Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains

Please cite this article in press as: Swanger et al., Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.10.002

ARTICLE Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains Sharon A. Swanger,1 Wenjuan Chen,1,7 Gordon Wells,2,8 Pieter B. Burger,2 Anel Tankovic,1 Subhrajit Bhattacharya,1 Katie L. Strong,1,2 Chun Hu,1 Hirofumi Kusumoto,1 Jing Zhang,1 David R. Adams,3 John J. Millichap,4 Slave´ Petrovski,5 Stephen F. Traynelis,1,6,* and Hongjie Yuan1,6,* Epilepsy and intellectual disability are associated with rare variants in the GluN2A and GluN2B (encoded by GRIN2A and GRIN2B) subunits of the N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel with essential roles in brain development and function. By assessing genetic variation across GluN2 domains, we determined that the agonist binding domain, transmembrane domain, and the linker regions between these domains were particularly intolerant to functional variation. Notably, the agonist binding domain of GluN2B exhibited significantly more variation intolerance than that of GluN2A. To understand the ramifications of missense variation in the agonist binding domain, we investigated the mechanisms by which 25 rare variants in the GluN2A and GluN2B agonist binding domains dysregulated NMDAR activity. When introduced into recombinant human NMDARs, these rare variants identified in individuals with neurologic disease had complex, and sometimes opposing, consequences on agonist binding, channel gating, receptor biogenesis, and forward trafficking. Our approach combined quantitative assessments of these effects to estimate the overall impact on synaptic and non-synaptic NMDAR function. Interestingly, similar neurologic diseases were associated with both gain- and loss-of-function variants in the same gene. Most rare variants in GluN2A were associated with epilepsy, whereas GluN2B variants were associated with intellectual disability with or without seizures. Finally, discerning the mechanisms underlying NMDAR dysregulation by these rare variants allowed investigations of pharmacologic strategies to correct NMDAR function.

Introduction N-methyl-D-aspartate receptors (NMDARs) are ligandgated cation channels activated by the excitatory neurotransmitter glutamate and the co-agonist glycine. Synaptic transmission mediated by NMDARs is critical for the formation and maturation of excitatory synapses and brain circuits.1 NMDARs are composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. GluN1 is expressed throughout the brain, whereas the GluN2 subunits (GluN2A-2D), encoded by GRIN2A2D (MIM: 138253, 138252, 138254, and 602717), have different developmental and regional expression profiles.2–5 The GluN2 subunits also have distinct pharmacological and biophysical properties,6–8 which suggests that distinct pathologies may result from dysregulation of different GluN2 subunits. NMDAR subunits comprise four semi-autonomous domains, including an extracellular amino-terminal domain, a clamshell-shaped agonist binding domain, a pore-forming transmembrane domain, and an intracellular carboxy-terminal domain.6 Agonist binding promotes clamshell closure, which is transduced into channel

opening by linker regions connecting the agonist binding domain to the transmembrane helices. Recently, numerous de novo mutations and rare variants in NMDARs have been associated with neurodevelopmental conditions including developmental delay, intellectual disability (MIM: 613970 and 614254), and epilepsy (MIM: 245570 and 616139).9–11 GRIN2A and GRIN2B are highly intolerant to genetic variation12 and have been identified as epilepsy-associated genes.13–16 Rare variants found in individuals with neurologic disease have been reported in all domains of GluN2A and GluN2B, but how these variants dysregulate NMDARs and whether they contribute to neurologic disease remains largely unknown. In this study, our goal was to elucidate connections between genetic variation, mechanisms of NMDAR dysregulation, and neurodevelopmental disorders. We investigated how 25 disease-associated rare variants in the GluN2A and GluN2B agonist binding domains altered NMDAR protein levels, localization, structure, and function. Our work demonstrates how disease-associated rare variants affect NMDAR biology through multiple mechanisms leading to complex, and sometimes conflicting, consequences on NMDAR function. To resolve this complex

1

Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA; 2Department of Chemistry, Emory University, Atlanta, GA 30322, USA; 3Undiagnosed Diseases Network, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA; 4Departments of Pediatrics and Neurology, Northwestern University Feinberg School of Medicine and Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL 60611, USA; 5Department of Medicine, The University of Melbourne, Austin Health and Royal Melbourne Hospital, Melbourne, VIC 3050, Australia; 6Center for Functional Evaluation of Rare Variants (CFERV), Emory University School of Medicine, Atlanta, GA 30322, USA 7 Present address: Department of Neurology, Xiangya Hospital, Central South University, Changsha 410013, China 8 Present address: Department of Biochemistry, Stellenbosch University, Stellenbosch 7602, South Africa *Correspondence: [email protected] (S.F.T.), [email protected] (H.Y.) http://dx.doi.org/10.1016/j.ajhg.2016.10.002. Ó 2016 American Society of Human Genetics.

The American Journal of Human Genetics 99, 1–20, December 1, 2016 1

Please cite this article in press as: Swanger et al., Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.10.002

combination of effects, we developed an approach that integrates measured parameters to estimate overall impact on NMDAR activity, and we applied these data to identify pharmacologic approaches for normalizing disrupted NMDAR function.

Materials and Methods Analysis of GRIN2A and GRIN2B Genetic Variation The background variation was estimated using the aggregated variant information from a cohort of 60,706 unrelated individuals, the Exome Aggregation Consortium (ExAC) dataset (release 0.3.1).17 We focused on protein-coding missense and synonymous single-nucleotide variants, and we restricted to variants that were judged to ‘‘PASS’’ the ExAC quality thresholds. The distribution of the missense and synonymous ExAC variants were plotted on the linear gene structure using lollipops-v.1.3.1, based on Uniprot accessions Q12879 (GRIN2A) and Q13224 (GRIN2B). GRIN2A and GRIN2B case missense variants were identified through two database searches and a review of the literature. First, a search for ‘‘pathogenic,’’ ‘‘likely pathogenic,’’ or ‘‘likely pathogenic/pathogenic’’ missense variants in ClinVar (accessed May 2016) was performed. Candidate case variants were required to be absent from ExAC and if also present in Human Gene Mutation Database (HGMD; see below) were required to be a concordant classification of ‘‘disease-causing mutation’’ (DM). This resulted in 34 ClinVar-based candidate pathogenic variants. Next, a search for DM-classified variants was performed based on HGMD (hgmd2016.1). Variants were required to be absent from ExAC and if also present in ClinVar were required to be concordant classification, as described above. This resulted in 39 HGMD-based candidate pathogenic variants, of which 25 were distinct from the ClinVar screen. A literature review revealed 20 additional variants,13,14,18–22 and some variants identified in ClinVar and HGMD were also found in the literature review.13–15,22–31 Finally, three additional variants identified herein were included. The 82 disease case variants in Table S1 were further dichotomized into two broad groups. First were the 41 candidate variants with sufficient segregation support in their source publication. To qualify, there had to be next-generation sequencing or Sanger sequencing support that the variant arose de novo in the affected individual, or for dominant familial epilepsies the variant showed familial segregation in at least four affected family members and no more than one presumed unaffected carrier. Second were the remaining 41 GRIN2A and GRIN2B variants that did not qualify for the above levels of segregation information. Only group 1 variants were included in the analyses presented in Table 1 and Figure 1.

not available), the Agilent Clinical Research Exome kit was used to target the exonic regions and flanking splice junctions of the genome. These targeted regions were sequenced simultaneously by massively parallel (NextGen) sequencing on an Illumina sequencing system with 2 3 100 bp paired-end reads. Bidirectional sequence was assembled, aligned to reference gene sequences based on human genome build GRCh37/UCSC hg19, and analyzed for sequence variants using a custom-developed analysis tool (Xome Analyzer). Proband 2 was referred for clinical WES (Ambry Genetics). Genomic DNA was isolated from whole blood of the patient, the patient’s mother, and the patient’s father. Samples were prepared using the SeqCap EZ VCRome 2.0 (Roche NimbleGen). Each DNA sample was sheared, adaptor ligated, PCR amplified, and incubated with the exome baits. Captured DNA was eluted and PCR amplified. Final quantified libraries were seeded onto an Illumina flow cell and sequenced using paired-end, 100 cycle chemistry on the Illumina HiSeq 2000 or HiSeq 2500. For proband 3, WES was performed by the pairedend pre-capture library procedure (Baylor College of Medicine, Medical Genetics Laboratories). Genomic DNA was isolated from blood, fragmented by sonication, ligated to the Illumina multiplexing PE adapters, and PCR amplified. The pre-capture library was enriched by hybridizing to biotin-labeled VCRome 2.1 in-solution Exome Probes. The post-capture DNA library was analyzed on an Illumina HiSeq platform for 100 bp paired-end reads. The DNA was also analyzed by a SNP-array (Illumina Human Exome12v1 array) and compared with the WES data to ensure sample identification and sequencing quality. Novel variants were confirmed by Sanger sequencing.

cDNA Mutagenesis and Cloning Human cDNA encoding the full-length WT GluN1-1a, GluN2A, and GluN2B proteins (GenBank: NP_015566, NP_000824, and NP_000825) were inserted in the pCI-Neo vector.32 Mutations were generated using the Quikchange method (Stratagene). Previously described rat GluN2BC1 and GluN2BC2 constructs were used in the experiments evaluating receptors with one or two mutant GluN2 subunits,33 and the p.Glu413Gly and p.Cys461Phe variants were made in the rat sequence by site-directed mutagenesis. b-lactamase constructs were made by fusing an oligonucleotide encoding the human GluN2B signal sequence and b-lactamase (GenBank: NC_005248.1 [3950..4810]; synthesized by Integrated DNA Technologies) with a PCR product encoding the mature polypeptide for human GluN2A or GluN2B, and an EcoRI-digested pCI-Neo plasmid using InFusion (Clontech). A plasmid encoding GFP-tagged rat GluN2B was obtained from Dr. Stefano Vicini34 and mutants were generated as described above. All mutagenesis and cloning products were verified by sequencing (Eurofins).

