Abstract：
Variants of the oxygen free radical scavenging enzyme superoxide dismutase-1 (SOD1) are associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These variants occur in roughly 20% of familial ALS cases, and 1% of sporadic ALS cases. Here, we identified a novel SOD1 variant in a patient in their 50s who presented with movement deficiencies and neuropsychiatric features. The variant was heterozygous and resulted in the isoleucine at position 149 being substituted with a serine (I149S). In silico analysis predicted the variant to be destabilizing to the SOD1 protein structure. Expression of the SOD1I149S variant with a C-terminal EGFP tag in neuronal-like NSC-34 cells resulted in extensive inclusion formation and reduced cell viability. Immunoblotting revealed that the intramolecular disulphide between Cys57 and Cys146 was fully reduced for SOD1I149S. Furthermore, SOD1I149S was highly susceptible to proteolytic digestion, suggesting a large degree of instability to the protein fold. Finally, fluorescence correlation spectroscopy and native-PAGE of cell lysates showed that SOD1I149S was monomeric in solution in comparison to the dimeric SOD1WT. This experimental data was obtained within 3 months and resulted in the rapid re-classification of the variant from a variant of unknown significance (VUS) to a clinically actionable likely pathogenic variant.
摘要：
氧自由基清除酶超氧化物歧化酶-1（SOD1）的变体与神经退行性疾病肌萎缩侧索硬化症（ALS）有关。这些变异发生在大约20%的家族性ALS病例中，和1％的散发性ALS病例。这里,我们在一名50多岁的患者中发现了一个新的SOD1变异体,该患者表现为运动缺陷和神经精神特征.该变体是杂合的并且导致位置149处的异亮氨酸被丝氨酸(I149S)取代。计算机模拟分析预测变体对SOD1蛋白结构不稳定。具有C末端EGFP标签的SOD1I149S变体在神经元样NSC-34细胞中的表达导致广泛的包涵体形成和降低的细胞活力。免疫印迹显示，对于SOD1I149S，Cys57和Cys146之间的分子内二硫化物完全减少。此外，SOD1I149S对蛋白水解消化高度敏感，表明蛋白质折叠存在很大程度的不稳定性。最后,荧光相关光谱和细胞裂解物的天然PAGE显示，与二聚体SOD1WT相比，SOD1I149S在溶液中是单体。该实验数据是在3个月内获得的，并导致将变异体从未知显著性变异体（VUS）快速重新分类为临床可行的可能致病变异体。
