Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ionotropic receptors mediating a slow, calcium-permeable component of excitatory synaptic transmission in the central nervous system. Variants in genes encoding NMDAR subunits have been associated with a spectrum of neurodevelopmental disorders. Here we report six novel GRIN2D variants and one previously-described disease-associated GRIN2D variant in two patients with developmental and epileptic encephalopathy. Methods: Each mutantation was introduced into a cDNA encoding human NMDAR GluN2D using the QuikChange protocol. cRNAs were synthesized from cDNA and injected into Xenopus laevis oocytes. Two-electrode voltage clamp current recordings of oocytes were performed to evaluate agonist potency, sensitivity to negative modulators (magnesium and protons), channel open probability, and the sensitivity to FDA-approved NMDAR channel blockers. Whole-cell voltage clamp current recordings were performed to evaluate response time course and channel open time. The influence of GRIN2D variants on receptor cell surface trafficking was evaluated using a β-lactamase reporter assay in transfected HEK293 cells Results: GRIN2D encodes for the GluN2D subunit protein; the GluN2D amino acids affected by the variants in this report are located in the pre-M1 helix, transmembrane domain M3, and the intracellular carboxyl terminal domain. Functional analysis in vitro reveals that the GluN2D-Leu670Phe, Ala675Thr and Ala678Asp substitutions confer significantly enhanced agonist potency, and/or increased channel open probability, while the GluN2D-Ser573Phe, Ser1271Phe and Arg1313Trp substitutions result in a mild increase of agonist potency, reduced sensitivity to endogenous protons, and decreased channel open probability. The GluN2D-Ser573Phe, Ala675Thr, and Ala678Asp substitutions significantly decrease current amplitude. GluN2D-Leu670Phe variant slows current response deactivation time course and increased charge transfer. All six variants result in decreased receptor surface trafficking. In addition, we evaluated a set of FDA-approved NMDAR channel blockers (memantine, dextromethorphan and ketamine) to rescue functional changes of mutant receptors. Conclusions: Overall, our findings further emphasize the importance of functional and biochemical validation for each individual variant. The combination of a rapidly growing array of genomic sequencing data with functional evaluation of specific molecular mechanisms and potential rescue pharmacology in vitro may provide a better understanding of disease mechanism, diagnoses, as well as choice of precision medicine for a subset of severe pediatric neurodevelopmental diseases.  Funding: No funding