Two-Electrode Voltage-Clamp Recordings Whole-Exome Sequencing Each referenced study participant provided written consent to an institutional review board-approved human subjects protocol at the site where their clinical information and specimens were obtained. Each protocol was reviewed to confirm that inclusion in the current study was within the scope of the associated consents. Proband 1 was referred for clinical microarray testing, subsequent clinical sequencing, deletion/duplication analysis of a 70-gene panel, and finally clinical whole-exome sequencing (WES; GeneDx). Using genomic DNA from the proband only (parents

2

NMDAR cRNA was transcribed in vitro using mMessage mMachine kit (Ambion) according to the manufacturer’s instructions, and 5–10 ng of cRNA was injected into Xenopus laevis oocytes obtained from Ecocyte. Oocytes were stored at 15
C– 19
C in Barth’s solution containing (in mM) 88 NaCl, 2.4 NaHCO3, 1 KCl, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, and 5 Tris/HCl (pH 7.4). Two-electrode voltage-clamp recordings were performed 2–5 days later in solution containing (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0.5 BaCl2, and 0.01 EDTA (pH 7.4). Current and voltage electrodes were filled with 3.0 M and 0.3 M KCl,

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Please cite this article in press as: Swanger et al., Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.10.002

respectively. Recordings were performed at room temperature with a holding potential of 40 mV. EC50 values were obtained by fitting concentration-response data with  . (Equation 1) Response ¼ 100% 1 þ ðEC50 =½agonistÞnH ; where EC50 is the agonist concentration that elicited the half maximal response and nH is the Hill slope. IC50 values for memantine were obtained by fitting the concentration-response data with .  Responseð%Þ ¼ ð100  minimumÞ 1 þ ð½concentration=IC50 ÞnH þ minimum; (Equation 2) where nH is the Hill slope, IC50 is the concentration that produces a half-maximal effect, and minimum is the degree of residual inhibition at a saturating concentration of memantine. The channel open probability (POPEN) evaluated by the degree of MTSEA (2-aminoethyl methanethiosulfonate hydrobromide; Toronto Research Chemicals) potentiation can be calculated by35 POPEN ¼ ðgMTSEA =gCONTROL Þ 3 ð1=potentiationÞ;

glutamate removal and reported as tFAST, tSLOW, a weighted tau (tW),33 and % tFAST.

b-Lactamase Assay HEK cells were seeded in 96-well plates and transiently transfected with cDNA encoding GluN1 and b-lac-GluN2A or b-lacGluN2B using Fugene6 (Promega). Cells treated with Fugene6 alone were used to determine background absorbance, and cells without GluN1 were a negative control for surface b-lactamase activity. NMDAR antagonists (200 mM APV and 200 mM 7-CKA) were added at the time of transfection. Eight wells were transfected for each condition; surface and total activities were measured in 4 wells each. After 24 hr, cells were washed with Hank’s Balanced Salt Solution (HBSS) supplemented with 10 mM HEPES, and then 100 mL of a 100 mM nitrocefin (Millipore) solution in HBSS with HEPES was added to the wells for measuring surface activity. To determine total activity, the cells were lysed by a 30 min incubation in 50 mL H2O prior to the addition of 50 mL of 200 mM nitrocefin. The absorbance at 468 nm was read every min for 30 min at 30
C using a microplate reader. The rate of increase in absorbance was determined from the slope of a linear fit of the data.

(Equation 3)

where potentiation is the current after MTSEA treatment divided by the current before treatment and g denotes the chord conductance measured before and after MTSEA treatment. Pregnenolone sulfate, tobramycin, and memantine were purchased from Sigma.

HEK Cell Culture and Whole-Cell Voltage-Clamp Recordings Human embryonic kidney-293 (HEK) cells (CRL 1573, ATCC) were plated on glass coverslips coated with 0.1 mg/mL poly-Dlysine and cultured at 37
C and 5% CO2 in DMEM/GlutaMax with 10% fetal bovine serum and 10 U/mL penicillin-streptomycin (GIBCO). The calcium phosphate method was used to co-transfect HEK cells with cDNA encoding GFP, GluN1, and GluN2 at a ratio of 5:1:1. The media was changed 4 hr after transfection and supplemented with NMDAR antagonists (200 mM D,L-2-amino-5-phosphonovalerate [APV] and 200 mM 7-chlorokynurenic acid [7-CKA]). After 24 hr, the cells were used for whole-cell voltage-clamp or outside-out patch recordings. Whole-cell recordings were performed at 23
C in recording solution containing (in mM) 150 NaCl, 10 HEPES, 30 D-mannitol, 3 KCl, 1.0 CaCl2, and 0.01 EDTA (pH 7.4). Recordings were made at a holding potential of 60 mV using an Axopatch 200B amplifier (Axon Instruments) and recording electrodes (3–5 MU) filled with (in mM) 110 D-gluconate, 110 CsOH, 30 CsCl, 5 HEPES, 4 NaCl, 0.5 CaCl2, 2 MgCl2, 5 BAPTA, 2 NaATP, and 0.3 NaGTP (pH 7.35). Rapid solution exchange was performed by lifting the cells from the coverslip and using a two-barrel theta glass pipette controlled by a piezoelectric translator (Burleigh Instruments and Siskyou). Open-tip solution exchange time was <1 ms. The data were acquired using Clampex (Axon Instruments). Current amplitude and deactivation time course were analyzed with Clampfit and ChanneLab (Synaptosoft). Series resistance was monitored throughout the recordings, and series resistance correction was performed offline using ChanneLab as previously described.36 Deactivation rates were determined by fitting a two-component exponential function to the current response time course after

Surface Protein Biotinylation and Western Blotting HEK cells were transiently transfected with NMDAR cDNA using Fugene6 (Promega) and processed for surface protein biotinylation 24 hr later as previously described.37 In brief, cells were incubated with 1 mg/mL biotin (Pierce) on ice for 20 min, and cell lysates were incubated with Neutravidin beads (Pierce) with rotation at 4
C for 2 hr. Total and pull-down fractions were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-GluN2A (1:2,500; Millipore cat# AB1555; RRID: AB_2112325), anti-GluN2B (1:1,000; Millipore cat# AB1557; RRID: AB_2112907), anti-transferrin receptor (TfR; 1:10,000; Invitrogen) as a control to ensure even biotinylation across samples, and anti-a-tubulin (1:200,000; Sigma) as a loading control. Secondary antibodies conjugated to horseradish peroxidase were detected with chemiluminescence. Film was imaged and quantified using ImageJ.

Molecular Dynamics A previously described homology model of GluN2B38 was prepared in VMD software39 for molecular dynamics in NAMD software.40 The system was parameterized with the CHARMM 27 forcefield41 assuming a neutral pH for ionizable residues. His405 and His703 were modeled in the neutral Nd-protonated state, while His486 in the L-glutamate binding site was modeled in the Nε-protonated form in order to maximize intra-molecular hydrogen bonding. The p.Glu413Gly mutant was built using the VMD mutagenesis module, and internal water molecules (TIP3 model) were placed using the VMD DOWSER module. Internal waters were retained if they superimposed with waters from existing agonist binding domain crystal structures (PDB: 2A5T, 2A5S, 3OEK, 3OEL, 3OEM, 3OEN). Solvation was completed by soaking the protein further using a 12 A˚ buffer resulting in an orthorhombic cell with approximate dimensions of 82 A˚ 3 69 A˚ 3 78 A˚. The system was neutralized with 0.15 mM NaCl. A 12 A˚ cut-off was used for non-bonded interactions, with a switching function applied from 10 A˚. Non-bonded interactions between atoms connected by three or fewer covalent bonds were excluded. Periodic boundary conditions were simulated with Particle Mesh

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Ewald Sums for long-range electrostatics using a grid spacing of 1 A˚. The solvent was first minimized for 1,000 steps followed by minimization of all atoms for 50 steps. The solvent was then relaxed using molecular dynamics for 150,000 steps of 1 fs each. Heating was applied from 60 K to a physiological temperature of 310 K in 5 K steps every 1,000 fs. A similar protocol was used to relax the entire system for 500,000 steps from 10 K to 310 K. Multiple time stepping was used for force evaluation, with short-range interactions evaluated every step and full electrostatic interactions every two steps. Data collection was carried out in the NPT ensemble with a 2 fs time-step at 310 K using a Langevin thermostat and at 1.01325 bar. Frames were collected every 1,000 steps (2 ps). Solvent accessibility of the L-glutamate ligand was determined using the ‘‘g_sas’’ module from GROMACS42 and independently with the protein tunnel finding program CAVER.43 A probe size of 1.4 A˚ was used in both cases. For CAVER, 100 ps snapshots of the protein and ligand were superimposed on the first frame. An average of the L-glutamate Ca coordinates were used as a starting point for the CAVER probe on each snapshot with the L-glutamate ligand excluded.

