Rapid classification of a novel ALS-causing I149S variant in superoxide dismutase-1.

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 SOD1^I149S 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 SOD1^I149S. Furthermore, SOD1^I149S 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 SOD1^I149S was monomeric in solution in comparison to the dimeric SOD1^WT. 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.