SCN1A基因内含子区突变对基因表达及钠通道功能和药物反应的影响及其临床相关性研究

SCN1A gene encode voltage-gated sodium channel type I (Nav1.1), which plays a critical role in brain functional activity and served as a target of some anti-epileptic drugs (AEDs). SCN1A is now the most relevant epilepsy gene. Epilepsy related SCN1A mutations can occur in intron with non-protein coding. Most of them were mutations in canonical splice junction, i.e. splice site mutation, which closely related with gene splicing and predominantly associated with severe phenotypes. However, we previously detected seven SCN1A intronic mutations in patients with epilepsy, in which not only splice site mutation, but also mutations located in variant splice motifs as well as in deep intronic area were indentified. It’s noted that the severity of phenotypes in these patients were various, and AEDs responses were obvious difference in them, even seizure aggravation by AEDs. The underlined mechanism is unknown and warrants further studies, due to the functional complexity of intron and unclear process in gene spicing. It is suspected whether mutations on the different site of SCN1A intron may result in different consequences of SCN1A splicing and severe or mild functional changes of Nav1.1 and are associated phenotype severity and AEDs response. In present study, recombinant expression vector and mutant plasmids will be constructed. The functional alterations of these mutants will be examined by using expressions of heterogonous cells in vitro, including detection of the consequent of SCN1A gene splicing through Minigene splicing assay and Nav1.1 protein expressed in cell membrane, as well as examination of electrophysiological properties of sodium channel by the whole cell patch clamp technique. Subsequently, different types of AEDs with different dosage will be administrated to each mutant to observe their electrophysiological properties changes of sodium channel. Comprehensive results of in vitro experiments will be analyzed to explore the relationship among genotypes, phenotypes, functional types as well as AEDs responses. This study will help understand the mechanism of epilepsy caused by SCN1A intronic mutation and disclose the effects of functional changes on the sodium channel function and AEDs reaction.

SCN1A基因编码I 型电压依赖型钠通道(Nav1.1)，Nav1.1临床上与癫痫关系密切，且是多种抗癫痫药物(AEDs)作用的靶点。已知癫痫有关的SCN1A突变可发生在非编码蛋白的内含子，以与基因剪切关系密切的固有剪切位点突变多见，多与严重表型相关。我们此前在癫痫患者中筛查到7个SCN1A内含子突变，除固有剪切位点外，还发现可变剪切位点，甚至深部内含子突变。这些突变的表型轻重不一，AEDs反应差异明显，存在AEDs加重发作。因内含子功能复杂，基因剪切机制未明，其潜在机制不清。因此，我们进行对照设计，通过质粒异源细胞体外表达，研究不同内含子突变对基因剪切、Nav1.1蛋白表达、亚分子定位和钠通道电生理的影响；给予不同类型和浓度AEDs处理，观察通道蛋白表达和电生理改变；综合数据，探讨其与临床的可能关系。结果将对揭示SCN1A内含子突变的致痫机制，对钠通道功能和AEDs疗效的影响有重要意义。

SCN1A基因编码Nav1.1钠通道，临床上与癫痫关系密切，是多种抗癫痫药物(AEDs)作用的靶点。已知癫痫有关的SCN1A突变可发生在非编码蛋白的内含子的不同区域，且表型轻重不一，AEDs反应差异明显，但其潜在机制不清。本研究经对照设计和构建重组质粒并异源细胞体外表达，minigene体外剪切分析、免疫蛋白印记、激光共聚焦和细胞膜片钳和不同类型AEDs干预等方法，研究SCN1A不同位置的内含子突变对mRNA剪切、Nav1.1蛋白表达、亚分子定位和钠通道电生理特性的影响；进一步探讨其基因型-功能型-表型-药物反应的可能关系。结果显示：1. 筛查出的6个SCN1A内含子突变，其中局灶性癫痫伴热性惊厥附加症（PEFS+）3个，位于可变剪切位点和深部内含子区， Dravet综合征（DS）2个，均位于固有剪切位点。不同区域突变引起外显子部分或完全丢失、内含子插入等不同形式的mRNA异常剪切，导致不同的Nav1.1蛋白截短；而突变导致正常mRNA表达量的改变与表型轻重呈正相关。2. 根据mRNA异常剪切结果重组构建的所有SCN1A突变体皆出现了相应的蛋白截短且都可在胞浆及胞膜上表达；突变体的Nav1.1蛋白在细胞膜的表达水平较野生型明显减少；PEFS+相关突变体与DS的相比，其截短的Nav1.1蛋白在细胞膜上的表达水平明显减少（p<0.05）。激光共聚焦可见突变体Nav1.1蛋白的细胞膜荧光表达较野生型减少（P<0.05）；PEFS+相关突变体荧光表达明显低于DS（P=0.002）。 3.各突变体在钠通道电流密度、电压依赖性激活、快/慢速失活、快/慢失活后恢复等电生理特性上均有不同程度的改变，PEFS+相关突变体的钠通道表现为功能丧失或功能增强，DS相关突变体则表现为部分或完全功能丧失。4.广谱AEDs丙戊酸钠、托吡酯，以及钠通道阻滞作用的卡马西平和拉莫三嗪干预后，各突变体的Nav1.1膜蛋白表达改变各异。本研究明确了SCN1A内含子突变的致病性，显示不同位置内含子突变对正常mRNA剪切形式及其表达量，以及Nav1.1蛋白表达、亚细胞定位和电生理功能均存在影响，且与临床表型严重程度相关，为阐明SCN1A内含子突变的致病性，及其基因型-表型和药物反应差异的潜在分子机制提供了有力证据。