Djavad Mowafaghian Centre for Brain Health and Department of Medicine, University of British Columbia, Vancouver, Canada.
Department of Neurobiology, Beijing Key Laboratory of Neural Regeneration and Repair, Beijing Laboratory of Brain Disorders (Ministry of Science and Technology), Beijing Institute for Brain Disorders, Capital Medical University, Beijing, China.
Mol Brain. 2019 Nov 10;12(1):92. doi: 10.1186/s13041-019-0513-9.
We report the identification of a de novo GABRA1 (R214C) variant in a child with epileptic encephalopathy (EE), describe its functional characterization and pathophysiology, and evaluate its potential therapeutic options. The GABRA1 (R214C) variant was identified using whole exome sequencing, and the pathogenic effect of this mutation was investigated by comparing wild-type (WT) α1 and R214C α1 GABA receptor-expressing HEK cells. GABA-evoked currents in these cells were recorded using whole-cell, outside-out macro-patch and cell-attached single-channel patch-clamp recordings. Changes to surface and total protein expression levels of WT α1 and R214C α1 were quantified using surface biotinylation assay and western blotting, respectively. Finally, potential therapeutic options were explored by determining the effects of modulators, including diazepam, insulin, and verapamil, on channel gating and receptor trafficking of WT and R214C GABA receptors. We found that the GABRA1 (R214C) variant decreased whole-cell GABA-evoked currents by reducing single channel open time and both surface and total GABA receptor expression levels. The GABA-evoked currents in R214C GABA receptors could only be partially restored with benzodiazepine (diazepam) and insulin. However, verapamil treatment for 24 h fully restored the function of R214C mutant receptors, primarily by increasing channel open time. We conclude that the GABRA1 (R214C) variant reduces channel activity and surface expression of mutant receptors, thereby contributing to the pathogenesis of genetic EE. The functional restoration by verapamil suggests that it is a potentially new therapeutic option for patients with the R214C variant and highlights the value of precision medicine in the treatment of genetic EEs.
我们报告了一例新发病例的 GABRA1（R214C）变异，该变异存在于患有癫痫性脑病（EE）的儿童中。我们对该变异进行了功能特征分析和病理生理学研究，并评估了其潜在的治疗选择。使用全外显子组测序发现了 GABRA1（R214C）变异，通过比较野生型（WT）α1 和 R214Cα1GABA 受体表达的 HEK 细胞，研究了该突变的致病性。使用全细胞、外部宏观贴片和细胞附着单通道贴片记录法记录这些细胞中的 GABA 诱发电流。使用表面生物素化测定法和 Western 印迹法分别定量 WTα1 和 R214Cα1 的表面和总蛋白表达水平的变化。最后，通过确定调节剂（包括地西泮、胰岛素和维拉帕米）对 WT 和 R214C GABA 受体的通道门控和受体转运的影响，探索了潜在的治疗选择。我们发现，GABRA1（R214C）变异通过减少单通道开放时间以及 WT 和 R214C GABA 受体的表面和总表达水平，降低了全细胞 GABA 诱发电流。R214C GABA 受体的 GABA 诱发电流只能通过苯二氮䓬（地西泮）和胰岛素部分恢复。然而，维拉帕米治疗 24 小时可完全恢复 R214C 突变受体的功能，主要是通过增加通道开放时间。我们得出结论，GABRA1（R214C）变异降低了通道活性和突变受体的表面表达，从而导致遗传 EE 的发病机制。维拉帕米的功能恢复表明，它是 R214C 变异患者潜在的新治疗选择，并强调了精准医学在治疗遗传 EE 中的价值。
