GRIN2A基因突变导致特发性全面性癫痫的机制及对抗癫痫药物反应性影响的研究

N-methyl-D-aspartate receptor (NMDAR) is one of ionotropic glutamate receptors, playing critical roles in excitatory synaptic transmission, plasticity and excitotoxicity in CNS. GRIN2A gene, encoding GluN2A subunit of NMDAR, was proved to be one of the pathogenic genes for idiopathic focal epilepsy and early onset epileptic encephalopathy. Our previous studies identified GRIN2A mutations in the patients with idiopathic generalized epilepsy (IGE), suggesting a pathogenic effect of GRIN2A mutations in IGE. However, the mechanism of the phenotypic variants is unclear to date. It has been demonstrated that GRIN2A mutations caused the functional changes of NMDAR and affected the trafficking of GluN2A through different pathways, which is possible one of factors causing different phenotypes. This project will select the novel GRIN2A mutations identified in IGEs, and analyze the changes of NMDAR function and neuronal excitability using the whole-cell clamp patch. The subcellular localization of GluN2A and the viability of neurons will be analyzed using immunofluorescence and Western blotting. It will reveal the relationship of genotype-phenotype-functional type and the molecular pathogenic mechanism of GRIN2A mutations in IGE. Our previous studies also indicated the patients with IGE had different responses to anti-epileptic drugs (AEDs). The different drug responses after GRIN2A mutations bring difficulties about the choice of AEDs in clinic. We will further explore the changes of NMDAR function and neuron viability using different AEDs, and analyze the influence of GRIN2A mutations on the sensitivity of AEDs, providing the evidence for the precise therapy in the patients with IGE and GRIN2A mutations.

N-甲基-D-天门冬氨酸受体（NMDAR）是神经元兴奋性突触传递的重要调节结构。已发现编码NMDAR的GluN2A亚基的GRIN2A基因突变与特发性局灶性癫痫和早发性癫痫性脑病有关。我们前期研究首次在特发性全面性癫痫（IGE）患者中发现GRIN2A基因突变，且患者对抗癫痫药的敏感性不同，提示GRIN2A突变可导致包括IGE在内的癫痫谱系疾病，突变后NMDAR功能和GluN2A转运的差异可能是表型和疗效不一的基础。本项目将选取IGE患者中新的GRIN2A基因突变，采用膜片钳研究突变后NMDAR功能和神经元兴奋性变化，采用免疫荧光和Western杂交观察GluN2A亚细胞定位，分析基因型-表型-功能型关系，揭示GRIN2A基因突变在IGE中的致病机理；通过观察GRIN2A突变后不同抗癫痫药作用下NMDAR功能变化，探讨突变对抗癫痫药物反应性的影响，为IGE的精准治疗提供新的理论依据。

已结题项目的研究结果为：(1)在313例特发性全面性癫痫（IGE）患者中发现3例携带未见报道的新GRIN2A错义突变：c.1770A>G/p.K590N，c.2636A>G/p.K879R，c.3199C>T/p.R1067W。基因负荷分析证实GRIN2A突变在IGE中的频率显著高于一般人群。晶体结构分析发现其改变了与周围残基的氢键数量。(2)膜片钳电生理分析显示GluN1/GluN2A-R1067W NMDARs电流密度比野生型高31%，GluN1/GluN2A-K590N和GluN1/GluN2A-K879R NMDARs电流密度无显著增加。三个突变体的Mg2+阻滞作用和谷氨酸效能、甘氨酸效能无明显改变。免疫荧光分析发现GluN2A-K879R和GluN2A-R1067W NMDARs表面/总相对密度显著增加。生物素化标记评估发现，GluN2A细胞表面表达显著增加。故GRIN2A突变引起IGE的机理为NMDAR功能增强。(3)基因型-表型-功能型关系研究发现GRIN2A表型轻重与突变的功能型无关，而与功能改变严重程度存在量-效关系：谷氨酸和甘氨酸效能增加5倍以上的突变，引起严重的癫痫性脑病；增加2倍以下的突变引起特发性癫痫。表型的严重程度与突变所在亚分子区域有关：位于GluN2A M2-M4的突变，氨基末端和配体结合区的突变表型轻。(4) 药效学研究发现，丙戊酸和拉莫三嗪可抑制NMDAR功能，纠正GRIN2A突变的功能增强。(5) 在致病基因筛选中，发现了BSN和CHD4为癫痫的新致病基因。. 项目已达到计划要点的研究目标。研究成果发现了GRIN2A的新表型，阐明了其导致IGE的机制和GRIN2A的基因型-表型-功能型的关系，发现了新的癫痫致病基因。对于诠释GRIN2A突变的致病机理、为IGE的精准诊治提供新的理论依据。已完成了人才培养目标，发表论文3篇。

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