焦磷酸测序定量检测γ珠蛋白基因启动子区甲基化

Quantitative Detection of Methylation of γ-globin Gene(HBG) Promoter by Pyrosequencing

目的:采用焦磷酸测序定量检测人体γ珠蛋白基因(HBG)启动子区甲基化。方法:收集30例经基因检测确诊为轻型β-地中海贫血(简称地贫)患者的全血DNA样本,查找γ珠蛋白基因的启动子区Cp G位点,设计PCR扩增引物及焦磷酸测序引物;DNA样本经亚硫酸氢盐转化后进行PCR扩增,对PCR产物作单链处理,在测序引物的引导下进行焦磷酸测序,对Cp G位点的甲基化程度进行定量分析。结果:30例轻型β-地贫患者的HBG启动子区共150个Cp G位点均得到了甲基化程度的定量结果,其中-53、-50、+5、+16及+49位点高甲基化分别有28例、24例、13例、0例及7例,中甲基化分别有2例、6例、17例、30例及23例,未检测到低甲基化。结论:焦磷酸测序可对HBG启动子Cp G位点的甲基化程度进行定量检测。

Objective： To establish quantitative detection of methylation of γ-globin gene（ HBG）promoter by pyrosequencing. Methods： The whole blood DNA samples from 30 patients with mild β-thalassemia diagnosed by gene detection were collected to find the Cp G site in the promoter region of γ-globin gene. PCR amplification primers and pyrosequencing primers were designed. DNA samples were amplified by PCR after transformation by hydrogen sulfite. The PCR products were treated with single strand,followed by pyrosequencing under the guidance of sequencing primers. The methylation degree of Cp G sites was quantitatively analyzed. Results： A total of 150 Cp G loci in HBG promoter region of 30 cases of mild thalassemia patients got quantitative results of methylation level. Among them,there were 28,24,13,0,7 cases of hypermethylation at-53,-50, ＋ 5、＋ 16 and ＋ 49 respectively; There were 2,6,17,30,23 cases of middle methylation at them respectively; No hypomethylation was detected for all of them. Conclusion： Pyrosequencing can be used to quantify the degree of methylation at the Cp G site of HBG promoter.