Outside-out Patch Recordings Outside-out patches were pulled from transfected HEK cells and single-channel recordings were performed at room temperature (23
C) with a holding potential of 80 mV using an Axopatch 200B amplifier (digitized at 40 kHz and filtered at 8 kHz). Recording electrodes were made using a vertical puller (Narishige P-10) from thick-walled glass pipettes (G150F-4, Warner Instruments), coated with Sylgard (Dow Corning), fire-polished to a resistance of 7–9 MU, and filled with the same internal solution used for whole-cell voltage-clamp recordings. The channel was activated by maximal agonist concentrations (1 mM glutamate and 50 mM glycine) in an external solution composed of (in mM) 150 NaCl, 10 HEPES, 3 KCl, 0.5 CaCl2, and 0.01 EDTA (pH 7.4). For analysis, the recordings were pre-filtered at 8 kHz (3 dB), digitized at 40 kHz, and analyzed using SCAN software (Dr. David Colquhoun, University College London). Open and shut duration histograms were generated with an imposed resolution for open time (53 ms) and shut time (31 ms) durations.

Dissociated Neuron Culture and Immunofluorescence Hippocampal neurons were isolated from E18 Sprague Dawley rat embryos of both sexes as described previously.44 All animal procedures were reviewed and approved by Emory IACUC. Cultured neurons were transfected with GFP-tagged GluN2B constructs at 10 days in vitro (DIV) using the calcium phosphate method as described previously37 and fixed 24 hr later with 4% paraformaldehyde in phosphate-buffered saline. Surface GFP was immunostained using chicken-anti-GFP (1:1,000, Aves Labs) antibodies on non-permeabilized cells and detected with a Cy3-anti-chicken secondary antibody (1:2,000, Jackson Immunoresearch). Images were acquired on an Olympus BX71 microscope and Orca ER cooled CCD camera (Hamamatsu). Within each experiment, samples from all groups were acquired in the same imaging session with identical acquisition settings. The fluorescence intensity of Cy3-labeled anti-GFP was measured in a 100 mm dendrite segment, and direct GFP fluorescence was measured in the soma and the same dendrite segment (ImageJ). Intensity measurements were normalized to region area and background subtracted. The experimenter was blinded during acquisition and analysis.

4

Equation for Calculating Charge Transfer Synaptic charge transfer was approximated as the product of the current amplitude and the mean weighted deactivation time constant (tW) after rapid removal of glutamate. The amplitude of the synaptic current is controlled by the receptor open probability (PO), surface protein levels (Surf), and relative response (Ragonist) in a given agonist concentration, which is given by Ragonist ¼ 1

 . 1 þ ðagonist EC50 =½agonistÞnH ;

(Equation 4)

where nH is the Hill slope, [glycine] is 3 3 106 M for RGly, and [glutamate] is 1 3 103 M and 1 3 107 M for RGlu,Synaptic and RGlu,Non-synaptic, respectively. We measured tW, PO, and Surf for mutant NMDARs and expressed them as a ratio to values determined for WT NMDARs (tmut/wt, POmut/wt, and Surfmut/wt). These ratios and the relative response in the given agonist concentration were used to calculate synaptic charge transfer according to Charge transferSynaptic ¼ tmut=wt 3 POmut=wt 3 Surfmut=wt 3 RGly 3 RGlu;Synaptic :

(Equation 5)

The relative fold-change in non-synaptic charge transfer in steady-state non-synaptic agonist concentrations was calculated from Charge transferNon-synaptic ¼ POmut=wt 3 Surfmut=wt 3 RGly 3 RGlu;Non-synaptic

(Equation 6)

where POmut/wt and Surfmut/wt are again the ratio of mutant to WT values.

Statistics Sample sizes were determined by a priori power analyses using G*Power 3.1.9.2 with a ¼ 0.05, power ¼ 0.8, and effect sizes ¼ 0.3–1.0.45 Effect sizes were estimated based on previously published data using similar methods or pilot data in the case of the b-lactamase assay, which had not been used to measure NMDAR protein levels prior to this study. Statistical analyses were performed in GraphPad Prism 6. Datasets were tested for normality using D’Agostino-Pearson test and homogeneity of variances using the Bartlett’s test. Datasets that failed homogeneity of variance test (a ¼ 0.01) and had unequal sample sizes were transformed as stated in the legends. The specific statistical tests used were reported in each figure and table legend. Significance level was set at 0.05 unless otherwise noted, and all tests were two-sided. p values for post hoc tests were adjusted for multiple comparisons using the correction stated in the legends. ANOVA F-statistics and p values for pairwise comparisons were reported in the legends, data tables, or Tables S16–S19.

Results Variation Intolerance of Protein Domains within GluN2A and GluN2B The intolerance to functional variation among GRIN2A and GRIN2B exons and conserved domains has been previously described using the sub-region variation intolerance score (subRVIS).46 To characterize the distribution of missense variation specifically across functional domains of GluN2A and GluN2B, namely the aminoterminal domain, agonist binding domain, linker regions,

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Please cite this article in press as: Swanger et al., Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.10.002 Table 1.

Analysis of Genetic Variation across Domains of GRIN2A and GRIN2B GRIN2A

GRIN2B ExAC Missense (n ¼ 382)

Case Missense (n ¼ 18)

ExAC Missense (n ¼ 238)

Case Missense (n ¼ 23)

Domain

Residues

#

%

#

%

p Value

Residues

#

%

#

%

p Value

Amino-Terminal Domain

23–404

101

26.44%

1

5.56%

0.05

27–404

52

21.85%

0

0%

0.01

Agonist-Binding Domain (ABD)

405–539

30

11.78%

2

27.78%

0.06

405–540

8

5.04%

6

56.52%

1.2 3 109*

661–801

15

662–802

4

540–555

0

541–557

0

8.70%

0.007

655–660

0

0

656–661

0

0

802–816

2

1

803–817

0

1

558–655

7

34.78%

8.5 3 106*

Linkers

7

3 0.52%

2

16.67%

0.0007*

6

7 0%

1

38.89%

2.5 3 10

818–838

1

2

11.11%

9.2 3 105*

839–1,484

166

69.75%

0

0%

1.2 3 1011*

7.85%

2

11.11%

0.6

405–540

8

3.36%

6

26.09%

0.0005*

3.93%

3

16.67%

0.04

662–802

4

1.68%

7

30.43%

4.3 3 106*

Transmembrane Domain

556–654

2.88%

817–837

4

Cytoplasmic Domain

838–1,469

223

58.38%

S1 ABD

405–539

30

S2 ABD

661–801

15

5

*

2

3.36%

5 3

Coordinates based on GRIN2A (GenBank: NM_000833.4; Uniprot: Q12879) and GRIN2B (GenBank: NM_000834.3; Uniprot: Q13224). Reported Fisher’s exact test p values are uncorrected; asterisk (*) denotes statistical significance using adjusted a ¼ 0.007 for comparisons within each gene.

transmembrane domain, and carboxy-terminal domain (Figure 1A), we examined the data from the ExAC Browser, which includes 60,706 exomes from unrelated individuals.17 We found that the agonist binding domain, linker regions, and transmembrane domain showed unusually low levels of missense variation in the general population despite the presence of synonymous variation (Figures 1B and 1C). These data suggest that, in general, missense variation occurring in these domains might be under greater negative selection than that occurring in the amino- and carboxy-terminal domains. To formally assess whether disease-associated variants were overrepresented in these regions, we compared the proportion of ExAC missense variants observed in each sub-region to the proportion of reported disease-associated missense variants in that sub-region (Table 1 and S1). The agonist binding and transmembrane domains as well as the linkers between these two domains had significantly greater proportions of disease-associated variants than expected based on the proportion of ExAC missense variants found in those regions (Table 1 and Figures 1B and 1C). Interestingly, when directly comparing the proportions of missense variation that occurred in the agonist binding domain, GRIN2B exhibited less variation tolerance than GRIN2A, with fewer of the population missense variants occurring in the agonist binding domain of GRIN2B (12/ 238 [5.0%] of ExAC GRIN2B missense variants) compared to GRIN2A (45/387 [11.8%] of ExAC GRIN2A missense variants; Fisher’s exact two-tailed p ¼ 0.004; Table 1 and Figure 2). In addition, functional variation even within the agonist binding domain was not evenly distributed.

The agonist binding domain is formed by two discontinuous segments of the polypeptide, referred to as S1 and S2 (Figures 2A and 2B), and the S2 region harbored less control missense variation (Table 1). The majority of functional variation within the agonist binding domain was observed in loops within S1 (Figures 2C and 2D), which are not well conserved across GluN2 subunits (Figure S1). In contrast, the regions of S1 and S2 forming the agonist binding cleft as well as adjacent helices in S2 showed minimal variation, consistent with these being highly conserved regions critical for receptor activation. Given these intriguing findings regarding the patterns of genetic variation in the agonist binding domain, we investigated the consequences of reported disease-associated rare variants in this domain on NMDAR protein levels, localization, structure, and function. Rare Variants in the Agonist Binding Domain Are Associated with Neurological Disease Twenty-five rare variants in the GluN2A and GluN2B agonist binding domains were investigated,13–16,20,23–25,47,48 including three variants identified by WES in this study (Table 2). Proband 1 had severe intractable epilepsy and global developmental delay, and a variant was identified in GRIN2A (GenBank: NM_000833.3; c.2054T>G [p.Val685Gly]). Parental samples were not available for testing. Variants in GRIN2B (GenBank: NM_000834) were identified in proband 2, who presented with intellectual disability and hypotonia (c.2087G>A [p.Arg696His]), and in proband 3, who exhibited intellectual disability and epilepsy (c.1306T>C [p.Cys436Arg]). These GRIN2B variants were not detected in the unaffected parents of proband 2 or proband 3,

