应用高分辨熔解曲线分析法检测遗传性球形红细胞增多症患者SLC4A1基因突变

Application of High Resolution Melting Curve Analysis in Detection of SLC4A1 Gene Mutation in Patients with Hereditary Spherocytosis

目的:探讨高分辨熔解(HRM)曲线分析法检测遗传性球形红细胞增多症(HS)患者SLC4A1基因D38A和K56E突变位点的可行性及其意义。方法:抽取23例HS患者外周血样本进行常规检测,提取基因组DNA,针对SLC4A1基因突变位点D38A和K56E设计引物,应用HRM法对所有样本进行突变分析,并通过DNA测序技术对HRM结果进行验证。结果:23例HS患者中,HRM法共检测出杂合突变型基因6例,其中包括D38A突变3例,K56E突变3例。DNA测序结果与HRM法检测结果一致。结论:应用HRM法能准确检测SLC4A1基因D38A和K56E突变位点,具有高效、快速、可靠等优点,不仅可用于临床辅助诊断,还有望成为确定HS患者基因突变谱的新方法。

Objective: To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS).Methods:Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique.Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed.The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology.Results:Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing.Conclusion: The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient,fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.