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

ABSTRACT

Background: 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. Aim of the Study: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

KEYWORDS:

• cholinergic nicotinic receptor
• chronic obstructive pulmonary disease
• high-resolution melting
• polymorphism

Acknowledgments

The authors gratefully acknowledge and thank everyone who participated in this study as well as its funders. In particular, we acknowledge Qicai Liu for implementing the trial. We acknowledge Elsevier for English Language Editing.

Declaration of interest

All of the authors declare the following: There is no support from any institution for the submitted work; there are no financial relationships with any institutions that might have an interest in the submitted work within the previous 3 years; and there is no other relationship or activity that might appear to have influenced the submitted work.

Ethical approval

The study protocol was approved by the medical ethics committee of the Guangzhou Institute of Respiratory Diseases.

Funding

This work was supported by National Natural Science Foundation of China (81170043) and the Science and Technology Project of Guangdong (2013B021800069). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Notes on contributors

Zhuxiang Zhao collected and monitored the data, planned the statistical analysis, analyzed the data and drafted and revised the manuscript. Yuming Zhou implemented the trial and conducted the intervention. Yujun Li, Changbing Jiang, and Dongxing Zhao collected blood samples and performed the data collection from patients and individuals. Zhaohui Liu and Ziwen Zhao monitored data collection, conducted the statistical analysis and revised the paper. Pixin Ran initiated and designed the study, monitored the data analysis, and revised the paper.