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

婴儿重症肌阵挛性癫痫（SMEI）是一种以热性惊厥为主要表现的恶性婴儿癫痫综合征。我们分析了SMEI患者和其他类型癫痫患者外周血细胞DNA中电压门控钠通道α1亚基（SCN 1A）、β1亚基（SCN 1B）和γ-氨基丁酸_A受体γ2亚基（GABRG 2）基因。在83%的SMEI患者中检测到SCN 1A基因突变，但其他类型的癫痫患者均未检测到。突变包括缺失、插入、错义和无义突变。我们在所有患者中均未发现任何SCN 1B和GABRG 2基因突变。我们克隆了野生型SCN 1A的cDNA，通过PCR突变法制备了突变型cDNA，并构建了在C端带有Lumio标签的SCN 1A cDNA表达质粒。将表达质粒转染人胚肾293细胞后，发现有几种突变形式定位于细胞膜上。结果表明，SCN 1A的突变体可能在细胞膜上存在野生型SCN 1A的情况下发挥显性负调控作用。我们的分析表明，这些异构体丢失了离子通道的门控区，并且我们检测到的许多突变发生在SCN 1A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负作用，像一种突变形式。

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.