Table_1_GRIN2A Variants Associated With Idiopathic Generalized Epilepsies.docx

Objective: The objective of this study is to explore the role of GRIN2A gene in idiopathic generalized epilepsies and the potential underlying mechanism for phenotypic variation. Methods: Whole-exome sequencing was performed in a cohort of 88 patients with idiopathic generalized epilepsies. Electro-physiological alterations of the recombinant N-methyl-D-aspartate receptors (NMDARs) containing GluN2A mutants were examined using two-electrode voltage-clamp recordings. The alterations of protein expression were detected by immunofluorescence staining and biotinylation. Previous studies reported that epilepsy related GRIN2A missense mutations were reviewed. The correlation among phenotypes, functional alterations, and molecular locations was analyzed. Results: Three novel heterozygous missense GRIN2A mutations (c.1770A > C/p.K590N, c.2636A > G/p.K879R, and c.3199C > T/p.R1067W) were identified in three unrelated cases. Electrophysiological analysis demonstrated R1067W significantly increased the current density of GluN1/GluN2A NMDARs. Immunofluorescence staining indicated GluN2A mutants had abundant distribution in the membrane and cytoplasm. Western blotting showed the ratios of surface and total expression of the three GluN2A-mutants were significantly increased comparing to the wild type. Further analysis on the reported missense mutations demonstrated that mutations with severe gain-of-function were associated with epileptic encephalopathy, while mutations with mild gain of function were associated with mild phenotypes, suggesting a quantitative correlation between gain-of-function and phenotypic severity. The mutations located around transmembrane domains were more frequently associated with severe phenotypes and absence seizure-related mutations were mostly located in carboxyl-terminal domain, suggesting molecular sub-regional effects. Significance: This study revealed GRIN2A gene was potentially a candidate pathogenic gene of idiopathic generalized epilepsies. The functional quantitative correlation and the molecular sub-regional implication of mutations helped in explaining the relatively mild clinical phenotypes and incomplete penetrance associated with GRIN2A variants.研究目的：本研究旨在探讨GRIN2A基因在特发性全面性癫痫（idiopathic generalized epilepsies）中的作用，以及表型变异的潜在分子机制。 研究方法：本研究对88例特发性全面性癫痫患者队列开展全外显子测序（whole-exome sequencing）。采用双电极电压钳记录（two-electrode voltage-clamp recordings），对携带GluN2A突变体的重组N-甲基-D-天冬氨酸受体（N-methyl-D-aspartate receptors, NMDARs）的电生理改变进行检测。通过免疫荧光染色（immunofluorescence staining）与生物素化（biotinylation）实验检测蛋白表达变化。回顾既往已报道的癫痫相关GRIN2A错义突变（missense mutations），并分析表型、功能改变与分子定位三者间的相关性。 研究结果：本研究在3例无关患者中鉴定出3种新型杂合错义GRIN2A突变：c.1770A > C/p.K590N、c.2636A > G/p.K879R及c.3199C > T/p.R1067W。电生理分析显示，R1067W突变可显著升高GluN1/GluN2A型NMDAR的电流密度（current density）。免疫荧光染色结果表明，GluN2A突变体在细胞膜与细胞质中均有丰富分布。蛋白质印迹实验显示，与野生型（wild type）相比，3种GluN2A突变体的膜表面与总蛋白表达比值均显著升高。对已报道的错义突变的进一步分析表明，携带严重功能获得性突变的患者易罹患癫痫性脑病（epileptic encephalopathy），而携带轻度功能获得性突变的患者则表现为轻型表型，提示功能获得性效应与表型严重程度之间存在定量相关性。位于跨膜结构域（transmembrane domains）附近的突变更易引发严重表型，而与失神发作相关的突变大多位于羧基末端结构域（carboxyl-terminal domain），这提示突变的分子亚区域效应。 研究意义：本研究证实GRIN2A基因可作为特发性全面性癫痫的潜在致病候选基因。突变的功能定量相关性及分子亚区域效应，有助于解释GRIN2A变异相关的相对轻微的临床表型与不完全外显率（incomplete penetrance）。