N-methyl-D-aspartate receptors (NMDARs or GluNRs) are excitatory glutamate receptors found throughout the brain, playing critical roles in neuronal development, synaptogenesis, plasticity, and in most processes of learning and memory (Paoletti et al., 2013; Lau and Zukin, 2007). The heterotetrametric receptor is assembled from seven different GRIN genes (Glutamate Receptor, Ionotropic, NMDA-kind) (Paoletti et al., 2013; Hansen et al., 2018), commonly by two glycine-binding GluN1-subunits (encoded by GRIN1 gene) combined with two glutamate- (GRIN2A-D) or glycine-binding (GRIN3A-B) subunits. GluN1 subunits are an obligatory subunit of all channel types and are therefore found throughout the brain and during all stages of life, whereas GluN2B- and GluN2D-subunits are particularly abundant during embryonic stages (Paoletti et al., 2013; Hansen et al., 2018; Xu and Luo, 2018). Conversely, GluN2A and GluN2C-subunits increase after birth, and GluN2A further replaces GluN2B during maturation of excitatory synapses (Paoletti et al., 2013; Lau and Zukin, 2007). This variety of subunits gives rise to a large combinatorial diversity of channel subtypes in neurons (Paoletti et al., 2013; Lau and Zukin, 2007) (but also glia [Verkhratsky and Kirchhoff, 2007]) with each channel-type displaying unique biophysical and pharmacological properties, and differential patterns (and timing) of expression (Paoletti et al., 2013; Hansen et al., 2018). Hence, owing to their essential roles in the brain, dysfunctional GluNRs are highly associated with a myriad of diseases of the brain (XiangWei et al., 2018; Yuan et al., 2015).

Since 2010, thousands of different GRIN variants have been discovered in pediatric patients (Xu and Luo, 2018; XiangWei et al., 2018; Myers et al., 2019), with the majority of mutations predominantly concentrated within the GRIN2A and −2B genes (46% and 38%, respectively) (XiangWei et al., 2018; Myers et al., 2019; García-Recio et al., 2021). To date, a small fraction of mutations have been functionally characterized, notably <15% reported for GRIN2B (Xu and Luo, 2018; XiangWei et al., 2018; Tang et al., 2020; Platzer et al., 2017) (see full list of mutation in Supplementary files 1 and 2). Pathogenic variants in GRIN genes cause severe encephalopathies, with complex and overlapping clinical pictures involving intellectual disabilities (ID), neurodevelopmental delays (DD), autism spectrum disorders (ASD), movement and language disorders, schizophrenia, seizures, epilepsy and more (XiangWei et al., 2018; Yuan et al., 2015; Myers et al., 2019). Currently, there are no specific treatments for different GRIN-diseases, rather patients receive supportive care and/or are specifically treated for the different co-morbidities of the disease (e.g. anti-seizure medication). Nevertheless, in the case of Gain-of-Function (GoF) mutations, there are several exploratory treatments with non-specific GluNR-blockers as primary candidates, notably the FDA-approved drug memantine (Pierson et al., 2014; Strehlow et al., 2019; Amador et al., 2020; Lipton, 2006). Loss-of-Function (LoF) mutations are harder to treat, as there are few channel openers, and even fewer subunit-selective (e.g. Tang et al., 2020; Mony et al., 2009; Costa et al., 2010; Perszyk et al., 2018; Warikoo et al., 2018). Moreover, none are FDA-approved (Wilkinson and Sanacora, 2019; Silva et al., 2019). A recent report suggests an alternative approach consisting of the use of L-serine for enhancing channel activity (Soto et al., 2019). L-serine is converted to D-serine; the co-agonist of the GluN1 subunit (Neame et al., 2019). This supplementation was shown to improve the condition of children with a mild GRIN2B LoF mutation (<sevenfold reduction in glutamate affinity) (Soto et al., 2019) and is currently under clinical trials (de Déu, 2020). However, it is worth noting that before treatments can be suggested (even if experimental), it is essential to understand the effects of the mutations on channel function (e.g. GoF vs LoF) because of the potential to worsen symptoms and provoke devastating effects (i.e. excessive channel activation, unwarranted cellular excitability, cytotoxicity, or neurodegeneration [Hardingham et al., 2002; Yan et al., 2020; Choi, 1987; Lipton and Rosenberg, 1994]) if, for example, an ‘opener’ is provided to treat a GoF mutation. These highlight the importance in curating and functionally characterizing each mutation before treatment(s) can be formulated (García-Recio et al., 2021).

Here, we provide functional characterization of two de novo GRIN2B mutations, occurring at the same residue (p.G689) in two pediatric patients; an Israeli patient with a G689C mutation and a Danish patient with G689S. The two patients have been diagnosed with a severe GRIN2B-encephalopathy, both showing similar—but also diverging—clinical symptoms (Table 1). We surveyed the literature and found that the G689C is a novel mutation, whereas the G689S variant has been noted in two previous patients though, to the best of our knowledge, characterization of the G689S-variant has not been reported (Platzer et al., 2017; Bosch et al., 2016).

We simulated the structures of the LBD with G689C or G689S and find the two variants to destabilize the glutamate-binding pocket; suggesting them to prompt LoF. Indeed, scrutiny of channel function showed that both variants show very strong reductions in glutamate potency, with G689S presenting a more drastic shift in EC₅₀ (G689C- ~1000-fold and G689S- ~2000-fold reduction). We also find that while G689C express poorly at membranes of cells (and therefore yields lower current amplitudes), G689S does not. The latter stands in contrast to the common notion that reduced glutamate potency correlates with reduced surface levels of the receptors (Swanger et al., 2016; She et al., 2012). Yet, despite differences in expression levels of the variants, both enhance the expression of GluN2Bwt-subunits and exert a very strong dominant-negative effect on receptor function. In primary neurons, these features translate into reduced synaptic NMDAR-dependent currents. We go on to explore channel potentiators (e.g. spermine), however, find that both variants fail to respond under physiological conditions. Scrutiny of the reasons behind this observation led us to discover that G689 is an essential residue in the elusive proton-sensor of the GluN2B subunit. Together, we describe two exceptional mutations in two patients exhibiting a diverging clinical picture. We demonstrate the two first cases showing LBD-mutations that exert a bona-fide dominant negative effect in oocytes and primary cultured neurons. Lastly, we estimate the effect of more than 5200 mutations on the stability of the LBD and these data help to explain why most LBD mutations instigate Loss-of-Function and further suggest that substitutions to glycine or serine are the most damaging to the LBD.

