Available online at www.sciencedirect.com
ScienceDirect NMDA receptor subunit mutations in neurodevelopmental disorders Nail Burnashev1,2,3 and Pierre Szepetowski1,2,3,4 N-Methyl-D-aspartate receptors (NMDARs) are glutamategated cation channels that are expressed throughout the brain and play essential role in brain functioning. Diversity of the subunits and of their spatio-temporal expression imparts distinct functional properties for the particular NMDAR in a particular brain region and developmental stage. Mutations in NMDARs may have pathological consequences and actually lead to various neurological disorders. Recent human genetic studies as highlighted here show the existence of multiple alterations in NMDARs subunits genes in several usual and common brain diseases, such as intellectual disability, autism spectrum disorders (ASD), or epilepsy. Relation of a particular mutation to the corresponding alteration of NMDARs function may provide an avenue to the targeted therapy for the pharmacological treatment of the disorders. Addresses 1 INSERM UMR_S901, Marseille, France 2 Mediterranean Institute of Neurobiology (INMED), Marseille, France 3 Aix-Marseille University, Marseille, France 4 French Epilepsy, Language and Development (EPILAND) Network, France Corresponding authors: Burnashev, Nail ([email protected]) and Szepetowski, Pierre ([email protected])
Current Opinion in Pharmacology 2015, 20:73–82 This review comes from a themed issue on Neurosciences Edited by Pierre Paoletti and Jean-Philippe Pin
modulatory subunits GluN2(A–D) or GluN3(A,B) to form either di-heterotetrameric or tri-heterotetrameric channels. All NMDAR subunits, as other glutamate receptors contain modular domains that are responsible for controlling distinct functions. An amino terminal domain (ATD) contributes to control of ion channel open probability and deactivation speeds and contains binding sites for subtype-specific allosteric modulator compounds, including zinc (GluN2A and 2B), ifenprodil (GluN2B), and polyamines (GluN2B). A ligand-binding domain (LBD) binds agonists, such as glutamate, glycine and antagonists to control ion channel opening. NMDARs are activated upon concurrent binding of glycine or D-serine to LBD of GluN1 and L-glutamate to LBD of GluN2 subunits [2,3]. Recent studies also implicate the ATD as a strong determinant of agonist efficacy [4], and interdomain interactions between the LBD and the ATD of the GluN2 subunit may be a central element in controlling channel opening upon agonist binding. The transmembrane domain (TMD) is formed by four hydrophobic segments (M1 to M4), with M2 partially entering the membrane to form the ion channel (Figure 1). An intracellular carboxyl terminal domain (CTD) associates with postsynaptic density (PSD) proteins, which in turn facilitates intracellular signaling. Important basic electrophysiological properties of NMDARs that allocate these receptors from other glutamate receptors include the use of co-agonist (glycine, D-serine) for activation, relatively slow current kinetics, voltage-dependent block by extracellular Mg2+, and high Ca2+ permeability.
http://dx.doi.org/10.1016/j.coph.2014.11.008 1471-4892/# 2014 Elsevier Ltd. All right reserved.
Introduction N-Methyl-D-aspartate receptors (NMDARs) are cation channels that are gated by the major excitatory neurotransmitter glutamate. NMDAR-mediated signaling is involved in normal development, plasticity, learning, memory and high cognitive functions. NMDARs play important role in temporal integration of neuronal network activity and long-term alterations in synaptic structure and function. There is long known association of NMDAR dysfunctions with various neurodevelopmental disorders and NMDARs might be targets for pharmacological treatment [1,2]. Typically NMDARs are composed of two obligatory GluN1 subunits and two www.sciencedirect.com
In general, much of the variation in function between NMDAR subtypes has been attributed to the identity of the GluN2 (GluN3) subunits in the channel assembly [5–7]. The GluN2 (GluN3) subunits have different temporal and spatial expression patterns in the brain and NMDAR subtypes also vary according to the cell types and subcellular localization. Different neuronal types usually express distinct combinations of NMDAR subunits thus providing a diversity of cell-specific and even input-specific synaptic responses. In addition to their postsynaptic localization, NMDARs are found at presynaptic, extrasynaptic and perisynaptic sites. Presynaptic NMDARs with different subunit composition expressed in axonal terminals may differentially modulate synaptic strength [1,2]. Although physiological function of extrasynaptic NMDARs is not fully understood, their activation by glutamate spillover may provide conditions for increased excitability and contribute to long-term synaptic changes [8]. In addition to the Current Opinion in Pharmacology 2015, 20:73–82
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Figure 1
(a)
Met1Thr Pro31fs Pro79Arg
N Phe183Ile Ile184Ser Trp198* Glu218* Cys231Tyr Ala243Val Arg370Trp Val375fs Cys436Arg Gly483Arg Arg504Trp Arg518His Pro522Arg
Phe139fs
Pro699Ser Met705Val Glu714Lys Ala716Thr Ala727Thr Asp731Asn Val734Leu
Ala290Val Gly295Ser Leu334* Val529fs Thr531Met Ser547del Ala548Thr
Ile694Thr Arg681* Lys669Asn Phe652Val Leu649Val
M1
M3
Lys772Glu Leu779fs Leu812Met Ile814Thr Met817Val
M4
M2 Ile904Phe Asp933Asn Tyr943*
Asp615Lys
Tyr1387* Asn976Ser
Asp1251Asn
(b)
N
C
Ser34fs
Gln711*
Thr268fs Cys456Tyr Cys461Phe Arg682Cys Arg540His Pro553Leu
M1
Leu825Val
M4
M3 M2
Trp559*
Ala636Pro Val618Gly Asn615Leu
C Current Opinion in Pharmacology
Location of GluN2A and of GluN2B mutations. (a) Mutations in GluN2A. (b) Mutations in GluN2B. The locations of nonsense, frameshift, splice-site and missense mutations found in NMDARs subunits are indicated in the schematics of the corresponding proteins. M1–M4 represent the four transmembrane domains of each subunit. Numbers indicate aminoacid positions.
neuronal expression, NMDARs are expressed in astrocytes, oligodendrocytes and microglia [9–11]. Taken together, it is not surprising that mutations in GRIN1, Current Opinion in Pharmacology 2015, 20:73–82
GRIN2A and GRIN2B, respectively, may cause somehow related and sometimes overlapping albeit clearly different spectrum of disorders. www.sciencedirect.com
NMDAR mutations in neurodevelopmental disorders Burnashev and Szepetowski 75
In the present review we will highlight recent studies on NMDAR subunit mutations in neurodevelopmental disorders and discuss their possible relations to alteration of NMDAR function and to clinical manifestations.
GRIN1 mutations GRIN1 encodes the GluN1 subunit of NMDARs. The gene maps at human chromosome 9q34 and spans more than 29 kb. Several alternatively splice variants with differences in expression patterns and in trafficking or electrophysiological properties of the corresponding isoforms have been described. The screening of 197 genes encoding ionotropic and metabotropic glutamate receptors and their known interacting proteins in a cohort of 95 sporadic patients with non-syndromic intellectual disability (ID), fished out one de novo GRIN1 missense mutation (p.Glu662Lys) in a patient with moderate ID [12]. Subsequent screening led to the identification of an in-frame 3 bp duplication (p.Ser560dup) in a patient with severe ID and partial complex epilepsy. As will be further examplified below, this and other types of comorbid associations are frequent in the context of neurodevelopmental disorders and likely reflect the existence of shared pathophysiological mechanisms at the intertwined molecular, cellular, electrophysiological, and developmental levels. Exome sequencing of 264 trios with epileptic encephalopathies (EEs) identified another de novo GRIN1 missense mutation (p.Tyr647Ser) in a girl with infantile spasms at age 4 months and who had subsequent profound delay [13]. In all three cases neuroimaging was normal. The p.Ser560dup mutation occurred just before the first transmembrane segment (M1) of GluN1. It drastically decreased the activity of the receptor when Xenopus oocytes were coinjected with mRNAs of GluN2B and either of wild-type or mutated GluN1. In contrast, p.Glu662Lys (located in the post-transmembrane segment M3) led to an increase in NMDAR-induced Ca2+ currents, which might be excitotoxic. The fact that two mutations with somehow opposite effects in vitro lead to overlapping or even to identical phenotypes represents an apparent paradox that has already been described in the case of another epilepsy gene (namely SCN1A mutations causing the syndrome of genetic epilepsy with febrile seizures plus) and that is also encountered in the case of GRIN2A mutations (see below). Nevertheless, whether the functional difference seen in the case of those two GRIN1 mutations underlies the more severe ID and the epileptic phenotype seen in the patient with p.Ser560dup remains elusive. Rather, this interpretation might well be an oversimplified view. As will be further seen, most if not all functional consequences of the human mutations found in NMDARs subunits have been studied using reconstituted receptors in vitro; whereas such tests are very useful and necessary to demonstrate the existence of functional differences between mutant and wild-type receptors, the type and extent of the pathophysiological www.sciencedirect.com
consequences that those mutations might actually have in vivo should be extrapolated with caution.
