Mutations in SLC6A1, encoding γ-aminobutyric acid (GABA) transporter 1 (GAT-1), have been recently associated with a spectrum of epilepsy syndromes, intellectual disability and autism in clinic. However, the pathophysiology of the gene mutations is far from clear. Here we report a novel SLC6A1 missense mutation in a patient with epilepsy and autism spectrum disorder and characterized the molecular defects of the mutant GAT-1, from transporter protein trafficking to GABA uptake function in heterologous cells and neurons. The heterozygous missense mutation (c1081C to A (P361T)) in SLC6A1 was identified by exome sequencing. We have thoroughly characterized the molecular pathophysiology underlying the clinical phenotypes. We performed EEG recordings and autism diagnostic interview. The patient had neurodevelopmental delay, absence epilepsy, generalized epilepsy, and 2.5-3 Hz generalized spike and slow waves on EEG recordings. The impact of the mutation on GAT-1 function and trafficking was evaluated by 3H GABA uptake, structural simulation with machine learning tools, live cell confocal microscopy and protein expression in mouse neurons and nonneuronal cells. We demonstrated that the GAT-1(P361T) mutation destabilizes the global protein conformation and reduces total protein expression. The mutant transporter protein was localized intracellularly inside the endoplasmic reticulum (ER) with a pattern of expression very similar to the cells treated with tunicamycin, an ER stress inducer. Radioactive 3H-labeled GABA uptake assay indicated the mutation reduced the function of the mutant GAT-1(P361T), to a level that is similar to the cells treated with GAT-1 inhibitors. In summary, this mutation destabilizes the mutant transporter protein, which results in retention of the mutant protein inside cells and reduction of total transporter expression, likely via excessive endoplasmic reticulum associated degradation. This thus likely causes reduced functional transporter number on the cell surface, which then could cause the observed reduced GABA uptake function. Consequently, malfunctioning GABA signaling may cause altered neurodevelopment and neurotransmission, such as enhanced tonic inhibition and altered cell proliferation in vivo. The pathophysiology due to severely impaired GAT-1 function may give rise to a wide spectrum of neurodevelopmental phenotypes including autism and epilepsy.

中文翻译：

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内质网保留和SLC6A1中与癫痫和自闭症相关的突变的降解。

最近，在SLC6A1中的编码γ-氨基丁酸（GABA）转运蛋白1（GAT-1）的突变与一系列癫痫综合征，智力障碍和自闭症有关。但是，基因突变的病理生理学尚不清楚。在这里，我们报告患有癫痫和自闭症谱系障碍患者的新型SLC6A1错义突变，并表征了突变GAT-1的分子缺陷，从转运蛋白运输到异源细胞和神经元中GABA的摄取功能。通过外显子组测序鉴定了SLC6A1中的杂合错义突变（从c1081C到A（P361T））。我们已经彻底表征了临床表型的分子病理生理学。我们进行了脑电图记录和自闭症诊断访谈。患者出现神经发育迟缓，癫痫发作，癫痫发作，EEG记录中出现2.5-3 Hz的尖峰和慢波。通过3 H GABA摄取，机器学习工具的结构模拟，活细胞共聚焦显微镜以及小鼠神经元和非神经元细胞中的蛋白表达，评估了突变对GAT-1功能和运输的影响。我们证明了GAT-1（P361T）突变破坏了全局蛋白构象并降低了总蛋白表达。突变的转运蛋白位于内质网（ER）的细胞内，其表达模式与用ER诱导剂衣霉素处理的细胞非常相似。放射性3H标记的GABA吸收试验表明，该突变使突变型GAT-1（P361T）的功能降低到与用GAT-1抑制剂处理的细胞相似的水平。综上所述，这种突变使突变体转运蛋白不稳定，这可能导致突变蛋白保留在细胞内并降低了总转运蛋白的表达，这可能是由于内质网过度降解所致。因此，这可能导致细胞表面功能转运蛋白数量减少，然后可能导致观察到的GABA吸收功能降低。因此，功能失常的GABA信号传导可能导致神经发育和神经传递改变，例如增强的滋补抑制和体内细胞增殖改变。由于严重损害GAT-1功能而引起的病理生理学可能会引起广泛的神经发育表型，包括自闭症和癫痫病。这可能导致突变蛋白保留在细胞内并降低了总转运蛋白的表达，这可能是由于内质网过度降解引起的。因此，这可能导致细胞表面功能转运蛋白数量减少，然后可能导致观察到的GABA吸收功能降低。因此，功能失常的GABA信号传导可能导致神经发育和神经传递的改变，例如增强的滋补抑制和体内细胞增殖的改变。由于严重损害GAT-1功能而引起的病理生理学可能会引起广泛的神经发育表型，包括自闭症和癫痫病。这可能导致突变蛋白保留在细胞内并降低了总转运蛋白的表达，这可能是由于内质网过度降解引起的。因此，这可能导致细胞表面功能转运蛋白数量减少，然后可能导致观察到的GABA吸收功能降低。因此，功能失常的GABA信号传导可能导致神经发育和神经传递的改变，例如增强的滋补抑制和体内细胞增殖的改变。由于严重损害GAT-1功能而引起的病理生理学可能会引起广泛的神经发育表型，包括自闭症和癫痫病。然后可能导致观察到的GABA吸收功能降低。因此，功能失常的GABA信号传导可能导致神经发育和神经传递的改变，例如增强的滋补抑制和体内细胞增殖的改变。由于严重损害GAT-1功能而引起的病理生理学可能会引起广泛的神经发育表型，包括自闭症和癫痫病。然后可能导致观察到的GABA吸收功能降低。因此，功能失常的GABA信号传导可能导致神经发育和神经传递的改变，例如增强的滋补抑制和体内细胞增殖的改变。由于严重损害GAT-1功能而引起的病理生理学可能会引起广泛的神经发育表型，包括自闭症和癫痫病。