The Identification And Characterization Of The Genetic Susceptibility Loci,EAF2 Rs200859609,for Idiopathic Pulmonary Fibrosis And Its Molecular Mechanism

Background and ObjectiveIdiopathic pulmonary fibrosis(IPF)is the most common pattern of idiopathic interstitial pneumonia with unknown cause and poor prognosis.No effective treatment exists except lung transplantation.The interaction between environmental risks and genetic predisposition has been recognized as a pathogenetic mechanism of IPF.Genetic factors play an important role in the pathogenesis of IPF.It is helpful to understand the pathogenesis of the disease through identifying the genetic susceptibility variants associated with disease phenotypes.Some genetic susceptibility variants associated with IPF have been identified in Western populations by high-throughput analysis.However,those variants can not account for all the pathogenesis of IPF and the distribution heterogeneity of some known IPF susceptibility variants in different ethnic populations exists.It is necessary to search the genetic susceptibility variants associated with IPF in Chinese population.Whole exome sequencing(WES)was performed on peripheral blood genomic DNA samples from 10 Han Chinese patients with classic IPF to screen out IPF susceptibility variants in Chinese population.The selected variant was then identified in larger populations.The function of the susceptibility genetic variant and the mechanism of its role in increasing the risk of IPF were further studied using the approaches of molecular biology and cell biology.Materials and Methods1.We performed WES on peripheral blood DNA samples from 10 Han Chinese IPF patients.The variants identified by exome sequencing were validated using Sanger sequencing and the IPF susceptibility variants were screened out by bioinformatics methods.2.A TaqMan assay was used to genotype IPF susceptibility loci discovered by WES,EAF2 rs200859609 in 1090 healthy controls and 881 subjects with interstitial pneumonia(IP),including 214 subjects with IPF,224 subjects with non-IPF idiopathic interstitial pneumonia(nIPF-IIP)and 443 subjects with connective tissue disease associated-IP(CTD-IP).Associations between the alleles and genotypes of rs200859609 and the risks of IP and its subgroups were analyzed by cohort analysis.Stratified analysis was used to observe if IPF susceptibility of rs200859609 was affected by age or gender.The Kaplan-Meier method was used to analyze if rs200859609 genotypes were associated with the survival of IPF patients.3.The localization and expression of EAF2 in the lung tissues of IPF patients and normal lung tissues were investigated by immunohistochemistry staining.SiRNA and overexpression vector of EAF2 were applied to regulate the expression level of EAF2 in alveolar epithelial cell line A549.Cell immunofluorescence and Western blot were utilized to observe the effect of EAF2 on epithelial-mesenchymal transition(EMT)and TGF-?/smad and Wnt/?-catenin signaling pathways in alveolar epithelial cells.Results1.We performed a preliminary screening using WES,then validate the candidate variants screened out by WES through Sanger sequencing,and selected out the IPF susceptibility variants with the help of bioinformatics methods.An IPF susceptibility single nucleotide variant in EAF2 gene exon 5,rs200859609(c.620C>T,p.S207F),was discovered for its ability to damage the function of EAF2 protein..2.Cohort analysis demonstrated that the allele T of EAF2 rs200859609 significantly increased the risk of IPF in Chinese population.Stratification analysis showed that the individuals with CT genotype had a more significantly increased risk of IPF in younger(?65 years)male individuals.Survival analysis indicated that the polymorphism of rs200859609 was an risk factor for the prognosis of IPF patients.3.EAF2 was mainly expressed in bronchial epithelial cells and alveolar epithelial cells in lung tissues.The expression of EAF2 in lung tissues obtained from IPF patients was significantly lower than that in normal lung tissues.EAF2 inhibited EMT of A549 alveolar epithelial cells.Besides directly reducing the expression of Wnt1 and Wnt10 in alveolar epithelial cells,EAF2 may inhibit TGF-?/smad and Wnt/?-catenin signaling pathways by other means.ConclusionWe identified and characterized an IPF susceptibility loci,EAF2 rs200859609,through exome-wide screening.EAF2 was then demonstrated to inhibit EMT during the process of lung fibrosis.Our findings provided a certain experimental basis for clarifying the mechanisms of IPF and looking for potential therapeutic targets.