575 mutant genotypes5.Colonic gene expression of HD5,HD6,lysozyme and SPLA2 in CD patients was not affected by NOD2 rs2066842 mutant genotypesConclusionsgenetic variations at rs3129891 and rs77005575 are associated with reduced expression of enteric ?-defensins in colonic IBD patients.