A gain-of-function GRIA2 variant associated with neurodevelopmental delay and seizures: Functional characterization and targeted treatment

Abstract

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) are ligand-gated cationic channels formed from combinations of GluA1-4 subunits. Pathogenic variants of GRIA1-4 have been described in patients with developmental delay, intellectual disability, autism spectrum disorder, and seizures, with GRIA2 variants typically causing AMPAR loss of function. Here, we identify a novel, heterozygous de novo pathogenic missense mutation in GRIA2 (c.1928 C>T, p.A643V, NM_001083619.1) in a 1-year-old boy with epilepsy, developmental delay, and failure to thrive. We made patch-clamp recordings to compare the functional and pharmacological properties of variant and wild-type receptors expressed in HEK293 cells, with and without the transmembrane AMPAR regulatory protein γ2. This showed GluA2 A643V-containing AMPARs to exhibit a novel gain of function, with greatly slowed deactivation, markedly reduced desensitization, and increased glutamate sensitivity. Perampanel, an antiseizure AMPAR negative allosteric modulator, was able to fully block GluA2 A643V/γ2 currents, suggesting potential therapeutic efficacy. The subsequent introduction of perampanel to the patient's treatment regimen was associated with a marked reduction in seizure burden, a resolution of failure to thrive, and clear developmental gains. Our study reveals that GRIA2 disorder can be caused by a gain-of-function variant, and both predicts and suggests the therapeutic efficacy of perampanel. Perampanel may prove beneficial for patients with other gain-of-function GRIA variants.

Keywords: AMPA receptor; GRIA disorder; GluA2; epilepsy; perampanel.

FIGURE 1

Clinical presentation and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid‐type glutamate receptor (AMPAR) gain of function with the GRIA2 A643V variant. (A) Electroencephalographic (EEG) findings. Progression is seen from normal EEG at 7 months of age (left) to hypsarrhythmia (middle) with infantile spasms (right) at 9 months of age. Hypsarrhythmia resolved with adrenocorticotropic hormone therapy, but frequent nocturnal asymmetric tonic seizures (right) developed at 14 months of age. (B) Sequence alignments highlighting the position of Ala643 (orange dot) in the third membrane region (M3; blue bar) of the AMPAR subunit. The surrounding region is completely conserved between all four AMPAR subunits, and the SYTANLAAF motif (pink box) is conserved throughout the iGluR superfamily. The gene sequences are from human GluA1 (NP_001107655.1), human GluA2 (NP_000817.5), human GluA3 (NP_015564.5), human GluA4 (NP_000820.4), human GluK2 (NP_068775.1), human GluN1 (NP_015566.1), human GluN2A (NP_000824.1), and human GluD2 (NP_001501.2). (C) Representative deactivating and desensitizing outside‐out patch responses (10 mmol·L^–1 glutamate, 1 and 500 ms, −60 mV; black bars) from HEK293 cells transfected with wild‐type (WT) GluA2(Q) (black) or GluA2(Q) A643V (red; superimposed). Right: pooled weighted time constant of deactivation (τ_{w, deact}) data for GluA2(Q) (n = 16) and GluA2(Q) A643V (n = 13) from 1‐ms glutamate applications, together with pooled desensitization time constant (τ_{w, des}) and residual current (steady‐state current at end of application divided by peak current; I _ss/I _peak) data for GluA2(Q) (n = 31) and GluA2(Q) A643V (n = 33) from 500‐ms glutamate applications. Box‐and‐whisker plots indicate the median (black line), the 25–75th percentiles (pale blue box), and the 10–90th percentiles (whiskers); filled circles are data from individual patches, and open circles indicate means. (D) Pooled normalized concentration–response curves for GluA2(Q) (n = 5) and GluA2(Q) A643V (n = 5). Symbols and error bars indicate mean values with SD, and the solid lines are fits to the Hill equation, yielding the indicated half‐maximal effective concentration (EC₅₀) values. Table 1 reports the statistical analysis of EC₅₀ values obtained from separate fits of the data from individual patches. (E) Representative deactivating and desensitizing responses (10 mmol·L^–1 glutamate, 1 and 500 ms, +60 mV; black bars) from heteromeric receptors in cells transfected with GluA1 and GluA2(R) (black) or GluA1 and GluA2(R) A643V (red; superimposed). Right: pooled τ_{w, deact} data for WT GluA1/GluA2(R) (n = 7) and GluA1/GluA2(R) A643V (n = 6), together with pooled τ_{w, des} and I _ss/I _peak data for GluA1/GluA2(R) (n = 9) and GluA1/GluA2(R) A643V (n = 5). Boxplots as in C. Indicated p‐values are from two‐sided approximate permutation t‐tests comparing WT and A643V variant (Table 1).

FIGURE 2

Functional and clinical impact of perampanel. (A) Representative GluA2(Q)/γ2 (black) and GluA2(Q) A643V/γ2 currents (red) with inhibition by 3 μmol·L^–1 perampanel (pink). (B) Concentration inhibition curves demonstrating decreased potency of perampanel against the A643V variant (n = 6 for GluA2[Q]/γ2 and 4 for GluA2[Q] A643V/γ2). Symbols and error bars indicate mean values with SD, and the solid lines are fits to the Hill equation, yielding the indicated half‐maximal inhibitory concentration (IC₅₀) values. Table 1 reports the statistical analysis of IC₅₀ values obtained from separate fits of the data from individual patches. (C) Crystal structure of a GluA2(Q) receptor with perampanel bound, showing the binding site (including F644, which interacts with the phenyl ring of perampanel) adjacent to A643 (Protein Data Bank: 5L1F ¹² ). (D) Number of parent‐reported daily seizures in the months before and after commencement of perampanel treatment (at 0 months). Colored bars and plots denote the timing of antiseizure medications. Phenobarbital (6 mg/kg daily) is currently being weaned, whereas the modified Atkins diet is being maintained. Following the introduction of perampanel, levetiracetam was gradually withdrawn. (E) Electroencephalograms (EEGs) at 30 months of age following treatment with perampanel for 3 months. The awake (left) and sleep (right) interictal patterns remain slow and disorganized, with multifocal spikes in sleep, consistent with an epileptic encephalopathy with ongoing risk for seizures.