GRIN2A mutations GRIN2A maps at human chromosome 16p13 and codes for the GluN2A subunit. More than 60 heterozygous GRIN2A mutations of various types have been found in the very last years in patients and families presenting with disorders of the epilepsy-aphasia spectrum. Rolandic epilepsy (RE) is the most frequent and so-called ‘benign’ form of this spectrum. There are long known relationships between RE and various comorbid conditions, such as cognitive and behavioral issues, or speech and/or language impairment (e.g. verbal dyspraxia). The continuous spike-and-waves during slow-wave sleep syndrome (CSWSS) and the Landau-Kleffner syndrome (LKS) — also known as ‘acquired’ epileptic aphasia — are two closely related epileptic EEs that represent more severe and less frequent forms of this continuum. All those syndromes share the association of usually unfrequent seizures with paroxysmal electroencephalographic (EEG) discharges activated during drowsiness and sleep, and with more or less severe neuropsychological deficits, such as the autistic regression seen in LKS patients. In contrast with RE, the genetic influence in LKS and in CSWSS has long remained controversial [14]. The search for genomic alterations that would influence LKS or CSWSS [15] has been a crucial step towards the identification of a first cause for this fascinating group of disorders situated at the crossroads between epileptic, cognitive, autistic and language disorders. Particularly the detection of one de novo partial microdeletion of GRIN2A in a girl with LKS [15] has represented a key starting point. Since then, the crucial and direct causal role of de novo or inherited GRIN2A mutations in LKS, in CSWSS, and in RE with verbal dyspraxia, has been demonstrated in three main parallel studies which each used a different screening strategy to reach similar conclusions [16,17,18]. In the first study [18], the screening of sporadic and familial cases with focal epilepsies and EEs of the epilepsy-aphasia spectrum led to the identification of 17 different GRIN2A mutations, in about 20% of all patients; particularly all undisputable familial cases had mutations in GRIN2A. GRIN2A defects occurred in patients with variable degree of phenotypic severity and were of various types (microdeletions, splice-site, nonsense, missense) (Table 1). The mutations were spaced throughout the different domains of the protein (Figure 1a) and no genotype–phenotype correlation appeared clearly. In a second study [16], next-generation sequencing of a candidate genes panel in numerous patients with various types of EEs, identified four inherited mutations (two splice-site and two missense) (Table 1 and Figure 1a) only in the subset of epilepsyaphasia families. No GRIN2A mutation was found in the rest of the cohort, which suggested that GRIN2A defects in EEs might be somehow restricted to the epilepsy-aphasia Current Opinion in Pharmacology 2015, 20:73–82
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Table 1 GRIN2A defects found in various neurodevelopmental disorders. CSWSS, continuous spike and waves during slow-wave sleep syndrome; CNVs, copy number variations; CTS, centrotemporal spikes; EE, epileptic encephalopathy; ESES, electrical status epilepticus during sleep; FS, febrile seizures; GTCS, generalized tonic-clonic seizures; ID, intellectual disability; IS, infantile spasms; LKS, Landau-Kleffner syndrome; NA, not available; ND, not determined; NMD, nonsense-mediated mRNA decay; pred, predicted; RE, Rolandic epilepsy; UTR, untranslated region. Chromosomal locations are given according to the February 2009 human reference sequence (hg19, build37) (http:// genome.ucsc.edu/) Mutation CNVs del chr16: 7 964 000–10 607 500 (hg19) del chr16: 8 992 500–9 992 500 (hg19) del chr16: 9 365 500–11 273 700 (hg19) del chr16: 9 809 522–9 856 618 (hg19) del chr16: 9 908 477–9 934 830 (hg19) del chr16: 9 850 000–9 900 000 (hg19) (precise coordinates NA) del chr16: 9 825 000–10 075 000 (hg19) (precise coordinates NA) del chr16: 10 227 121–10 354 862 (hg19) del chr16: 10 250 000–10 275 000 (hg19) (precise coordinates NA) dup chr16: 10 075 000–10 225 000 (hg19) (precise coordinates NA) Translocation t(16;17)(p13;q11)
Nonsense p.Trp198* p.Gln218*
p.Leu334* p.Arg681* p.Tyr943* p.Tyr1387* Splice-site c.1007+1G>A c.1007+1G>A c.1007+1G>A c.1007+1G>A c.1007+1G>A c.1007+1G>T c.1123 2A>G
c.2007+1G>A
Phenotype Pseudo-Lennox syndrome, various epileptic seizures, ESES, severe ID, no speech, dysmorphism RE, moderate ID, delayed speech, dysmorphism Myoclonic seizures, severe ID, no speech, dysmorphism, CTS RE + mild ID
Consequences
Refs
Total microdeletion, no product
[21]
Partial microdeletion, p.Lys592fs (pred)
[21]
Total microdeletion, no product (pred)
[21]
Partial microdeletion, p.Arg865fs (pred)
[20]
LKS (proband), LKS + verbal dyspraxia (brothers) Atypical benign partial epilepsy
Partial microdeletion, p.Phe670fs (pred)
[18]
ND
[17]
RE
ND
[17]
CSWSS + verbal dyspraxia (three brothers)
Partial microdeletion, no product (pred)
[18]
RE
ND
[17]
CSWSS
Intronic; disruption of regulatory element?
[17]
FS, GTCS, severe ID (proband), GTCS + learning difficulties (father, aunt), seizures + severe ID (cousin)
Gene disruption
[24]
Atypical benign partial epilepsy Moderate ID + CTS (proband), FS + focal seizures + learning difficulties (mother), infantile seizures + learning difficulties (grandmother) CSWSS (proband), Panayiotopoulos syndrome (brother), partial epilepsy (father) LKS (proband) CSWSS (proband), FS + CTS (sister), RE (father) CSWSS (proband), childhood epilepsy (mother, uncle)
Truncated or no product NMD, no product
[17] [24]
Truncated or no product
[17]
Truncated or no product Truncated or no product
[17] [17]
Loss of phosphorylation-dependent regulation by Src kinases (pred)
[18]
RE + verbal dyspraxia (proband + six relatives) CSWSS (proband + father) LKS Atypical benign partial epilepsy (proband), uncharacterized epilepsy (two half brothers) LKS (proband), RE (half sister) CSWSS (proband), CTS (brother) CSWSS (proband + brother), CSWSS + dysphasia (aunt), LKS (uncle), RE + dysphasia (grand-mother), RE (mother, cousin) CSWSS (proband), uncharacterized epilepsy (father)
p.Phe139fs (pred)
[16]
p.Phe139fs (pred) p.Phe139fs (pred) p.Phe139fs (pred)
[16] [17] [17]
p.Phe139fs (pred) p.Phe139fs (pred) p.Val375fs (pred)
[17] [17] [18]
ND
[17]
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NMDAR mutations in neurodevelopmental disorders Burnashev and Szepetowski 77
Table 1 (Continued ) Mutation Indels p.Pro31fs p.Val529fs p.Ser547del p.Leu779fs Missense p.Met1Thr p.Pro79Arg p.Phe183Ile p.Ile184Ser p.Cys231Tyr p.Ala243Val p.Ala290Val p.Gly295Ser p.Arg370Trp p.Cys436Arg p.Gly483Arg p.Arg504Trp p.Arg518His p.Arg518His p.Pro522Arg p.Thr531Met
p.Ala548Thr p.Asp615Lys p.Leu649Val
p.Phe652Val p.Lys669Asn p.Ile694Thr p.Pro699Ser p.Met705Val p.Glu714Lys p.Ala716Thr p.Ala727Thr p.Asp731Asn p.Val734Leu p.Lys772Glu p.Leu812Met
p.Ile814Thr p.Met817Val p.Ile904Phe p.Asp933Asn p.Asn976Ser p.Asn976Ser p.Asp1251Asn
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Phenotype
Consequences
Refs
RE (proband, brother, father) RE (proband, brother, mother) CSWSS (proband), CTS (sister) CSWSS
Frameshift Frameshift Frameshift Frameshift
[17] [17] [17] [17]
LKS (proband, sister), seizures + speech/ language impairment (father) CSWSS (proband), RE (mother, uncle, grandmother) RE CSWSS LKS (proband), CTS (brother, sister) RE + learning difficulties RE RE RE Atypical benign partial epilepsy CSWSS + dysphasia (proband), RE + dysphasia (sister) CSWSS (proband, brother) CSWSS (proband), RE (brother), verbal dyspraxia (father) LKS Early-onset seizures + severe ID + no speech CSWSS (proband), intermediate epilepsyaphasia (brothers), learning difficulties + speech/language impairment (mother) LKS IS, myoclonies, severe ID Epileptic seizures, severe ID, dysplastic corpus callosum, severe feeding impairment, hypothyroidy, dysmorphism CSWSS
ND
[16]
ND
[17]
ND ND
[17] [18] [17] [17] [17] [18] [17] [17] [18]
CSWSS LKS RE RE (proband, brother, sister) CSWSS RE + verbal dyspraxia (proband + six relatives), verbal dyspraxia (cousin) RE RE + verbal dyspraxia (proband), verbal dyspraxia (mother) RE (proband, brother) Atypical benign partial epilepsy Early-onset EE + profound developmental delay
