© 2013 Nature America, Inc. All rights reserved. Michael Michael S Hildebrand Meng-Han Tsai Nail Burnashev authors contributed equally to this work. Correspondence should be addressed to H.C.M. ( of Neurology and Neurosurgery, The Children’s Hospital at Westmead, Sydney, New South Wales, Australia. Wellington, New Zealand. EPILAND Network on Epilepsy, Language and Development, Marseille, France. Scientifique (UMRS) 901, Marseille, France. 4 University of Melbourne, Austin Health, Melbourne, Victoria, Australia. 1 in motor aphasia; present focal occur with epileptic seizures acquired delay language isolated or had normal previously were who children (LKS), syndrome Landau-Kleffner In awakening. on remits largely ( ous the which continu virtually shows EEG in sleep movement) eye (rapid non-REM (CSWS) sleep slow-wave during wave and spike ous have overlapping a manifestations EEG notable of signature continu with Twosyndromes manifestations. EEG and clinical and onset of comprise a range of syndromes electroclinical with agescharacteristic impairment and to behavioral cognitive contribute that activity epileptiform abundant and seizures by ized are a character group of disorders severe encephalopathies Epileptic the diagnostic this knowledge, spikes probands GRIN2A aphasia families segregated We epileptic delineate implicated Rare developmental a severe Epilepsy-aphasia Gemma L Carvill disorders GRIN2A Nature Ge Nature Received 20 November 2012; accepted 18 July 2013; published online 11 August 2013; Mediterranean Institute of Neurobiology (INMED), Marseille, France. Department of Pediatrics, Division of Genetic Medicine, University of Washington, Seattle, Washington, USA.

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during during slow-wave sleep (ECSWS), previous development is delayed in wave and spike continuous with encephalopathy epileptic of drome In in the syn contrast, controlled. 70% of and easily are cases usually of autosomal dominant rolandic epilepsy with speech dyspraxia dyspraxia speech with epilepsy rolandic dominant autosomal syndrome of the with transmission dominant autosomal with family a reported we 1995, In difficulties. language or speech and epilepsy of rolandic inheritance monogenic with reported been have families four far,only So spectrum. epilepsy-aphasia the of disorders the for BECTS with children some in noted been has dyspraxia oral of subtle presence The decline. cognitive show not do children affected and CSWS, of pattern synchronous bilaterally ous spikes that are by activated but sleep do not show the continu almost seizures. The typical EEG shows unilateral or bilateral centrotemporal and in occurs normal who children present with motor focal rolandic childhood in syndrome epilepsy focal common most the is BECTS with centrotemporal spikes (BECTS) at epilepsy the mild end of childhood the spectrum benign and middle the in IEAD end, severe the ECSWS with atLKS, with a along spectrum as falling conceptualized (IEAD) disorder epilepsy-aphasia mediate inter an of indicative as regarded be should it whether or CSWS as diagnosable is sleep non-REM of <85% during activity epileptiform that may in severity. fluctuate There is bilateral debate about whether difficulties or learning language substantial have they yet sleep, slow for occur not do abnormalities EEG their because usually characteristics, clinical or EEG of basis the on ECSWS and LKS for polymicrogyria. perisylvian as such are often normal or may show a malformation of cortical development motor impairment is types is usual. Regression more with language, global, behavior and half of children, affected and epilepsy with refractory multiple seizure Until recently, there has been scant evidence for a genetic etiology etiology for a genetic evidence scant Until been has recently, there criteria the do not who meet are patients there practice, In clinical [email protected] 7 INSERM UMRS 663, Paris Descartes University, Paris, France. d o i : 1 2 0 1 , . 10 , , Lynette G Sadleir 1 0 , 3 12 8 3 , Natalia Lozovaya / , n 13 g 12 2 . 2 & Heather C Mefford 2 Florey Institute, Melbourne, Victoria, Australia. . . Magnetic resonance imaging (MRI) brain studies , , Samuel F Berkovic 7 2 Department of Medicine, Epilepsy Research Centre, 2 7 ) or I.E.S. ( 9 , , Samantha J Turner [email protected] 4 – 7 , Nadine Bruneau 11 3 2 T.Y. Nelson Department . These disorders can be be can disorders These . , , Jay Shendure 1 4 . , 13 s r e t t e l 8 ). French 13 2 ≥ These , 10 85% of of 85% 4 3 – , , 6 , 8 3  , - - - . © 2013 Nature America, Inc. All rights reserved. notypic spectrum of of spectrum notypic phe the delineate to sought we Therefore, emerged. have relations disability cor disability, no and clear genotype-phenotype intellectual epilepsy intellectual for with role a support strongly observations these individuals Although of cohort sequencing novo de encephalopathies epileptic early-onset severe a and members family in EEG three or abnormal epilepsy with segregating mutation nonsense a mutations: pathogenic 2 detected disability and/or intellectual EEG abnormal or epilepsy with probands 127 in screening epileptogenesis. in role a have to attrac an candidate it tive making brain, mammalian the in transmission tatory exci mediates that channel ion neurotransmitter-gated a receptor, the of subunit (GluN2A) NR2A included that deletions 16p13 with reported were phenotypes dysmorphic complex with children three gene, one containing tion ECSWS An studies. (CNV) of and LKS in of with was noted a rare excess CNVs probands cohort variant number copy from come has etiology for a genetic evidence recent Furthermore, described. forms familial rare the by supported is etiology genetic a steroids, high-dose with resolution their on based partly basis, immune an have syndromes probands of relatives in phenotype common most the being seizures febrile with likely, most is inheritance plex com with that suggests probands disorders with spectrum relatedness epilepsy-aphasia or of BECTS degrees three to up with bers mem family of Investigation effect. major of genes for support little described. been have types additional no disability, intellectual mild with relatives had female who seizures and rolandic polymicrogyria perisylvian with proband unrelated an in mutation fied with a gain-of-glycosylation lepsy, oral and speech dyspraxia and intellectual disability, was epi identi rolandic X-linked with presenting family, fourth a Conversely, reported dysphasia and and with epilepsy generalized focal manifestations was more was reported phenotype recently similar (ADRESD) Total Progressive myoclonicepilepsies seizures Epilepsy ofinfancywithmigratingfocal Ohtahara syndrome Lennox-Gastaut syndrome Dravet syndrome Febrile infection-relatedepilepsysyndrome Symptomatic generalizedepilepsies Epilepsy withmyoclonic-atonicseizures Infantile spasms Epileptic encephalopathies(other) Focal epilepsy, symptomaticfocalepilepsy Epilepsy-aphasia syndromes mutations T s r e t t e l  encephalopathy. epileptic with individuals of able able 1

