Ⅰ型电压依赖型钠通道基因突变嵌合体的检测

Detection of mosaic mutation in SCN1A

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。

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.