RE Refractory seizures + global developmental delay RE (proband, father), FS + CTS (brother) LKS Atypical benign partial epilepsy CSWSS RE (proband), absence epilepsy (father)
Impaired zinc inhibition ND ND ND ND ND ND Increased open state duration + decreased close state duration See above ND Increased open state duration
[18] [18]
ND Loss of magnesium block ND
[18] [24] [22]
Increased open state duration + decreased close state duration ND ND ND ND ND ND
[18]
ND ND
[17] [17]
ND ND Increased potency of glutamate and glycine, decreased magnesium block, reduced proton sensitivity, reduced zinc inhibition, increased open probability ND ND
[17] [17] [23]
ND ND ND ND ND
[17] [18] [17] [17] [18]
[38] [22] [16]
[18] [18] [17] [17] [17] [18]
[17] [25]
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syndromes. In the third study [17], various heterozygous GRIN2A mutations (Table 1 and Figure 1a) were also found in about 8% of patients with either of RE, LKS or CSWSS. Mutations were more frequent in the more severe phenotypes. Since then, several studies have identified additional GRIN2A defects in patients with disorders of the epilepsy-aphasia spectrum [19,20]. A few other GRIN2A heterozygous defects (Table 1) were reported in patients presenting with various and more severe forms of neurodevelopmental disorders that did not belong to the epilepsy-aphasia spectrum but that showed partially overlapping clinical features, variably associating dysmorphia, ID, epileptic manifestations that included seizures of the Rolandic area and nocturnal paroxysms or refractory seizures without electroclinical seizure during sleep, behavioral impairment, profound developmental delay [21,22,23,24,25]. All those findings demonstrate that the phenotypic spectrum of GRIN2A mutations expands beyond the epilepsy-aphasia core group of disorders. Moreover, GRIN2A was also identified recently as a modifying genetic factor influencing Parkinson’s disease in inverse association with caffeine intake [26,27]. On the one hand, the existence of GRIN2A microdeletions and of nonsense mutations argued for haploinsufficiency effects. This might lead to a relative excess in GluN2B subunits which in turn would lead to NMDARs opening longer, resulting in temporarily hyperactive NMDARs. Also, pro-survival GluN2A-mediated signaling is involved in normal development [28] of inhibitory interneurons and those subunits are heavily expressed in parvalbumin (PV)-positive interneurons [29]. Thus loss of function of GluN2A may lead to undeveloped inhibitory network. Moreover, disruption of NMDAR signaling specifically in PV-positive interneurons may lead to a looser inhibitory control of excitatory principal neurons. All these would increase overall excitability of the neuronal networks and promote proepileptic effect. On the other hand, other mutations show gain-of-function effects, at least in vitro. ‘Gain-offunction’ in excitatory neurons due to subunit overexpression and/or to slowing down of the kinetics of NMDAR-mediated currents would also favor overexcitability due to increased excitatory drive and higher extent of temporal integration of sensory signals in thalamocortical circuits. Several studies of the functional consequences of specific GluN2A missense mutations have indeed made the overall pathophysiological picture(s) complicated and heterogeneous, and hence are worth being mentioned with more details. It should be emphasized that the differences in the consequences of the mutations described below, might at least partly reflect the variability in the battery of electrophysiological evaluations that were performed in each specific case. Current Opinion in Pharmacology 2015, 20:73–82
The p.Ala243Val found in a patient with RE and learning difficulties [17] occurred in the Zn2+ binding domain and led to an impaired inhibition of mutant NMDAR currents by low concentrations of Zn2+ in vitro. This suggested that, in vivo, there might be a reduced highaffinity Zn2+-mediated inhibition that in turn would lead to increased susceptibility to channel activation and to increased calcium influx. The p.Arg518His and the p.Thr531Met mutations that were found in typical epilepsy-aphasia families occurred in the extracellular LBD of GluN2A. They were predicted to modify NMDAR gating and kinetic properties by affecting ligand binding. As a matter of fact, the durations of channel open and closed states as calculated by single-channel recordings, were significantly modified when the corresponding mutant heteromeric GluN1/GluN2A-p.Arg518His NMDAR was reconstituted in HEK cells [18]. Similarly, p.Thr531Met led to a shift in NMDAR kinetics with a significant increase in the mean duration of the open state [16]. Whereas the p.Pro522Arg mutation is expected to have similar electrophysiological effects, it was found in a patient with a much more severe phenotype that falls outside the epilepsy-aphasia spectrum (Table 1) [22]. p.Asp615Lys occurred near the apex of the pore-lining reentrant loop (the M2-loop) of GluN2A — a site known to strongly influence Mg2+ block. Reconstituted NMDARs with wild-type GluN1 and mutant p.Asp615Lys-GluN2A showed a dramatic loss of the Mg2+ block and a decrease in Ca2+ permeability, whereas co-expression of wild-type and mutant GluN2A with wild-type GluN1 had intermediate effects. The loss of Mg2+ inhibition of NMDAR activity likely results in increased current flow through mutant-GluN2A-containing NMDARs, leading to excessive excitatory drive. Again this is in sharp contrast with the haploinsufficiency effects of other GRIN2A defects as mentioned above. A de novo mutation (p.Phe652Val) (Figure 1a) yielded qualitatively similar results as those obtained for channels with other mutations situated right within the LBD (see above) [18]. Hence the Phe652 residue might well represent a new site for the modulation of NMDA receptor kinetics. This may also be postulated in the case of the Leu812 residue which also led to mutant channels that open more often (see below). Moreover, p.Phe652Val was found in a patient with typical CSWSS, whereas another missense mutation situated only three aminoacids apart, p.Leu649Val, was detected in a patient with severe ID, dysplastic corpus callosum, epileptic seizures, severe feeding impairment, hypothyroidy, and mild facial dysmorphisms [22]. p.Leu812Met was located in the linker region between the ligand-binding and transmembrane domains. This mutation had multiple effects on the properties of the corresponding GluN1/GluN2A mutant receptors expressed in Xenopus oocytes or in HEK cells [23] (Table 1). The overall hyperactivation seen in that case might well drive neuronal hyperexcitability and possibly neuronal excitotoxic loss — and hence might www.sciencedirect.com
NMDAR mutations in neurodevelopmental disorders Burnashev and Szepetowski 79
lead to epileptic seizures. However and as already discussed just above in the case of p.Asp615Lys, heterozygous loss-of-function GRIN2A genomic alterations can lead to as severe phenotypes with early onset and similar clinical manifestations. Based on in vitro experiments showing that memantine, an NMDA receptor open channel blocker, could reduce GluN2A-p.Leu812Met-related NMDAR hyperactivity [30], this drug was added to the patient’s current medication and led to decreased seizure frequency and improved interictal EEG recordings, whereas it had no effect on his cognitive ability. The same authors also reported that dextromethorphan, another NMDAR antagonist, was effective in vitro in the case of the p.Asp615Lys mutation but not in the case of p.Leu812Met. The close p.Met817Val mutation was not studied at the functional level; yet interestingly, the antiepileptic drug topiramate decreased the frequency and duration of the seizures in the corresponding patient [25], whereas it was ineffective in the aforementioned patient who had inherited another (p.Val506Ala)
missense mutation [19]. Together with the various pathophysiological mechanisms and consequences associated with the different types of GRIN2A mutations identified so far, the differential responses of several GluN2Amutated NMDARs to various antiepileptic drugs suggest that electrophysiological testing of GluN2A mutations in vitro might be useful before the choice of the appropriate targeted medication.
