To validate and apply a high-throughput functional assay for the evaluation of ATP1A2 variants associated with epilepsy.

Heterozygous pathogenic variants in ATP1A2, which encodes the astrocyte-predominant α2 subunit of the Na⁺/K⁺-ATPase, cause a range of childhood- and adult-onset neurological disorders including polymicrogyria, developmental-epileptic encephalopathy, and hemiplegic migraine. Functional assays could increase the yield of clinical testing for ATP1A2-related disorders by resolving nondiagnostic “variant of uncertain significance” (VUS) results. Here, we validate a high-throughput complementation assay and use it to characterize three novel epilepsy-associated ATP1A2 variants.

The 96-well plate-format complementation assay was evaluated using thirteen known-pathogenic or known-benign controls and successfully discriminated pathogenic from benign variants. Three patient VUS were tested using the assay and in preliminary results, one variant, associated with medication-refractory absence epilepsy, showed a possible decrease in cell survival.

High-throughput functional validation of Na⁺/K⁺-ATPase VUS may reduce diagnostic uncertainty in the neurology clinic. We present a practical high-throughput assay validated with pathogenic and benign controls. We plan to continue expanding the pool of controls to quantify the performance of this test. Surprisingly, preliminary testing of patient VUS suggested pathogenicity of an ATP1A2 variant associated with isolated medication-refractory absence epilepsy, further supporting the occurrence of presentations mimicking “idiopathic”/genetic generalized epilepsies within the ATP1A2 disease spectrum.