FULL-LENGTH ORIGINAL RESEARCH

GRIN1 mutations cause encephalopathy with infantileonset epilepsy, and hyperkinetic and stereotyped movement disorders *†Chihiro Ohba, ‡Masaaki Shiina, §Jun Tohyama, ¶Kazuhiro Haginoya, #Tally Lerman-Sagie, **Nobuhiko Okamoto, #Lubov Blumkin, #Dorit Lev, ††Souichi Mukaida, ‡‡Fumihito Nozaki, §§Mitsugu Uematsu, ¶¶Akira Onuma, *Hirofumi Kodera, *Mitsuko Nakashima, *Yoshinori Tsurusaki, *Noriko Miyake, †Fumiaki Tanaka, ###Mitsuhiro Kato, ‡Kazuhiro Ogata, *Hirotomo Saitsu, and *Naomichi Matsumoto Epilepsia, 56(6):841–848, 2015 doi: 10.1111/epi.12987

SUMMARY

Chihiro Ohba, a neurologist, is interested in genetic factors causing neurodevelopmental disorders.

Objective: Recently, de novo mutations in GRIN1 have been identified in patients with nonsyndromic intellectual disability and epileptic encephalopathy. Whole exome sequencing (WES) analysis of patients with genetically unsolved epileptic encephalopathies identified four patients with GRIN1 mutations, allowing us to investigate the phenotypic spectrum of GRIN1 mutations. Methods: Eighty-eight patients with unclassified early onset epileptic encephalopathies (EOEEs) with an age of onset A) with a mutant allele frequency of 16% (in DNA of blood leukocytes) was detected in one patient. Three mutations were located in the transmembrane domain (3/4, 75%), and one in the extracellular loop near transmembrane helix 1. All the mutations were predicted to impair the function of the NMDA receptor. Significance: Clinical features of de novo GRIN1 mutations include infantile involuntary movements, seizures, and hand stereotypies, suggesting that GRIN1 mutations cause encephalopathy resulting in seizures and movement disorders.

Accepted March 4, 2015; Early View publication April 10, 2015. *Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan; †Department of Clinical Neurology and Stroke Medicine, Yokohama City University, Yokohama, Japan; ‡Department of Biochemistry, Graduate School of Medicine, Yokohama City University, Yokohama, Japan; §Department of Pediatrics, Epilepsy Center, Nishi-Niigata Chuo National Hospital, Niigata, Japan; ¶Department of Pediatric Neurology, Takuto Rehabilitation Center for Children, Sendai, Japan; #Metabolic Neurogenetic Clinic, Wolfson Medical Center, Holon, Israel; **Department of Medical Genetics, Osaka Medical Center, Research Institute for Maternal and Child Health, Osaka, Japan; ††Department of Pediatric Neurology, National Hospital Organization Utano Hospital, Kyoto, Japan; ‡‡Department of Pediatrics, Shiga Medical Center for Children, Shiga, Japan; §§Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan; ¶¶Department of Pediatrics, Ekoh-Ryoikuen, Sendai, Japan; and ###Department of Pediatrics, Faculty of Medicine, Yamagata University, Yamagata, Japan Address correspondence to Hirotomo Saitsu and Naomichi Matsumoto, Department of Human Genetics, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. E-mails: [email protected] and [email protected] Wiley Periodicals, Inc. © 2015 International League Against Epilepsy

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842 C. Ohba et al. KEY WORDS: GRIN1, Encephalopathy, Neurotransmitter disorders, Seizure, Movement disorders.

N-methyl-D-aspartate (NMDA) receptors are glutamategated ion channels, and are essential to synaptic function in the brain.1–3 In fact, dysfunction of NMDA receptors is associated with a spectrum of neuropsychiatric disorders such as Alzheimer’s disease, Huntington’s disease, depression, schizophrenia, stroke, traumatic brain injury, and epilepsy.2–4 NMDA receptors have seven different subunits: one GluN1 subunit encoded by GRIN1; four GluN2 subunits (GluN2A, GluN2B, GluN2C, and GluN2D) encoded by GRIN2A, GRIN2B, GRIN2C, and GRIN2D, respectively; and two GluN3 subunits (GluN3A and GluN3B) encoded by GRIN3A and GRIN3B, respectively.2,3 NMDA receptors are assembled as heterotetramers, and are typically composed of two copies of GluN1, and GluN2, or a mixture of GluN2 and GluN3.2,5 The GluN1 subunit contains four domains: the extracellular amino-terminal domain (ATD); the extracellular ligand-binding domain (LBD), which consists of two segments of amino acids termed S1 and S2; the transmembrane domain (TMD); and an intracellular carboxyl-terminal domain (CTD).2,3,5 Recently, de novo mutations in GRIN1, which encodes the GluN1 subunit,3 were identified in two patients with nonsyndromic intellectual disability,6 and in one patient with epileptic encephalopathy,7 indicating that the GluN1 subunit is indispensable for proper brain function. However, a comprehensive phenotypic spectrum of GRIN1 mutations remains to be determined. In this study, whole exome sequencing (WES) of patients with unclassified early onset epileptic encephalopathies (EOEEs) revealed four patients with de novo GRIN1 mutations. These patients had severe developmental delay, seizures, and hyperkinetic and stereotyped movement disorders, delineating the phenotype of the GRIN1-related disorder.