GRIN2B mutations GRIN2B is located onto human chromosome 12p13 and codes for the GluN2B subunit. Two de novo translocation events disrupting GRIN2B were identified in patients with variable degree of ID, behavioral issues and EEG abnormalities [24]. Four more heterozygous de novo mutations (two splice-site, one frameshift and one missense) were also found in patients with mild to moderate ID (Table 2 and Figure 1b). The screening of various cohorts of patients by next-generation sequencing approaches also detected GRIN2B as a gene the defects
Table 2 GRIN2B defects found in various neurodevelopmental disorders. EEG, electroencephalogram; ID, intellectual disability; ND, not determined Mutation Translocation t(9;12)(p23;p13)
Phenotype
Consequences
Refs
Moderate ID, behavioral issues, abnormal EEG Severe ID, behavioral issues, abnormal EEG
Gene disruption
[24]
Gene disruption
[24]
Inversion inv(12)(p13q21)
Autism
Gene disruption
[34]
Nonsense p.Trp559* p.Gln711*
Autism Autism
Truncated or no product Truncated or no product
[31] [33]
Splice-site c.411+1G>A c.2172 2A>G c.2351 2A>G c.2351 2A>G
Moderate ID, behavioral issues Autism Moderate ID, behavioral issues Mild ID, autism
ND ND ND ND
[24] [31] [24] [32]
Indels p.Ser34fs p.Thr268fs
Autism Moderate ID, behavioral issues
Frameshift Frameshift
[31] [17]
Autism Lennox-Gastaut syndrome, autistic features ID, focal epilepsy
ND ND
[31] [13]
Decreased magnesium block, increased calcium permeability ND
[35]
Decreased magnesium block, increased calcium permeability Decreased magnesium block, increased calcium permeability ND
[35]
ND ND
[24] [37]
t(10;12)(q21;p13)
Missense p.Cys456Tyr p.Cys461Phe p.Arg540His p.Pro553Leu p.Asn615Leu
Severe ID, hypotonia, no speech, dysmorphism West syndrome
p.Val618Gly
West syndrome
p.Ala636Pro
Mild ID, myoclonia, abnormal EEG, behavioral issues Mild ID, behavioral issues Autism
p.Arg682Cys p.Leu825Val
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[22]
[35] [39]
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of which might cause disorders of the autism spectrum: several splice-site, nonsense, frameshift and missense mutations have been identified [31,32,33]. The sequencing of balanced chromosomal abnormalities in a series of autistic patients identified an inversion at chromosome 12 that disrupted GRIN2B [34]. A missense mutation in the extracellular LBD (p.Arg540His) (Figure 1b) was also found in a patient with ID and focal epilepsy [35]. Quite surprisingly, the mutation caused a decrease in Mg2+-dependent inhibition and an increase in Ca2+ permeability in vitro, whereas the expected consequence on glutamate binding was not observed. Despite this and similar findings (see below) indicating possible gain-of-function effects for the few missense mutations that were studied in Xenopus laevis oocytes, most GRIN2B alterations identified so far in those disorders (Table 2) are expected to have haploinsufficiency effects, indicating that GluN2B loss-of-function likely contributes to ID and to autism. The significant excess of rare GRIN2B missense mutations found in large cohorts of patients with either autism or schizophrenia [36] further emphasizes the influence of GRIN2B on psychiatric disorders. The clinical spectrum of GRIN2B mutations has been expanded to more severe phenotypes in the context of EEs of various types: a de novo missense mutation was found in a patient with severe ID, absent speech, hypotonia and dysmorphia [22]; another de novo missense mutation (p.Cys461Phe) was detected in a patient with Lennox-Gastaut syndrome who experienced absence epilepsy, developmental delay, and autistic features [13]. More de novo missense mutations were found in two patients with West syndrome [35], which is a distinct EE characterized by infantile spasms and EEG abnormalities known as hypsarrhythmia. Those two mutations (p.Asn615Leu, p.Val618Gly) lied in close vicinity: they both occurred in the ion channel-forming re-entrant loop (Figure 1b). Consistently, in vitro analyses demonstrated a decreased Mg2+ block and an increase in Ca2+ permeability that appeared more dramatic than in the case of the p.Arg540His mutation mentioned just above. In contrast with GluN2A, no deleterious mutation has been identified in the C-terminal part of the protein so far.
GRIN2C,2D,3A,3B mutations Only a few truncating mutations of GRIN2C, GRIN3A or GRIN3B have been detected in patients with autism or with schizophrenia but the identification of those or other truncating mutations in control individuals has challenged their direct causal role [37]. Rather, genetic variations in those subunits might well represent genetic risk factors conferring susceptibility to neurodevelopmental disorders. Indeed, a significant excess in potentially detrimental GRIN3B variants has been found in autistic or schizophrenic patients [36].
Concluding remarks The aforementioned heterogeneity and plasticity in the subunit composition of the NMDARs, the developmental Current Opinion in Pharmacology 2015, 20:73–82
and regional variability in the respective expressions of those subunits in the brain, the broad range of the physiological properties and functions associated with such an NMDARs diversity, and the consequences of NMDARs hyper-activity and hypo-activity [2], were all predictive of an expected corresponding pleiotropy in the phenotypic spectrum their mutations in humans would be associated with. Thanks to the advent of novel massive parallel sequencing technologies, of whole-genome analyses, of bioinformatics tools, all being coupled with the accompanying completion of the human genome sequence, recent human genetic studies have indeed highlighted the existence of multiple heterozygous alterations in NMDARs subunits genes in several usual and common brain diseases, such as ID, ASD, or epilepsy. However what underlies the huge variability in the phenotypes caused by mutations in NMDARs remain unclear. The developmental changes in the number and composition of synaptic NMDARs, such as the classical 2B/2A switch, likely explain (at least partly) that mutations in GRIN2A and GRIN2B lead to different phenotypes. Also, that neighboring missense mutations give rise to clearly different phenotypes may be due to differences in their respective modes of action (i.e. lossof-function, gain-of-function, or dominant-negative) and to the variable degree of their functional consequences. More puzzling is the existence of the intrafamilial variability that is seen with a subset of the inherited NMDARs subunits mutations. The existence of multiple potentially pathogenic genomic alterations in a large number of patients with EEs of the epilepsy-aphasia spectrum suggest that genetic variations of this and of other types might participate in the variable presentation seen within family members who inherited one given GRIN2A mutation. There is indeed significant enrichment in genomic variations encompassing autism-related cell adhesion genes in patients with EEs of this spectrum [15]. Obviously variations in genes encoding other NMDARs subunits, in genes controlling their respective expressions or modulating their functional properties, and generally in genes possibly influencing the development and the maturation of the glutamatergic system may all play a modifying role — notwithstanding the obvious participation of environmental factors, especially at critical developmental periods. Obviously the huge interfamilial and intrafamilial variability and the difficult-topredict degree of severity associated with specific mutations, particularly in the case of GRIN2A, inherently raises important practical issues in the fields of genetic counseling and of future therapeutic avenues.
Conflict of interest statement Nothing declared.
Acknowledgements We thank Dr N Bruneau for her help in figure drawing. This work was supported by ANR (Agence Nationale de la Recherche) grant ‘EPILAND’ www.sciencedirect.com
NMDAR mutations in neurodevelopmental disorders Burnashev and Szepetowski 81
(ANR-2010-BLAN-1405 01) with EuroBiomed label, by the European Union Seventh Framework Programme FP7/2007-2013 under the project DESIRE (grant agreement n8602531), and by INSERM (Institut National de la Sante´ et de la Recherche Me´dicale).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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genomic dissection makes the link with autism. Epilepsia 2012, 53:1526-1538. First evidence for a de novo GRIN2A deletion in a patient with epilepsyaphasia; also detected a significant enrichment of genomic defects encompassing autism-associated cell adhesion genes in epilepsy-aphasia patients. 16. Carvill GL, Regan BM, Yendle SC, O’Roak BJ, Lozovaya N, Bruneau N, Burnashev N, Khan A, Cook J, Geraghty E et al.: GRIN2A mutations cause epilepsy-aphasia spectrum disorders. Nat Genet 2013, 45:1073-1076. In this study that focused on a broad spectrum of various epileptic encephalopathies, four inherited mutations were identified only in the subset of the patients that corresponded to the epilepsy-aphasia phenotypes. 17. Lemke JR, Lal D, Reinthaler EM, Steiner I, Nothnagel M, Alber M, Geider K, Laube B, Schwake M, Finsterwalder K et al.: Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nat Genet 2013, 45:1067-1072. The screening of hundreds of patients with focal epilepsy of the epilepsyaphasia spectrum identified numerous GRIN2A mutations in about 8% of the patients. 18. Lesca G, Rudolf G, Bruneau N, Lozovaya N, Labalme A, Boutry Kryza N, Salmi M, Tsintsadze T, Addis L, Motte J et al.: GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction. Nat Genet 2013, 45:1061-1066. Starting from the screening of sporadic and familial cases with focal epilepsies and EEs of the epilepsy-aphasia spectrum, this study led to the identification of 17 different GRIN2A inherited or de novo mutations of various types in about 20% of all patients. 19. DeVries SP, Patel AD: Two patients with a GRIN2A mutation and childhood-onset epilepsy. Pediatr Neurol 2013, 49:482-485. 