Functional analysis of a de novo GRIN2A missense mutation associated with early-onset epileptic encephalopathy

Abstract

NMDA receptors (NMDAR), ligand-gated ion channels, play important roles in various neurological disorders, including epilepsy. Here we show the functional analysis of a de novo missense mutation (L812M) in a gene encoding NMDAR subunit GluN2A (GRIN2A). The mutation, identified in a patient with early-onset epileptic encephalopathy and profound developmental delay, is located in the linker region between the ligand-binding and transmembrane domains. Electrophysiological recordings revealed that the mutation enhances agonist potency, decreases sensitivity to negative modulators including magnesium, protons and zinc, prolongs the synaptic response time course, and increases single channel open probability. The functional changes of this amino acid apply to all other NMDAR subunits, suggesting an important role of this residue on the function of NMDARs. Taken together, these data suggest that the L812M mutation causes over-activation of NMDARs and drives neuronal hyperexcitability. We hypothesize that this mechanism underlies the patient’s epileptic phenotype as well as cerebral atrophy.

Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases

“…The profound increase in the current produced by this mutation seems likely to drive aberrant excitation and potentially contribute to neuronal loss and consequently the patients' clinical symptoms. In subsequent years, a large number of missense mutations and deletions/truncations (.100) have been identified through whole exome and genome sequencing (reviewed by Soto et al, 2014;Burnashev and Szepetowski, 2015) and are scattered across all domains in NMDA receptor subunits (Supplemental Table S2; Tables 2 and 3) Myers et al, 2011;Tarabeux et al, 2011;de Ligt et al, 2012;O'Roak et al, 2012;Carvill et al, 2013b, DeVries andPatel, 2013;Epi4K and Epilepsy Phenome/ Genome Project, 2013;Freunscht et al, 2013;Lemke et al, 2013Lemke et al, , 2014Lesca et al, 2013;Adams et al, 2014;Andreoli et al, 2014;Fromer et al, 2014;Kenny et al, 2014;Pierson et al, 2014;Redin et al, 2014;Venkateswaran et al, 2014;Yuan et al, 2014;Burnashev and Szepetowski, 2015;Ohba et al, 2015;Turner et al, 2015). More recently, several case-control studies have isolated de novo and inherited mutations in the GRIN2A gene in patients diagnosed with different forms of epilepsy, including continuous spike-and-waves during slow-wave sleep syndrome, epileptic encephalopathy, Landau-Kleffner syndrome, and Rolandic epilepsy (Endele et al, 2010;Carvill et al, 2013b;Lemke et al, 2013;Lesca et al, 2013; reviewed by Burnashev and Szepetowski, 2015).…”

“…For example, of more than 100 published mutations in NMDA receptor subunits, functional data are reported for only 12 (Endele et al, 2010;Hamdan et al, 2011;Carvill et al, 2013b;Lemke et al, 2013Lemke et al, , 2014Lesca et al, 2013;Adams et al, 2014;Pierson et al, 2014;Yuan et al, 2014). The lack of functional information for de novo mutations in genes with a strong genetic link to disease underscores a pressing need.…”

“…(A) Location of mutant L812M (green space fill) and possible van der Waals interaction with the adjacent GluN1 subunit pre-M1 helix (purple) and SYTANLAAF (yellow) of the NMDA receptor gate as predicted from the homomeric GluA2 structure. The GluN2A(L812M) mutation changes the pharmacology and channel properties of NMDA receptors and shows increased glutamate potency (B), prolonged deactivation time course (C), and increased open probability (D) with triheteromeric NMDA receptors, with 0, 1, or 2 copies of the L812M mutation in each complex (B-D reproduced from Yuan et al, 2014). (E) GluN2A(L812M) modestly reduces the sensitivity to the FDA-approved drug memantine.…”

“…In addition, some mutations alter protein trafficking or RNA stability, leading to changes in the level and location of receptors that reach the neuronal plasma membrane (e.g., Macdonald and Kang, 2012;see below). By changing critical channel properties, surface expression, or localization, mutations can alter neuronal signaling, which leads to changes in brain function that can underlie patient symptoms (e.g., Macdonald et al, 2010;Pierson et al, 2014;Yuan et al, 2014). Adaptive neurobiological consequences of perturbations in neuronal function also occur secondary to the effects of the mutations, and thus functionally relevant mutations can alter circuitry to create neuropathological situations that subsequently persist and drive symptoms independent of the initial insult (e.g., Jefferys and Whittington, 1996).…”

Important Role of Translational Science in Rare Disease Innovation, Discovery, and Drug Development

“…For example, a boy with uncontrolled seizures was diagnosed with specific GRIN2A mutations that caused enhanced agonist potency for the NMDA receptor subunit GluN2A. 13 The in vitro demonstration of inhibition of this type of hyperexcitability using memantine allowed for targeted therapy with this child, with the potential to benefit other individuals with similar GRIN2A mutations.…”