Two de novo GluN2B mutations affect multiple NMDAR-functions and instigate severe pediatric encephalopathy
Abstract
The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at excitatory synapses. Mutations affecting receptor function often lead to devastating neurodevelopmental disorders. We have identified two toddlers with different heterozygous missense mutations of the same, and highly conserved, glycine residue located in the ligand-binding-domain of GRIN2B: G689C and G689S. Structure simulations suggest severely impaired glutamate binding which we confirm by functional analysis. Both variants show three-orders of magnitude reductions in glutamate EC50, with G689S exhibiting the largest reductions observed in GRIN2B (~2000-fold). Moreover, variants multimerize with, and upregulate, GluN2Bwt-subunits, thus engendering a strong dominant-negative effect on mixed channels. In neurons, overexpression of the variants instigates suppression of synaptic GluNRs. Lastly, while exploring spermine potentiation as a potential treatment, we discovered that the variants fail to respond due to G689's novel role in proton-sensing. Together, we describe two unique variants with extreme effects on channel function. We employ protein-stability measures to explain why current (and future) LBD mutations in GluN2B primarily instigate Loss-of-Function.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Raw data is deposited at Dryad (doi:10.5061/dryad.1jwstqjv3)
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Two de novo GluN2B mutations affect multiple NMDAR-functions and instigate severe pediatric encephalopathyDryad Digital Repository, doi:10.5061/dryad.1jwstqjv3.
Article and author information
Author details
Funding
Israel Science Foundation (1096/17)
- Shai Berlin
Teva Pharmaceutical Industries (PR783187)
- Shai Kellner
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All Experiments were approved by the Technion Institutional Animal Care and Use Committee (permit SB, no. IL-129-09-17).
Copyright
© 2021, Kellner et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Methods: To study accessibility and reproducibility in cardiovascular research reporting, we screened 639 randomly selected articles published in 2019 in three top cardiovascular science publications: Circulation, the European Heart Journal, and the Journal of the American College of Cardiology (JACC). Of those 639 articles, 393 were empirical research articles. We screened each paper for accessible and reproducible research practices using a set of accessibility criteria including protocol, materials, data, and analysis script availability, as well as accessibility of the publication itself. We also quantified the consistency of open research practices within and across cardiovascular study types and journal formats.
Results: We identified that fewer than 2% of cardiovascular research publications provide sufficient resources (materials, methods, data, and analysis scripts) to fully reproduce their studies. Of the 639 articles screened, 393 were empirical research studies for which reproducibility could be assessed using our protocol, as opposed to commentaries or reviews. After calculating an accessibility score as a measure of the extent to which an article makes its resources available, we also showed that the level of accessibility varies across study types with a score of 0.08 for Case Studies or Case Series and 0.39 for Clinical Trials (p = 5.500E-5) and across journals (0.19 through 0.34, p = 1.230E-2). We further showed that there are significant differences in which study types share which resources.
Conclusion: Although the degree to which reproducible reporting practices are present in publications varies significantly across journals and study types, current cardiovascular science reports frequently do not provide sufficient materials, protocols, data, or analysis information to reproduce a study. In the future, having higher standards of accessibility mandated by either journals or funding bodies will help increase the reproducibility of cardiovascular research.
Funding: Authors Gabriel Heckerman, Arely Campos-Melendez, and Chisomaga Ekwueme were supported by an NIH R25 grant from the National Heart, Lung and Blood Institute (R25HL147666). Eileen Tzng was supported by an AHA Institutional Training Award fellowship (18UFEL33960207).