GENETIC, FUNCTIONAL AND BIOCHEMICAL ANALYSIS OF <EM>GRIN</EM> GENE VARIANTS IDENTIFIED IN A COHORT OF CZECH PEDIATRIC PATIENTS

NMDARs mediate excitatory neurotransmission in the central nervous system and are essential for learning and memory. Dysfunction of NMDARs caused by GRIN variants is associated with epilepsy, neurodevelopmental, and neuropsychiatric disorders. However, only a limited subset of GRIN variants has been functionally characterized. Here, we report functional analyses of additional GRIN variants identified through routine diagnostic practice. To date, nearly thirty GRIN1 and GRIN2A/B variants have been detected in Czech and Slovak pediatric patients diagnosed with developmental delay (68%), intellectual disability (56%), and epilepsy (56%). The variant spectrum is dominated by missense (76%), but also includes nonsense, frameshift, and indel variants distributed across all receptor domains. In silico prediction classified approximately one-third of variants as pathogenic and another third as likely pathogenic. We focused on biochemical and functional characterization of selected missense variants: GluN2B-L612M, and GluN2B-A827V (variants of uncertain significance) located in the transmembrane domain (TMD), and GluN2A-F756Y (uncertain significance), GluN2B-T691A (likely pathogenic) located in the agonist-binding domain (ABD). Variant subunits were co-expressed with wild-type GluN1 in HEK293 cells, and receptor properties were examined using patch-clamp electrophysiology and immunofluorescence microscopy. Dose–response analysis revealed reduced glutamate affinity for the ABD variants GluN2A-F756Y and GluN2B-T691A. Analysis using the open-channel blocker MK-801 showed that the TMD variant GluN2B-L612M increased channel open probability. Finally, reduced whole-cell current density and surface expression indicated impaired trafficking of the GluN2B-A827V variant. Together, these data support gain-of-function classification for GluN2B-L612M and loss-of-function effects for the remaining variants, providing functional evidence to guide personalized therapeutic strategies.