逆转录-PCR-变性梯度凝胶电泳法检测葡萄糖-6-磷酸脱氢酶基因缺陷女性携带者

Genetic diagnosis for female carriers of glucose-6-phosphate dehydrogenase deficiency by RT-PCR-DGGE

目的探讨应用逆转录-PCR-变性梯度凝胶电泳法(RT-PCR-DGGE)对葡萄糖-6-磷酸脱氢酶(G6PD)基因缺陷女性携带者人群进行分子诊断的可行性。方法对54例G6PD基因缺陷可疑携带女性的血液标本,应用外周血提取总RNA并逆转录成cDNA;针对已报道的中国人群17种突变位点,以cDNA为模版,分段设计6对DGGE引物,PCR扩增、DGGE筛查分型,必要时进行测序验证。结果54例可疑标本共检出1例1024C/T、20例1376G/T、12例1388G/A,共检出杂合突变标本33例,总检出率为61.1%。结论RT-PCR-DGGE对G6PD基因缺陷女性携带者人群具有较高的检出率,具有临床诊断意义。

Objective To study the feasibility of genetic diagnosis for female carriers of human glucose-6-phosphate dehydrogenase（G6PD）deficiency by reverse transcriptase-PCR-denaturing gradient gel electrophoresis（RT-PCR-DGGE）.Methods Blood samples were collected from suspected 54 female carriers of G6PD deficiency.Total RNAs of peripheral blood were prepared and reverse-transcripted into cDNA.Design of 6 primer pairs for DGGE was based on 17 mutation sites of G6PD cDNA described in the Chinese population.Mutations in the coding region of G6PD gene were screened and genotyped by combination of PCR-DGGE and DNA sequencing.Results One case of 1024C/T,20 cases of 1376G/T and 12 cases of 1388G/A were detected in the 54 samples.The total detection rate was 66.1%（33/54）.Conclusions Heterozygous mutation rate in female carriers of G6PD deficiency detected by RT-PCR-DGGE is high.RT-PCR-DGGE is value of clinical diagnosis for G6PD-deficiency female carriers.