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Figure 1. Genetic Variation across Functional Domains of GluN2A and GluN2B (A) The domain structure of a GluN2 subunit is depicted by a linear representation and a drawing. Abbreviations and colors are as follows: ATD, amino-terminal domain (green); ABD, agonist binding domain (blue); TMD, transmembrane domain including the M1, M3, and M4 helices and the M2 re-entrant loop (orange); linker regions (white); and CTD, carboxy-terminal domain (black). The residue numbers correspond to the GluN2A sequence. (B and C) Control synonymous variants (gray) and control missense variants (blue) were ascertained from the ExAC population; this included 387 distinct missense and 283 distinct synonymous variants for GluN2A (B) and 248 distinct missense and 291 distinct synonymous variants for GluN2B (C). Missense variants from disease cases (red) were compiled as described in Material and Methods. Variants were plotted on the linear protein structure from (A).

indicating that these were probably de novo occurrences. Additional WES results and clinical testing for these individuals are described in Table S2. Altogether, 17 rare variants in GluN2A were identified in individuals with seizure disorders with or without developmental delay, and one variant was identified in individuals with schizophrenia. All individuals with the GluN2B variants studied here exhibited intellectual disability and, in some cases, seizures (Tables S2 and S3). Of the 25 variants identified in individuals with neurologic disease, 20 occurred at sites conserved across GluN2 subunits, 2 had conserved charge, and 3 were not

6

conserved (Figure S1). All GRIN2B variants and 15 out of 18 GRIN2A variants occurred at sites where no missense variants were found in the ExAC database (Figures 2E and 2F and Table S4). The p.Val452Met variant in GRIN2A was previously identified in individuals with schizophrenia; however, this variant occurs at a non-conserved site and was also observed 36 times in the ~45,000 nonpsychiatric ExAC sub-population samples. Collectively, this suggests that the p.Val452Met variant is unlikely to be disease causing. Interestingly, the GluN2A-p.Ala716Thr variant is reported in a single individual in ExAC, and the read data provided from this single observation suggest the

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Figure 2. Distribution of Missense Variation within the Agonist Binding Domains of GluN2A and GluN2B (A) A model illustrates the tetrameric structure of the human NMDAR,62,63 with two GluN1 subunits and two GluN2 subunits. The semiautonomous domain structure is highlighted for one GluN2 subunit by distinct colors for the amino-terminal domain (green), agonist binding domain (blue), and transmembrane domain (orange). The second GluN2 subunit is shown in magenta. (B) The S1 (blue) and S2 (cyan) segments contribute to the structure of the GluN2 agonist binding domain; an expanded view of the boxed region containing the agonist binding domain is shown to the right. (C and D) The agonist binding domain was colored using a heatmap representing the number of missense variants at each residue detected in the ExAC database. The distribution of missense variation is shown as surface renderings (left) and two angles of a cartoon rendering (right), with L-glutamate shown in cyan. (E and F) Linear diagrams of missense variation in the S1 and S2 segments using the same color scale as in (C). The circles show the locations of the 25 rare variants studied herein. The variants represented by gray circles were present in the ExAC database, whereas those represented by black circles were not.

possibility that it is a somatic mutation with only 24% of the 54 reads at the site supporting the variant (binomial exact test under a 50% allelic ratio expectation, p ¼ 0.0002). All GRIN2B variants studied herein occurred de novo, whereas only four GRIN2A variants were confirmed as de novo or had strong segregation support. The remaining GRIN2A variants showed modest segregation support (Table S1). Altogether, this genetic variation analysis suggests that, although all of these rare variants were identi-

fied in individuals with neurologic disorders, they have varying probabilities of conferring a risk of disease, and thus, may have different levels of functional pathogenicity for NMDARs. Rare Variants in the Agonist Binding Domain Alter Glutamate Potency To study NMDAR function, each variant was introduced into recombinant human GluN2A or GluN2B cDNA. We

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Pharmacological and Biophysical Properties of Rare Variants in the Agonist Binding Domain

Receptor

Genotype

Phenotype

Glutamate EC50, mM (n)a

Glycine EC50, mM (n)a

tw, ms (n)b

Amplitude, Peak, pA/pF (n)b

WT GluN2A-WT

–

–

3.4 5 0.11 (51)

1.2 5 0.05 (76)

56 5 3.6 (47)

136 5 19 (41)

GluN2A-p.Cys436Arg

c.1306T>C

epi

2.3 5 0.10 (9)*

1.8 5 0.10 (14)*

—

0.04 5 0.02 (6)*

GluN2A-p.Val452Met

c.1354G>A

SCZ

1.0 5 0.13(10)*

1.1 5 0.11 (12)

70 5 6.4 (7)

152 5 55 (6)

GluN2A-pGly483Arg

c.1447G>A

epi

54 5 6.3 (10)*

1.5 5 0.12 (16)

20 5 3.0 (10)*

30 5 11 (10)*

GluN2A-p.Arg504Trp

c.1510C>T

epi

2.8 5 0.18 (6)

0.96 5 0.04 (4)

89 5 8.7 (12)*

59 5 13 (14)

GluN2A-p.Val506Ala

c.1517T>C

epi

2.0 5 0.18 (6)*

1.1 5 0.11 (12)

81 5 13 (7)

67 5 34 (7)

GluN2A-p.Arg518His

c.1553G>A

epi

–

d d

–

d

–

d

c

c

0.19 5 0.07 (5)*

c

–

GluN2A-p.Thr531Met

c.1592C>T

epi

–

–

0.09 5 0.03 (6)*

GluN2A-p.Lys669Asn

c.2007G>T

epi

1.1 5 0.49 (6)*

0.31 5 0.05 (10)*

239 5 30 (13)*

143 5 40 (11)

GluN2A-p.Val685Gly

c.2054T>C

epi

270 5 11 (10)*

1.5 5 0.14 (6)

24 5 2.7 (10)*

7.1 5 3.1 (8)*

GluN2A-p.Ile694Thr

c.2081T>C

epi

9.8 5 0.37 (12)*

0.94 5 0.13 (9)

45 5 3.7 (11)

57 5 15 (8)*

GluN2A-p.Pro699Ser

c.2095C>T

epi

2.2 5 0.33 (6)*

1.3 5 0.23 (8)

52 5 4.7 (7)

98 5 51 (7)

GluN2A-p.Met705Val

c.2113A>G

epi

5.7 5 0.14 (8)*

1.0 5 0.15 (8)

57 5 9.1 (6)

34 5 19 (5)

GluN2A-p.Glu714Lys

c.2140G>A

epi

3.0 5 0.27 (8)

1.2 5 0.08 (13)

66 5 9.0 (5)

114 5 51 (7)

GluN2A-p.Ala716Thr

c.2146G>A

epi

20 5 1.9 (10)*

1.3 5 0.09 (11)

32 5 3.6 (13)*

63 5 15 (14)

GluN2A-p.Ala727Thr

c.2179G>A

epi

5.1 5 0.37 (10)*

1.4 5 0.09 (8)

50 5 4.2 (6)

83 5 24 (6)

GluN2A-p.Asp731Asn

c.2191G>A

epi

6,418 5 278 (7)*

1.5 5 0.26 (10)

–

0.22 5 0.16 (5)*

GluN2A-p.Val734Leu

c.2200G>C

epi

5.1 5 0.80 (8)*

1.3 5 0.10 (12)

30 5 2.3 (11)*

69 5 24 (12)

GluN2A-p.Lys772Glu

c.2314A>G

epi

4.8 5 0.17 (10)*

1.3 5 0.09 (10)

47 5 5.6 (13)

55 5 22 (13)*

GluN2B-WT

–

–

1.5 5 0.07 (57)

0.38 5 0.03 (47)

570 5 23 (35)

41 5 5.0 (31)

GluN2B-p.Glu413Gly

c.1238A>G

ID

79 5 5.3 (12)*

0.32 5 0.02 (8)

20 5 1.3 (9)*

3.3 5 1.3 (8)*

GluN2B-p.Cys436Arg

c.1306T>C

ID & epi

–d

–d

–c

0.05 5 0.02 (6)*

c

c

GluN2B-p.Cys456Tyr

c.1367G>A

ID

0.39 5 0.03 (14)*

1.0 5 0.05 (7)*

–

0.03 5 0.01 (6)*

GluN2B-p.Cys461Phe

c.1382G>T

ID & epi

169 5 9.0 (14)*

0.15 5 0.007 (8)*

28 5 1.8 (6)*

4.2 5 1.0 (6)*

GluN2B-p.Arg540His

c.1619G>A

ID & epi

0.64 5 0.07 (19)*

0.25 5 0.02 (16)*

1,157 5 130 (7)*

35 5 12 (7)

GluN2B-p.Arg682Cys

c.2044C>T

ID

0.91 5 0.05 (6)*

0.22 5 0.01 (7)*

729 5 85 (6)*

35 5 8.3 (6)

GluN2B-p.Arg696His

c.2087G>A

ID

0.33 5 0.07 (8)*

0.44 5 0.01 (7)

2,079 5 165 (8)*

10 5 3.6 (7)*

Abbreviations are as follows: epi, epilepsy; ID, intellectual disability; SCZ, schizophrenia. *p < 0.05, ANOVA, post hoc Dunnett’s test versus WT; see Table S16 for F-statistics and p values. a Two-electrode voltage-clamp recordings from oocytes. b Whole-cell voltage-clamp recordings from HEK cells with 1.5 s glutamate application; data failed homogeneity of variances test and were square root-transformed; statistical analyses were performed on transformed data. c Deactivation was not measured due to the small current amplitude. d Current response < 50 nA at 1,000 mM glutamate and 100 mM glycine.

