Abstracts

Rationale: Pedigree analysis and genome wide sequencing have identified over 280 missense mutations in the SCN1A gene that result in epilepsy including Genetic Epilepsy with Febrile Seizures Plus (GEFS+) and Dravet Syndrome (DS). The large variety of phenotypes associated with SCN1A mutations and differential responsiveness to anticonvulsant therapies suggests that clinical differences depend on the location of the mutation and the specific amino acid change. The K1270T mutation in SCN1A locus was identified in a large family with GEFS+ in which affected individuals exhibited afebrile and/or febrile seizures. Our previous work showed that SCN1AK1270T mutation in a Drosophila knock-in model causes a temperature-sensitive gain-of-function change in the voltage-dependence of sodium current deactivation and heat induced seizures (Sun et al, 2012). However, Drosophila have only one sodium ion channel gene in contrast to nine genes in humans. To analyse the effect of K1270T mutation in Scn1a in the mammalian brain we generated a transgenic mouse model. Methods: Mice carrying a mutation equivalent to the human K1270T mutation ( Scn1aK1259T ; henceforth, referred to as Scn1aKT) were generated using CRISPR/Cas9. The Scn1aKT/+ mutation was backcrossed for >5 generations into two genetic backgrounds used previously to model SCN1A mutations; C57BL/6J and 129X1/SvJ. Animals within each line were mated to generate homozygous, heterozygous mutant and wild-type (control) litter mates for all experiments. To determine if this mutation causes a dominant seizure phenotype in mice, seven days of continuous (24h/7d) video- EEG monitoring was performed on 3-4 month old wild-type and mutant litter mates. In addition, whole-cell patch-clamp recordings will be obtained from neurons in slices and acutely dissociated cells to assess firing properties, synaptic activity and sodium currents in excitatory and inhibitory neurons in the heterozygous mutant mice compared to wild-type littermates. Results: Animals homozygous for the mutation had spontaneous behavioral seizures and died by post-natal day 21. In contrast, longevity of heterozygous mutants is at least 20 weeks, similar to their wild-type litter mates (on-going assay). Heterozygous mice on the 129X1/SvJ background exhibited spontaneous electrographic seizures (n=2); no seizure activity was observed in wild-type litter mates monitored in parallel (n=2). Experiments are underway to evaluate mutants on the C57BL/6J strain as well as effects of increased temperature on seizure duration and frequency.  Conclusions: Heterozygous mice with Scn1aKT/+ mutation develop spontaneous electrographic seizures and homozygous mice have a shortened lifespan, recapitulating key features of epilepsy disorders. Future studies delving into the neuronal firing properties and sodium current dynamics would help us to understand the cellular mechanism of K1270T GEFS+ mutation. Key effects of the mutation that are preserved across species are most likely to reflect cellular mechanisms contributing to the disease phenotype in humans. Funding: O'Dowd (NS083009)Hunt (NS085046, NS096012)