title: An efficient method to genotype the polymorphisms of cholinergic nicotinic receptor subunit genes and their associations with COPD onset risk.
abstract: Single-nucleotide polymorphisms (SNPs) in the cholinergic nicotinic receptor subunit genes on chromosome 15q25.1, including CHRNA3, CHRNB4 and CHRNA5, are well-established biomarkers of chronic obstructive pulmonary disease (COPD) and lung cancer. Thus, there is great demand for a rapid, easy and inexpensive method to detect these variations for purpose of risk prediction in large populations.
  
  
  
  The aim of this study was to establish an accurate and efficient method for genotyping CHRN SNPs and testing their association with age at onset of COPD in Chinese population as well as the clinical stage in COPD patients.
  
  
  
  We designed a method to specifically genotype 5 SNPs of CHRN genes based on a modified high-resolution melt (HRM) method and then validated the genotyping results by direct sequencing of 120 samples. We further used the HRM method to genotype these 5 SNPs in 1,013 COPD patients.
  
  
  
  Requiring little time, few material costs and only a simplified protocol, the modified HRM method could accurately distinguish the genotypes of CHRN SNPs, demonstrating kappa coefficients >0.96 based on the results from direct sequencing. Furthermore, the data showed that the GG genotype of SNP rs56218866 was associated with a significantly earlier age of COPD onset than A (AA+AG) genotypes (61.0 ± 8.93 vs. 67.8 ± 9.88; P = 0.031), which was not found for the other SNPs. No significant association was observed between the COPD stages and any of the above SNPs.
  
  
  
  A simple, rapid and efficient HRM method was introduced for CHRN SNP genotyping and a suggestion that the SNP rs56218866A>G is associated with early-onset COPD in a Chinese population was found.
authors.affiliation: a The First Affiliated Municipal Hospital , Guangzhou Medical University , Guangzhou , Guangdong , China.
authors.forename: Zhuxiang
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: b The State Key Laboratory of Respiratory Diseases , Guangzhou Institute of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University , Guangzhou , Guangdong , China.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: a The First Affiliated Municipal Hospital , Guangzhou Medical University , Guangzhou , Guangdong , China.
authors.forename: Yujun
authors.initials: Y
authors.lastname: Li
authors.affiliation: b The State Key Laboratory of Respiratory Diseases , Guangzhou Institute of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University , Guangzhou , Guangdong , China.
authors.forename: Changbin
authors.initials: C
authors.lastname: Jiang
authors.affiliation: b The State Key Laboratory of Respiratory Diseases , Guangzhou Institute of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University , Guangzhou , Guangdong , China.
authors.forename: Dongxing
authors.initials: D
authors.lastname: Zhao
authors.affiliation: a The First Affiliated Municipal Hospital , Guangzhou Medical University , Guangzhou , Guangdong , China.
authors.forename: Ziwen
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: a The First Affiliated Municipal Hospital , Guangzhou Medical University , Guangzhou , Guangdong , China.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Liu
authors.affiliation: b The State Key Laboratory of Respiratory Diseases , Guangzhou Institute of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University , Guangzhou , Guangdong , China.
authors.forename: Pixin
authors.initials: P
authors.lastname: Ran
mesh_terms: D017668:Age of Onset; D000368:Aged; D017046:Cost Savings; D005260:Female; D060005:Genotyping Techniques / Q000191:economics; D006801:Humans; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D029424:Pulmonary Disease, Chronic Obstructive / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000235:genetics*
chemical_list: D011978:Receptors, Nicotinic
keywords: cholinergic nicotinic receptor; chronic obstructive pulmonary disease; high-resolution melting; polymorphism