first assessed glutamate and glycine potency by generating concentration-response curves for NMDARs expressed in Xenopus oocytes. Eleven variants reduced glutamate potency, with fitted EC50 values ranging between 2-fold and >1,000-fold higher than WT receptors (Table 2 and Figures 3A and 3B). Nine variants enhanced glutamate potency, with the most notable being a 4.5-fold decrease in glutamate EC50 value by the GluN2B-p.Arg696His variant (Table 2 and Figure 3C). Because these were heterozygous variants, native NMDARs could contain 0, 1, or 2 copies of the mutant GluN2 subunit. Receptors with a single mutant

8

GluN2 subunit were tested for two variants using engineered NMDAR cDNA that controls the subunit stoichiometry of NMDARs at the cell surface.33,49 One copy of GluN2B-p.Glu413Gly increased the glutamate EC50 value (21 5 1.1 mM) over WT receptors (0.9 5 0.08 mM), but not to the extent of receptors with two GluN2Bp.Glu413Gly subunits (82 5 6.2 mM; Figure 3D). Receptors containing 0, 1, or 2 copies of GluN2B-p.Cys461Phe yielded similar results (EC50 values: 1.5 5 0.15 mM, 61 5 0.08 mM, and 290 5 13 mM, respectively). These data suggest that many rare variants in the agonist binding

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Figure 3. GluN2A and GluN2B Agonist Binding Domain Rare Variants Alter Glutamate Potency and NMDAR Deactivation Time Course (A–D) Concentration-effect data and fitted curves for glutamate, in the presence of 30 mM glycine, were plotted as a percentage of the maximal current response for (A–C) recombinant human NMDARs and (D) rat NMDARs with zero (closed circle), one (half-filled circle), or two (open circle) copies of GluN2B-p.Glu413Gly (p.E413G) expressed in oocytes. Data are mean 5 SEM. (E–G) Representative whole-cell recordings of current responses to 1 mM glutamate applications (1.5 s, black bars) in the presence of 30 mM glycine are shown for HEK cells transiently expressing human NMDARs. In (E), the waveforms in the boxed region (left) are normalized and expanded (right). In (F), the response of GluN2B-WT and -p.Glu413Gly are superimposed as raw current (left) and as normalized waveforms (right). In (G), the GluN2B-WT and -p.Arg696His (p.R696H) recordings were for different durations but are shown at the same timescale. The dashed line illustrates the baseline.

domain markedly decreased glutamate potency, and a single mutant GluN2 subunit can have significant effects on NMDAR function.

harboring rare deleterious variants may have altered kinetics, which could affect the strength and time course of synaptic transmission.

Rare Variants in the Agonist Binding Domain Alter NMDAR Deactivation Rates NMDARs mediate the slow, calcium-permeable component of excitatory neurotransmission. Therefore, the rate of NMDAR deactivation after glutamate removal contributes to the time course of excitatory neurotransmission.50 WT and mutant NMDAR deactivation rates were measured using whole-cell recordings from HEK cells expressing recombinant human NMDARs. A rapid perfusion apparatus was used to apply and remove glutamate, and deactivation rates were calculated by fitting a two-component exponential function to current responses after glutamate removal (Figures 3E–3G). Overall, mutant receptors with reduced glutamate potency had accelerated deactivation rates compared to WT, and those with enhanced glutamate potency had prolonged deactivation rates (Table 2 and Table S5). Indeed, glutamate potency and deactivation rate should be related, because agonist dissociation occurs during NMDAR deactivation after rapid agonist removal.51,52 Scatterplots of glutamate EC50 values versus deactivation time constants illustrate this relationship (Figure S2). Thus, synaptic NMDARs

Decreased Glutamate Potency Correlates with Reduced NMDAR Surface Levels The number of receptors at the cell surface controls the overall impact of NMDAR signaling. Since agonist binding regulates forward trafficking of several receptor classes, including NMDARs,53–56 we hypothesized that mutant receptors with reduced agonist potency would have altered NMDAR localization. Cell surface and total protein levels were measured using a b-lactamase reporter assay in HEK cells expressing GluN2A or GluN2B tagged extracellularly with b-lactamase (b-lac-GluN2A and b-lac-GluN2B), which cleaves the cell-impermeable chromogenic substrate nitrocefin.57 Nitrocefin absorbance increased over time for cells expressing GluN1 and b-lac-GluN2A or b-lac-GluN2B, whereas cells lacking the obligate GluN1 subunit showed no significant increase (Figure S3). All samples had high b-lactamase activity after cell lysis, demonstrating the fidelity of this medium-throughput assay for measuring NMDAR surface levels. The ratio of surface-to-total protein levels, a measure of forward trafficking efficiency, and total protein levels were

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Figure 4. Rare Variants Disrupt NMDAR Trafficking and Glutamate Binding (A and B) Representative plots of nitrocefin absorbance (O.D.) versus time are shown for HEK cells expressing WT or mutant b-lac-GluN2. GluN1 was present in all conditions except control cells. The slopes of O.D. versus time were averaged (n ¼ 3–5 independent experiments) and graphed as percentages of WT for the ratio of surface/total (white bars) and total (gray bars). Data are mean 5 SEM. Data were analyzed by ANOVA with post hoc Dunnett’s tests compared to WT (surface/total ratio, *p < 0.01; total, yp < 0.01; see Table S17 for F-statistics and p values. (C) Surface/total ratios were plotted versus fold change in glutamate EC50 values for the variants. (D) The GluN2A agonist binding domain shows the location of variants that reduced agonist potency (L-glutamate, cyan). (E) An expanded view of the glutamate binding pocket depicts interactions (yellow dashed lines) between glutamate and nearby residues or water molecules. (F) Surface renderings of the GluN2B-WT and -p.Glu413Gly agonist binding domains (upper D1 lobe, gray; lower D2 lobe, yellow) generated with the MSMS algorithm using a water-sized probe with L-glutamate in blue. Black arrows highlight edges of the solvent-accessible pocket. (G) Exit tunnels (green) for a water-sized probe were predicted for GluN2B-WT and -p.Glu413Gly from snapshots of 100 ns trajectory using CAVER. Black arrows are positioned at the ends of exit tunnels for GluN2B-p.Glu413Gly.

compared between WT receptors and rare variants that decreased glutamate potency. Surface-to-total ratios and/ or total protein levels were found to be reduced for all of these variants, except GluN2A-p.Val734Leu (Figures 4A, 4B, and S4). Surface protein biotinylation confirmed the b-lactamase results for GluN2A-p.Val685Gly and GluN2B-p.Cys461Phe (Figure S5). Furthermore, glutamate EC50 values and surface protein levels were negatively

correlated (r ¼ 0.7, p ¼ 0.027), which is consistent with rare variants that reduce agonist potency also reducing receptor protein levels. A scatterplot illustrated that mutant receptors with low glutamate potency always had low surface levels, but variants that slightly reduced potency (<2-fold) exhibited varied surface levels (Figure 4C), suggesting that additional mechanisms alter receptor protein levels.

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Figure 5. Agonist Binding Domain Mutations Disrupt NMDAR Trafficking in Dendrites of Cultured Neurons (A) Representative images of cortical neurons transiently expressing GFP-tagged GluN2B-WT, -p.Glu413Gly, or -p.Cys461Phe and immunostained for surface GFP (top row: GFP signal; bottom: anti-GFP, Cy3). Scale bar represents 10 mm. (B and C) The ratios of (B) dendritic Cy3 fluorescence intensity to dendritic GFP fluorescence intensity and (C) GFP fluorescence intensity in distal dendrites (>100 mm from soma) to somatic GFP fluorescence intensity were graphed as a percentage of WT. Individual data points are shown with the mean 5 SEM (n ¼ 8–10 cells from two independent cultures). Data were analyzed by ANOVA and post hoc Dunnett’s tests; (B) F(2,22) ¼ 8.49, p ¼ 0.002, *p ¼ 0.006, yp ¼ 0.002; (C) F(2,21) ¼ 21.47, p < 0.001, *p < 0.001, yp < 0.001.

