Clinical and functional consequences of GRIA variants in patients with neurological diseases.

AMPA receptors are members of the glutamate receptor family and mediate a fast component of excitatory synaptic transmission at virtually all central synapses. Thus, their functional characteristics are a critical determinant of brain function. We evaluate intolerance of each GRIA gene to genetic variation using 3DMTR and report here the functional consequences of 52 missense variants in GRIA1-4 identified in patients with various neurological disorders. These variants produce changes in agonist EC50, response time course, desensitization, and/or receptor surface expression. We predict that these functional and localization changes will have important consequences for circuit function, and therefore likely contribute to the patients' clinical phenotype. We evaluated the sensitivity of variant receptors to AMPAR-selective modulators including FDA-approved drugs to explore potential targeted therapeutic options.

Opportunities for Precision Treatment of GRIN2A and GRIN2B Gain-of-Function Variants in Triheteromeric N-Methyl-D-Aspartate Receptors.

N-methyl-D-aspartate receptors (NMDARs) are tetrameric assemblies of two glutamate N-methyl-D-aspartate receptor subunits, GluN1 and two GluN2, that mediate excitatory synaptic transmission in the central nervous system. Four genes (GRIN2A-D) encode four distinct GluN2 subunits (GluN2A-D). Thus, NMDARs can be diheteromeric assemblies of two GluN1 plus two identical GluN2 subunits, or triheteromeric assemblies of two GluN1 subunits plus two different GluN2 subunits. An increasing number of de novo GRIN variants have been identified in patients with neurologic conditions and with GRIN2A and GRIN2B harboring the vast majority (> 80%) of variants in these cases. These variants produce a wide range of effects on NMDAR function depending upon its subunit subdomain location and additionally on the subunit composition of diheteromeric versus triheteromeric NMDARs. Increasing evidence implicates triheteromeric GluN1/GluN2A/GluN2B receptors as a major component of the NMDAR pool in the adult cortex and hippocampus. Here, we explore the ability of GluN2A- and GluN2B-selective inhibitors to reduce excess current flow through triheteromeric GluN1/GluN2A/GluN2B receptors that contain one copy of GRIN2A or GRIN2B gain-of-function variants. Our data reveal a broad range of sensitivities for variant-containing triheteromeric receptors to subunit-selective inhibitors, with some variants still showing strong sensitivity to inhibitors, whereas others are relatively insensitive. Most variants, however, retain sensitivity to non-selective channel blockers and the competitive antagonist D-(-)-2-amino-5-phosphonopentanoic acid. These results suggest that with comprehensive analysis, certain disease-related GRIN2A and GRIN2B variants can be identified as potential targets for subunit-selective modulation and potential therapeutic gain. SIGNIFICANCE STATEMENT: Triheteromeric NMDA receptors that contain one copy each of the GluN2A and GluN2B subunits show intermediate sensitivity to GluN2A- and GluN2B-selective inhibitors, making these compounds candidates for attenuating overactive, GRIN variant-containing NMDA receptors associated with neurological conditions. We show that functional evaluation of variant properties with inhibitor pharmacology can support selection of a subset of variants for which GluN2 subunit-selective agents remain effective inhibitors of variant-containing triheteromeric NMDA receptors.

De novo mutations in GRIN1 cause extensive bilateral polymicrogyria.

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria.

A de novo loss-of-function GRIN2A mutation associated with childhood focal epilepsy and acquired epileptic aphasia.

OBJECTIVE: N-methyl-D-aspartate receptors (NMDAR) subunit GRIN2A/GluN2A mutations have been identified in patients with various neurological diseases, such as epilepsy and intellectual disability / developmental delay (ID/DD). In this study, we investigated the phenotype and underlying molecular mechanism of a GRIN2A missense mutation identified by next generation sequencing on idiopathic focal epilepsy using in vitro electrophysiology. METHODS: Genomic DNA of patients with epilepsy and ID/DD were sequenced by targeted next-generation sequencing within 300 genes related to epilepsy and ID/DD. The effects of one missense GRIN2A mutation on NMDAR function were evaluated by two-electrode voltage clamp current recordings and whole cell voltage clamp current recordings. RESULTS: We identified one de novo missense GRIN2A mutation (Asp731Asn, GluN2A(D731N)) in a child with unexplained epilepsy and DD. The D731N mutation is located in a portion of the agonist-binding domain (ABD) in the GluN2A subunit, which is the binding pocket for agonist glutamate. This residue in the ABD is conserved among vertebrate species and all other NMDAR subunits, suggesting an important role in receptor function. The proband shows developmental delay as well as EEG-confirmed seizure activity. Functional analyses reveal that the GluN2A(D731N) mutation decreases glutamate potency by over 3,000-fold, reduces amplitude of current response, shortens synaptic-like response time course, and decreases channel open probability, while enhancing sensitivity to negative allosteric modulators, including extracellular proton and zinc inhibition. The combined effects reduce NMDAR function. SIGNIFICANCE: We identified a de novo missense mutation in the GRIN2A gene in a patient with childhood focal epilepsy and acquired epileptic aphasia. The mutant decreases NMDAR activation suggesting NMDAR hypofunction may contribute to the epilepsy pathogenesis.