高分辨率融解曲线技术检测冠心病患者IL-1β和IL-1Ra基因多态性

High-resolution melting technology for detecting genetic polymorphisms of IL-1β and IL-1Ra in patients with coronary artery disease

目的为探讨白细胞介素1β（IL-1β）和白细胞介素1受体拮抗剂（IL-IRa）基因单核苷酸多态性（SNP）与冠心病（CHD）易感及其血清学指标的相关性，应用高分辨率融解曲线分析（HRM）技术建立IL-1β和IL-1Ra基因SNP的分子诊断方法。方法建立IL-1B-511C〉T、IL-1β-31C〉T、IL-1β＋3954C〉T和IL-1RNT〉C这4个SNP位点的PCR-HRM检测方法，对260例CHD患者和274例健康对照进行比较研究，分析其与CHD易感及血清学指标的关系。结果上述4个SNP位点未发现与CHD易感有统计学差异，性别分层后发现男性IL-1β．31C〉T位点和女性IL-1β＋3954C〉T位点的等位基因频率，在病例组和对照组间存在差异；单倍型分析发现IL-1β-511T／IL-1β-31C／IL-1β＋3954C／IL-1RNT（TCCT）单倍型增加CHD发病风险（OR=1．217，95％CI：0．950—1．559，P=0．123），CTCC和玳T单倍型降低cHD发病风险（OR=0．612，95％CI：0．376～0．994，P=0．046；OR=0．365，95％CI：0．154～0．862，P=0．017）；病例组血清学指标的相关性分析未发现其与SNP位点之间有关联性。结论应用HRM技术建立的4个SNP位点PCR—HRM检测方法经测序验证和临床标本检测均能正确基因分型，实现了快速基因分型的均相检测。IL-1β-31C〉T、IL-1β＋3954C〉T位点和单倍型CTCC和TTCT与中国兰州地区汉族人群CHD易感性有关。

Objective To develop a method of high-resolution melting curve analysis （HRM） for investigating the association of single nucleoUde polymorphisms （SNPs） of IL-1β and IL-1Ra genes with susceptibility to coronary heart disease （CHD） and serological indexes in CHD. Methods PCR-HRM assays were established for detecting four SNPs （ IL-1β-511C 〉 T, IL-1β-31C 〉 T, IL-1β ＋ 3954C 〉 T and IL-1 RNT 〉 C） in 260 patients with CHD and 274 healthy controls. A case-control study was performed for analyzing the relationships of the four SNPs with CHD risk and serological indexes. Results No significant statistical difference was observed between the four SNPs and the susceptibility to CHD, however, the result of gender stratification showed that there was the statistical differences in the male IL-1β-31C 〉 T and the female IL-1β ＋3954C 〉 T allele frequencies （P = 0. 039, 0. 032, respectively）. The result of haplotype analysis of the four SNPs showed that haplotype of IL-1β-5 It T/IL-1β-31 C IL-1β ＋ 3954 C/IL-I RN T （ TCCT ） had a significant correlation with obviously increased CHD risk, but without statistical difference （ OR = 1. 217, 95% CI： 0.950 - 1. 559, P = 0.123）, while those of CTCC and TTCT had significant correlations with decreased CHD risk （ OR = 0.612, 95% CI： 0.376 - 0.994, P = 0.046; OR = 0.365, 95%C/： 0.154-0. 862, P=0.017）. No association was found between the four SNPs and the serological indexes in CHD subjects. Conclusion The developed PCR-HRM detection assays based on HRM technology has been verified by direct sequencing and clinical specimen detection to be a rapid and homogeneous detection method for genotyping of the four SNPs. The SNPs of IL-1B-31C 〉 T, IL-1B ＋ 3954C 〉 T and the haplotypes of CTCC and TTCT are associated with the susceptibility to CHD in Chinese Han population of Lanzhou region.