20. Dimassi S, Labalme A, Lesca G, Rudolf G, Bruneau N, Hirsch E, Arzimanoglou A, Motte J, de Saint Martin A, Boutry-Kryza N et al.: A subset of genomic alterations detected in rolandic epilepsies contains candidate or known epilepsy genes including GRIN2A and PRRT2. Epilepsia 2014, 55:370-378. 21. Reutlinger C, Helbig I, Gawelczyk B, Subero JI, Tonnies H, Muhle H, Finsterwalder K, Vermeer S, Pfundt R, Sperner J et al.: Deletions in 16p13 including GRIN2A in patients with intellectual disability, various dysmorphic features, and seizure disorders of the rolandic region. Epilepsia 2010, 51:1870-1873. 22. de Ligt J, Willemsen MH, van Bon BW, Kleefstra T, Yntema HG, Kroes T, Vulto-van Silfhout AT, Koolen DA, de Vries P, Gilissen C et al.: Diagnostic exome sequencing in persons with severe intellectual disability. N Engl J Med 2012, 367:1921-1929. 23. Yuan H, Hansen KB, Zhang J, Pierson TM, Markello TC, Fajardo KV, Holloman CM, Golas G, Adams DR, Boerkoel CF et al.: Functional analysis of a de novo GRIN2A missense mutation associated with early-onset epileptic encephalopathy. Nat Commun 2014, 5:3251. This article nicely shows that one given specific GRIN2A missense mutation exerts multiple effects on various properties of reconstituted NMDARs in vitro. 24. Endele S, Rosenberger G, Geider K, Popp B, Tamer C, Stefanova I, Milh M, Kortum F, Fritsch A, Pientka FK et al.: Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes. Nat Genet 2010, 42:1021-1026. 25. Venkateswaran S, Myers KA, Smith AC, Beaulieu CL, Schwartzentruber JA, Majewski J, Bulman D, Boycott KM, Dyment DA: Whole-exome sequencing in an individual with severe global developmental delay and intractable epilepsy identifies a novel, de novo GRIN2A mutation. Epilepsia 2014, 55:e75-e79. 26. Yamada-Fowler N, Fredrikson M, Soderkvist P: Caffeine interaction with glutamate receptor gene GRIN2A: Parkinson’s disease in Swedish population. PLOS ONE 2014, 9:e99294. 27. Hamza TH, Chen H, Hill-Burns EM, Rhodes SL, Montimurro J, Kay DM, Tenesa A, Kusel VI, Sheehan P, Eaaswarkhanth M et al.: Genome-wide gene–environment study identifies glutamate Current Opinion in Pharmacology 2015, 20:73–82
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and autism spectrum disorders. Mol Psychiatry 2014, 19: 872-879. 34. Talkowski ME, Rosenfeld JA, Blumenthal I, Pillalamarri V, Chiang C, Heilbut A, Ernst C, Hanscom C, Rossin E, Lindgren AM et al.: Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Cell 2012, 149:525-537. 35. Lemke JR, Hendrickx R, Geider K, Laube B, Schwake M, Harvey RJ, James VM, Pepler A, Steiner I, Hortnagel K et al.: GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy. Ann Neurol 2014, 75:147-154. 36. Myers RA, Casals F, Gauthier J, Hamdan FF, Keebler J, Boyko AR, Bustamante CD, Piton AM, Spiegelman D, Henrion E et al.: A population genetic approach to mapping neurological disorder genes using deep resequencing. PLoS Genet 2011, 7:e1001318. 37. Tarabeux J, Kebir O, Gauthier J, Hamdan FF, Xiong L, Piton A, Spiegelman D, Henrion E, Millet B, Fathalli F et al.: Rare mutations in N-methyl-D-aspartate glutamate receptors in autism spectrum disorders and schizophrenia. Transl Psychiatry 2011, 1:e55. 38. Conroy J, McGettigan PA, McCreary D, Shah N, Collins K, ParryFielder B, Moran M, Hanrahan D, Deonna TW, Korff CM et al.: Towards the identification of a genetic basis for LandauKleffner syndrome. Epilepsia 2014, 55:858-865. 39. Freunscht I, Popp B, Blank R, Endele S, Moog U, Petri H, Prott EC, Reis A, Rubo J, Zabel B et al.: Behavioral phenotype in five individuals with de novo mutations within the GRIN2B gene. Behav Brain Funct 2013, 9:20.
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ScienceDirect NMDA receptor subunit mutations in neurodevelopmental disorders Nail Burnashev1,2,3 and Pierre Szepetowski1,2,3,4 N-Methyl-D-aspartate receptors (NMDARs) are glutamategated cation channels that are expressed throughout the brain and play essential role in brain functioning. Diversity of the subunits and of their spatio-temporal expression imparts distinct functional properties for the particular NMDAR in a particular brain region and developmental stage. Mutations in NMDARs may have pathological consequences and actually lead to various neurological disorders. Recent human genetic studies as highlighted here show the existence of multiple alterations in NMDARs subunits genes in several usual and common brain diseases, such as intellectual disability, autism spectrum disorders (ASD), or epilepsy. Relation of a particular mutation to the corresponding alteration of NMDARs function may provide an avenue to the targeted therapy for the pharmacological treatment of the disorders. Addresses 1 INSERM UMR_S901, Marseille, France 2 Mediterranean Institute of Neurobiology (INMED), Marseille, France 3 Aix-Marseille University, Marseille, France 4 French Epilepsy, Language and Development (EPILAND) Network, France Corresponding authors: Burnashev, Nail ([email protected]) and Szepetowski, Pierre ([email protected])
Current Opinion in Pharmacology 2015, 20:73–82 This review comes from a themed issue on Neurosciences Edited by Pierre Paoletti and Jean-Philippe Pin
modulatory subunits GluN2(A–D) or GluN3(A,B) to form either di-heterotetrameric or tri-heterotetrameric channels. All NMDAR subunits, as other glutamate receptors contain modular domains that are responsible for controlling distinct functions. An amino terminal domain (ATD) contributes to control of ion channel open probability and deactivation speeds and contains binding sites for subtype-specific allosteric modulator compounds, including zinc (GluN2A and 2B), ifenprodil (GluN2B), and polyamines (GluN2B). A ligand-binding domain (LBD) binds agonists, such as glutamate, glycine and antagonists to control ion channel opening. NMDARs are activated upon concurrent binding of glycine or D-serine to LBD of GluN1 and L-glutamate to LBD of GluN2 subunits [2,3]. Recent studies also implicate the ATD as a strong determinant of agonist efficacy [4], and interdomain interactions between the LBD and the ATD of the GluN2 subunit may be a central element in controlling channel opening upon agonist binding. The transmembrane domain (TMD) is formed by four hydrophobic segments (M1 to M4), with M2 partially entering the membrane to form the ion channel (Figure 1). An intracellular carboxyl terminal domain (CTD) associates with postsynaptic density (PSD) proteins, which in turn facilitates intracellular signaling. Important basic electrophysiological properties of NMDARs that allocate these receptors from other glutamate receptors include the use of co-agonist (glycine, D-serine) for activation, relatively slow current kinetics, voltage-dependent block by extracellular Mg2+, and high Ca2+ permeability.
http://dx.doi.org/10.1016/j.coph.2014.11.008 1471-4892/# 2014 Elsevier Ltd. All right reserved.
Introduction N-Methyl-D-aspartate receptors (NMDARs) are cation channels that are gated by the major excitatory neurotransmitter glutamate. NMDAR-mediated signaling is involved in normal development, plasticity, learning, memory and high cognitive functions. NMDARs play important role in temporal integration of neuronal network activity and long-term alterations in synaptic structure and function. There is long known association of NMDAR dysfunctions with various neurodevelopmental disorders and NMDARs might be targets for pharmacological treatment [1,2]. Typically NMDARs are composed of two obligatory GluN1 subunits and two www.sciencedirect.com
In general, much of the variation in function between NMDAR subtypes has been attributed to the identity of the GluN2 (GluN3) subunits in the channel assembly [5–7]. The GluN2 (GluN3) subunits have different temporal and spatial expression patterns in the brain and NMDAR subtypes also vary according to the cell types and subcellular localization. Different neuronal types usually express distinct combinations of NMDAR subunits thus providing a diversity of cell-specific and even input-specific synaptic responses. In addition to their postsynaptic localization, NMDARs are found at presynaptic, extrasynaptic and perisynaptic sites. Presynaptic NMDARs with different subunit composition expressed in axonal terminals may differentially modulate synaptic strength [1,2]. Although physiological function of extrasynaptic NMDARs is not fully understood, their activation by glutamate spillover may provide conditions for increased excitability and contribute to long-term synaptic changes [8]. In addition to the Current Opinion in Pharmacology 2015, 20:73–82
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Figure 1
(a)
Met1Thr Pro31fs Pro79Arg
N Phe183Ile Ile184Ser Trp198* Glu218* Cys231Tyr Ala243Val Arg370Trp Val375fs Cys436Arg Gly483Arg Arg504Trp Arg518His Pro522Arg
Phe139fs
Pro699Ser Met705Val Glu714Lys Ala716Thr Ala727Thr Asp731Asn Val734Leu
Ala290Val Gly295Ser Leu334* Val529fs Thr531Met Ser547del Ala548Thr
Ile694Thr Arg681* Lys669Asn Phe652Val Leu649Val
M1
M3
Lys772Glu Leu779fs Leu812Met Ile814Thr Met817Val
M4
M2 Ile904Phe Asp933Asn Tyr943*
Asp615Lys
Tyr1387* Asn976Ser
Asp1251Asn
(b)
N
C
Ser34fs
Gln711*
Thr268fs Cys456Tyr Cys461Phe Arg682Cys Arg540His Pro553Leu
M1
Leu825Val
M4
M3 M2
Trp559*
Ala636Pro Val618Gly Asn615Leu
C Current Opinion in Pharmacology
Location of GluN2A and of GluN2B mutations. (a) Mutations in GluN2A. (b) Mutations in GluN2B. The locations of nonsense, frameshift, splice-site and missense mutations found in NMDARs subunits are indicated in the schematics of the corresponding proteins. M1–M4 represent the four transmembrane domains of each subunit. Numbers indicate aminoacid positions.
neuronal expression, NMDARs are expressed in astrocytes, oligodendrocytes and microglia [9–11]. Taken together, it is not surprising that mutations in GRIN1, Current Opinion in Pharmacology 2015, 20:73–82
GRIN2A and GRIN2B, respectively, may cause somehow related and sometimes overlapping albeit clearly different spectrum of disorders. www.sciencedirect.com
NMDAR mutations in neurodevelopmental disorders Burnashev and Szepetowski 75
In the present review we will highlight recent studies on NMDAR subunit mutations in neurodevelopmental disorders and discuss their possible relations to alteration of NMDAR function and to clinical manifestations.