Here, we describe, and characterize, two de novo heterozygous GRIN2B loss-of-function (LoF) variants—G689C and G689S—in two pediatric patients (Figure 1a). Both patients display severe DD, ID, dyskinetic movements, and speech impairment (Table 1); four very well-documented phenotypes observed in GRIN2B patients (Myers et al., 2019; Platzer et al., 2017). We also find that the G689S-patient exhibits epileptic seizures, consistent with the two previously documented G689S-patients (Platzer et al., 2017). Interestingly, the G689C-patient does not show seizures, but the clinical likelihood of this young patient to develop epileptic seizures remains relatively high (Myers et al., 2019; Platzer et al., 2017; Devinsky et al., 2018). Along with the known comorbidities of GRIN2B patients, here we report several phenotypes that deviate from reported symptoms. The G689S patient displays hypertonia, rather than hypotonia (Myers et al., 2019; Platzer et al., 2017), as is also displayed by the G689C patient. Moreover, while movement disorders are common in GRIN patients, the G689C patient (but not the G689S patient) is also reported to exhibit hypermobility/hyperflexibility. The reason for this divergence between these two synonymous mutations is poorly understood (as both mutations are highly analogous and instigate similar LoF). However, it is highly common for GRIN-patients with similar (or even identical) mutations to show diversifying symptoms (Myers et al., 2019; García-Recio et al., 2021; Platzer et al., 2017; Strehlow et al., 2019; Supplementary file 2) or, inversely, for patients with different mutations (even in different GRINs) to display overlapping symptoms (Myers et al., 2019). These phenotypes are likely to involve additional genetic and environmental factors. In the case of the G689S patient, we do find (by whole-exome sequencing; WES) two additional variants of unknown/uncertain significance (VUS), specifically the SLC6A8 (creatine Transporter) and CACNA1A (voltage-gated calcium Channel) genes. However, whether these are directly involved or implicated in the disease remains unknown. Nonetheless, no VUS, pathogenic variants, nor aberrations in the Chromosomal Microarray Analysis (CMA) were detected in screens of the G689C patient (Table 1). Resultantly, we and others suggest that patients' symptoms cannot serve as primary diagnostic measures for GRIN-related encephalopathies (e.g. [Zehavi et al., 2017]). Indeed, no formal diagnostic criteria for GRIN2B-related neurodevelopmental disorder have been established (Platzer and Lemke, 1993; revised 2021).

An additional puzzling observation shows that the severity of phenotypes does not correlate with the magnitude of the effect of the mutation on channel function (Tang et al., 2020). In fact, despite the critical LoF effect of the mutations described here, the clinical phenotypes resemble many other GRIN2B LoF mutations (Supplementary file 1; Swanger et al., 2016; Bell et al., 2018). For instance, GluN2B-E413G inflicts a much smaller reduction in glutamate potency (EC₅₀ = 79 ± 5.3 μM), though manifests with severe phenotypes as described here for the two variants. This dissonance is reflected in a recent GRIN2B study (consisting of a cohort of 86 patients), in which they found no clear association between: (1) effect of mutation (GoF or LoF), (2) extent of effect (e.g. shift in EC₅₀), and (3) localization of the mutations in the subunit, with the clinical outcomes (Platzer et al., 2017). The only significant correlation obtained was between variant class (i.e. missense or truncation mutation) and intellectual outcome (mild to moderate moderate vs severe ID); with truncation carriers tending to present mild/moderate intellectual disability. However, it is not really unexpected for these symptoms to appear in GRIN2B cases, owing to the subunit’s very early (embryonic) expression pattern (Paoletti et al., 2013; Platzer et al., 2017; Myers et al., 2019). In contrast, analysis of a larger cohort of GRIN2A patients (n = 248) finds only two distinct phenotypes that could be associated with the location and effect (gain or loss—but not size of effect) of the mutations (Strehlow et al., 2019). More precisely, the authors show that: (1) mutations in TMDs and connecting linkers yield GoF (although to very different extents) and these may be associated with broad developmental and epileptic encephalopathy phenotypes. (2) Mutations in the two large extracellular domains (ATD, LBD) typically instigate LoF and are better related with speech abnormalities and seizures, with mild to no ID (Strehlow et al., 2019). These observations highlight the need for larger cohorts in order to establish better correlations (if any).

Despite these associations, it remains challenging to infer how, and to what extent, a mutation may affect receptor function or its expression solely based on clinical symptoms or even based on the location of the mutation within the protein (XiangWei et al., 2018). In our case, in silico scrutiny of the G689 residue shows it to reside at the lower lobe of the LBD of the GluN2B subunit (S2 domain)—at the base of the glutamate entry tunnel—virtually lining the glutamate binding pocket (Figure 1b and Figure 1—figure supplement 1). When the location of the residue is combined with structure simulations of the variants (Figure 1c,d, Figure 1—figure supplement 1b and Figure 1—figure supplement 2), estimation of protein stability (ΔΔG; Figure 1—figure supplement 4), and considering that most GluN2B mutations in LBD reduce glutamate potency (Supplementary file 1 and 2; XiangWei et al., 2018; Tang et al., 2020; Platzer et al., 2017; Swanger et al., 2016; Vyklicky et al., 2018), it was safe to assume that the variants would yield LoF. However, these could not have predicted the extent of the effect, explicitly >1000-fold and ~2000-fold by G689C and G689S, respectively (Figure 2, Figure 8—figure supplement 1 and Table 2). Potential reasons why we could not anticipate these large shifts are likely because they represent two very extreme cases (only second to another potent GRIN2A mutation D731N; ~6000-fold reduction [Swanger et al., 2016]) and because of the very few characterized LBD-mutations in GRIN2B (~20 mutations, See Supplementary file 1 and 2). With these limitations in mind, we decided to explore the functional outcome of any possible mutation within the LBD (consisting of ~280 a.a.); yielding a collection of 5282 substitutions. This large number made it impractical for us to employ structure simulations, thereby motivating us to proceed with ΔΔG estimations (Pires et al., 2014). We calculated ΔΔG for 5282 substitutions within the LBD of GluN2B (Supplementary file 3, closed circles) and immediately note that most of the substitutions yield a negative outcome on protein stability (ΔΔG < −0.5; Supplementary file 3a, red region). Substitutions with a stabilizing nature appeared in a much smaller fraction of cases (~20%) (Supplementary file 3a, b -blue). We explored the relationship between characterized LBD mutations with our ΔΔG estimates, however there are too few characterized mutations (18 mutations) to address this (Supplementary file 3c). However, we can observe that LoF mutations correlate better with more negative ΔΔG, whereas GoF mutations are associated with less negative ΔΔG (albeit this is based on two out of three characterized mutations) (Supplementary file 3c). To examine whether there are locations within the LBD that might be more vulnerable for mutagenesis, we estimated each residue’s relative contribution to the stability of the LBD (by averaging the ΔΔG of 19 substitutions for each residue, see Materials and methods) and plotted these on the structure. Unfortunately, we do not observe negative hot-spots nor did the 18 characterized mutations show a pattern in the LBD (Supplementary file 3d). However, the data does suggest that stabilizing mutations are likely to locate on the outer layers of the LBD (Supplementary file 3e, blue and dashed blue circle). Indeed, these estimations encompassed all three GoF mutations. Interestingly, and perhaps with the most predictive nature, this analysis emphasizes that mutations resulting in a glycine or a serine—no matter the residue they replace in the LBD—are likely to be the most damaging (Supplementary file 3f). This is highly reasonable as these amino acids are very small (Figure 1—figure supplement 4), especially glycine, and their incorporation in-lieu of other (larger) residues should be very destabilizing. Interestingly, the same may apply inversely, namely removal of glycine and its substitution by other (larger) residue should be highly disfavorable for receptor function. In support, glycine residues are suggested to serve as essential hinges in GRIN2B and their mutagenesis causes severe channel dysfunction (Amin et al., 2018), including the two cases described here.

