Abstracts

This abstract has been invited to present during the Basic Science Poster Highlights poster session

Rationale: Voltage-gated sodium channel (VGSC) b1 subunits play critical roles in regulating neuronal excitability. b1 subunits are multifunctional. They modulate sodium and potassium currents, function as cell adhesion molecules, and regulate gene transcription following regulated intramembrane proteolysis. Biallelic variants in SCN1B have been reported in patients with early infantile developmental and epileptic encephalopathy (DEE), with clinical features overlapping Dravet syndrome (DS). Our goal is to understand whether SCN1B DEE patient variants are functionally null, or whether some SCN1B functions remain. A recessive variant, SCN1B-R89C, was found to be homozygous in 2 children of a non-consanguineous family. One child mirrored aspects of DS whereas the other exhibited a milder phenotype. We also identified an unrelated patient homozygous for SCN1B-R89C with a clinically more severe phenotype than DS. Variable penetrance for SCN1B-R89C homozygosity in patients suggests interplay with other genes, complicating our ability to understand SCN1B variant-specific effects in patient-derived neurons. Here, we used CRISPR/Cas9 gene editing to knock-in SCN1B-p.R89C to the mouse Scn1b locus. We then back-crossed to the C57Bl/6J background to allow comparisons between Scn1b^R/R, Scn1b^R/C, and Scn1b^CC littermates with Scn1b^-/- and Scn1b^+/+ mice to determine whether this variant was loss- or gain-of-function (LOF or GOF).

Methods: Brains were collected from P15-16 mice to assess WT vs. mutant b1 protein levels by western blot. A cohort of mice was tested for thermal seizure susceptibility at postnatal day (P) 15. We also used a controlled hyperthermia protocol to induce a febrile seizure in Scn1b^CC pups at P15 and then assess the development of spontaneous seizures compared to uninduced pups using continuous infrared video recording.  A subset of mice was implanted with EEG electrodes for recording from P60-P90. Seizures and interictal background were assessed manually by an experienced reader.

Results: Scn1b^R/R mice had normal body weights and a SUDEP rate of approximately 20%, suggesting that the SCN1B-p.R89C variant does not result in complete Scn1b LOF. b1 subunit protein expression levels were comparable in Scn1b^R/R, Scn1b^R/C, and Scn1b^CC brains. However, the complexity of b1 protein glycosylation differed between Scn1b^R/R and Scn1b^CC homogenates. Scn1b^CC mice were more susceptible to hyperthermia-induced seizures at P15 compared to Scn1b^R/R littermates. Seizure onset, frequency, and duration were similar in Scn1b^CC mice that had been induced with an early febrile seizure vs. those that did not undergo hyperthermia. Video EEG recording detected epileptic discharges in Scn1b^CC, but not in Scn1b^R/R, Scn1b^R/C mice, that coincided with convulsive seizures and myoclonic jerks. The seizure pattern in Scn1b^CC mice was diurnal, occurring with higher frequency during the dark cycle.

Conclusions: This new mouse model suggests that the SCN1B-p.R89C variant does not result in complete SCN1B LOF. Scn1b^CC mice may more accurately model human SCN1B-linked DEE than Scn1b^-/- mice and thus will be valuable in understanding disease mechanisms and developing novel therapeutics.

Funding: R37 NS076752 to LLI