Abstracts

Rationale: De novo mutations are major contributors to the development of childhood epileptic encephalopathies (CEEs) such as Dravet syndrome, infantile spasms, and Lennox-Gastaut syndrome (LGS) (Carvill et al 2013; O’Brien et al 2013; Epi4K Consortium 2013). CEEs comprise some of the most severe and pharmaco-resistant classes of epilepsy (Chiron and Dulac 2011). Mutations in the SCN8A gene have recently been shown to be causative for cases of infantile spasm, LGS, and CEE (Veeramah et al 2012; O’Brien et al 2013). CEEs often lead to intellectual and physical disabilities in later life as well as high rates of SUDEP (Sudden Unexpected Death in Epilepsy). These mutations have been shown to result in both gain-of-function and loss-of-function mutations (Veeramah et al 2012; Blanchard et al 2015). The goal of this research project is to use patient-derived cells to characterize electrophysiology alterations caused by SCN8A mutations associated with early-onset epilepsy and develop a novel platform for identifying effective pharmacological agents for this debilitating disease.Methods: SCN8A, which encodes for the voltage-gated sodium channel is expressed in both the PNS and CNS, where it is the most abundant Nav channel. We have identified two CEE patients with missense mutations in SCN8A (Arg1872>Leu and Val1592>Leu). Fibroblasts from the patients’ skin biopsies were reprogrammed into induced pluripotent stem cells (iPSCs). These iPSCs have subsequently been differentiated by two different protocols into neurons expressing cortical excitatory and peripheral markers respectively. These cells are being used to first deduce the effect the Nav1.6 mutations have on electrophysiological properties of the neurons, particularly sodium current density and persistent sodium current. We hypothesize that these mutations will increase both of these measures leading to hyperexcitability of the neurons as has been shown in the majority of other SCN8A mutations in mouse models and heterologous systems. We have also used a multielectrode array platform (Axion Biosystems) to measure spontaneous activity.Results: Cortical neurons from three patient lines all show increased presistent sodium current and spontaneous activity compared to human iPSC-derived neuronal control lines.Conclusions: Future studies will involve culturing cortical and peripheral neurons, as well as cardiac myocytes, on MEAs to test known antiepileptic drugs and screen drug libraries for amelioration of hyperexcitable phenotypes. This work will shed light on SCN8A CEE seizure and SUDEP mechanisms and should provide lead compounds for clinical testing.