Another elusive feature for prediction is expression of variants, especially when the mutations do not lead to truncation (García-Recio et al., 2021). It has been previously shown that high glutamate affinity is associated with proper surface trafficking (She et al., 2012; Horak et al., 2014; Wang et al., 2015; Kenny et al., 2009). Thus, it can be assumed that since most LBD mutations yield reduction in EC₅₀, most should also show reduced expression levels. This assumption correctly predicts the effect of the G689C mutation (Figure 3a,b), but completely fails to explain the robust expression of the G689S variant with an even larger reduction in EC₅₀ (Figures 2 and 3, Figure 8—figure supplements 1 and 2). This assumption (She et al., 2012; Vyklicky et al., 2018) further neglects to explain how both low-affinity variants enhance the expression of the wt subunit (Figures 3c and 4 and Figure 4—figure supplement 1). Thus, our results describe two novel cases in which intracellular glutamate-binding is not essential for proper trafficking of GluN2B-subunits to the membrane and cautions the use of glutamate affinity for predicting expression levels and/or functional effect. Together, the functional outcome of our observations suggests that during normal synaptic transmission, tri-heteromeric receptors assembled from GluN2Bwt and mutant variants, are non-responsive to normal neurotransmission (Figures 4 and 8; Lisman et al., 2007). In fact, regular neurotransmission does not typically saturate wildtype receptors (Ishikawa et al., 2002; Nimchinsky et al., 2004). Thus, we suggest that the severe clinical phenotypes observed for both variants arise from a complex combination of: (1) severe LoF of channel consisting of two copies of the variants, (2) poor trafficking to membrane, (3) co-assembly and exertion of dominant-negative effect over native GluN2Bwt-containing receptors.

With the intention to provide a potential treatment, we examined whether spermine would potentiate the currents of the variants. Of note, we focused on spermine as it is highly GluN2B-selective (Mony et al., 2011), unlike other potentiators (e.g. tobramycin, pregnenolone-sulfate; PS or D-serine) that can exert non-specific effects on other GluN-subunits or even other glutamate-receptors (Tang et al., 2020; Swanger et al., 2016; Stoll et al., 2007; Masuko et al., 1999). Secondly, spermine can cross the blood-brain-barrier to certain extents (BBB [Shin et al., 1985; Diler et al., 2002]), and could potentially be administered orally or intraperitoneally (e.g. Okumura et al., 2016; Guerra et al., 2016). Third, it is inexpensive. Importantly, previous reports showed that spermine acted on LoF GluN2B variants in a similar fashion as they do on GluN2Bwt-subunits (Swanger et al., 2016). However, and strikingly, we found that G689C- and G689S-containing channels poorly respond to the reagent under physiological conditions; with the G689S-mutant even undergoing strong inhibition by spermine (Figure 5a,b). We go on to demonstrate that reduced spermine-sensitivity stems from the variants’ reduced pH-sensitivity (Figure 6 and Figure 5—figure supplement 2) (but not disrupted binding domain; Figure 5—figure supplement 1). This suggests that the G689 residue is directly involved in proton-sensing in the GluN2B subunit (Low et al., 2003; Banke et al., 2005; DeCoursey, 2018). This feature adds another layer of complexity to the growing list of effects exerted by these unique mutations. These observations suggest that though spermine (or potentially other GluN2B-positive allosteric modulators [Mony et al., 2009; Burnell et al., 2019; Zhu and Paoletti, 2015]) may be useful in other cases of GluN2B-LoF mutations, it is not suitable for treating G689C- or G698S-induced deficiencies at the synapse.

We similarly tested another suggested potentiator, namely L- (but de facto D)-serine (Soto et al., 2019). D-serine was suggested to act as potentiator owing to its ability to increase currents of a mild LoF GRIN2B variant (showing ~seven-fold reduction in EC₅₀). We applied 100 μM D-serine onto receptors activated by sub- or saturating glycine concentrations (Figure 7), but D-serine enhanced the currents of the variants solely when glycine concentrations were sub-saturating (see glycine EC₅₀, Figure 2—figure supplement 2). Thus, it appears that D-serine does not act as a classic potentiator (i.e. [Tang et al., 2020; Mony et al., 2009; Burnell et al., 2019; Hackos et al., 2016]). Instead, it is an equipotent ligand for the GluN1a subunit and the observed increases in currents (up to 60%) are obtained by saturation of GluN1 (glycine or D-serine; EC₅₀: ~0.7 [Traynelis et al., 2010]). Regardless the exact definition of the mechanism by which D-serine augments the currents, under physiological conditions increase in the extracellular D-serine concentration would likely lead to further opening of the channels, as resting glycine (and D-serine) extracellular concentrations may be sub-saturating; 1–5 μM (Zhang et al., 2018; Hashimoto et al., 1995; Lazarewicz et al., 1992). However, and even in cases where GluN1a subunits are not fully saturated by glycine, the G689C or G689S mutations require very high (mM) glutamate concentrations for opening and, therefore, increase in D-serine (via L-serine supplementation) is ineffective (Figure 7c,d). We therefore do not recommend the use of L-serine in this, or other extreme LoF mutations, as L-serine may acts on the obligatory GluN1-subunit found in all receptor combinations and possibly induce side effects.

