Molecular Mechanism Of SNPs In The 1q43 Region Associated With Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia(BPD)is a common chronic disease of lung,which is characterized by initial lung injury,and its incidence is negatively correlated with birth weight and gestational age of the newborn.With the rapid development of medical technology,the incidence of BPD is increasing due to the improving survival rate of preterm infants.A large amount of clinical and molecular research focus on reducing the incidence of BPD.However,the incidence failed to decrease significantly,which constituted a very difficult problem in clinical practice.BPD is mainly induced by premature birth,immature lung development,mechanical ventilation,genetics,infection,and malnutrition.Among them,genetic factors account for more than 50%.Single nucleotide polymorphisms(SNP)is the most common mutation in human heritable variations,accounting for more than 90%of the known genetic polymorphisms.Using genome-wide association study(GWAS),it was found that the SNP marker rs2105146 in the human genome 1q43 region locating the intron of CEP170(centronsomal protein 170KD)was closely associated with BPD.Nevertheless,GWAS also has certain limitations because the rs2105146 is only a tag SNP and the true causal SNP might be not tag SNP,but the ones in linkage disequilibrium(LD)with it.Moreover,GWAS cannot reveal the pathogenic mechanism of the causal mutations.This study utilized allele-specific expression(ASE)to directly determine whether the CEP 170 expression was influenced by cis-regulatory factor SNP based on GWAS.Chromatin conformation capture(3C),dual-luciferase reporter gene assay,chromatin immunoprecipitation(ChIP),and overexpression of transcription factors were used to investigate the true pathogenic site and pathogenic mechanism of BPD in the human genome 1q43 region.The main results were as follows:(1)A total of 71 SNPs were found in LD rs2105146 in the IBS(Iberian Population in Spain)population based on the 1000 Genomes Project data.Then the further analysis on the East Asian populations revealed that CHB(Han Chinese in Beijing)and JPT(Japanese in Tokyo)populations also possesses similar LD patterns.Among these SNPs,rs6701379 was located in the 3'-untranslated region(3'UTR)of the CEP170 gene,while the others in the non-coding regions.(2)ASE analysis on 3'UTR SNP rs6701379 showed that 12 of the 48 lung tissue samples were heterozygous individuals,and the expression level of the A allele is significantly lower than that of the G(P=0.000002).These results indicated that rs6701379 or the SNPs in LD with it could be cis-regulatory element for CEP 170.(3)3C was utilized to screen the interacting genomic fragment(s)with the CEP 170 promoter region.A total of 4 genomic fragments were observed with CEP 170 promoter,and 8 SNPs(rs6429410,rs12406630,rs12406654,rs200997665,rs151302646,rs75388769,rs74651335,and rs1766026)were locating within these four segments might regulate CEP 1 70 expression through enhancer model.(4)Experiments including constructing pGL3-promoter-SNP recombinant plasmids,site-directed mutagenesis,and dual-luciferase assay proved that 5 sites including rs1766026,rs12406654,rs200997665,rs75388769,and rs151302646 were functional mutations,all of which were locating in the non-coding region and might regulate gene expression through enhancer patterns.(5)ChIPwas utilized and it was observed that rs1766026,rs12406654,rs200997665,and rs151302646 were locating within the binding region of the transcription factors CUX1(cut like homeobox 1),TBX20(T-box transcription factor 20),HNF4A(hepatocyte nuclear factor 4 alpha)and CUX1,repectively.(6)By constructing and transfecting the transcription factor pEGFP-N 1 overexpression vector and dual-luciferase assay,it could be proposed that the transcription factors CUX1,TBX20,HNF4A and CUX1 could reguate the expression of target gene.Among them,TBX20 possessed a negative correlation with gene expression and could inhibit gene expression.Our effort explored the regulatory mechanism of the SNPs in 1q43 region on BPD susceptibility gene CEP 170,which provide more insight into the occurrence,development mechanism and individualized treatment of BPD.The identification of functional sites shed might light on diagnose,prevent and treatment of BPD.In addition,it could provide an ideal example for how to utilize ASE and 3C-qPCR to identify functional regions in human genome.