GRIN2A mutation and early-onset epileptic encephalopathy: personalized therapy with memantine

Abstract

Objective: Early-onset epileptic encephalopathies have been associated with de novo mutations of numerous ion channel genes. We employed techniques of modern translational medicine to identify a disease-causing mutation, analyze its altered behavior, and screen for therapeutic compounds to treat the proband.

Methods: Three modern translational medicine tools were utilized: 1) high-throughput sequencing technology to identify a novel de novo mutation; 2) in vitro expression and electrophysiology assays to confirm the variant protein's dysfunction; and 3) screening of existing drug libraries to identify potential therapeutic compounds.

Results: A de novo GRIN2A missense mutation (c.2434C>A; p.L812M) increased the charge transfer mediated by NMDA receptors containing the mutant GluN2A-L812M subunit. In vitro analysis with NMDA receptor blockers indicated that GLuN2A-L812M-containing NMDARs retained their sensitivity to the use-dependent channel blocker memantine; while screening of a previously reported GRIN2A mutation (N615K) with these compounds produced contrasting results. Consistent with these data, adjunct memantine therapy reduced our proband's seizure burden.

Interpretation: This case exemplifies the potential for personalized genomics and therapeutics to be utilized for the early diagnosis and treatment of infantile-onset neurological disease.

Figure 1

Proband and neuroimaging. (A) The proband was nondysmorphic. (B–D) MRI of the brain at 9 years of age (B-Axial T1, C-Sagittal T1, D-Axial FLAIR) showed progressive cerebral atrophy and a thin corpus callosum associated with subtle abnormalities of the white matter including hypomyelination of the terminal zones and the temporal lobes.

Figure 2

Genetic and protein changes of GRIN2A and GluN2A. (A) Family pedigree and genotypes reveal a de novo mutation (affected proband is indicated by arrow; parentage was confirmed by SNP array; data not shown). (B) Schematic representation of GluN2A subunit (asterisk notes the position of the L812M mutation). The L812 residue is highly conserved across vertebrate species. (C) A homology model of GluN2A subunit built from the GluA2 crystallographic data and shown as space fill. The red asterisk in the cartoon depicting the domain arrangement of an individual subunit shows the position of L812M in the linker region between the ligand-binding domain (S2, LBD) and the transmembrane domain (M4).

Figure 3

Functional analysis of GluN1/GluN2A receptors, GluN1/GluN2A-N615K receptors, and GluN1/GluN2A-L812M receptors. (A) The composite glutamate (in the presence of 100 μmol/L glycine) and (B) glycine (in the presence of 100 μmol/L glutamate) concentration-response curves indicate an increased agonist potency in GluN1/GluN2A-L812M compared to wild-type and GluN1/GluN2A-N615K NMDA receptors. Fitted EC₅₀ values are given in μmol/L in the symbol legend.

Figure 4

Screening of NMDAR antagonists and personalized therapy. (A) Composite concentration-response curves for memantine inhibition of GluN1/GluN2A-L812M and wild-type current responses to 100 μmol/L glutamate and glycine determined by TEVC recordings from Xenopus oocytes. Memantine inhibits the wild-type and the mutant receptors with IC₅₀ values of 4.6 μmol/L and 12 μmol/L, respectively. (B) Adjunct-AED treatment with memantine reduced seizure frequency. Seizures per week were compared against AED treatment. Lacosamide and rufinamide were fully weaned at weeks 50 and 58, respectively. Memantine was titrated up to its full dosage over weeks 50–55; valproate dosing remained unchanged. The average number of episodes and associated standard deviations over the full course of the study are represented by the labeled dashed lines. (C–D) Routine EEG segments before (C) and after (D) memantine treatment. Both demonstrate a poorly organized, diffusely slow background. However, the right frontal-predominant spike-wave discharges recorded in the prememantine EEG were not present in the postmemantine EEG.