In summary, we have systematically characterized two unique mutations occurring at the same residue of the GRIN2B gene in two patients. The variants exert a strong dominant-negative effect over wt-subunits, leading to reduced potency of mixed channels and reduced synaptic GluNR-currents (and compensation by AMPARs [Sutton et al., 2006]). To make things worse, the variants are resistant to protons, thereby limiting the use of spermine. We provide an assessment of the stability of the LBD for all possible mutations within the LBD. These exemplify the vulnerability of the LBD to mutagenesis, particularly for insertion (but also deletions) of glycine and serine. Together, our study complements ongoing efforts invested in characterizing GRIN variants (appearing faster than can be functionally tested, Supplementary file 2), provide insights concerning the structure-function relationship of GluN2B and underscore the need for new, more potent, GluN2B-specific channel openers.

All data generated or analysed during this study are included in the manuscript and supporting files. Raw data is deposited at Dryad (https://doi.org/10.5061/dryad.1jwstqjv3).

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Acceptance summary:

This interesting study characterizes NMDA receptor mutants in regard to ion channel physiology and to functional consequences from clinical impairments of two pediatric cases. A strength of the study is that missense mutations are analyzed with respect to ion channel receptor function. The mechanism of a dominant negative effect on NMDA receptor function is proposed. A combination of modeling and electrophysiological methods is used to support the key conclusions of the paper.

Decision letter after peer review:

Thank you for submitting your article "Two de novo GluN2B mutations affect multiple NMDAR-functions and instigate severe pediatric encephalopathy" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Huda Zoghbi as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this letter to help you prepare a revised submission. Detailed comments are below.

Essential revisions:

The study was considered to be well done and to be of interest. Positive comments were also raised about the nature and details of the findings. Several major reservations were raised. There have been many characterizations of NMDAR variants that have been published. Given the two missense mutations in the agonist binding site of GluN2B, the conclusions should be strengthened by additional pharmacological or translational insights to delineate the novelty and impact of the results.

1) Either a novel insight into the function of NMDARs or a new clinical insight would greatly enhance the impact of the manuscript. There are many papers being generated on this topic that draw correlations between functional characterizations and clinical phenotypes. We are not certain that they provide new insights into the etiology of the disease phenotype or potential treatment options.

2) One point to address is the nature of the dominant negative mechanism. A discussion of significance should make clear the novelty of the dominant negative mechanism of suppression of receptor currents by the mutations studied here. How is this mechanism distinguished from other disability-causing mutations that have been reported elsewhere. The NMDA receptor does not open unless all four LBDs are occupied. Therefore, the incorporation of one mutant NR2B into the hetero-tetramer can block function. So mutant NR2B Is dominant over wild type NR2B, in the same tetramer. Is this novel or are most NMDA receptor mutations like this?

Reviewer #1:

NMDA receptors are glutamate-gated ion channels that make significant contributions to a variety of brain functions. Recently, de novo missense mutations have been identified in genes encoding NMDA receptor subunits and are often associated with severe clinical phenotypes. How these missense mutations lead to clinical phenotypes is unknown and one way to address this is to characterize their effect on receptor function. In the present manuscript, the authors characterize the effect of two missense mutations at a glycine in the agonist binding pocket of the GluN2B subunit. The authors find that the mutations have different effects on receptor function and propose that these differences may contribute to variations in their associated clinical phenotypes.

The manuscript is a positive characterization of two missense mutations at a site in the agonist binding pocket of the GluN2B subunit. The data are quite reasonable and are displayed appropriately. The general conclusions are appropriate.

Potential impact of the manuscript. While a nice characterization, many of these have been done and it is not clear that the results in the present manuscript provide a high impact insight. Perhaps it is there but the authors need to do a better job of demonstrating that they have made a significant new discovery about NMDA receptors, either in terms of structure-function or clinical aspects. Many characterizations of missense mutations in NMDA receptors have been done and have provided new insights into how receptors carry out their biological function (e.g., Odgen et al., 2017, PLoS Genetics, Amin et al., 2018, Nat Comm; Fedele et al., 2018, Nat Comms) or how they might function at synapse (e.g., Liu et al., 2017 J. Neurosci.). Other pathways are available to provide impact, e.g., providing some new therapeutic insight or potentially how they affect signaling in vivo but these are absent in the present version.

Again, perhaps there is buried in the manuscript some new insight but at present it feels like a number of observations (e.g., that has a very strong effect on glutamate potency or have effects on pH sensing) with no clear advance in terms of mechanism. Also in terms of relating the observed effects of the missense mutations on receptor function to clinical phenotypes is always challenging and is really just speculation unless a large number are done.

Reviewer #2:

This is a well-written manuscript in which the authors have identified two patients with missense mutations in the GRIN2B gene, substituting a highly conserved glycine residue. They find the mutations produce marked reductions in glutamate potency, one showing the largest reduction in EC50 reported for GRIN2B-mutations (~2000-fold reduction). Variants readily multimerize with the GluN2Bwt- subunits, promote their surface expression and exert a dominant- negative effect on receptor function. Interestingly, one variant entirely fails to respond to spermine, a specific GluN2B-potentiator, at physiological pH. The manuscript provides important contribution to the understanding of functional consequences of two specific mutations of the GRIN2B gene. As such the manuscript is a great example of how to integrate knowledge on basic receptor function and personalized medicine. This is indeed achieved by combining sophisticated modeling and electrophysiological techniques that have permitted addressing of important aspects of receptor function which has been investigated in depth by a large variety of experiments that were well executed. Because of the clinical relevance of the study, the research and clinical community should learn from this manuscript and be interested in it. This reviewer could not identify any weakness in the manuscript.

Reviewer #3:

Kelner et al. have studied the effects of naturally occurring mutations in the ligand binding domain of the GluN2B subunit of the NMDA receptor. Two mutations at the same residue, G689S and G698C, were identified in two pediatric patients with severe intellectual disability and delayed neural development. The authors describe the clinical manifestations of these mutations and investigate the molecular and physiological consequences through expression in oocytes and mammalian cells.

