2025 American Academy of Neurology Abstract Website

Objective:

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

Background:

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.

Design/Methods:

Deidentified genotype/phenotype information were collected on patients with ATP1A2 variants evaluated in our institution's pediatric epilepsy clinic. Three ATP1A2 missense VUS were tested for pathogenicity using a 96-well plate-format cell-based complementation assay measuring the ability of ATP1A2 to replace endogenous Na⁺/K⁺-ATPase, which is essential for cell survival. The ATP1A2 coding sequence was cloned into pcDNA with two variants (Q116R/N127D) that confer partial resistance to an Na⁺/K⁺-ATPase inhibitor, ouabain. Expression constructs containing variants of interest were transfected into HEK293 cells, endogenous Na⁺/K⁺-ATPases were inhibited using ouabain, and cell viability was quantified by tetrazolium-dye reduction.

Results:

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.

Conclusions:

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.