Functional Characterization of a De Novo SCN2A Mixed Variant Linked to Early Infantile Developmental and Epileptic Encephalopathy

Background and Objectives – Pathogenic variants in the SCN2A gene, encoding the α-subunit type 2 of the voltage-gated sodium channel NaV1.2, cause a phenotypic spectrum including 4 major disorders as benign familial infantile seizures, developmental and epileptic encephalopathy, intellectual disability, and autism. Gain-of-function variants resulting phenotypes may be treated with sodium channel blockers, while loss-of-function (LoF) conditions are non-respondent. We focused on the effects of the pathogenic SCN2A variant c.4976C>T (p.A1659V) found in heterozygosity in 3 patients affected by DEE non responsive to SCB. We functionally investigated this previously uncharacterized SCN2A variant.Methods – Three individuals with the SCN2A c.4976C>T (p.A1659V) variant were studied. This variant was detected by next-generation sequencing (NGS). The nucleotide substitution was inserted by site-directed mutagenesis in a stabilized SCN2A plasmid encoding NaV1.2. Expression and functional characterization of the NaV1.2 A1659V variant was performed in HEK293 cells by western blotting, confocal microscopy, and patch clamp electrophysiology.Results – The same de novo pathogenic SCN2A variant was detected in 3 patients with DEE characterized by early onset, severe ID, and seizures unresponsive to SCB. In 2 patients, the variant is in a mosaic state. The NaV1.2 A1659V variant did not affect channel protein expression while exhibiting significant effects on its function as shown by the reduced Na+ currents, a shift of the activation curve toward more negative potentials, a shift of the inactivation curve to more negative voltages, and slower kinetics of inactivation compared with native NaV1.2 in HEK293 cells. Simulations suggested that the variant increases excitability in neurons.Discussion – These results revealed the multifaceted functional effect of A1659V variant on channel activity and highlighted the complex genotype-phenotype correlation underlying significant clinical and pharmacological variability in SCN2A-related encephalopathies.