The GluN2B crystal structure suggested that the mutations would occlude glutamate binding, and indeed, while the EC50 (glutamate concentration for half maximal current) of the wt was in the μM range, the EC50 was 1000 x higher for 2B-G689S and 2000 x higher for 2B-G698C, with both in the millimolar range. This indicates reduced affinity of glutamate for the mutant receptors leading to loss of function, although at saturating glutamate concentrations (10mM), Imax for the mutants and wt were similar.

Experiments in 293 cells showed that surface expression of GluN2B-G689S was similar to wt, and GluN2B2B-G689C was 45% of this value. Both variants increased the surface expression of wt subunits, indicating that both mutants could multimerize with wt and GluN1a to form surface-expressed heterotetramers. Expression of increasing levels of mutant with an excess of wt progressively depressed currents and increased KD values, supporting co-assembly of mutant and wt into heterotetramers resulting in loss of function through a strong dominant negative effect on glutamate affinity, but a much lower effect on co-assembly and surface expression.

Analysis of dose-response curves indicated that co-expression of wt and mutant led to the formation of two receptor populations, one wt and the other incorporating a mutant receptor.

In an attempt to find a pharmacologic treatment that overcame the diminished currents of the mutants, the authors treated oocytes expressing hetero-tetramers with spermine, a GluN2B-specific potentiator. However under conditions for which spermine potentiates wt receptors, spermine poorly potentiated the mutants. Furthermore, decreasing the pH, which greatly increased spermine potentiation of wt, only modestly increased mutant receptor currents in oocytes. Experiments with a competitive antagonist of spermine indicated that the spermine binding site was intact in the mutants. Further, the mutants were more resistant to proton inhibition, which suggested that residue 689 is part of the proton sensing domain.

Other experiments showed that while D-serine, another possible potentiator, increased currents of wt and of the mutants, this increase was eliminated in the presence of the co-agonist, glycine, indicating D-serine simply augments filling the glycine site in GluN1a, rather than acting as a true potentiator.

Finally, the current reduction by GluN2B-G689C seen in oocytes was reproduced in the mammalian 293 cell line, confirming the relevance of the oocyte studies.

The paper presents a comprehensive analysis of the changes in physiological and cell biological properties of two GluN2B subunits with LBD mutations that are associated with intellectual disability and delayed development, and in one case, epileptic seizures. The data support a mechanism of dominant negative inhibition of NMDA receptor function, in which the mutant subunit assembles with wt GluN1a and wt GluN2B to form tri-heterotetrametric receptors that traffic to the plasma membrane but fail to conduct because they have very low affinity for glutamate.

In addition to the graphic presentation of the data in Figures, the data is also presented numerically in Tables in a comprehensive form. Also summarized in Tables are the clinical assessments of the two proband patients from whom the mutants were isolated.

The patient with the G689S mutation also had mutations in SLC6A8 gene (sodium- and chloride-dependent creatine transporter 1) and CACNA1A gene (Calcium Voltage-Gated Channel Subunit Alpha1A). It is not known whether these mutations contributed to the clinical phenotype. Similar data are not available for the G689C mutation patient.

The paper provides considerable insight into the mechanism by which GluN2B mutations exert a strong, dominant negative effect on NMDA receptor function. The paper does not address how these mutations affect function in neurons, either in vivo or in vitro.

This article presents a set of well-chosen and well executed experiments that supports a dominant-negative mode of action of mutant NMDA receptor GluN2B subunits that cause severe intellectual disability.

Although not necessary for this submission or for drawing the main conclusions of the article, experiments in neurons, either in vivo in genetically modified mice, or in cultured neurons, would enhance this report. Experiments in oocytes and heterologous cells, although providing valuable information about receptor assembly, trafficking and electrophysiological function, cannot take into account factors that arise only in vivo in a neuronal context. These include the developmental patterns of expression of the various NMDA receptor subunits and binding proteins, and neuron-specific interactions including ones at synapses. Also, the effects of the mutations on synaptic plasticity, such as LTP/LTD, cannot be assessed.

The conclusions of a number of the experiments depend on expressing wt and mutant receptors at comparable or other predetermined levels, and this can be affected by the quality of oocyte-injected mRNA, differences in expression efficiency and protein stability etc. Indeed, the mutant receptors are not expressed at the surface with the same efficiency and have differences in expression and/or stability. The authors should discuss more fully how these parameters were addressed.

The authors discuss the effects of the mutations on the stereochemistry of the ligand binding site occupied by glutamate. But because the mutations occlude binding glutamate, it would be interesting to consider the LBD structure of the mutants in the unliganded state, if the appropriate crystal structure were available. This might suggest a pharmacologic (small molecule) means for closing the clamshell and opening the pore of the mutant, even in the absence of bound glutamate.

The speculation about the effects of water access on deactivation kinetics (p 5), which turned out to be incorrect, interrupts the flow of the Results section and might be considered better in the Discussion.

The manuscript has a significant number of typographical errors and a large number of grammatical errors or non-colloquial phrasings. It should be revised carefully by a native English speaker.

Essential revisions:

The study was considered to be well done and to be of interest. Positive comments were also raised about the nature and details of the findings. Several major reservations were raised. There have been many characterizations of NMDAR variants that have been published. Given the two missense mutations in the agonist binding site of GluN2B, the conclusions should be strengthened by additional pharmacological or translational insights to delineate the novelty and impact of the results.

1) Either a novel insight into the function of NMDARs or a new clinical insight would greatly enhance the impact of the manuscript. There are many papers being generated on this topic that draw correlations between functional characterizations and clinical phenotypes. We are not certain that they provide new insights into the etiology of the disease phenotype or potential treatment options.

We thank the reviewers for highlighting the issue of novelty. We now realize to have clearly downplayed the importance of our findings. We would therefore like emphasize that our study not only provides description and characterization of two novel GRIN2B variants, it also provides several new insights into NMDAR function, dominant effect of variants over tri-heteromeric channels and effect in neurons. These are now specifically highlighted throughout the text.

