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

<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 &gt;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&gt;G is associated with early-onset COPD in a Chinese population was found.

<b>研究背景：</b> 15号染色体q25.1区域胆碱能烟碱型受体亚基基因（包括<i>CHRNA3</i>、<i>CHRNB4</i>和<i>CHRNA5</i>）的单核苷酸多态性（Single-nucleotide polymorphisms, SNPs）是已被广泛证实的慢性阻塞性肺疾病（chronic obstructive pulmonary disease, COPD）与肺癌生物标志物。因此，亟需开发一种快速、简便且低成本的检测方法，以用于大人群的疾病风险预测。<b>研究目的：</b> 本研究旨在建立一种准确高效的<i>CHRN</i>基因单核苷酸多态性基因分型方法，并验证其在中国人群中与慢性阻塞性肺疾病发病年龄及患者临床分期的关联。<b>材料与方法：</b> 本研究基于改良的高分辨率熔解（high-resolution melt, HRM）技术，设计了一种可特异性分型<i>CHRN</i>基因5个单核苷酸多态性位点的方法，随后通过对120份样本的直接测序验证了该基因分型结果。进一步使用该HRM方法对1013名慢性阻塞性肺疾病患者的上述5个SNP位点进行基因分型。<b>研究结果：</b> 该改良HRM方法耗时少、耗材成本低且操作流程简便，可准确区分<i>CHRN</i>基因SNP的基因型，经直接测序验证的Kappa系数均大于0.96。此外，数据显示，与A等位基因携带者（AA+AG基因型）相比，SNP rs56218866的GG基因型与慢性阻塞性肺疾病更早的发病年龄显著相关（61.0±8.93 vs 67.8±9.88；<i>P</i>=0.031），这一关联在其余SNP位点中未被观察到。未观察到慢性阻塞性肺疾病临床分期与上述任一SNP位点存在显著关联。<b>研究结论：</b> 本研究建立了一种简便、快速且高效的<i>CHRN</i>基因SNP分型HRM方法，并发现SNP rs56218866A>G与中国人群慢性阻塞性肺疾病早发存在关联。