Study On The Molecular Mechanism Of The Association Between Llql3.5 And 5q31.1 Regions Of The Human Genome And Asthma

Asthma is a human lung disease,also known as bronchial asthma.It is essentially a chronic airway inflammation and caused by a variety of cell and cell ? Lar components.The main symptoms of asthma are shortness of breath,chest tightness,wheezing and coughing.Due to automobile exhaust,factory waste air and other harmf u L gas emissions which induce more and more serious air pollution,the accelerated pace of people's lives and the bad habits of life,the incidence of asthma had increased yearly,with the treand of younder onset age.Some young children suffer from severe asthma,which brings pain and burden to their lives and families.Although the incidence of asthma in China is lower than that in some developed countries,the mortality rate is very high,reaching 36.7 per 100,000 people and ranking first in the world.In short,asthma not only causes great damage to human health,but also brings a lot of inconvenience to people's livesand a great economic burden to the society,which deserves our attention.A number of studies have found that the heritability of asthma is as high as 80%,thus demonstrating that genetic effects play a very important role in the onset of asthma.There are now a number of genome-wide association studies(GWAS)in the scientific community to explore the underlying hereditary nature of asthma.GWAS have identified gene targets for the development of asthma,which explained some of the genetic properties of asthma and greatly deepen our understanding for the pathogenesis of asthma.However,GWAS only discovers genetic markers and cannot explain the pathogenesis of the mutation.The significance of this research:Based on the research of GWAS,we wo ? Ld analyze the 1000 genomes project data and identify the SNPs in strong linkage disequilibrium(LD)as potential functional SNPs for asthma.By utilizing functional genomics approaches,the real causal SNPs and their mechanism co ? Ld be disclosed.This study wo ? Ld shed more light on the genetic mechanism of asthma and provide more insight into the screen,preventation and therapy of asthma.Research content and res ? Lts:Through analyzing 1000 genomes project data,the SNPs in LD with rs7130588 and rs1295686 co ? Ld be identified.The luciferase plasmid containing the nearby region of these SNPs wo ? Ld be constructed and the plasmid with mutation at these sites wo ? Ld be generated by mutagenesis.Through transfecting the plasmids into lung cell,the influence of these mutation in enhancer activity wo ? Ld be obtained.Through chromosome conformation capture(3C),the interaction between the enhancer containing these SNPs and nearby gene promoters co? Ld be identified.Further Chromatin immunoprecipitation(ChIP)wo ? Ld be utilized to scrutinize the related transcript factor binding these SNPs.In the 11q13.5 region in human genome,only one SNP,rs6592645 presented strong LD with rs7130588(r2=1).The dual luciferase assay indicated that the two alleles displayed similar luciferase expression(P=0.487).In contrast,the T allele of rs6592645 presented-4.2-fold luciferase expression than that of C(P=0.000125),which indicated that the causal SNP in 11q13.5 was not rs7130588 but rs6592645.The 3C res ? Lt indicated that the nearby protein-coding genes failed to display interaction with this enhancer and were not the reg ? Lation target of this enhancer.The ChIP res ? Lt indicated that transcript factor TCF3(transcription factor 3)co ? Ld bind the surrounding region of rs6592645.In 5q31.1,there were four SNPs,rs20541,rs1295685,rs848 and rs847,in LD with rs 1295686.The dual luciferase asssay indicated that rs 1295686 and rs20541 failed to alter relative enhancer activity(P=0.93 and 0.15,respectively).In contrast,G allele of rs1295685,C of rs848 and C of rs847co ? Ld reduce the relative luciferase expression?90%(P=3.75 × 10-13),?97%(P=4.14 × 10-14)and?97%(=3.73 × 10-14),which indicated that these three SNPs were causal SNPs for asthma.3C suggested that the enhancer containing these three SNPs interacted with the promoter of one long non-coding RNA(IncRNA),TH2LCRR(T helper type 2 locus control region associated RNA).The lncRNA co ? Ld reg ? Late the cytokine expression,which migh further reguate the immune response and induce asthma.By ChIP,the transcript factors,TCF3,USF1(upstream transcription factor 1)and HNF4A(hepatocyte nuclear factor 4 alpha)were identifed to bind the region surrounding rs 1295685,rs848 and rs847,respectively.The purpose of this study:identification of the causal SNPs and its molec ? Lar mechanism of astham.The innovation of the article:Based on GWAS,this experiment has further identified the causal SNPs and the pathogenic mechanism for 11q13.5 and 5q31.1.Conclusion:The functional site of 11q13.5 causing asthma is rs6592645,which co? Ld alter binding affinity of transcript factor TCF3,regualte enhancer activity and further influence asthma susceptibility.The causal SNPs in 5q31.1 were rs1295685,rs848 and rs847,which co ? Ld bind transcript factors TCF3,USF1 and HNF4A,respective,regualte the expression of TH2LCRR and further influence asthma risk.