Briefly, the novelties we report are:

1. We describe two mutations that lead to the most extreme cases of reduction in glutamate potency of the GluN2B subunit (and only second to a GRIN2A mutation). We therefore expected these mutations to mirror effects of mutations that completely abolish expression of the subunit (e.g., truncation mutations¹). This would suggest that the disease could be associated with cases of haploinsufficiency (as commonly suggested in literature^2–4). Instead, we observe the opposite. First G689S expresses as well as the native subunit and, although G689C expresses less, both variants significantly reduce glutamate potency of mixed channels (Figures 2 and 3). Additionally, both variants promote the expression of 2Bwt subunits (Figure 3). In neurons, the variants affect synaptic currents (significantly alter frequency and amplitude of miniature EPSCs; Figure 8 and Figure 8—figure supplement 2). These are best explained by a very strong dominant-negative effect (and see next reply specifically addressing the novelty of the dominant negative effect).

2. We provide new insights pertaining to glutamate-affinity and expression levels. Briefly, high affinity is not essential for proper membrane trafficking. This is very different than most LoF mutations in the LBD and argues against classification of the latter as haploinsufficiency (now explicitly noted in page 11). In fact, we deem this as a major issue as the medical community often confuses between these terms (For an explicit example see our reply for query #2).

3. We discover that the G689 residue is involved in proton sensing of the GluN2B-subunit.

4. We present new simulations showing that residues that protrude towards the glutamate and shift its position are likely behind the significant decrease in glutamate-affinity (Figure 1 and Figure 1—figure supplement 1).

5. Moreover, we present ∆∆G predictions for ALL possible mutations in the LBD (consisting of 5282 mutations; we provide the scripts and graphical analysis in Suppl. File 3). These suggest that most substitutions (~60%) within the LBD destabilize its structure; providing plausible explanation why most LBD mutation lead to reduced affinity to glutamate (i.e, LoF). The data also show that substitutions to glycines or serines are the most destabilizing. Interestingly, the same may apply inversely, namely removal of glycine and its substitution by other (larger) residues should be highly disfavorable for receptor function. In support, glycine residues are suggested to serve as essential hinges in GRIN2B and their mutagenesis causes severe channel dysfunction⁵, including the two cases shown here (pages 18-19)

Per the second part of the question (potential treatment), we would first like reiterate that this was our primary goal and the reason behind the scrutiny of specific and non-specific channel potentiators (spermine and D-serine, respectively). These failed to enhance receptor function (but provided valuable insights into proton sensitivity). Notably, whereas most therapeutics target GoF mutations, and therefore employ channel blockers (most commonly FDA-approved pan-NMDAR blocker memantine, e.g., ^3,6–10, pages 3-4)., there are very few openers (e.g., ^11–13) and even fewer GluN2B-selective.

Nevertheless, to try and develop a potential treatment, we wanted to explore gene editing techniques, specifically base editing¹⁴ (see details below). We examined all possible substitutions that can be made:

WT – p.689 – GGC,

G689C – p.689 – TGC

G689S – p.689 – (AGC)

C•G to T•A base editors (CBEs)-

G689C – p.689 – TGT = maintains Cysteine mutation

G689S – p.689 – AGT = maintains Serine mutation

A•T to G•C base editors (ABEs)

G689C – p.689 – TGC – none-modifiable

G689S – p.689 – GGC – Glycine **restores to normal**

C•G to G•C base editors¹⁵

G689C – p.689 – TGG – modifies to Tryptophan (W)

G689S – p.689 – AGG – modifies to Arginine (R)

Whereas only G689S can be restored to normal (i.e., glycine) by using A•T to G•C base editors (ABEs), G689C cannot. G689C can only be modified to tryptophan using C•G to G•C base editors¹⁵ (which would produce an arginine if applied onto G689S). We produced both of these variants and tested in oocytes and find that both yield very low expression levels/complete loss of function. We would be happy to include these results, even if negative should the reviewers deem it interesting.

Lastly, we address the comment about genotype-phenotype correlations by several ways. First, we provide an extensive analysis of the literature, collating all reported GRIN2B mutations (using PubMed, ClinVar and CFERV databases; described in text- page 17 and shown in Suppl. File 2), as well as focus on LBD mutations (Suppl. Table 1). Unfortunately, there are too few LBD mutations with reported characterizations (<20), and not all mutations show a clear binary effect (e.g., I655F mutation acts both as LoF mutation – instigating ~3-fold reduction in glutamate EC₅₀, as well as a GoF mutation –by reducing Mg²⁺-sensitivity) from which we can draw any firm conclusions (Suppl. File 3). We also examined several correlations with the various clinical phenotypes though fail to find any clear associations. These observations are not completely unexpected (and the reason why we have initially left them out of the manuscript), as similar analyses have been previously performed and they all provide a very limited genotype-phenotype associations (e.g., ^2,7,16; see below for specific details). Resultantly, there is lack of formal diagnostic criteria based on clinical phenotype for GRIN2B-related disorders (¹⁷). We now note all these in discussion (pages 16-17), in particular the current consensus regarding the strong evidence that mutations in GRIN2B are more associated with developmental delays and autism spectrum disorder, whereas GRIN2A more associated with epilepsy (² and “Clinical and genetic spectrum of GRIN2A and GRIN2B variants.” Dr. Johannes Lemke. University of Leipzig. Presented at 2019 CFERV Conference on GRIN Variants. Atlanta. 2019).

Details:

A recent analysis of the largest GRIN2B patient cohort (86 patients) found no clear association between: (1) effect of mutation (GoF or LoF), (2) extent of effect (how much gain or loss compared to wt), (3) localization of the mutations and clinical phenotype (⁶ and Clinical and genetic spectrum of GRIN2A and GRIN2B variants.” Dr. Johannes Lemke. University of Leipzig. Presented at 2019 CFERV Conference on GRIN Variants. Atlanta. 2019). The only significant correlation obtained was between variant class (i.e., missense or truncation) and intellectual outcome (mild to moderate moderate vs severe ID) (Fisher’s exact test, p=0.0079) with truncation carriers tending to present mild/moderate intellectual disability. This is not completely unexpected as this appears to be the only recurring theme for GRIN2B mutations (as we have mentioned in the text) owing to its embryonic expression. With regard to GRIN2A, analysis of a larger cohort (248 patients) found only two distinct phenotype groups that could be explained by the location and effect (gain or loss – but not size of effect) of the mutations⁷. More precisely, the authors show that most mutations in the TMDs (and there are 4 TMDs) or linkers are associated with broad developmental and epileptic encephalopathy phenotypes and these mutations are also typically associated with GoF of the receptors (though to very different extents), whereas mutations in the two large extracellular domains (amino terminal domain and ligand binding domain) are associated with speech abnormalities and/or seizures with mild to no ID only; and these typically instigate LoF⁷ (these are now mentioned in page 17).