Methods Patients Eighty-eight patients with unclassified EOEEs with an age of onset A mutation were amplified using template DNA from blood, saliva, hair roots, and nails of the patient. After adaptor ligation and indexing using a Nextera DNA Sample Preparation Kit (Illumina), PCR products were sequenced on a MiSeq (Illumina) with 150-bp paired-end reads. Image analysis, base calling, and demultiplexing were performed by sequence control real-time analysis using CASAVA software v1.8 (Illumina). Data processing, variant calling, and variant detection were performed as described for WES.8 Structural analysis The effect of mutations on NMDA receptors was examined by mapping altered amino acids onto the crystal struc-

843 Phenotypic Spectrum of GRIN1 Mutations ture of a rat NMDA receptor (PDB code 4PE5)5 using PyMOL. Standard protocol approvals, registrations, and patient consents The experimental protocols were approved by the institutional review board of Yokohama City University School of Medicine and Yamagata University Faculty of Medicine. Written informed consent was obtained from all individuals and/or their families in compliance with relevant Japanese regulations. Permission for presenting photographs and videos was obtained from the parents.

Results Identification of GRIN1 mutations De novo missense GRIN1 mutations were identified in four patients with unclassified EOEEs (Fig. 1). Of interest, a “de novo” mosaic mutation (c.1923G>A) was detected in patient 4, in which the mutant allele frequency was 16% (15/96 alleles) based on WES read counts. We confirmed mosaicism in DNA of blood leukocytes, saliva, hair roots, and nails from the patient by deep sequencing of PCR products (Table S1: mutant allele frequency range 13.4–19.7%). Three mutations were located in the transmembrane domain (3/4, 75%), and the other was located in the extracellular loop near transmembrane helix 1 (1/4, 25%) (Fig. 1A). All mutations substituted evolutionarily conserved amino acids (Fig. 1A), and SIFT, Polyphen2, and Mutation Taster predicted that all the mutations would be highly damaging to the structure of GluN1 (Table S2). Structural consideration of mutational effects To evaluate effects of the identified missense mutations on NMDA function in terms of protein structure, we mapped the mutation sites on the crystal structure of a rat NMDA receptor (PDB code 4PE5).5 This crystal structure of the NMDA receptor is arranged as a dimer of the GluN1-GluN2B heterodimer, and all the identified mutations were mapped in the GluN1 TMD, which contains four helices M1–M4 (Fig. 1B and 1C). The position of the Asp552Glu mutation was mapped in the pre-M1 region, which bridges the LBD and the TMD, and possibly plays a role in transmitting information about ligandinduced structural changes in the LBD to the TMD.10–12 Thus, it is plausible that the Asp552Glu mutation has an impact on NMDA receptor function. The Met641 and Asn650 mutations are located in the pore-lining M3 helix (Fig. 1C). The former residue faces toward the ion pore (Fig. 1C), and thus the Met641Ile mutation would alter the gating property of the ion channel. The latter residue is located within the highly conserved SYTANLAAF motif (Fig. 1C), which is crucial to the gating function of the NMDA receptor, and thus the Asn650Lys muta-

