葡萄糖-6-磷酸脱氢酶缺乏症基因诊断方法的建立  

A new method of genetic diagnosis for glucose-6-phosphate dehydrogenase deficiency

作　　者：吴彤华 朱元昌 陈春媚 蔡靖 林奇 张晓庆 曾勇 尹彪 

机构地区：[1]深圳中山泌尿外科医院生殖医学中心,深圳中山生殖与遗传研究所,深圳市围着床期生殖免疫重点实验室,广东深圳518045

出　　处：《临床检验杂志》2014年第3期188-192,共5页Chinese Journal of Clinical Laboratory Science

基　　金：深圳市科技计划项目(201202200)

摘　　要：目的建立有效可行的高通量葡萄糖．6一磷酸脱氢酶（glucose-6-phosphatedehydrogenase，G6PD）基因诊断新技术。方法建立单管三重PCR扩增G6PD基因外显子2—13目的片段，构建双管25重SNaPshot反应体系检测25个已报道的中国人群的G6PD基因突变位点。用20份已知基因型的样品进行重复性试验。对60例未知样品同时用多重SNaPshot基因分型技术和DNA测序法进行双盲试验。结果建立的多重SNaPshot技术可同时检测25个G6PD基因突变位点，且可区分正常、女性杂合子与男性半合子／女性纯合子，有效检出复合突变。批内及批间的重复性均达到100％。60例未知样本检出23例突变，SNaPshot基因分型结果与测序结果完全一致，特异性和准确性均为100％。结论建立的单管三重PCR结合双管25重SNaP—shot技术是一种有效可行的G6PD缺乏症基因诊断的新方法。Objective To establish an effective and feasible high-throughput assay for genetic diagnosis of glucose-6-phosphate dehy- drogenase （G6PD）. Methods A single-tube triplex PCR was performed to amplify the target fragments of exon 2 to 13 of G6PD gene, and two-tube 25 plex SNaPshot system was established to detect 25 mutation sites in G6PD gene that have been reported in Chinese population. Repeatability was tested in 20 samples with known genotype. A double-blind trial for 60 unknown samples was performed by using multiplex SNaPshot assay and DNA sequencing simultaneously. Results The established multiplex SNaPshot genotyping as- say was able to detect the 25 mutation sites of G6PD gene simultaneously and may definitely distinguish the genotype of C,6PD gene in- cluding normality, heterozygotes in female and hemizygotes in male/homozygotes in female clearly. It was also valid to detect compound mutations. The repeatabilities of both intra-assay and inter-assay of the method were 100 percent. Among the 60 unknown samples 23 mutational cases were found out. All the genotyping results of SNaPshot assay were in complete concordance with the direct DNA se- quencing, and both specificity and accuracy reached 100 percent. Conclusion The established one-tube triplex PCR combined with two-tube 25plex SNaPshot may be a new feasible and effective assay for genetic diagnosis of G6PD deficiency.

关 键 词：葡萄糖-6-磷酸脱氢酶 基因突变 单管多重聚合酶链反应 多重SNaPshot技术 基因诊断 

分 类 号：R394.3[医药卫生—医学遗传学]