Heterozygous gain-of-function and mixed-type Kcna2 variants recapitulate SUDEP and increased cortical Npy-Npy1r...

Variants in the KCNA2 gene are associated with developmental and epileptic encephalopathy and are classified as loss-of-function, gain-of-function (GoF), or mixed-type based on electrophysiological characteristics. Although Kcna2 knockout mice exhibit epilepsy, the pathogenic mechanisms underlying GoF and mixed-type variants remain poorly understood. This study compared GoF and mixed-type models to investigate their underlying molecular mechanisms.

CRISPR-edited heterozygous mice carrying R297Q or L290R variants were generated. Seizure activity was monitored and characterized using simultaneous video-EEG-ECG recordings. Cortical single-cell transcriptomic analysis was performed to demonstrate pathogenic mechanisms and associated regulatory pathways.

R297Q and L290R heterozygous mice developed spontaneous lethal seizures within 1-2 months, with median survival times of 52 and 49 days, respectively. No overt motor deficits or brain malformations were observed. Video-EEG-ECG monitoring of R297Q mice revealed frequent Racine stage 2-3 seizures, with the most severely affected mouse exhibiting head nodding almost every minute, while stage 4-5 seizures were less common. All monitored mice eventually experienced lethal stage 5 seizures, characterized by postictal generalized EEG suppression followed by bradycardia and cardiac arrest, producing a terminal pattern that resembled human sudden unexpected death in epilepsy (SUDEP). Interictal ECG analysis demonstrated increased skipped beats and elevated heart rate variability, suggesting autonomic dysfunction. Single-cell transcriptomic analysis showed that R297Q selectively affected specific neuronal subtypes while L290R induced widespread changes, yet both variants converged on synaptic pathway disruption. In addition, enhanced NPY signaling was identified in both models, reflected in upregulation of Npy1r.

GoF and mixed-type KCNA2 pathogenic variants converged on a seizure-induced premature death phenotype that recapitulated SUDEP and a shared cortical Npy-Npy1r signaling pathway. Enhanced Npy-Npy1r signaling may represent a potential therapeutic target for KCNA2-related epilepsies.