GRIN1 mutations GRIN1 encodes the GluN1 subunit of NMDARs. The gene maps at human chromosome 9q34 and spans more than 29 kb. Several alternatively splice variants with differences in expression patterns and in trafficking or electrophysiological properties of the corresponding isoforms have been described. The screening of 197 genes encoding ionotropic and metabotropic glutamate receptors and their known interacting proteins in a cohort of 95 sporadic patients with non-syndromic intellectual disability (ID), fished out one de novo GRIN1 missense mutation (p.Glu662Lys) in a patient with moderate ID [12]. Subsequent screening led to the identification of an in-frame 3 bp duplication (p.Ser560dup) in a patient with severe ID and partial complex epilepsy. As will be further examplified below, this and other types of comorbid associations are frequent in the context of neurodevelopmental disorders and likely reflect the existence of shared pathophysiological mechanisms at the intertwined molecular, cellular, electrophysiological, and developmental levels. Exome sequencing of 264 trios with epileptic encephalopathies (EEs) identified another de novo GRIN1 missense mutation (p.Tyr647Ser) in a girl with infantile spasms at age 4 months and who had subsequent profound delay [13]. In all three cases neuroimaging was normal. The p.Ser560dup mutation occurred just before the first transmembrane segment (M1) of GluN1. It drastically decreased the activity of the receptor when Xenopus oocytes were coinjected with mRNAs of GluN2B and either of wild-type or mutated GluN1. In contrast, p.Glu662Lys (located in the post-transmembrane segment M3) led to an increase in NMDAR-induced Ca2+ currents, which might be excitotoxic. The fact that two mutations with somehow opposite effects in vitro lead to overlapping or even to identical phenotypes represents an apparent paradox that has already been described in the case of another epilepsy gene (namely SCN1A mutations causing the syndrome of genetic epilepsy with febrile seizures plus) and that is also encountered in the case of GRIN2A mutations (see below). Nevertheless, whether the functional difference seen in the case of those two GRIN1 mutations underlies the more severe ID and the epileptic phenotype seen in the patient with p.Ser560dup remains elusive. Rather, this interpretation might well be an oversimplified view. As will be further seen, most if not all functional consequences of the human mutations found in NMDARs subunits have been studied using reconstituted receptors in vitro; whereas such tests are very useful and necessary to demonstrate the existence of functional differences between mutant and wild-type receptors, the type and extent of the pathophysiological www.sciencedirect.com
consequences that those mutations might actually have in vivo should be extrapolated with caution.
GRIN2A mutations GRIN2A maps at human chromosome 16p13 and codes for the GluN2A subunit. More than 60 heterozygous GRIN2A mutations of various types have been found in the very last years in patients and families presenting with disorders of the epilepsy-aphasia spectrum. Rolandic epilepsy (RE) is the most frequent and so-called ‘benign’ form of this spectrum. There are long known relationships between RE and various comorbid conditions, such as cognitive and behavioral issues, or speech and/or language impairment (e.g. verbal dyspraxia). The continuous spike-and-waves during slow-wave sleep syndrome (CSWSS) and the Landau-Kleffner syndrome (LKS) — also known as ‘acquired’ epileptic aphasia — are two closely related epileptic EEs that represent more severe and less frequent forms of this continuum. All those syndromes share the association of usually unfrequent seizures with paroxysmal electroencephalographic (EEG) discharges activated during drowsiness and sleep, and with more or less severe neuropsychological deficits, such as the autistic regression seen in LKS patients. In contrast with RE, the genetic influence in LKS and in CSWSS has long remained controversial [14]. The search for genomic alterations that would influence LKS or CSWSS [15] has been a crucial step towards the identification of a first cause for this fascinating group of disorders situated at the crossroads between epileptic, cognitive, autistic and language disorders. Particularly the detection of one de novo partial microdeletion of GRIN2A in a girl with LKS [15] has represented a key starting point. Since then, the crucial and direct causal role of de novo or inherited GRIN2A mutations in LKS, in CSWSS, and in RE with verbal dyspraxia, has been demonstrated in three main parallel studies which each used a different screening strategy to reach similar conclusions [16,17,18]. In the first study [18], the screening of sporadic and familial cases with focal epilepsies and EEs of the epilepsy-aphasia spectrum led to the identification of 17 different GRIN2A mutations, in about 20% of all patients; particularly all undisputable familial cases had mutations in GRIN2A. GRIN2A defects occurred in patients with variable degree of phenotypic severity and were of various types (microdeletions, splice-site, nonsense, missense) (Table 1). The mutations were spaced throughout the different domains of the protein (Figure 1a) and no genotype–phenotype correlation appeared clearly. In a second study [16], next-generation sequencing of a candidate genes panel in numerous patients with various types of EEs, identified four inherited mutations (two splice-site and two missense) (Table 1 and Figure 1a) only in the subset of epilepsyaphasia families. No GRIN2A mutation was found in the rest of the cohort, which suggested that GRIN2A defects in EEs might be somehow restricted to the epilepsy-aphasia Current Opinion in Pharmacology 2015, 20:73–82
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Table 1 GRIN2A defects found in various neurodevelopmental disorders. CSWSS, continuous spike and waves during slow-wave sleep syndrome; CNVs, copy number variations; CTS, centrotemporal spikes; EE, epileptic encephalopathy; ESES, electrical status epilepticus during sleep; FS, febrile seizures; GTCS, generalized tonic-clonic seizures; ID, intellectual disability; IS, infantile spasms; LKS, Landau-Kleffner syndrome; NA, not available; ND, not determined; NMD, nonsense-mediated mRNA decay; pred, predicted; RE, Rolandic epilepsy; UTR, untranslated region. Chromosomal locations are given according to the February 2009 human reference sequence (hg19, build37) (http:// genome.ucsc.edu/) Mutation CNVs del chr16: 7 964 000–10 607 500 (hg19) del chr16: 8 992 500–9 992 500 (hg19) del chr16: 9 365 500–11 273 700 (hg19) del chr16: 9 809 522–9 856 618 (hg19) del chr16: 9 908 477–9 934 830 (hg19) del chr16: 9 850 000–9 900 000 (hg19) (precise coordinates NA) del chr16: 9 825 000–10 075 000 (hg19) (precise coordinates NA) del chr16: 10 227 121–10 354 862 (hg19) del chr16: 10 250 000–10 275 000 (hg19) (precise coordinates NA) dup chr16: 10 075 000–10 225 000 (hg19) (precise coordinates NA) Translocation t(16;17)(p13;q11)
Nonsense p.Trp198* p.Gln218*
p.Leu334* p.Arg681* p.Tyr943* p.Tyr1387* Splice-site c.1007+1G>A c.1007+1G>A c.1007+1G>A c.1007+1G>A c.1007+1G>A c.1007+1G>T c.1123 2A>G
c.2007+1G>A
Phenotype Pseudo-Lennox syndrome, various epileptic seizures, ESES, severe ID, no speech, dysmorphism RE, moderate ID, delayed speech, dysmorphism Myoclonic seizures, severe ID, no speech, dysmorphism, CTS RE + mild ID
Consequences
Refs
Total microdeletion, no product
[21]
Partial microdeletion, p.Lys592fs (pred)
[21]
Total microdeletion, no product (pred)
[21]
Partial microdeletion, p.Arg865fs (pred)
[20]
LKS (proband), LKS + verbal dyspraxia (brothers) Atypical benign partial epilepsy
Partial microdeletion, p.Phe670fs (pred)
[18]
ND
[17]
RE
ND
[17]
CSWSS + verbal dyspraxia (three brothers)
Partial microdeletion, no product (pred)
[18]
RE
ND
[17]
CSWSS
Intronic; disruption of regulatory element?
