Strehlow et al. describe the largest cohort to date of individuals with GRIN2A-related disorders. The results reveal two phenotypic subgroups associated with different classes of variants affecting distinct domains of the GluN2A protein with different functional consequences. The findings will help predict outcomes in newly diagnosed individuals.Alterations of the N-methyl-d-aspartate receptor (NMDAR) subunit GluN2A, encoded by GRIN2A, have been associated with a spectrum of neurodevelopmental disorders with prominent speech-related features, and epilepsy. We performed a comprehensive assessment of phenotypes with a standardized questionnaire in 92 previously unreported individuals with GRIN2A-related disorders. Applying the criteria of the American College of Medical Genetics and Genomics to all published variants yielded 156 additional cases with pathogenic or likely pathogenic variants in GRIN2A, resulting in a total of 248 individuals. The phenotypic spectrum ranged from normal or near-normal development with mild epilepsy and speech delay/apraxia to severe developmental and epileptic encephalopathy, often within the epilepsy-aphasia spectrum. We found that pathogenic missense variants in transmembrane and linker domains (mis(TMD+Linker)) were associated with severe developmental phenotypes, whereas missense variants within amino terminal or ligand-binding domains (mis(ATD+LBD)) and null variants led to less severe developmental phenotypes, which we confirmed in a discovery (P = 10(6)) as well as validation cohort (P = 0.0003). Other phenotypes such as MRI abnormalities and epilepsy types were also significantly different between the two groups. Notably, this was paralleled by electrophysiology data, where mis(TMD+Linker) predominantly led to NMDAR gain-of-function, while mis(ATD+LBD) exclusively caused NMDAR loss-of-function. With respect to null variants, we show that Grin2a(+/) cortical rat neurons also had reduced NMDAR function and there was no evidence of previously postulated compensatory overexpression of GluN2B. We demonstrate that null variants and mis(ATD+LBD) of GRIN2A do not only share the same clinical spectrum (i.e. milder phenotypes), but also result in similar electrophysiological consequences (loss-of-function) opposing those of mis(TMD+Linker) (severe phenotypes; predominantly gain-of-function). This new pathomechanistic model may ultimately help in predicting phenotype severity as well as eligibility for potential precision medicine approaches in GRIN2A-related disorders.