tion would functionally affect the ion channel. Gly815 in the M4 helix faces toward the lipid layer; therefore, upon Gly815Arg mutation, the positively charged side chain of the mutant Arg residue would protrude into the lipid layer, possibly affecting the conformation around the M4 helix. Given a previous report demonstrating that a Gly815Ala mutation in GluN1 abolished agonist-evoked currents,11 the Gly815Arg mutation is likely to impair the ion channel function of the NMDA receptor. Clinical features of patients with GRIN1 mutations The clinical information of patients with GRIN1 mutations is summarized in Table 1, and their facial appearance and EEG and MRI findings are shown in Figure 2. Mild facial dysmorphism such as an elongated face and deep set eyes can be recognized in four patients (Fig. 2A–D). Initial symptoms appeared within 3 months of birth and included hyperkinetic movements in two patients (2/4, 50%), and seizures in two patients (2/4, 50%). All patients developed different types of seizures during first year of life including spasms, and myoclonic and partial seizures. EEG studies of these patients showed only exhibited nonspecific focal and diffuse epileptiform abnormality, and never showed suppression-burst or hypsarrhythmia during infancy (Fig. 2E, F). In two cases (patients 1 and 4), epileptic seizures were confirmed by video-EEG monitoring. Seizures were controlled in one patient with phenobarbital and in another with zonisamide, but were uncontrolled in the two other patients. All patients showed severe developmental delay, which was initially observed before onset of seizures in two patients (patients 1 and 2), and control of seizures did not restore developmental milestones (patients 2 and 3). Therefore, it might be more appropriate to state that patients with GRIN1 mutations showed encephalopathy with seizures. Hyperkinetic movements such as fragmentary myoclonus, chorea, and dyskinesia were observed in three patients, and one remaining patient (patient 4) showed peculiar involuntary movements in which he groaned with his head swaying and breath holding. In addition, abnormal dystonic eye movements resembling oculogyric crises were observed in two patients (patients 1 and 3), leading to a suspicion of monoamine neurotransmitter disorders; however, examination of neurotransmitter levels in cerebrospinal fluid in three patients showed no abnormalities. Hand stereotypies were observed in three patients. Stereotyped movement of hands, arms, and legs with transient upper eye deviation in patient 1 are shown in the Videos S1 and S2. Patient 2 showed hand-washing, hand-wringing, and hand-mouthing stereotypical movements, bruxism, and inappropriate laughter and crying. In addition, patient 4 demonstrated a sleep disorder, hyperventilation, and no purposeful hand skills: Rett syndrome was, therefore, suspected in the two patients (patients 2 and 4). It is interesting to note that these two patients also showed postnatal microcephaly. All patients were bedridden (at ages of 5–14 years). Brain MRI showed Epilepsia, 56(6):841–848, 2015 doi: 10.1111/epi.12987

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Figure 1. (A) Schematic presentation of GRIN1 and evolutionary conservation of amino acids substituted by GRIN1 mutations. (B) The crystal structure of a rat NMDA receptor tetramer composed of two GluN1 (colored cyan and light cyan) and two GluN2B subunits (magenta and pink) is shown. The residues at the mutation sites are shown as red spheres in one GluN1 subunit. The highly conserved SYTANLAAF motif in the TMD is colored yellow. (C) Close-up views of the TMD parallel to the membrane (left) and along the pore axis from the extracellular side (right). Epilepsia ILAE

thin corpus callosum and ventriculomegaly in three patients (3/4, 75%), respectively, cerebral atrophy in two patients (2/ 4, 50%), and cerebellar atrophy in one patient. Case reports are available in the Supporting Information Data S1. Epilepsia, 56(6):841–848, 2015 doi: 10.1111/epi.12987

Discussion Our study demonstrates that de novo GRIN1 mutations cause infantile-onset encephalopathy with hyperkinetic and

845 Phenotypic Spectrum of GRIN1 Mutations Table 1. Clinical features of patients with GRIN1 mutations Patient 1

Patient 2

Patient 3

Patient 4

7 yr Male c.1656C>G (p.Asp552Glu) Deep set eyes, elongated face, pointed chin Bedridden At birth Bilateral eyelid myoclonus at birth Spasma at 11 mo GTCS at 3 yr 7mo

5 yr Male c.1950C>G (p.Asn650Lys)

7 yr 10 mo Female c.2443G>C (p.Gly815Arg) Elongated face

14 yr Male c.1923G>A (p.Met641Ile)

Interictal EEG

Focal spikes at 6 mo Diffuse high-amplitude sharp wave or sharp and slow waves at 6 yr

Focal epileptic activity in the right centrotemporal area at 7 mo, No epileptic activity at 4 yr

