Ⅰ型电压依赖型钠通道基因突变嵌合体的检测

目的:寻找鉴定Ⅰ型电压依赖型钠通道基因(SCN1A)突变嵌合体的有效方法,降低癫痫伴热性惊厥附加症的再发风险.方法:抽取携带已知突变c.5768A＞ G/p.Q1923R的患儿及无突变的患儿母亲的外周血全基因组DNA,将患儿的DNA与母亲的DNA以1∶0、1∶1、1∶3、1∶7、1∶15、1∶31及0∶1混合,对包含该突变位点的片段进行PCR扩增后,进行变性高效液相色谱(dHPLC)技术分析、常规测序及焦磷酸测序(pyrosequencing).结果:常规测序仅能检测出1∶0及1∶1混合样本的突变,而dHPLC及焦磷酸测序均能检测出低至1∶15混合样本的突变,且后者能对突变进行定量.结论:利用dHPLC结合焦磷酸测序的方法能有效快速地检测出低至3％左右的突变,可以避免绝大多数SCN1A基因嵌合突变的漏检.

Detection of mosaic mutation in SCN1A

Objective Finding a rapid and effective method to detect the mosaic mutations in sodium channel(Nav) α-subunit type 1 gene(SCN1A),then to decrease the recurrent risk of epilepsy with febrile seizures plus(EFS+).Methods Genomic DNA from peripheral blood were extracted in patients with the mutation c.5768A > G /p.Q1923R and their unaffected mothers without the mutation.Then the DNA of the patients and their mothers were mixed by the proportion of 1:0,1:1,1:3,1:7,1;15,1:31,and 0:1.The fragment including the mutation site was amplified by PCR and then detected by denaturing high-performance liquid chromatography(dHPLC),routine sequencing and pyrosequencing.Results The mutation of the mixed samples of the proportion of 1:0 and 1:1 could be detected by routine sequencing,whereas the mutation of the mixed samples of the lowest proportion of 1:15 could be detected by dHPLC and pyrosequencin.And quantitative analysis of mutations could be performed by pyrosequencing.Conclusions The lowest amount of 3% of mutations could be detected by dHPLC combined with pyrosequencing,avoiding the missed detection of the majority of mosaic mutation in SCN1A.