GluN2B-p.Glu413Gly Reduces Glutamate Potency by Altering Glutamate Binding Most rare variants that severely reduced glutamate potency occurred at residues within the glutamate binding pocket (Figure 4D), suggesting that these variants could directly impact glutamate binding. Indeed, several residues interact with glutamate through electrostatic interactions or water-mediated hydrogen bonds (Figure 4E).58–60 As a representative variant, GluN2B-p.Glu413Gly was used to model how disease-associated rare variants influenced glutamate binding. A molecular dynamics model predicted a larger solvent-accessible area for GluN2Bp.Glu413Gly (17 A˚2) compared to GluN2B-WT (14 A˚2; Figure 4F). Analysis of primary exit tunnels from the binding site for a water-sized probe predicted a larger number of overlapping exit tunnels for GluN2B-p.Glu413Gly (n ¼ 1,783) than for GluN2B-WT (n ¼ 269; Figure 4G). These data suggest that water can more readily compete for atomic contacts and that glutamate has more paths to exit the cleft in GluN2B-p.Glu413Gly, consistent with the observed reduced potency and accelerated deactivation rate. Glutamate potency could also be affected by altered transduction of agonist binding into channel opening (i.e., gating). To evaluate this, we measured channel open probability by applying maximal concentrations of agonists and the thiol-modifying reagent MTSEA to NMDARs containing GluN1-p.Ala652Cys expressed in oocytes. MTSEA-modified Cys652 locks the channel pore open, making the magnitude of MTSEA potentiation inversely proportional to open probability.35,61 Rare variants near the glutamate binding pocket did not significantly alter NMDAR open probability (Table S6). Furthermore,

single-channel recordings in outside-out patches that contained GluN1/GluN2B-WT or -p.Glu413Gly showed no significant differences in conductance or mean open time (Figure S6 and Table S7), suggesting that ion permeation and stability of the open state were unaffected. Open probability could not be measured for GluN2A-p.Arg518His, -p.Thr531Met, -p.Val685Gly, or -p.Asp731Asn because the low glutamate potency rendered it impossible to achieve saturating glutamate concentrations (EC50 > 3 mM). Single-channel recordings of GluN1/GluN2A-p.Arg518His and -p.Thr531Met receptors in outside-out patches showed infrequent channel openings (Table S8). Combined with our data showing reduced protein levels and the lack of macroscopic current responses, these data are consistent with GluN1/GluN2A-p.Arg518His and -p.Thr531Met receptors having reduced function compared to WT. Altogether, these data suggest that most rare variants in the glutamate binding pocket reduce agonist potency by altering glutamate atomic contacts, not channel gating. Rare Variants Disrupt NMDAR Trafficking in Cultured Neurons Trafficking of mutant NMDARs with reduced glutamate potency was also evaluated in dissociated rat cortical neurons transfected with N-terminal GFP-tagged GluN2B-WT, -p.Glu413Gly, or -p.Cys461Phe. Neurons were immunostained for surface GFP and imaged by wide-field fluorescence microscopy (Figure 5A). Ratios of surface-to-total GFP fluorescence within a 100 mm dendritic segment for GluN2B-p.Glu413Gly and -p.Cys461Phe were reduced to 50% 5 6.9% and 43% 5 4.6% of WT levels (100% 5 16%), respectively (Figure 5B). We reasoned that if forward

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trafficking were reduced, then total protein levels should be reduced in distal dendrites. Total GFP fluorescence in dendrites (>100 mm from the soma) relative to somatic GFP fluorescence for GluN2B-p.Glu413Gly and -p.Cys461Phe were reduced to 43% 5 3.5% and 36% 5 7.4% of WT (100% 5 9.3%; Figure 5C), suggesting that agonist binding domain variants can reduce NMDAR levels at their functional site in neuronal dendrites. Rare Variants at Subunit Interfaces Reduce NMDAR Protein Levels Several rare variants enhanced glutamate potency but had reduced macroscopic current amplitudes (Table 2). The b-lactamase reporter assay revealed reduced surface-tototal ratios and total protein levels for many of these variants when expressed in HEK cells (Figures 6A and S7). We hypothesized that reduced protein levels and forward trafficking may be due to disrupted protein folding and/ or receptor assembly. Three rare variants occurred at conserved Cys residues that form disulfide bonds predicted to stabilize ‘‘loop 1’’ at the heterodimer interface and facilitate GluN1-GluN2 dimerization.60,62–64 These variants reduced NMDAR surface localization to control levels in HEK cells without GluN1 (Figures 6B–6D). In addition, the GluN2A-p.Lys772Glu variant decreased NMDAR surface levels to ~20% of WT (Figure 4A) but modestly reduced glutamate potency (Table 2). Lys772 resides in a charged region at the dimer interface and the charge-switching substitution to Glu may hinder inter-subunit interactions (Figure S8).58,63 Thus, it is possible that NMDARs harboring rare variants at subunit interfaces do not fold and/or assemble properly, leading to reduced protein levels and receptor trafficking.

Figure 6. Rare Variants Have Opposing Consequences on NMDAR Biology (A) The surface-to-total ratios and total levels of b-lac-GluN2A and b-lac-GluN2B were measured using the b-lactamase reporter assay. Data were normalized to WT b-lac-GluN2A or b-lac-GluN2B and were analyzed by one-way ANOVA with post hoc Dunnett’s tests compared to WT (*p < 0.05 surface/total, yp < 0.05 total). Data are mean 5 SEM. (B) Absorbance values (O.D.) for surface b-lactamase activity in cells expressing GluN1/b-lac-GluN2B constructs and control cells (without GluN1) were plotted versus time (n ¼ 3–4 independent experiments). (C) NMDAR surface rendering shows the GluN2 amino-terminal domain (ATD, green), agonist binding domain (ABD, blue), transmembrane domain (orange), and ‘‘loop 1’’ within the ABD (magenta). (D) The boxed region from (C) is shown and depicts the disulfide bonds between Cys429-Cys456 and Cys436-Cys457 and the location of ‘‘loop 1’’ at the GluN2 ABD-ATD interface and heterodimer interface with GluN1. See Table S18 for F-statistics and p values.

Rare Variants Near the Transmembrane Domain Linkers Affect Channel Gating Agonist binding is transduced into channel opening via three linkers connecting the lower clamshell lobe, referred to as D2 (Figure S1), to the transmembrane helices. We hypothesized that variants in D2 that modestly reduced potency and resided outside the glutamate binding pocket may alter open probability. MTSEA potentiation showed that GluN2A-p.Met705Val, -p.Ile694Thr, and -p.Ala727Thr reduced open probability compared to WT receptors (Table S6). We also postulated that variants that enhanced both glutamate and glycine potency (Table 2) may alter gating. MTSEA potentiation was unaffected by rare variants that enhanced only glutamate potency, whereas three of the four variants that enhanced potency of both agonists altered open probability (Table S9). Variants located near the linkers had notable effects, with a 2-fold enhancement by GluN2B-p.Arg540His and a 4-fold decrease by GluN2Ap.Lys669Asn (Figure S9). Therefore, variants in D2 may affect agonist potency via altering transduction of agonist binding into gating, rather than agonist association or dissociation.

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Please cite this article in press as: Swanger et al., Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains, The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.10.002 Table 3. Predicted Synaptic and Non-synaptic Functional Changes of Mutant NMDARs Relative to WT

GluN2A-WT GluN2A-p.Val452Met

a

Synaptic Charge Transfer

Non-synaptic Charge Transfer

1.0

1.0

1.4 8.5 3 10

GluN2A-pGly483Arg GluN2A-p.Arg504Trp

3.7 2

1.6 3 102

0.26

0.19

a

2.0

2.4

GluN2A-p.Lys669Asn

1.2

GluN2A-p.Val506Ala

0.87 2

1.5 3 103

GluN2A-p.Val685Gly

4.2 3 10

GluN2A-p.Ile694Thr

0.35

0.15

GluN2A-p.Pro699Ser

0.76

1.3

GluN2A-p.Met705Val

0.28

0.17

GluN2A-p.Glu714Lys

0.96

0.92

GluN2A-p.Ala716Thra

0.08

0.02

GluN2A-p.Ala727Thr

0.32

GluN2A-p.Asp731Asn

1.3 3 10

GluN2A-p.Val734Leu

0.49

0.24 2

5.1 3 105 0.61

2

4.8 3 102

GluN2A-p.Lys772Glu

5.7 3 10

GluN2B-WT

1.0

1.0

GluN2B-p.Glu413Gly

3.4 3 103

2.0 3 103

GluN2B-p.Cys456Tyr

0.06

0.21 3

1.0 3 103

GluN2B-p.Cys461Phe

5.0 3 10

GluN2B-p.Arg540His

1.0

1.2

GluN2B-p.Arg682Cys

1.1

1.4

GluN2B-p.Arg696His

1.9

2.4

The values were calculated by Equations 4, 5, and 6 and indicate the fold difference in synaptic and non-synaptic function for mutant NMDARs relative to the corresponding WT GluN2A- and GluN2B-containing NMDARs (set as 1.0). a Variant was observed in the ExAC population samples (p.Val452Met ¼ 36 ExAC carriers; p.Val506Ala ¼ 3 p.Val506Leu ExAC carriers; p.Ala716Thr ¼ 1 ExAC carrier).

Estimating the Impact of Rare Variants on NMDAR Function Genetic variants identified in individuals with neurologic disorders had multifaceted, and often conflicting, consequences on NMDAR biology, rendering the interpretation of their overall impact on NMDAR activity and neuronal function challenging. We reasoned that estimating the effect of rare variants on two key modes of NMDAR function (synaptic and non-synaptic responses) would allow us to more accurately predict effects on neuronal function. To accomplish this, we developed a formula that combines measured parameters to estimate changes in synaptic and non-synaptic NMDAR charge transfer for variants relative to WT receptors (see Material and Methods for equations). Synaptic charge transfer can be estimated as the integral of an exponential function approximating the NMDAR synaptic time course, which is the product of the amplitude and deac-