[17]
FS, GTCS, severe ID (proband), GTCS + learning difficulties (father, aunt), seizures + severe ID (cousin)
Gene disruption
[24]
Atypical benign partial epilepsy Moderate ID + CTS (proband), FS + focal seizures + learning difficulties (mother), infantile seizures + learning difficulties (grandmother) CSWSS (proband), Panayiotopoulos syndrome (brother), partial epilepsy (father) LKS (proband) CSWSS (proband), FS + CTS (sister), RE (father) CSWSS (proband), childhood epilepsy (mother, uncle)
Truncated or no product NMD, no product
[17] [24]
Truncated or no product
[17]
Truncated or no product Truncated or no product
[17] [17]
Loss of phosphorylation-dependent regulation by Src kinases (pred)
[18]
RE + verbal dyspraxia (proband + six relatives) CSWSS (proband + father) LKS Atypical benign partial epilepsy (proband), uncharacterized epilepsy (two half brothers) LKS (proband), RE (half sister) CSWSS (proband), CTS (brother) CSWSS (proband + brother), CSWSS + dysphasia (aunt), LKS (uncle), RE + dysphasia (grand-mother), RE (mother, cousin) CSWSS (proband), uncharacterized epilepsy (father)
p.Phe139fs (pred)
[16]
p.Phe139fs (pred) p.Phe139fs (pred) p.Phe139fs (pred)
[16] [17] [17]
p.Phe139fs (pred) p.Phe139fs (pred) p.Val375fs (pred)
[17] [17] [18]
ND
[17]
Current Opinion in Pharmacology 2015, 20:73–82
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NMDAR mutations in neurodevelopmental disorders Burnashev and Szepetowski 77
Table 1 (Continued ) Mutation Indels p.Pro31fs p.Val529fs p.Ser547del p.Leu779fs Missense p.Met1Thr p.Pro79Arg p.Phe183Ile p.Ile184Ser p.Cys231Tyr p.Ala243Val p.Ala290Val p.Gly295Ser p.Arg370Trp p.Cys436Arg p.Gly483Arg p.Arg504Trp p.Arg518His p.Arg518His p.Pro522Arg p.Thr531Met
p.Ala548Thr p.Asp615Lys p.Leu649Val
p.Phe652Val p.Lys669Asn p.Ile694Thr p.Pro699Ser p.Met705Val p.Glu714Lys p.Ala716Thr p.Ala727Thr p.Asp731Asn p.Val734Leu p.Lys772Glu p.Leu812Met
p.Ile814Thr p.Met817Val p.Ile904Phe p.Asp933Asn p.Asn976Ser p.Asn976Ser p.Asp1251Asn
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Phenotype
Consequences
Refs
RE (proband, brother, father) RE (proband, brother, mother) CSWSS (proband), CTS (sister) CSWSS
Frameshift Frameshift Frameshift Frameshift
[17] [17] [17] [17]
LKS (proband, sister), seizures + speech/ language impairment (father) CSWSS (proband), RE (mother, uncle, grandmother) RE CSWSS LKS (proband), CTS (brother, sister) RE + learning difficulties RE RE RE Atypical benign partial epilepsy CSWSS + dysphasia (proband), RE + dysphasia (sister) CSWSS (proband, brother) CSWSS (proband), RE (brother), verbal dyspraxia (father) LKS Early-onset seizures + severe ID + no speech CSWSS (proband), intermediate epilepsyaphasia (brothers), learning difficulties + speech/language impairment (mother) LKS IS, myoclonies, severe ID Epileptic seizures, severe ID, dysplastic corpus callosum, severe feeding impairment, hypothyroidy, dysmorphism CSWSS
ND
[16]
ND
[17]
ND ND
[17] [18] [17] [17] [17] [18] [17] [17] [18]
CSWSS LKS RE RE (proband, brother, sister) CSWSS RE + verbal dyspraxia (proband + six relatives), verbal dyspraxia (cousin) RE RE + verbal dyspraxia (proband), verbal dyspraxia (mother) RE (proband, brother) Atypical benign partial epilepsy Early-onset EE + profound developmental delay
RE Refractory seizures + global developmental delay RE (proband, father), FS + CTS (brother) LKS Atypical benign partial epilepsy CSWSS RE (proband), absence epilepsy (father)
Impaired zinc inhibition ND ND ND ND ND ND Increased open state duration + decreased close state duration See above ND Increased open state duration
[18] [18]
ND Loss of magnesium block ND
[18] [24] [22]
Increased open state duration + decreased close state duration ND ND ND ND ND ND
[18]
ND ND
[17] [17]
ND ND Increased potency of glutamate and glycine, decreased magnesium block, reduced proton sensitivity, reduced zinc inhibition, increased open probability ND ND
[17] [17] [23]
ND ND ND ND ND
[17] [18] [17] [17] [18]
[38] [22] [16]
[18] [18] [17] [17] [17] [18]
[17] [25]
Current Opinion in Pharmacology 2015, 20:73–82
78 Neurosciences
syndromes. In the third study [17], various heterozygous GRIN2A mutations (Table 1 and Figure 1a) were also found in about 8% of patients with either of RE, LKS or CSWSS. Mutations were more frequent in the more severe phenotypes. Since then, several studies have identified additional GRIN2A defects in patients with disorders of the epilepsy-aphasia spectrum [19,20]. A few other GRIN2A heterozygous defects (Table 1) were reported in patients presenting with various and more severe forms of neurodevelopmental disorders that did not belong to the epilepsy-aphasia spectrum but that showed partially overlapping clinical features, variably associating dysmorphia, ID, epileptic manifestations that included seizures of the Rolandic area and nocturnal paroxysms or refractory seizures without electroclinical seizure during sleep, behavioral impairment, profound developmental delay [21,22,23,24,25]. All those findings demonstrate that the phenotypic spectrum of GRIN2A mutations expands beyond the epilepsy-aphasia core group of disorders. Moreover, GRIN2A was also identified recently as a modifying genetic factor influencing Parkinson’s disease in inverse association with caffeine intake [26,27]. On the one hand, the existence of GRIN2A microdeletions and of nonsense mutations argued for haploinsufficiency effects. This might lead to a relative excess in GluN2B subunits which in turn would lead to NMDARs opening longer, resulting in temporarily hyperactive NMDARs. Also, pro-survival GluN2A-mediated signaling is involved in normal development [28] of inhibitory interneurons and those subunits are heavily expressed in parvalbumin (PV)-positive interneurons [29]. Thus loss of function of GluN2A may lead to undeveloped inhibitory network. Moreover, disruption of NMDAR signaling specifically in PV-positive interneurons may lead to a looser inhibitory control of excitatory principal neurons. All these would increase overall excitability of the neuronal networks and promote proepileptic effect. On the other hand, other mutations show gain-of-function effects, at least in vitro. ‘Gain-offunction’ in excitatory neurons due to subunit overexpression and/or to slowing down of the kinetics of NMDAR-mediated currents would also favor overexcitability due to increased excitatory drive and higher extent of temporal integration of sensory signals in thalamocortical circuits. Several studies of the functional consequences of specific GluN2A missense mutations have indeed made the overall pathophysiological picture(s) complicated and heterogeneous, and hence are worth being mentioned with more details. It should be emphasized that the differences in the consequences of the mutations described below, might at least partly reflect the variability in the battery of electrophysiological evaluations that were performed in each specific case. Current Opinion in Pharmacology 2015, 20:73–82
The p.Ala243Val found in a patient with RE and learning difficulties [17] occurred in the Zn2+ binding domain and led to an impaired inhibition of mutant NMDAR currents by low concentrations of Zn2+ in vitro. This suggested that, in vivo, there might be a reduced highaffinity Zn2+-mediated inhibition that in turn would lead to increased susceptibility to channel activation and to increased calcium influx. The p.Arg518His and the p.Thr531Met mutations that were found in typical epilepsy-aphasia families occurred in the extracellular LBD of GluN2A. They were predicted to modify NMDAR gating and kinetic properties by affecting ligand binding. As a matter of fact, the durations of channel open and closed states as calculated by single-channel recordings, were significantly modified when the corresponding mutant heteromeric GluN1/GluN2A-p.Arg518His NMDAR was reconstituted in HEK cells [18]. Similarly, p.Thr531Met led to a shift in NMDAR kinetics with a significant increase in the mean duration of the open state [16]. Whereas the p.Pro522Arg mutation is expected to have similar electrophysiological effects, it was found in a patient with a much more severe phenotype that falls outside the epilepsy-aphasia spectrum (Table 1) [22]. p.Asp615Lys occurred near the apex of the pore-lining reentrant loop (the M2-loop) of GluN2A — a site known to strongly influence Mg2+ block. Reconstituted NMDARs with wild-type GluN1 and mutant p.Asp615Lys-GluN2A showed a dramatic loss of the Mg2+ block and a decrease in Ca2+ permeability, whereas co-expression of wild-type and mutant GluN2A with wild-type GluN1 had intermediate effects. The loss of Mg2+ inhibition of NMDAR activity likely results in increased current flow through mutant-GluN2A-containing NMDARs, leading to excessive excitatory drive. Again this is in sharp contrast with the haploinsufficiency effects of other GRIN2A defects as mentioned above. A de novo mutation (p.Phe652Val) (Figure 1a) yielded qualitatively similar results as those obtained for channels with other mutations situated right within the LBD (see above) [18]. Hence the Phe652 residue might well represent a new site for the modulation of NMDA receptor kinetics. This may also be postulated in the case of the Leu812 residue which also led to mutant channels that open more often (see below). Moreover, p.Phe652Val was found in a patient with typical CSWSS, whereas another missense mutation situated only three aminoacids apart, p.Leu649Val, was detected in a patient with severe ID, dysplastic corpus callosum, epileptic seizures, severe feeding impairment, hypothyroidy, and mild facial dysmorphisms [22]. p.Leu812Met was located in the linker region between the ligand-binding and transmembrane domains. This mutation had multiple effects on the properties of the corresponding GluN1/GluN2A mutant receptors expressed in Xenopus oocytes or in HEK cells [23] (Table 1). The overall hyperactivation seen in that case might well drive neuronal hyperexcitability and possibly neuronal excitotoxic loss — and hence might www.sciencedirect.com
NMDAR mutations in neurodevelopmental disorders Burnashev and Szepetowski 79
lead to epileptic seizures. However and as already discussed just above in the case of p.Asp615Lys, heterozygous loss-of-function GRIN2A genomic alterations can lead to as severe phenotypes with early onset and similar clinical manifestations. Based on in vitro experiments showing that memantine, an NMDA receptor open channel blocker, could reduce GluN2A-p.Leu812Met-related NMDAR hyperactivity [30], this drug was added to the patient’s current medication and led to decreased seizure frequency and improved interictal EEG recordings, whereas it had no effect on his cognitive ability. The same authors also reported that dextromethorphan, another NMDAR antagonist, was effective in vitro in the case of the p.Asp615Lys mutation but not in the case of p.Leu812Met. The close p.Met817Val mutation was not studied at the functional level; yet interestingly, the antiepileptic drug topiramate decreased the frequency and duration of the seizures in the corresponding patient [25], whereas it was ineffective in the aforementioned patient who had inherited another (p.Val506Ala)
missense mutation [19]. Together with the various pathophysiological mechanisms and consequences associated with the different types of GRIN2A mutations identified so far, the differential responses of several GluN2Amutated NMDARs to various antiepileptic drugs suggest that electrophysiological testing of GluN2A mutations in vitro might be useful before the choice of the appropriate targeted medication.
