Functional Analysis of the mutated ion-channel gene in severe myoclonic epilepsy in infancy

婴儿期严重肌阵挛性癫痫（SMEI）是一种伴有热性癫痫发作的恶性婴儿期癫痫综合征。我们分析了SMEI患者和其他类型癫痫患者外周血dna中电压门控钠通道α1-亚基（SCN1A）基因、β1-亚基（SCN1B）基因和γ-氨基丁酸_A受体γ2-亚基（GABRG2）基因。在83%的SMEI患者中检测到SCN1A基因突变，尽管没有其他类型的癫痫患者。突变包括缺失、插入、错义和无义突变。我们在所有患者中未发现任何SCN1B和GABRG2基因突变。我们的数据表明，SCN1A突变与SME显著相关（p<0.0001）。克隆野生型SCN1A cDNA，通过pcr诱变制备突变型cDNA，构建c端带lumio标签的SCN1A cDNA表达质粒。将表达质粒转染人胚胎肾293细胞，发现部分突变形式定位在细胞膜上。结果表明，当野生型SCN1A存在于细胞膜上时，突变型SCN1A可能发挥显性负性作用。当我们克隆表达系统的cDNA时，我们在人脑组织中发现了两种不同的SCN1A mRNA亚型。我们的分析显示，这些同工型丢失了离子通道的门区，并且我们检测到的许多突变发生在SCN1A区域。这些结果表明，替代异构体也可能对野生型钠离子通道起负作用，就像一种突变形式一样。

Severe myoclonic epilepsy in infancy(SMEI) is a malignant infant-onset epileptic syndrome with febrile seizures. We analyzed the voltage-gated sodium channel α1-subunit (SCN1A) gene, β1-subunit (SCN1B) gene and γ-aminobutyric acid _A receptor γ2-subunit (GABRG2) gene in DNAs from peripheral blood cells of patients with SMEI and patients with other types of epilepsy. Mutations of the SCN1A gene were detected in 83% of the patients with SMEI, although none with other types of epilepsy. The mutations included deletion, insertion, missense and nonsense mutations. We could not find any mutations of the SCN1B and GABRG2 genes in all patients. Our data suggested that the SCN1A mutations were significantly correlated with SME (p<0.0001).We cloned the wild type SCN1A cDNA, made the mutant type cDNAs by PCR-based mutagenesis, and constructed the SCN1A cDNA expression plasmids with Lumio-tag at the C-terminal region. When the expression plasmids were transfected into human embryonic kidney 293 cells, we found that some kinds of mutant forms are localized on cell membrane. The result suggests that the mutant forms of SCN1A may function dominant-negatively in the presence of the wild type SCN1A on the cell membrane.We found two alternative isoforms of SCN1A mRNA in human brain tissues, when we were cloning the cDNA for the expression system. Our analyses revealed that the isoforms loss the gate region of ion-channel, and that many mutations we detected had occurred in the region of SCN1A. These results suggest that the alternative isoforms also may negatively function for wild type of sodium ion-channel, like as a kind of mutant form.