tivation time constant (estimated by tW). Amplitude is influenced by channel open probability, receptor surface levels, and agonist potency. Deactivation rate is not a component of non-synaptic charge transfer: non-synaptic receptors are activated by ambient glutamate as opposed to phasic glutamate release, which occurs at synapses. Experimental glutamate and glycine EC50 values were used to calculate fractional responses at ambient glycine concentration (3 mM) and either synaptic (1 mM) or non-synaptic (100 nM) glutamate concentrations. Receptor properties were expressed relative to WT and used to calculate the fold change in synaptic and non-synaptic charge transfer (Table 3). Our analyses demonstrated several important points regarding the impact of genetic variants on NMDAR activity. First, functional enhancements could compensate for moderately reduced surface localization, such as GluN2B-p.Arg540His, but not severe reductions, such as GluN2B-p.Cys456Tyr. Second, due to the low glutamate concentration, changes in agonist potency impacted nonsynaptic function more than synaptic function. Third, the synergistic relationship between receptor properties suggests that robust changes could result from modest alterations to multiple parameters. We assessed the reliability of calculated synaptic charge transfer by measuring charge transfer for NMDAR responses to brief synaptic-like applications of saturating glutamate in HEK cells (Figure S10). Overall, relative changes in measured charge transfer, determined as the integral of the response time course (Table S10), were similar to predicted values, supporting this form of analysis. A caveat of experimental charge transfer is that the measurements are confounded by highly variable receptor levels across cells. Our calculated charge transfer approach measures inherent receptor properties and identifies the mechanistic underpinnings of altered synaptic and non-synaptic NMDAR function in a tractable system. Pharmacologic Modulation of Mutant NMDARs Our determination that rare variants altered synaptic and non-synaptic NMDAR charge transfer implied that these effects could contribute to neurological symptoms. To explore strategies for rectifying receptor dysfunction, we evaluated how positive or negative modulators affected select rare variants. Memantine, a well-tolerated NMDAR channel blocker approved for treatment of Alzheimer disease, was tested on GluN2B-p.Arg696His, which was predicted to enhance synaptic and non-synaptic function, as well as GluN2A-p.Lys669Asn, which may enhance synaptic function. Concentration-effect curves showed that memantine inhibited both variants with similar potency and efficacy as WT receptors (Figure 7A and Table S11). Thus, given brain concentrations of 0.5–1 mM,65 memantine could partially mitigate enhanced NMDAR function caused by these variants, which may be beneficial if persistent gain-of-function contributes to neurologic disease symptoms. The neurosteroid pregnenolone sulfate is a positive allosteric modulator at GluN1/GluN2A and GluN1/GluN2B

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S12). Pregnenolone sulfate potentiated the loss-of-function GluN1/GluN2B-p.Glu413Gly and -p.Cys461Phe receptors similarly to WT (Figure 7C and Table S12). In addition, pregnenolone sulfate prolonged the NMDAR deactivation time course for responses to brief, synapticlike glutamate applications (5 ms) in HEK cells. The GluN1/GluN2A-p.Val685Gly deactivation rate was prolonged from 15 5 1.2 ms to 26 5 1.4 ms, which significantly enhanced measured charge transfer (Figure 7E and Table S13). Pregnenolone sulfate increased charge transfer by 2-fold for GluN2B-WT and -p.Glu413Gly and 6.5-fold for GluN2B-p.Cys461Phe (Figure 7F and Table S14). Two additional positive allosteric modulators, spermine and FDA-approved tobramycin, potentiated GluN1/GluN2BWT, -p.Glu413Gly, and -p.Cys461Phe current responses in oocytes (Figure 7D and Table S15). However, their effects on NMDAR responses in HEK cells were complex as these modulators potentiated response amplitude, but accelerated deactivation time course (Table S14).68 These data suggest that further work evaluating positive allosteric modulators as a strategy for enhancing mutant NMDAR function could be beneficial.

Discussion

Figure 7. Modulation of NMDARs with Agonist Binding Domain Mutations by FDA-Approved Drugs (A–D) Two-electrode voltage-clamp recordings were used to measure NMDAR current responses to maximal glutamate (100 mM for GluN2B-p.Arg696His; 1–3 mM for GluN2A-p.Val685Gly and -p.Asp731Asn and for GluN2B-p.Glu413Gly and -p.Cys461Phe) and glycine (100 mM) in the presence of increasing concentrations of NMDAR modulator. Concentration-effect data were plotted as a percentage of the current response to glutamate and glycine without the NMDAR modulator. Data are mean 5 SEM. (A) The maximum inhibition and log(IC50) values for memantine at GluN1/GluN2B-WT and -p.Arg696His were compared by t tests. See Table S11 for values and statistics. (B–D) The maximum potentiation and log(EC50) values for pregnenolone sulfate (PS) or tobramycin were compared by one-way ANOVA and post hoc Dunnett’s tests. (E and F) Representative whole-cell recordings of current responses to rapid and brief glutamate applications (5 ms duration, black arrows) are shown for HEK cells transiently expressing human NMDARs in the presence of pregnenolone sulfate or vehicle. See Tables S12–S15 for values and statistics on positive modulators.

receptors.66,67 Concentration-effect curves showed that the maximum potentiation for WT GluN1/GluN2A receptors was 3-fold, whereas loss-of-function GluN1/GluN2Ap.Val685Gly and -p.Asp731Asn receptors were potentiated ~5-fold and ~9-fold, respectively (Figure 7B and Table

The list of known disease-associated rare variants in NMDAR genes is rapidly expanding, and mechanistic data are necessary to link genetic variants with underlying disease pathologies and improve therapeutic strategies. In this study, we identified mechanisms by which rare variants in the GluN2A and GluN2B agonist binding domains dysregulate NMDARs, including alterations to agonist binding, channel gating, receptor biogenesis, and forward trafficking. Together, these mechanisms lead to complex, and sometimes opposing, effects on NMDAR activity, which establishes the importance of analyzing many facets of NMDAR biology. We demonstrate a strategy to integrate these distinct parameters and estimate the overall impact on synaptic and non-synaptic NMDAR function. Through comprehensive biochemical and functional assessments, we determined that both gain- and loss-of-function variants in the same gene were associated with similar neurologic disorders, emphasizing that underlying mechanisms of NMDAR dysregulation cannot be inferred from disease symptoms alone. Although previous studies noted that gain-of-function and truncation or frameshift mutations were associated with similar outcomes,14,48 none had performed a comprehensive analysis that provided a complete picture of expression, trafficking, and function of NMDARs. Furthermore, we identified differences and similarities between NMDAR variants in GluN2A and GluN2B that may be relevant for modulating NMDAR function and understanding disease mechanisms. Our findings hold important implications for broadly relating genetic variation to disease as well as understanding the role of NMDAR variants in neurological disorders.

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Mechanisms of NMDAR Dysregulation Our data allow several conclusions to be drawn about structural features mediating the effects of genetic variants. Many disease-associated rare variants reside within the highly conserved glutamate binding cleft and altered the atomic contacts for glutamate. Some mutated residues were known structural determinants of glutamate binding.58,59,69–72 Consistent with these reports, rare variants that occurred at these residues severely reduced glutamate potency. These rare variants also accelerated NMDAR deactivation, which sets the time course of the slow, calciumpermeable component of excitatory postsynaptic currents. Altering the synaptic time course could impact total synaptic charge transfer as well as spike timing-dependent synaptic mechanisms.73 Furthermore, reduced glutamate potency correlated with decreased surface and total NMDAR levels, probably because agonist binding is required for forward trafficking to prevent dysfunctional receptors from reaching the cell surface.55,56,74–78 Importantly, diseaseassociated rare variants restricted NMDAR trafficking in cultured neurons as well. Thus, variation within the glutamate binding cleft can lead to severe loss of function through cumulative effects on glutamate potency, deactivation rate, protein levels, and forward trafficking. Rare variants at GluN1-GluN2 interfaces may dysregulate NMDARs through disruption of tertiary structure. For instance, missense mutations at Cys residues that form disulfide bonds reduced NMDAR protein levels and forward trafficking. These disulfide bonds probably facilitate folding of structural features at the heterodimer interface, including ‘‘loop 1,’’ which is predicted to assist tetramer assembly via interactions with helix G in GluN1.58,60,62,63 Similarly, GluN1/GluN2A-p.Lys772Glu receptors had low surface levels but only modestly reduced glutamate potency. Lys772 resides in a charged region of the dimer interface that interacts with the GluN1 agonist binding and amino-terminal domains.58,63 Therefore, it is plausible that NMDAR protein levels and forward trafficking could be reduced by altered tertiary structure and receptor assembly, despite the receptors having relatively normal or even enhanced functional properties. Rare variants at the interface of the D1 and D2 clamshell lobes may also dysregulate NMDARs by affecting tertiary structure. The GluN2A-p.Gly483Arg variant is in a conserved region of D1 that makes cross-cleft interactions with helices F and G in D2 to stabilize the agonist-bound closed-cleft conformation.59,79,80 Perhaps, substituting a large, polar Arg for the small, nonpolar Gly disrupted crosscleft interactions, leading to reduced potency. Conversely, the GluN2A-p.Pro699Ser and GluN2B-p.Arg696His variants in the complementary part of D2 enhanced glutamate potency, perhaps by stabilizing the closed-cleft conformation.60,80,81 Rare variants in helices F and G also altered open probability, which is consistent with reports implicating these helices in channel gating.81 Lastly, NMDAR open probability was affected by rare variants at Lys669 and Arg540, which reside at junctions with the linkers that

