NIPA2基因突变以及染色体15q11.2微缺失在儿童失神癫痫中的致病机制研究

Childhood absence epilepsy (CAE) is one of the most common idiopathic generalized epilepsies (IGEs). CAE is believed a complex or polygenic genetic disease. To date, there are only a few genes associated with CAE. The pathogenic recurrent copy number variation has been found in idiopathic generalized epilepsies including CAE recently. But the pathogenesis mechanism is still unclear for those genetic variation identified in CAE, including the variations in the susceptic genes and the recurrent pathogenic CNVs. In our previous study, we found three 15q11.2 and one 15q13.3 microdeletions in our 200 patients, not in 200 controls. NIPA2, a highly conserved gene in the region of 15q11.2 microdeletion we found, have been identified two missence mutaitons and one small deletion in our CAE patients without 15q11.2 microdeletions, which not found in 700 controls. Therefore we first identified that NIPA2 locaded in 15q11.2, encoding a selective magnesium transporter, is a susceptible gene of CAE. Therefore, in this study, we will further investigate the pathogenesis of the NIPA2 mutations and 15q11.2 microdeletions found in CAE using the primary culture of rat embryo cortical neuron transfected with wild type and mutant genes by modified HSV virus, as well as with the shRNA for NIPA2 and othe susceptic genes located in the 15q11.2. We will also foung whether there is any novel mutation or functional SNP in another allele of all four highly conserved genes expressed in the brain which locate in 15q11.2 in the patients with 15q11.2 microdeletion. If we can confirm the pathogenesis of genetic variations in NIPA2, we will hopefully indicate the new mechanism of CAE, functional abnormalities of magnesium transporter. We may also clarify the different roles for different genes located in 15q11.2 microdeletion, and also the mutation and/or functional SNP will play the role in the difference of the phenotype with 15q11.2 microdeletion. These results will help us to understand the pathogenesis of CAE, even IGEs more deeply, and may give us a clue of the new target for developing the new anti-epileptic strategy.

儿童失神癫痫（childhood absence epilepsy，CAE）是特发性全面性癫痫的主要类型，遗传模式复杂，可能存在多个致病/易感基因，迄今仅在很少部分病人中发现数个相关基因。近年国际上新报道了数个CAE相关联的致病性基因组片段拷贝数变异（CNV），包括15q11.2, 15q13.3等（发生率约1%），但对于这些遗传变异导致CAE的机制尚不清楚。我们前期研究发现中国CAE病人中也存在15q11.2微缺失（1.5%），此区域内的NIPA2基因（编码一种高选择性Mg2+转运体）在其他CAE病人中存在点突变和小缺失。本课题拟构建野生型及突变体病毒载体转染神经元模型，结合膜片钳、Confocal等技术研究NIPA2基因突变及15q11.2微缺失的致病机制，以进一步证实NIPA2基因是CAE的新致病/易感基因，从而首次确定特发性癫痫新致病机制-镁离子转运异常，并初步探索CNV致病机制。

儿童失神癫痫（childhood absence epilepsy，CAE）是遗传性全面性癫痫的主要类型，遗传模式复杂，存在多个致病/易感基因(如NIPA2基因)。本课题组通过对儿童失神易感基因的筛查及基因突变功能研究，结果发现：1、儿童失神癫痫基因NIPA2的突变可导致NIPA2蛋白不能正常分布在细胞膜上。2、NIPA2基因突变可导致细胞内镁离子浓度的降低。3、 NIPA2基因突变对神经存活率无显著影响。4、 基于NIPA2基因突变对镁离子浓度的影响，本课题组在CA3区海马神经元的生物模拟电脑模型上发现，细胞内低镁（0.1 mM）只是轻度减少了BK通道的激活，并没有显著地改变输入阻抗和神经元的放电频率，在NMDA受体生物模拟电脑模型上，细胞内低镁情况下NMDA受体通道电流增强，突触后电位幅度增加；基于大鼠脑片膜片钳实验，结果发现丘脑、皮层以及海马的神经元在细胞内低镁的情况下都能产生显著的fEPSPs，在应用CPP（NMDA受体阻断剂）后，fEPSPs的幅度明显降低。5、NIPA2-mRNA的表达观察：生后1天至生后8天，NIPA2-mRNA表达量是逐渐降低的，从生后第8天开始NIPA2-mRNA表达量呈逐渐升高的趋势。6、NIPA2蛋白的总蛋白及膜蛋白表达量的观察：转染NIPA2突变体的大鼠皮层神经元细胞的NIPA2总蛋白表达量与转染野生型质粒的细胞相比无明显差异；转染突变体的神经元细胞中NIPA2膜蛋白的表达量低于转染野生型的细胞。7、发现NIPA2蛋白作为细胞膜上的镁离子转运体，可能是通过功能性二聚体结构发挥作用。8、应用二代测序技术检测了100例儿童失神癫痫患者DNA标本，结果发现1例SCN1A基因de novo杂合突变，1例 CLCN2基因de novo杂合突变，1例SCN8A基因de novo杂合突变。9、构建成功了NIPA2基因敲除小鼠模型，并发现通过行为学实验证实NIPA2基因敲除小鼠产生癫痫样发作的阈值较野生型小鼠的显著下降。通过本项目的研究，本课题组初步揭示了NIPA2基因突变所致的功能改变及可能的导致癫痫的生物学机制，并发现如SCN1A基因等新的儿童失神癫痫易感基因，为以后的儿童失神癫痫易感基因研究提供线索和理论依据。