2) One point to address is the nature of the dominant negative mechanism. A discussion of significance should make clear the novelty of the dominant negative mechanism of suppression of receptor currents by the mutations studied here. How is this mechanism distinguished from other disability-causing mutations that have been reported elsewhere. The NMDA receptor does not open unless all four LBDs are occupied. Therefore, the incorporation of one mutant NR2B into the hetero-tetramer can block function. So mutant NR2B Is dominant over wild type NR2B, in the same tetramer. Is this novel or are most NMDA receptor mutations like this?

We agree with the reviewers that the dominant-negative effect should be further discussed, as it is unique as it diverges from the effect of other reported mutations. We now provide these details in pages 10-11 and in the discussion. We highlight the fact that a dominant-negative effect in GRINs is not commonly reported. In support, only a handful of reports (three) explicitly describe or note a dominant-negative effect for three GRIN variants (GRIN1¹⁸, GRIN2A¹⁹ and GRIN2B²⁰). In two reports (^18,19) the authors interpret reduced current amplitudes (~50%) as an indication for dominant negative effect, whereas the third shows that a single copy of GluN2B‐N616K produces a dominant reduction in Mg²⁺-block similar to channels including two copies of the variant²⁰. However, most reports examining other GRIN mutations do not describe dominance, even though we expect the least functional subunit to be the limiting factor in mixed channels. For instance, a recent report examining eight different GRIN variants (M2-pore mutations) shows that mixed-channels exhibit very mild reduction in Mg²⁺ IC₅₀, with values corresponding to values of the wt channels²⁰. Another report examining mixed-channels containing GluN2Awt and 2A-P552R²¹ shows that, whereas 2A-P552R significantly alters stability of the pore when it is found in two copies per channel, it fails to do so when mixed with GluN2Awt. Very similar observations are reported for mixed channels bearing 2Bwt and the GluN2B-E413G variant in which the EC₅₀ is not dominated by the low affinity subunit ¹⁶. Thus, while a dominant negative effect is somewhat intuitive – as all LBDs of NMDARs need to be liganded for full channel opening^22–24 – it is not commonly observed in GRINs, especially not LBD mutations of GRIN2B.

Notably, following an extensive re-review of the literature, we found two additional reviews (specifically^25,26) that cite the reports mentioned above, and notice that they also cite additional ones. The reviews interpret the findings of the added reports as proof for dominant negative. However, close scrutiny of the added citations shows that the original reports do not show a dominant negative effect, nor suggest it in their original manuscripts Chen et al. ²⁶ refers readers to references #5,6-11, 13,16 and 18; Poot M²⁵, refers to Lemke 2016 and Platzer 2017. Thus, we still maintain that we show a unique dominant negative effect.

Significance of dominant negative findings – Our findings diverge from reports that examined the effect of variants on mixed channels (i.e., channel made of one copy of a wt-subunit mixed with another copy of the variant). First, previous reports reach this conclusion by relying on a single observation whereby mixed channels exhibit reduced (~50%) current amplitudes. However, as reduced currents can result from multiple reasons, such as expression. This motivated us to address this by multiple means. Indeed, we do not rely on a single DNA-mixture and current amplitude, rather explore different channel stoichiometries (by employing a highly established method of mRNA titrations, e.g., ^27–29) and assess current amplitudes, expression levels and, importantly, glutamate potency. These ultimately lead to the likely interpretation that mixed channels that exhibit normal current amplitudes (supported by expression assays, Figure 3) contain the wt subunits, but the very reduced EC₅₀ results from the limiting effect of the variants. We go on to confirm that our results do not reflect two distinct populations of channels (Figure 4). Notably, this is different than previous reports (see ¹⁶). Thus, we do not simply suggest a dominant negative, we explore it by numerous means and provide strong evidence to support it. Finally, though severe LoF are commonly equated to cases of haploinsufficiency (e.g., by null mutations and early stop codons), we show that this is not the case for these two variants. We now provide these details in the discussion.

Reviewer #1:

NMDA receptors are glutamate-gated ion channels that make significant contributions to a variety of brain functions. Recently, de novo missense mutations have been identified in genes encoding NMDA receptor subunits and are often associated with severe clinical phenotypes. How these missense mutations lead to clinical phenotypes is unknown and one way to address this is to characterize their effect on receptor function. In the present manuscript, the authors characterize the effect of two missense mutations at a glycine in the agonist binding pocket of the GluN2B subunit. The authors find that the mutations have different effects on receptor function and propose that these differences may contribute to variations in their associated clinical phenotypes.

The manuscript is a positive characterization of two missense mutations at a site in the agonist binding pocket of the GluN2B subunit. The data are quite reasonable and are displayed appropriately. The general conclusions are appropriate.

Potential impact of the manuscript. While a nice characterization, many of these have been done and it is not clear that the results in the present manuscript provide a high impact insight. Perhaps it is there but the authors need to do a better job of demonstrating that they have made a significant new discovery about NMDA receptors, either in terms of structure-function or clinical aspects. Many characterizations of missense mutations in NMDA receptors have been done and have provided new insights into how receptors carry out their biological function (e.g., Odgen et al., 2017, PLoS Genetics, Amin et al., 2018, Nat Comm; Fedele et al., 2018, Nat Comms) or how they might function at synapse (e.g., Liu et al., 2017 J. Neurosci.). Other pathways are available to provide impact, e.g., providing some new therapeutic insight or potentially how they affect signaling in vivo but these are absent in the present version.

Again, perhaps there is buried in the manuscript some new insight but at present it feels like a number of observations (e.g., that has a very strong effect on glutamate potency or have effects on pH sensing) with no clear advance in terms of mechanism. Also in terms of relating the observed effects of the missense mutations on receptor function to clinical phenotypes is always challenging and is really just speculation unless a large number are done.

We thank the review for the positive comments on our manuscript and understand his/her comment about list of observations. We would like to emphasize that we have not simply tested random features, rather the results of one observation led us to test the next. More specifically, our hypothesis regarding lower glutamate potency (based on location of mutation, simulations and literature³) led to the assessment of glutamate affinity (but not other features related to other domains, such as amino terminal domain). The smaller currents motivated evaluation of expression. Lastly, our aspirations to find a potential treatment led us to explore channel potentiators, which in turn led us to uncover the variants’ diminished proton-sensitivity and to elaborate on the agonistic actions of D-serine, rather than potentiator.

