Investigation On A Functional SNP Upstream Of ADRB2with Gene Expression And Correlation With Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD), one of common respiratory diseases in old people, is characterized by sustainability airflow limitation. Tobacco smoking has been proven to be the predominant environmental factor for COPD, however, only10-20%of severe smokers suffered from COPD. This phenomenon, together with the familial clustering in patients of COPD, suggested that genetic factors might play an important role in the development of COPD.The β2-adrenergic receptor (ADRB2) is a member of the G protein coupled receptors family, and is widely expressed in different lung tissues. Recent studies have reported that two non-synonymous SNPs of the ADRB2, Argl6Gly (rs1042713) and Glu27Gln (rs1042714), which could cause amino acid substitution in the ADRB2encoding area, were associated with COPD. However, there was no consistent result. Moreover, the role of most genetic polymorphisms, especially those of the non-coding ones, on ADRB2gene expression regulation and COPD has not been evaluated.In order to investigate the association between ADRB2genetic polymorphisms and COPD comprehensively, methods including real-time fluorescent quantitative PCR (RT-qPCR), SNaPshot technology, dual luciferase assay, and chromatin immunoprecipitation (ChIP) were adopted in this study. The results indicated that:(1) there was a significant reduced expression of ADRB2in COPD patients tissues (P=0.033) in comparison with normal smokers;(2) six tag-SNPs (MAF≥5%), including Argl6Gly and Glu27Gln, were selected in East Asian populations from1000genome, and further genotyped in274COPD patients and292controls from southwest Chinese population. The rs12654778-A allele, of a SNP located～654bp upstream of ADRB2, indicated significant association with risk for COPD in genotype compared with normal controls (33.9%,55.1%and10.9%for GG, GA and AA, respectively in case;42.8%,42.8%and14.4%for GG, GA and AA, respectively in control; P=0.014);(3) subsequently, a dual luciferase assay revealed that the rsl2654778-A allele reduced the transcriptional activity of the luciferase reporter gene by approximately26%, compared with the rs12654778-G allele (P=0.003);(4) ChIP assays demonstrated that rs12654778modulated the binding affinity with transcript factor, neurofibromin1 (NF-1), which could bind with ADRB2promoter region specifially, compared with IgG protein (P=0.0021);(5) In addition, meta-analyses for Arg16Gly and Glu27Gln were undertaken and it was found that there was no correlation between these two SNPs and COPD in Asia or Caucasian populations, whatever in the allele, domoninant, recessive and additive genetic modes.In summary, our results obtained from RNA expression, genotype and function analysis indicated that, the rsl2654778-A allele was proved to reduce the expression of ADRB2in the lung tissue of COPD, and further contributed to the onset of COPD in the East Asian population.