Response to therapy Head circumference

Refractory 0.8 SD at birth, 1.8 SD at 7 yr

Controlled 1 SD at birth, 3 SD at 2 yr, 4.5 SD at 3.5 yr, 5 SD at 5 yr

Abnormal eye movements Pyramidal symptoms

Oculogyric crisis like

–

Oculogyric crisis like

Spastic quadriplegia with exaggerated tendon reflex and clonus

Brisk tendon reflex with flexor plantar response

Ankle clonus, patellar tendon reflex was exaggerated

Movement disorders

Fragmentary myoclonus, chorea, dyskinesia, hand stereotypies

Fragmentary myoclonus (fingers and upper limbs), choreatic movement

Autonomic disturbance Intellectual disability MRI findings

–

Dyskinesia, hand-washing, hand-wringing, hand mounting stereotypical movements, choreatic movements of legs and tongue at 5 yr Severe obstructive sleep apnea Severe Thin CC, mild ventriculomegaly, mild cerebellar atrophy Irritability, inappropriate crying and laughter, Severe feeding problems, Bruxism

Hip subluxation

Age Sex Mutation Dysmorphic features Current status Age at onset Initial symptoms Seizure types

Other notes

Severe Cerebral cortex atrophy, ventriculomegaly, thin CC Failure to thrive at 7 yr Bruxism

Deep set eyes, elongated face, Metopic ridge Bedridden 2–3 mo Dyskinesia, DD, feeding problems at 2–3 mo Complex partial seizure at 7 mo

Bedridden 3 mo Seizure, DD at 2.5 mo Myoclonic seizures at 2.5 mo

High amplitude spike-waves in left or right central to mid-central areas at 2.5 mo, Diffuse irregular spike-waves bursts and focal spikes and polyspikes in the right frontal area at 1 yr, Multifocal spikes, spike-waves in the bilateral frontal region, right and left central regions at 8 yr Controlled 0.8 SD at birth, 0.7 SD at 6 yr

– Severe Thin CC, mild ventriculomegaly

Deep set eyes, and others (see Data S1) Bedridden 2 mo Seizure, feeding problems at 2 mo Breath-holding attacka at 2 mo, Abnormal eye movement with tonic posture unilateral limbsa at 6 mo Diffuse spike-wave and polyspike-wave bursts at 3 mo, Focal spike or spike-waves in the bilateral frontopolar area at 13 yr

Refractory 1.3 SD at birth, 2.8 SD at 6 mo, 3.2 SD at 1 yr 1 mo, 3.4 SD at 2 yr 5 mo, 4.3 SD at 12 yr – Brisk tendon reflex in both legs with flexor plantar response in the left Stereotyped movements of fingers. Peculiar involuntary movements groaning with head swaying and breath held Sleep disorder, hyperventilation Severe Cerebral atrophy

Laughs without reason, no purposeful hand skills

yr, years; mo, months; DD, developmental delay; GTCS, generalized tonic–clonic seizure; CC, corpus callosum. a Epileptic seizures were confirmed by ictal EEG with video monitoring.

Epilepsia, 56(6):841–848, 2015 doi: 10.1111/epi.12987

846 C. Ohba et al. A

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D

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E

F

G

H

I

J

K

L

stereotyped movement disorders. Of note, two patients showed dystonic eye movements resembling oculogyric crises; therefore, we suspected monoamine neurotransmitter disorders. In addition, extrapyramidal symptoms, sleep abnormalities, lack of eye pursuit, and feeding problems, which are common in the four patients with GRIN1 mutations, overlap with clinical features of monoamine metabolism disorders.13,14 On the other hand, two patients were suspected as having Rett syndrome, since they showed RettEpilepsia, 56(6):841–848, 2015 doi: 10.1111/epi.12987

Figure 2. (A, B) Facial photographs of patient 3 at 7 years of age showing elongated face. (C, D) Facial photographs of patient 4 at 12 years of age showing dysmorphic features, including deep set eyes, upslanted palpebral fissures, epicanthal folds, bulbous nose, short philtrum, wide set teeth, bilateral prominent maxilla, and low set and large ears. Sleep EEG of patient 3 at 2 months (E) and 7 months (F) of age. (G, H) T2-weighted axial (G) and sagittal (H) images of patient 1 at 5 years of age showed cerebral atrophy especially in the frontal lobe, enlargement of lateral ventricles and thin corpus callosum. (I–K) T2weighted axial (I, J) and T1-weighted sagittal (K) images of patient 3 at 5 months (I) and 5 years of age (J, K) showed no obvious brain abnormalities at 5 months, but mildly enlarged lateral ventricle and thin corpus callosum at 5 years. (L) T2weighted axial brain MRI of patient 4 at 10 years of age showed cerebral atrophy. Epilepsia ILAE

