An efficient method to genotype the polymorphisms of cholinergic nicotinic receptor subunit genes and their associations with COPD onset risk

Abstract

<p><b>Background:</b> Single-nucleotide polymorphisms (SNPs) in the cholinergic nicotinic receptor subunit genes on chromosome 15q25.1, including <i>CHRNA3</i>, <i>CHRNB4</i> and <i>CHRNA5</i>, 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. <b>Aim of the Study:</b> The aim of this study was to establish an accurate and efficient method for genotyping <i>CHRN</i> SNPs and testing their association with age at onset of COPD in Chinese population as well as the clinical stage in COPD patients. <b>Materials and Methods:</b> We designed a method to specifically genotype 5 SNPs of <i>CHRN</i> 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. <b>Results:</b> Requiring little time, few material costs and only a simplified protocol, the modified HRM method could accurately distinguish the genotypes of <i>CHRN</i> 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; <i>P</i> = 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. <b>Conclusion:</b> A simple, rapid and efficient HRM method was introduced for <i>CHRN</i> SNP genotyping and a suggestion that the SNP rs56218866A>G is associated with early-onset COPD in a Chinese population was found.</p