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N-methyl-d-aspartate receptors (NMDARs) play important roles in brain development and neurological disease. We report two individuals with similar dominant de novo GRIN1 mutations (c.1858 G>A and c.1858 G>C; both p.G620R). Both individuals presented at birth with developmental delay and hypotonia associated with behavioral abnormalities and stereotypical movements. Recombinant NMDARs containing the mutant GluN1-G620R together with either GluN2A or GluN2B were evaluated for changes in their trafficking to the plasma membrane and their electrophysiological properties. GluN1-G620R/GluN2A complexes showed a mild reduction in trafficking, a ~2-fold decrease in glutamate and glycine potency, a strong decrease in sensitivity to Mg2+ block, and a significant reduction of current responses to a maximal effective concentration of agonists. GluN1-G620R/GluN2B complexes showed significantly reduced delivery of protein to the cell surface associated with similarly altered electrophysiology. These results indicate these individuals may have suffered neurodevelopmental deficits as a result of the decreased presence of GluN1-G620R/GluN2B complexes on the neuronal surface during embryonic brain development and reduced current responses of GluN1-G620R-containing NMDARs after birth. These cases emphasize the importance of comprehensive functional characterization of de novo mutations and illustrates how a combination of several distinct features of NMDAR expression, trafficking and function can be present and influence phenotype.

The N-methyl-d-aspartate receptor (NMDAR), a ligand-gated ionotropic glutamate receptor, plays important roles in normal brain development and a wide range of neurologic disorders, including epilepsy. Here, we evaluate for the first time the functional properties of a de novo GRIN2A missense mutation (p.M817V) in the pre-M4 linker in a child with profound global developmental delay and refractory epilepsy. Electrophysiologic recordings revealed that the mutant GluN2A(M817V)-containing receptors showed enhanced agonist potency, reduced sensitivity to endogenous negative inhibitors (Mg2+, proton, and zinc), prolonged synaptic-like response time course, increased single-channel mean open time, and increased channel open probability. These results suggest that the gain-of-function M817V mutation causes overactivation of NMDAR and drives neuronal hyperexcitability, which may contribute to the patient's observed epileptic phenotype. Molecular modeling of the closed channel conformation reveals that this mutation weakens the interaction between GluN2 transmembrane helix M4 and two GluN1 transmembrane helices, and increases atomic fluctuation or movement of the pre-M1 region of GluN1 subunit, suggesting a mechanism by which channel function is enhanced. The functional changes of this mutation on agonist potency occur when the mutation is introduced into all other GluN2 subunits, suggesting a conserved role of this residue in control of NMDAR function through interactions of membrane spanning GluN2 and GluN1 helices. A number of NMDAR-targeted drugs including U.S. Food and Drug Association-approved NMDAR channel blockers were evaluated for their ability to inhibit receptors containing GluN2A(M817V) as a first step to exploring the potential for rescue pharmacology and personalized medicine.

Epilepsy and intellectual disability are associated with rare variants in the GluN2A and GluN2B (encoded by GRIN2A and GRIN2B) subunits of the N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel with essential roles in brain development and function. By assessing genetic variation across GluN2 domains, we determined that the agonist binding domain, transmembrane domain, and the linker regions between these domains were particularly intolerant to functional variation. Notably, the agonist binding domain of GluN2B exhibited significantly more variation intolerance than that of GluN2A. To understand the ramifications of missense variation in the agonist binding domain, we investigated the mechanisms by which 25 rare variants in the GluN2A and GluN2B agonist binding domains dysregulated NMDAR activity. When introduced into recombinant human NMDARs, these rare variants identified in individuals with neurologic disease had complex, and sometimes opposing, consequences on agonist binding, channel gating, receptor biogenesis, and forward trafficking. Our approach combined quantitative assessments of these effects to estimate the overall impact on synaptic and non-synaptic NMDAR function. Interestingly, similar neurologic diseases were associated with both gain- and loss-of-function variants in the same gene. Most rare variants in GluN2A were associated with epilepsy, whereas GluN2B variants were associated with intellectual disability with or without seizures. Finally, discerning the mechanisms underlying NMDAR dysregulation by these rare variants allowed investigations of pharmacologic strategies to correct NMDAR function.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated cation channels that mediate excitatory synaptic transmission. Genetic mutations in multiple NMDAR subunits cause various childhood epilepsy syndromes. Here, we report a de novo recurrent heterozygous missense mutation—c.1999G>A (p.Val667Ile)—in a NMDAR gene previously unrecognized to harbor disease-causing mutations, GRIN2D, identified by exome and candidate panel sequencing in two unrelated children with epileptic encephalopathy. The resulting GluN2D p.Val667Ile exchange occurs in the M3 transmembrane domain involved in channel gating. This gain-of-function mutation increases glutamate and glycine potency by 2-fold, increases channel open probability by 6-fold, and reduces receptor sensitivity to endogenous negative modulators such as extracellular protons. Moreover, this mutation prolongs the deactivation time course after glutamate removal, which controls the synaptic time course. Transfection of cultured neurons with human GRIN2D cDNA harboring c.1999G>A leads to dendritic swelling and neuronal cell death, suggestive of excitotoxicity mediated by NMDAR over-activation. Because both individuals’ seizures had proven refractory to conventional antiepileptic medications, the sensitivity of mutant NMDARs to FDA-approved NMDAR antagonists was evaluated. Based on these results, oral memantine was administered to both children, with resulting mild to moderate improvement in seizure burden and development. The older proband subsequently developed refractory status epilepticus, with dramatic electroclinical improvement upon treatment with ketamine and magnesium. Overall, these results suggest that NMDAR antagonists can be useful as adjuvant epilepsy therapy in individuals with GRIN2D gain-of-function mutations. This work further demonstrates the value of functionally evaluating a mutation, enabling mechanistic understanding and therapeutic modeling to realize precision medicine for epilepsy.

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 N-methyl-D-aspartate receptors (NMDAs) 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.

The N-methyl-d-aspartate receptor (NMDAR), a ligand-gated ionotropic glutamate receptor, plays important roles in normal brain development and a wide range of neurologic disorders, including epilepsy. Here, we evaluate for the first time the functional properties of a de novo GRIN2A missense mutation (p.M817V) in the pre-M4 linker in a child with profound global developmental delay and refractory epilepsy. Electrophysiologic recordings revealed that the mutant GluN2A(M817V)-containing receptors showed enhanced agonist potency, reduced sensitivity to endogenous negative inhibitors (Mg2+, proton, and zinc), prolonged synaptic-like response time course, increased single-channel mean open time, and increased channel open probability. These results suggest that the gain-of-function M817V mutation causes overactivation of NMDAR and drives neuronal hyperexcitability, which may contribute to the patient’s observed epileptic phenotype. Molecular modeling of the closed channel conformation reveals that this mutation weakens the interaction between GluN2 transmembrane helix M4 and two GluN1 transmembrane helices, and increases atomic fluctuation or movement of the pre-M1 region of GluN1 subunit, suggesting a mechanism by which channel function is enhanced. The functional changes of this mutation on agonist potency occur when the mutation is introduced into all other GluN2 subunits, suggesting a conserved role of this residue in control of NMDAR function through interactions of membrane spanning GluN2 and GluN1 helices. A number of NMDAR-targeted drugs including U.S. Food and Drug Association–approved NMDAR channel blockers were evaluated for their ability to inhibit receptors containing GluN2A(M817V) as a first step to exploring the potential for rescue pharmacology and personalized medicine.