Abstracts

Rationale: KCNQ2 encephalopathy (Q2E) was first described in patients with profound global developmental disability after a neonatal presentation with refractory seizures, burst-suppression EEG background, and clinical encephalopathy akin to Ohtahara syndrome. However, some neonatal-onset KCNQ2 patients have milder neonatal epilepsy and subsequent moderate to severe developmental disabilities. Construct and face valid murine models of Q2E may help to better understand these complex patients and guide therapy development.

Methods: We reviewed clinical data on a child bearing a KCNQ2 p.G256W variant. We introduced G256W into recently obtained cryoEM structures. We made recordings from transfected Chinese hamster ovary cells. The JAX Rare and Orphan Disease Center generated G256W knock-in mice via CRISPR/Cas9, as well as a strain with a neighboring seven base deletion (c.760_767DelAAAAGGG/p.Glu254ValFs19, “E254VFs”), serving a model for stop-gained variants that usually lead to self-limited outcomes.

Results: Two unrelated children with de novo heterozygous G256W variants, neonatal seizures, and developmental disability were identified. For one, further phenotype was unavailable; for the other, neonatal seizures resolved easily, gross motor milestones were reached with moderate delay, but difficulties in language, social, and cognitive domains, autistic-like features, and breakthrough seizures persisted. The cryoEM structure showed Gly256 in a tight loop at the extracellular extreme of the pore domain, facing water. In silico mutation to G256W introduces a bulky hydrophobic side chain near the selectivity filter. In patch clamp recordings, G256W was nonconducting if expressed alone, and had dominant-negative effects if co-expressed with WT subunits, reducing current density by 3.3 and 2.5-fold in homomeric and heteromeric channels, respectively. Homozygous G256W mice die within hours of birth. Heterozygous G256W (G256W/+) mice show no premature mortality, but experience spontaneous generalized seizures as early as P10 (figure). EEG background continuity emerged normally in P9-P12 G256W/+ mice; no electrographic neonatal seizures were observed. Hippocampal cDNA sequencing demonstrated that E254VFs mRNA is spliced normally, resulting in in-frame premature termination codons, and nonsense-mediated mRNA decay was predicted. Indeed, in adult E254VFs/+ mice, Kcnq2 mRNA was ~30% lower than in control cortex, hippocampus, or cerebellum. Kcnq3 mRNA was unaltered in Kcnq2 E254VFs/+ mice. Unexpectedly, Kcnq3 mRNA was increased (16%, p=0.009) in G256W/+ hippocampus relative to WT. Western blots of P14 whole brain lysate showed that E254VFs/+, but not G256W/+ mice, had reduced Kcnq2 protein relative to WT. Preliminary open field tests of adult mice suggested increased activity and heightened anxiety-like behavior in G256W/+ mice.

Conclusions: We have analyzed a model of “moderate severity” Q2E. We have begun to elucidate links between the variant’s molecular consequences, epilepsy, and development.

Funding: Please list any funding that was received in support of this abstract.: RO1 NS49119, The Jack Pribaz Foundation, KCNQ2 Cure Alliance, CURE (ECC); AES and Wishes for Elliott (TA), KO8 NS NS096029 (AM).