transduce agonist binding into channel opening. Notably, the S1-M1 and M3-S2 linkers and neighboring regions in S1 and S2 had synonymous variation but lacked missense variation in the ExAC population. Moreover, disease case variants were over-represented in these regions, indicating that functional variation therein is likely to be deleterious. Integrating the Mechanisms of Dysregulated NMDAR Biology Investigating the mechanisms underlying NMDAR dysregulation revealed significant complexity in NMDAR functional genomics relevant for understanding and treating disease. For instance, rare variants affected multiple aspects of NMDAR biology resulting in conflicting consequences. Thus, evaluating one receptor property in isolation can provide misleading or incomplete conclusions. Our approach emphasizes the measurement and consideration of multiple aspects of synaptic and non-synaptic NMDAR function. Some rare variants affected synaptic and nonsynaptic charge transfer differently, which has important implications for disease pathology because synaptic and non-synaptic NMDARs have distinct roles in neuronal excitability. Furthermore, modest effects on individual parameters may have a robust cumulative impact on receptor function, and reduced receptor protein levels can negate functional enhancements. These findings underscore the need for a comprehensive analysis of NMDAR activity to discern the impact of genetic variants. Our data combined with previously published reports suggest that similar disease phenotypes can result from both enhanced and reduced NMDAR function. In support of this, epilepsy is associated with at least four loss-of-function and two gain-of-function GluN2A variants studied herein as well as several reported gain-of-function GluN2A variants.13,14,16,48,49 Likewise, gain- and loss-of-function GluN2B variants are both linked to intellectual disabilities, which is consistent with reported GluN2B transmembrane domain variants.24,48 It is conceivable that either enhanced or reduced NMDAR function could lead to intellectual disability and epilepsy because both increases and decreases in excitatory synapse strength and number are evident in single-gene disorders associated with intellectual disability and epilepsy.82 Furthermore, GluN2A and GluN2B are expressed in both excitatory and inhibitory neurons in the brain;3,83–85 therefore, the impact NMDAR variants have on the balance of excitation and inhibition in brain circuits will be influenced by the functions (i.e., postsynaptic, non-synaptic, presynaptic) and expression levels of GluN2A and GluN2B in different neuron types. Additionally, compensatory mechanisms and variation in other genes could affect brain development and lead to paradoxical effects on circuit function. Interestingly, whether a variant occurs in GluN2A or GluN2B appears to be a key factor in disease phenotype. For example, the GluN2A-p.Cys436Arg, -p.Val685Gly, and -p.Asp731Asn variants were identified in individuals

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with severe intractable epilepsy, general tonic clonic seizures, and developmental delay.13,24 The equivalent or mechanistically similar variants in GluN2B, namely p.Cys436Arg, p.Glu413Gly, and p.Cys461Phe, were associated with severe intellectual disability and, in the case of GluN2B-p.Cys461Phe, absence seizures.15,23 GluN2B expression is higher than GluN2A during embryonic and early postnatal development,2,3 which could contribute to more severe intellectual disability resulting from GluN2B dysfunction. Furthermore, GluN2A and GluN2B have distinct roles during synaptogenesis and cannot functionally substitute for one another.34,86–90 Animal models will be necessary to understand how brain circuits respond to NMDAR variants in GluN2A versus GluN2B or with reduced versus enhanced function. However, an important message from these in vitro data is that disease phenotype alone cannot predict how the underlying NMDARs have been dysregulated. Intolerance of GluN2A and GluN2B to Variation Evaluating genetic variation across the functional domains of GluN2A and GluN2B revealed interesting connections between intolerance to variation, NMDAR dysregulation, and neurologic disease. Our findings established that the agonist binding domain, transmembrane domain, and associated linker regions have an exceptionally low tolerance for functional variation. Remarkably, most diseaseassociated rare variants occurred in short stretches in these domains wherein missense variation was absent and synonymous variation was high in the ExAC control population. Furthermore, our study demonstrated that GRIN2B is clearly less tolerant to functional variation in the agonist binding domain than GRIN2A, whereas the linker regions and transmembrane domain of both genes are highly intolerant. This is supported by the paucity of inherited GRIN2B variants in the agonist binding domain, the robust consequences of all GRIN2B variants on NMDAR activity, and the severe disability and developmental delay in all individuals with de novo GRIN2B variants in the agonist binding domain. These observations are consistent with the dominant expression of GluN2B-containing NMDARs during rapid cortical synaptogenesis in late embryonic and early postnatal development91 and their key role in neuronal development and synaptic plasticity.92,93 The wide spectrum of neurologic phenotypes and functional consequences associated with both de novo and inherited GRIN2A variants suggest that missense variants in the agonist binding domain of GRIN2A are, on average, less likely to cause severe disease than GRIN2B. Elucidating these distinctions in genetic intolerance between different NMDAR genes as well as small regions within one gene may be important for deciphering which rare variants identified in a genome are relevant to the individual’s disease and, thus, may facilitate determining appropriate therapeutic targets. Surprisingly, several GRIN2A variants that caused dramatic changes in glutamate potency were inherited

from individuals with mild intellectual disability or verbal dyspraxia but not severe neurologic disease as observed in the individuals described herein. These GRIN2A variants probably affect neurologic function, but other genetic factors must also contribute to the severe disease phenotypes in these individuals. Perhaps altered glutamate binding to GluN2A can be compensated for by one normal allele, whereas variants affecting NMDAR biogenesis, such as p.Cys436Arg (studied herein) or channel gating, such as p.Leu812Met49 and p.Pro552Arg22 (and unpublished data), are more likely to be deleterious. In contrast, all GRIN2B variants studied herein occurred de novo and were associated with severe developmental delay, intellectual disability, and, in some cases, seizures, including GRIN2B variants that reduced glutamate potency. Perhaps altered agonist potency at GluN2B is more deleterious due to GluN2B being required during synapse formation when connections are weak and non-synaptic NMDAR function is critical.94–96 Interestingly, our calculated charge transfer showed small or no changes for two gain-of-function GluN2B variants that occurred in particularly intolerant regions of the agonist binding domain. This result may suggest that altering a specific parameter, such as deactivation time course, can be deleterious even if overall charge transfer is not significantly affected. Future studies on the functional consequences of missense variation in a control population may help elucidate which parameters are most relevant for disease. Approaches for Regulating NMDAR Activity Therapeutic strategies for correcting NMDAR function are limited. The pan-NMDAR channel blocker memantine inhibited NMDARs containing the gain-of-function GluN2A-p.Leu812Met variant and may have modest clinical utility in a limited subset of individuals.49,97 However, GluN1/GluN2A-p.Asn615Lys receptors were insensitive to memantine,97 demonstrating the necessity for personalized therapies. Herein, memantine inhibited GluN1/GluN2Ap.Lys669Asn and GluN1/GluN2B-p.Arg696His receptors similarly to WT, suggesting that memantine levels reached in vivo might attenuate function of these mutant receptors. However, the enhanced potency of memantine at GluN2Dcontaining NMDARs in interneurons may reduce inhibition as an unanticipated consequence, which could have confounding effects.85,98 As for rectifying loss-of-function variants, enhancing NMDAR activity carries the risk of engaging excitotoxic mechanisms. However, modulators with properties similar to pregnenolone sulfate, which can modulate deactivation time course to a greater extent than current amplitude, may be useful. Our data demonstrate the proof of principle that NMDARs with rare variants can be positively and negatively modulated and provide impetus for continued development of clinically available NMDAR modulators. An alternative strategy for rare variants that disrupt tertiary structure and reduce receptor protein levels is to develop pharmacological chaperones, which

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could assist protein folding and NMDAR assembly. Pharmacological chaperones have enhanced protein stability and forward trafficking of other ion channels, including ATPsensitive potassium channels and cystic fibrosis transmembrane conductance regulator.99–101 A substantial portion of disease-associated NMDAR variants appear to cause protein folding or assembly defects, and perhaps these variants would benefit more from facilitated biogenesis than modulation at the cell surface. In summary, the consequences of rare variants can be multifaceted and conflicting, which highlights the importance of a thorough and comprehensive analysis of NMDAR biology to understand disease mechanisms. Moreover, our findings suggest that enhanced circuit excitability, as occurs in seizures, is not necessarily indicative of enhanced NMDAR activity. Conversely, the cognitive impairments associated with developmental delay or intellectual disabilities are not solely suggestive of reduced NMDAR activity. Our study is consistent with disease phenotype being more closely associated with which GluN2 subtype is affected as opposed to the direction of change in receptor activity. Moreover, severe neurologic disease was more strongly linked with functional variation in the agonist binding domain of GRIN2B compared to GRIN2A. Altogether, this study elucidated relationships between genetic variants, NMDAR dysfunction, and disease phenotypes fundamental to understanding NMDAR functional genomics and advancing personalized therapies for individuals with rare variants. Supplemental Data Supplemental Data include 10 figures and 19 tables and can be found with this article online at http://dx.doi.org/10.1016/j. ajhg.2016.10.002.

Acknowledgments The authors would like to thank the Exome Aggregation Consortium and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at the ExAC website. The authors also thank Dr. Dennis Liotta for generous support of this study. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (R01HD082373 to H.Y.), the National Center for Advancing Translational Sciences of the NIH (UL1TR000454 to H.Y.), the National Institute of Neurological Disorders and Stroke (F32NS086361 to S.A.S. and NS036654 to S.F.T.), and the Xiangya-Emory Medical Schools Visiting Student Program (to W.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. S.F.T. is co-founder of NeurOp Inc., a consultant of Janssen Pharmaceuticals, Pfizer, and NeurOp Inc., and a PI on a research grant from Janssen to Emory University. S.P. serves on the advisory board of Pairnomix. Received: August 20, 2016 Accepted: October 3, 2016 Published: November 10, 2016

Web Resources ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/ ExAC Browser, http://exac.broadinstitute.org/ ExAC sub-populations, ftp://ftp.broadinstitute.org/pub/ExAC_release/ release0.3/subsets/ Human Gene Mutation Database, http://www.hgmd.org/ Lollipops v.1.3.1, https://github.com/pbnjay/lollipops/releases OMIM, http://www.omim.org/ RCSB Protein Data Bank, http://www.rcsb.org/pdb/home/ home.do RRID, https://scicrunch.org/resources SCAN, http://www.ucl.ac.uk/Pharmacology/dcpr95.html UniProt, http://www.uniprot.org/

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