Apolipoprotein M gene single nucleotide polymorphisms discovery in patients with chronic obstructive pulmonary disease and determined by the base-quenched probe technique

Background: It has been reported increased serum apoM levels seen in the patients suffered from obstructive sleep apnoea and chronic obstructive pulmonary diseases. In the present, we further examine the prevalence of apoM gene SNPs in COPD patients. And a new method base-quenched probe technique is established.

Methods: In the present study, we first used the Roche 454 GS Junior high-throughput sequencer to analyze 6 COPD samples and 6 control samples, in these samples we found 3 interesting SNPs (rs805264, rs707922 and rs707921) and then we designed primers and probes to establish a simple and quick screening method that is a base-quench probe technique and the genotype was confirmed by melting curves. With this new technique, we further determined 252 COPD samples and 248 normal subjects were applied as controls.

Results: A total of 19 high-confidence mutations were detected in the Roche 454, 6 mutations among them were not been reported in NCBI, but the mutation frequency was <20%. Four mutations occurred only in COPD patients, rs751064723 is located in the first exon of transcript 1 and the rest are located in either apoM gene promoter or intron region. The results of melting curve showed that the wild-type and homozygous mutants of rs805264, rs707922 and rs707921 presented melting valley at two different melting temperatures, and the results were consistent with those of DNA sequencing (K=1, P=0.000).

Conclusions: The detection of apoM gene SNPs laid the foundation for the study of the relationship between COPD and apoM, and the base-quenched probe technique is simple, economic and accurate, and it is suitable for a large number of apoM genotyping studies.