like stereotypic hand movements and one of the features of Rett syndrome such as bruxism, inappropriate crying and laughter, hyperventilation, sleep abnormalities, and no purposeful hand skills. These findings indicate that GRIN1 mutations cause a recognizable disorder, in which clinical features overlap with encephalopathy with epilepsy, monoamine neurotransmitter disorders, and Rett syndrome. Key features for suspecting GRIN1 mutations seem to be infantile-onset seizures with hyperkinetic and stereotyped

847 Phenotypic Spectrum of GRIN1 Mutations movements, and occasional abnormal eye movements resembling oculogyric crisis. Patient 4 showed somatic mosaicism, with 13.4–19.7% of alleles (26.8–39.4% of cells) carrying the mutation. In previous reports, ratios of mutated cells in patients with Dravet syndrome arising from SCN1A mutations ranged from 0.04% to 85%.15 In addition, we have previously reported a somatic mosaic SCN2A mutation in 36% of blood leukocytes in a patient with Ohtahara syndrome.16 Therefore, it is likely that mosaic GRIN1 mutation in patient 4 caused epileptic encephalopathy with extrapyramidal symptoms. Given the phenotypic similarities between the GRIN1 mutations and monoamine metabolism disorders, it is interesting to note that NMDA receptors are known to interact with dopamine receptors at multiple levels.17 It is well known that interaction between the carboxyl tails of the GluN1 receptor and the D1 dopamine receptor facilitates D1 receptor trafficking to the cell surface, and inhibits D1 receptor internalization.18,19 Therefore, GRIN1 mutations are likely to affect function of both NMDA and D1 receptors, leading to aberrations in both glutamatergic and dopaminergic neurons. Based on this hypothesis, it is possible that extrapyramidal symptoms caused by GRIN1 mutations can be alleviated by anti-parkinsonism agents. The mouse model of GRIN1 deficiency demonstrates some of the human phenotype. Grin1neo/neo mice, which have reduced NMDA receptor function, demonstrate repetitive behaviors.20–22 Furthermore, Grin1D481N/K483Q mice, which show marked NMDA receptor hypofunction, exhibit increased stereotypic behavior.23 Considering that hand stereotypies were observed in three patients with GRIN1 mutations, these results suggest that impaired NMDA receptor function may manifest with stereotypic movements in humans. Given that two of the four patients with GRIN1 mutations were suspected as having Rett syndrome, it is interesting to note the diminished prevalence of GluN1 and GluN2 in MeCP2-null synaptic membranes,24 which suggest that impaired NMDA receptor function may also be involved in the pathogenesis of Rett syndrome. In conclusion, we found four novel GRIN1 mutations in patients showing encephalopathy with infantile-onset epilepsy, and hyperkinetic and stereotyped movement disorders. These data shed light on the understanding of phenotypic spectrum of de novo GRIN1 mutations.

Acknowledgments We are grateful to the patients and their families for their participation in this study. We thank Nobuko Watanabe, Kiyomi Masuko, Sayaka Sasamoto, and Mai Satoh for their excellent technical assistance. This work was supported by the Ministry of Health, Labour and Welfare of Japan; the Japan Society for the Promotion of Science [a Grant-in-Aid for Scientific Research (B) (25293085, 25293235), a Grant-in-Aid for challenging Exploratory Research (26670505), a Grant-in-Aid for Scientific Research (A) (13313587), a Grant-in-Aid for Scientific Research (C) (24591500, 26330331)]; the Takeda Science Foundation; the fund for Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in

the Project for Developing Innovation Systems from the Japan Science and Technology Agency; the Strategic Research Program for Brain Sciences (11105137); and a Grant-in-Aid for Scientific Research on Innovative Areas (Transcription Cycle) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (12024421).

Disclosure None of the authors has any conflict of interest to disclose. We confirm that we have read the journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Read counts in patient 4 with a mosaic mutation in GRIN1 (c.1923G>A). Table S2. Prediction of mutation pathogenicity. Data S1. Case reports. Video S1. Patient 1 showed continuous choreoathetotic movement of both hands and arms with transient upward eye deviation. Video S2. Patient 1 showed continuous hyperkinetic movement of both hands, arms, and legs.