Abstracts

Rationale: SCN2A encodes for the Nav1.2 channel, located on chromosome 2q24.3. Nav1.2 is one of the most relevant neuronal sodium channels and it is expressed in excitatory and glutamatergic neurons [Menezes et al, 2020], with a fundamental role in the initiation and conduction of action potentials [Meisler et al, 2021]. Haploinsufficiency in the SCN2A gene is involved in a wide range of epileptic phenotypes [Oyrer et al, 2018]. Pathogenic SCN2A variants are generally de novo germline mutations and no cases of mosaicism have been described, except for a case where paternal germline mosaicism resulted in developmental and epileptic encephalopathy (DEE) in half siblings [Zerem et al, 2014].

Methods: Two patients with seizure onset within the first three days of life and resistant to multiple anti-seizure medications (including sodium channel blockers) were subjected to next-generation sequencing (NGS) from blood. The analysis identified a SCN2A: c.4976C >T; p. Ala1659Val with a low-level mosaicism of 15%, confirmed on both patients’ fibroblasts. The p.Ala1659Val variant was inserted in a stabilized SCN2A plasmid by site-directed mutagenesis. Expression and functional characterization of the p.Ala1659Val variant were performed in Hek293 cells by western blotting, confocal microscopy, and patch clamp. Patients’ fibroblasts were cultured to reprogram them into induced pluripotent stem cells (iPSCs).

Results: No effect of the p.Ala1659Val variant on channel expression was noted, whilst a reduction of the peak current and a shift of half activation voltage towards more negative values was observed thus confirming that the selected variant is detrimental to the brain’s electrophysiological function. Then to get iPSC clones derived from patients, fibroblasts were transduced with Sendai Virus to induce pluripotency. Around 50 iPSC clones were obtained. Each clone was characterized to discern between wild-type (WT) and heterozygous ones. The best clones, both WT and mutated, will be differentiated into glutamatergic neurons through neurogenin protocol to test them on MicroElectrode Assay (MEA). Mutated and isogenic neurons will be plated alone and with different grades of mosaicism on MEA and will serve to investigate the impact of sodium channel mosaicisms on brain function.

Conclusions: This study provides valuable insights into the SCN2A p.Ala1659Val variant's functional consequences, highlighting the utility of heterologous cell and iPSC-derived neuronal models in understanding complex neurological disorders.

Funding: This work was supported by Bando Fondazione Epilessia LICE 2023 attributed to the presenting Author