测序法筛选慢性阻塞性肺疾病尼古丁受体CHRNB4基因外显子单核苷酸多态性

DNA sequencing for screening single nucleotide polymorphisms of nicotine receptor gene exon CHRNB4 in chronic obstructive pulmonary disease patients

目的:寻找中国南方汉族人群尼古丁受体(CHRNB4)基因功能区与慢性阻塞性肺疾病(COPD)疾病相关的单核苷酸多态性位点(SNP)。方法:在广州地区抽取按照慢性阻塞性肺疾病全球创议(GOLD)诊断标准(扩张后FEV1/FVC<70%)确诊的汉族COPD患者120例,按年龄(±3岁)与病例频数匹配原则选取非COPD对照者120例,病例组来自广州医科大学附属市一人民医院住院及门诊患者,对照组来自门诊志愿者。抽取受试者外周血提取基因组DNA,利用Pubmed(http://www.ncbi.nlm.nih.gov/gene/)查寻CHRNB4的全基因组序列,以Oligo6引物设计软件设计包括全外显子序列在内的测序引物5对,焦磷酸法DNA测序,Bio Edit分析软件对测序序列进行比对及基因分型分析。结果:通过第一代测序,快速筛选出中国汉族人群CHRNB4外显子区3个SNP,分别为rs56218866、rs6743072、rs1948。其中rs56218866可引起氨基酸位点改变。结论:通过第一代测序,可以简单、快速筛选出中国南方汉族人群与尼古丁受体CHRNB4外显子功能区和COPD易感性相关的SNP位点。

Objective：To identify the single nucleotide polymorphisms （SNP ）on functional gene domains of nicotine receptor （CHRNB4）associated with susceptibility of chronic obstructive pulmonary disease （COPD） in southern Chinese Han population. Methods：Included in this study were 120 Han patients with COPD in Guangzhou diagnosed according to Global Initiative for Chronic Obstructive Lung Disease （GOLD ） （post-bronchodilator FEV1/FVC〈70%）. Based on matching for age （±3 years）and number of cases,120 non-COPD subjects were recruited as the control group. All COPD subjects were inpatients or outpatients at the Guangzhou First Municipal People′s Hospital Affiliated to Guangzhou Medical University,and the control group consisted of clinic volunteers. The study subjects received phlebotomy for extraction of genomic DNA. The complete genome sequence of CHRNB4 was retrieved from Pubmed （http：//www. ncbi. nlm. nih. gov/gene/）. Five pairs of primers containing full-length of DNA sequences of the exon were designed by using Oligo6 primer design software. Pyrophosphate DNA method was used for the sequencing. Bio Edit software was used for the sequence comparison and genotyping analysis. Results：First-generation sequencing quickly identified three SNPs at thenbsp;CHRNB4 exon of Chinese Han population：rs56218866,rs6743072,and rs1948. Of these,only rs56218866 resulted in a change in amino acids. Conclusion：First-generation sequencing can be simple and quick in screening the SNP at the functional domains of nicotine receptor CHRNB4 exon associated with susceptibility to COPD in southern Chinese Han population.