GRIN2B mutations GRIN2B is located onto human chromosome 12p13 and codes for the GluN2B subunit. Two de novo translocation events disrupting GRIN2B were identified in patients with variable degree of ID, behavioral issues and EEG abnormalities [24]. Four more heterozygous de novo mutations (two splice-site, one frameshift and one missense) were also found in patients with mild to moderate ID (Table 2 and Figure 1b). The screening of various cohorts of patients by next-generation sequencing approaches also detected GRIN2B as a gene the defects
Table 2 GRIN2B defects found in various neurodevelopmental disorders. EEG, electroencephalogram; ID, intellectual disability; ND, not determined Mutation Translocation t(9;12)(p23;p13)
Phenotype
Consequences
Refs
Moderate ID, behavioral issues, abnormal EEG Severe ID, behavioral issues, abnormal EEG
Gene disruption
[24]
Gene disruption
[24]
Inversion inv(12)(p13q21)
Autism
Gene disruption
[34]
Nonsense p.Trp559* p.Gln711*
Autism Autism
Truncated or no product Truncated or no product
[31] [33]
Splice-site c.411+1G>A c.2172 2A>G c.2351 2A>G c.2351 2A>G
Moderate ID, behavioral issues Autism Moderate ID, behavioral issues Mild ID, autism
ND ND ND ND
[24] [31] [24] [32]
Indels p.Ser34fs p.Thr268fs
Autism Moderate ID, behavioral issues
Frameshift Frameshift
[31] [17]
Autism Lennox-Gastaut syndrome, autistic features ID, focal epilepsy
ND ND
[31] [13]
Decreased magnesium block, increased calcium permeability ND
[35]
Decreased magnesium block, increased calcium permeability Decreased magnesium block, increased calcium permeability ND
[35]
ND ND
[24] [37]
t(10;12)(q21;p13)
Missense p.Cys456Tyr p.Cys461Phe p.Arg540His p.Pro553Leu p.Asn615Leu
Severe ID, hypotonia, no speech, dysmorphism West syndrome
p.Val618Gly
West syndrome
p.Ala636Pro
Mild ID, myoclonia, abnormal EEG, behavioral issues Mild ID, behavioral issues Autism
p.Arg682Cys p.Leu825Val
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[22]
[35] [39]
Current Opinion in Pharmacology 2015, 20:73–82
80 Neurosciences
of which might cause disorders of the autism spectrum: several splice-site, nonsense, frameshift and missense mutations have been identified [31,32,33]. The sequencing of balanced chromosomal abnormalities in a series of autistic patients identified an inversion at chromosome 12 that disrupted GRIN2B [34]. A missense mutation in the extracellular LBD (p.Arg540His) (Figure 1b) was also found in a patient with ID and focal epilepsy [35]. Quite surprisingly, the mutation caused a decrease in Mg2+-dependent inhibition and an increase in Ca2+ permeability in vitro, whereas the expected consequence on glutamate binding was not observed. Despite this and similar findings (see below) indicating possible gain-of-function effects for the few missense mutations that were studied in Xenopus laevis oocytes, most GRIN2B alterations identified so far in those disorders (Table 2) are expected to have haploinsufficiency effects, indicating that GluN2B loss-of-function likely contributes to ID and to autism. The significant excess of rare GRIN2B missense mutations found in large cohorts of patients with either autism or schizophrenia [36] further emphasizes the influence of GRIN2B on psychiatric disorders. The clinical spectrum of GRIN2B mutations has been expanded to more severe phenotypes in the context of EEs of various types: a de novo missense mutation was found in a patient with severe ID, absent speech, hypotonia and dysmorphia [22]; another de novo missense mutation (p.Cys461Phe) was detected in a patient with Lennox-Gastaut syndrome who experienced absence epilepsy, developmental delay, and autistic features [13]. More de novo missense mutations were found in two patients with West syndrome [35], which is a distinct EE characterized by infantile spasms and EEG abnormalities known as hypsarrhythmia. Those two mutations (p.Asn615Leu, p.Val618Gly) lied in close vicinity: they both occurred in the ion channel-forming re-entrant loop (Figure 1b). Consistently, in vitro analyses demonstrated a decreased Mg2+ block and an increase in Ca2+ permeability that appeared more dramatic than in the case of the p.Arg540His mutation mentioned just above. In contrast with GluN2A, no deleterious mutation has been identified in the C-terminal part of the protein so far.
GRIN2C,2D,3A,3B mutations Only a few truncating mutations of GRIN2C, GRIN3A or GRIN3B have been detected in patients with autism or with schizophrenia but the identification of those or other truncating mutations in control individuals has challenged their direct causal role [37]. Rather, genetic variations in those subunits might well represent genetic risk factors conferring susceptibility to neurodevelopmental disorders. Indeed, a significant excess in potentially detrimental GRIN3B variants has been found in autistic or schizophrenic patients [36].
Concluding remarks The aforementioned heterogeneity and plasticity in the subunit composition of the NMDARs, the developmental Current Opinion in Pharmacology 2015, 20:73–82
and regional variability in the respective expressions of those subunits in the brain, the broad range of the physiological properties and functions associated with such an NMDARs diversity, and the consequences of NMDARs hyper-activity and hypo-activity [2], were all predictive of an expected corresponding pleiotropy in the phenotypic spectrum their mutations in humans would be associated with. Thanks to the advent of novel massive parallel sequencing technologies, of whole-genome analyses, of bioinformatics tools, all being coupled with the accompanying completion of the human genome sequence, recent human genetic studies have indeed highlighted the existence of multiple heterozygous alterations in NMDARs subunits genes in several usual and common brain diseases, such as ID, ASD, or epilepsy. However what underlies the huge variability in the phenotypes caused by mutations in NMDARs remain unclear. The developmental changes in the number and composition of synaptic NMDARs, such as the classical 2B/2A switch, likely explain (at least partly) that mutations in GRIN2A and GRIN2B lead to different phenotypes. Also, that neighboring missense mutations give rise to clearly different phenotypes may be due to differences in their respective modes of action (i.e. lossof-function, gain-of-function, or dominant-negative) and to the variable degree of their functional consequences. More puzzling is the existence of the intrafamilial variability that is seen with a subset of the inherited NMDARs subunits mutations. The existence of multiple potentially pathogenic genomic alterations in a large number of patients with EEs of the epilepsy-aphasia spectrum suggest that genetic variations of this and of other types might participate in the variable presentation seen within family members who inherited one given GRIN2A mutation. There is indeed significant enrichment in genomic variations encompassing autism-related cell adhesion genes in patients with EEs of this spectrum [15]. Obviously variations in genes encoding other NMDARs subunits, in genes controlling their respective expressions or modulating their functional properties, and generally in genes possibly influencing the development and the maturation of the glutamatergic system may all play a modifying role — notwithstanding the obvious participation of environmental factors, especially at critical developmental periods. Obviously the huge interfamilial and intrafamilial variability and the difficult-topredict degree of severity associated with specific mutations, particularly in the case of GRIN2A, inherently raises important practical issues in the fields of genetic counseling and of future therapeutic avenues.
Conflict of interest statement Nothing declared.
Acknowledgements We thank Dr N Bruneau for her help in figure drawing. This work was supported by ANR (Agence Nationale de la Recherche) grant ‘EPILAND’ www.sciencedirect.com
NMDAR mutations in neurodevelopmental disorders Burnashev and Szepetowski 81
(ANR-2010-BLAN-1405 01) with EuroBiomed label, by the European Union Seventh Framework Programme FP7/2007-2013 under the project DESIRE (grant agreement n8602531), and by INSERM (Institut National de la Sante´ et de la Recherche Me´dicale).
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