Abstracts

Rationale: De novo pathogenic variants in the KCNA2 gene, which encodes the voltage-gated K+ channel Kv1.2, were recently described as a cause of developmental and epileptic encephalopathy (DEE). We previously reported a case of a probable SUDEP-Plus in a child with KCNA2-related encephalopathy. To model KCNA2 DEE in human cortical neurons and explore disease mechanisms, we generated “virtual patient” and isogenic control human induced pluripotent stem cell (iPSC) lines using CRISPR gene editing to insert a variant associated with DEE and sudden unexpected death in epilepsy (SUDEP). We also expressed the same KCNA2 variant in human embryonic kidney (HEK) cells.

Methods: We used CRISPR gene editing of a control human iPSC line to generate a virtual patient line carrying the KCNA2 variant, p.Asp379Gly (GAC >GGC): c. 1136 A >G in exon 3. We also expressed this variant or wild-type KCNA2 in HEK cells. Whole-cell voltage-clamp recordings were made to measure potassium currents in HEK cells transfected with the variant or a control plasmid. KCNA2 DEE and isogenic control iPSCs were differentiated into excitatory cortical-like induced neurons (iNeurons) using viral expression of dox-inducible Neurogenin-2. Current clamp recordings of action potentials were made, and calcium imaging was performed using viral RCaMP and an Incucyte device.

Results: Potassium current recordings in HEK cells revealed much smaller currents in cells expressing the variant than the wild-type construct. Both CRISPR mutant and control iPSCs generated iNeurons with normal morphology that expressed mature forebrain glutamatergic neuron markers. Whole-cell recordings made from iNeurons expressing the heterozygotes KCNA2 variant and isogenic control iPSC lines revealed increased numbers of spontaneous action potentials in mutant cells. Calcium imaging showed increased network bursting rate and more irregular firing in mutant neurons.

Conclusions: These results suggest that the KCNA2 variant p.Asp379Gly causes a loss of Kv1.2 channel function leading to neuronal network hyperexcitability and seizures. Our study provides a novel in vitro cell platform to understand KCNA2 DEE mechanisms and identify precision therapies.

Funding: Please list any funding that was received in support of this abstract.: NIH HL149363.