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

Background and ObjectivesPathogenic variants in the SCN2A gene, encoding the alpha-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. MethodsThree 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. ResultsThe 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. DiscussionThese 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.