Reviewer #3:

[…] Although not necessary for this submission or for drawing the main conclusions of the article, experiments in neurons, either in vivo in genetically modified mice, or in cultured neurons, would enhance this report. Experiments in oocytes and heterologous cells, although providing valuable information about receptor assembly, trafficking and electrophysiological function, cannot take into account factors that arise only in vivo in a neuronal context. These include the developmental patterns of expression of the various NMDA receptor subunits and binding proteins, and neuron-specific interactions including ones at synapses. Also, the effects of the mutations on synaptic plasticity, such as LTP/LTD, cannot be assessed.

We now provide results from cultured rat hippocampal neurons in which we have overexpressed 2B, G689C or G689S (Figure 8 and Figure 8—figure supplement 2, page 15). Briefly, overexpression of the variants causes a strong reduction in synaptic GluNR-events: neurons overexpressing the variants showed a strong reduction in the frequency of mini_NMDAR, without any effect on the frequencies of mini_AMPAR. Moreover, overexpression of G689S (but not G689C) reduces amplitudes of mini_NMDAR, with a concomitant increase in the amplitude of mini_AMPAR. Lastly, mini_NMDARs from neurons overexpressing the variants display faster deactivation kinetics than control. Together, these results demonstrate that expression of the variants in hippocampal neurons prompts a pronounced effect on synaptic GluNRs. The reduction in the frequency of mini_NMDAR – in combination with the unaffected frequency of mini_AMPAR – rules-out loss of excitatory synapses (as may be instigated by other variants, e.g., GluN2B-S1413L³¹). Moreover, G689S dual effect on amplitudes of both mini-types suggests that the ‘silencing’ of GluNRs (by the dominant negative effect) induces compensatory mechanism that drive increases in synaptic GluARs³². Lastly, acceleration of τ_off due to overexpression of the variants reveals that the remaining minis are more associated with the faster deactivating GluN2A subunits³³. Notably, all of these effects are highly consistent with our dominant negative observations (see Figure 4) and with results obtained from animal model bearing a GluN2B LoF mutation³⁴.

The conclusions of a number of the experiments depend on expressing wt and mutant receptors at comparable or other predetermined levels, and this can be affected by the quality of oocyte-injected mRNA, differences in expression efficiency and protein stability etc. Indeed, the mutant receptors are not expressed at the surface with the same efficiency and have differences in expression and/or stability. The authors should discuss more fully how these parameters were addressed.

We now elaborate on these in methods. Briefly, mRNA quality is systematically assessed by electrophoresis and diluted mRNA is aliquoted, and stored at -80 degrees (as previously reported^28,35). Thus, the same batch of mRNA serves in multiple experiments. Moreover, we also note that we do not record from dying/unhealthy oocytes (assessed by appearance, resting potentials and leak currents). All experiments are repeated multiple times. In the particular case of the mixed groups experiments (Figure 4), the experiments themselves serve as control for mRNA quality. All groups are injected with the same GluN1a mRNA. Moreover, every experiment consists of multiple groups, including control groups that express the variants or wt subunits on their own, not to mention the groups in which one mRNA is excessively expressed over the other. (e.g., G689C mRNA >> wt mRNA; 16:1). In cases these failed to provide any current, we did not analyze the experiment. However, as the reviewer rightly notes, when the variants are mixed with the 2Bwt-subunit, expression levels may vary – but only in the case of the G689C subunit (G689S expresses as well as 2Bwt). In mixed groups, we obtain large currents in all groups despite the differing mRNA stoichiometries. In the case of G689C, this is indicative of the expression of the wt subunits, but not in the case of G689S. The dramatic drop in EC₅₀ (and complete lack of two populations assessed by various fitting modes), demonstrates that there are no purely-2Bwt channels, rather most are mixed channels. Of note, this is one particular strength in our work over previous reports, namely employing mRNA titration for controlling protein levels is a (e.g., ^28,29,35) as we could determine a bona-fide dominant negative effect that does not solely rely on current amplitudes (i.e., ^18,19), which is prone to yield errors owing to the limitations mentioned by the reviewer.

The authors discuss the effects of the mutations on the stereochemistry of the ligand binding site occupied by glutamate. But because the mutations occlude binding glutamate, it would be interesting to consider the LBD structure of the mutants in the unliganded state, if the appropriate crystal structure were available. This might suggest a pharmacologic (small molecule) means for closing the clamshell and opening the pore of the mutant, even in the absence of bound glutamate.

We agree with the reviewer, but there are no crystal structures of this LBD (or other subunits) without a ligand. However, we now provide better simulations (along PDBs) of the LBD with the mutations (Figure 1).

The speculation about the effects of water access on deactivation kinetics (p 5), which turned out to be incorrect, interrupts the flow of the Results section and might be considered better in the Discussion.

We have re-written this part. We describe the general outcomes of the simulations, without going into detail about the holes, to lessen interruption.

The manuscript has a significant number of typographical errors and a large number of grammatical errors or non-colloquial phrasings. It should be revised carefully by a native English speaker.

The text has been substantially revised, edited and corrected by a native English speaker, as suggested. We hope this version reads better.

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Author details

1. Shai Kellner

   Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   Data curation, Software, Formal analysis, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Abeer Abbasi

   Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   Data curation, Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

3. Ido Carmi

   Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Ronit Heinrich

   Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   Data curation, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

5. Tali Garin-Shkolnik

   Clalit health services, Tel Aviv, Israel

   Contribution

   Resources

   Competing interests

   No competing interests declared

6. Tova Hershkovitz

   Genetics Institute, Rambam medical center, Haifa, Israel

   Contribution

   Resources

   Competing interests

   No competing interests declared

7. Moshe Giladi

   Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

8. Yoni Haitin

   Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

9. Katrine M Johannesen

   1. Department of Epilepsy Genetics and Personalized Treatment, the Danish Epilepsy Centre, Dianalund, Denmark
   2. Institute for Regional Health Services, University of Southern Denmark, Odense, Denmark

   Contribution

   Resources

   Competing interests

   No competing interests declared

10. Rikke Steensbjerre Møller

    1. Department of Epilepsy Genetics and Personalized Treatment, the Danish Epilepsy Centre, Dianalund, Denmark
    2. Institute for Regional Health Services, University of Southern Denmark, Odense, Denmark

    Contribution

    Resources

    Competing interests

    No competing interests declared

11. Shai Berlin

    Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

    Contribution

    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    shai.berlin@technion.ac.il

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-5153-4876

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