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missense mutations were recently reported in a large exome exome large a in reported recently were mutations missense 1 E 0 7 , including in , LKS a proband a with with dele including 16p13 single . A causal gene has not been implicated in these families. families. these in implicated been not has gene causal A . pileptic encephalopathy pileptic cohort encephalopathy screened for 5 . An additional three-generational family with a notably a notably with family three-generational . additional An de novo de GRIN2A SRPX2 missense mutation in an individual with with individual an in mutation missense GRIN2A mutations by screening a large cohort cohort large a screening by mutations variants in epilepsy-aphasia pheno epilepsy-aphasia in variants SRPX2 ( N GRIN2A N -methyl- M mutation _ 0 Total 0 519 0 1 3 34 17 12 85 85 84 87 50 44 1 8 , 6 7 8 6 9 . . d N 3 . . Finally, a family with 1 . GRIN2A -aspartate (NMDA) (NMDA) -aspartate 2 3 . Furthermore, two two Furthermore, . 8 . GRIN2A 3 . Besides . an Besides ) GRIN2A 1 0 . Furthermore, . Furthermore, GRIN2A encodes the the encodes GRIN2A mutations mutation mutation 4 0 0 0 0 0 0 0 0 0 0 0 4 SRPX2

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( thusrepresenting frameshiftaalteration, p.Phe139Ilefs*15 (predicted) in the removal of 593 exonic nucleotides from the mature transcript inand silico tion( identical haplotype in these families, suggestingnucleotide varianta (SNV)common flanking this founder muta splice donor site. Genotyping conserved of microsatellite highly a makers affecting variant and c.1007+1G>Aa rare same single- the carried not present in 6,500 control exomes (see URLs). Two families (A and C) dominantmanner( familymembersshowedvarianteachthat autosomalsegregated an in was confirmed by Sanger sequencing. Segregation analysis in additional across (>25×) coverage Methods) (Online exceptions minor with previously described as sequencing molecular next-generation and PCR using multiplex highly (MIPs), probes inversion sequence flanking of bp 5 and exons all quenced including epilepsy, with associated genes study of a As larger part ( encephalopathies epileptic of range a with probands 519 encephalopathy with continuous spike and wave in slow-wave sleep. in slow-wave wave and spike continuous with encephalopathy epileptic ESCWS, disorder; aphasia epilepsy intermediate IEAD, syndrome; Landau-Kleffner LKS, dyspraxia; speech with epilepsy rolandic dominant autosomal ADRESD, disorders. syndrome epilepsy-aphasia with families 1 Figure on effects detrimental have to likely is codon start translation the mediated decay of the mutant transcript ( monoallelicvariant,wild-typeexpressionsuggestingthe nonsense- of We(III-1).detected C familyfrom one and III-5) and (II-5 A family individuals, in the RNA transcripts of three affectedSNV (rs61753382), individuals,encompassed by the commontwo haplotype from in affected SupplementaryTable 1 III III II II I I We identified four probands with of analysis sequence high-throughput Weperformed We detected a p.Met1Thr variant in family B. The alteration of of alteration The B. family in variant p.Met1Thr a detected We Family A: 2 1 Family C: –/– 2 1 SupplementaryFig. 1 to cause skipping of exon 4 during pre-mRNA splicing, resulting Unclassified epilepsy Febrile seizures ECSW IEAD LKS ADRESD

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© 2013 Nature America, Inc. All rights reserved. events) ( events) (9.1 channels wild-type the to compared (36.7 channels mutant the for state open the of duration mean the in increase fourfold a with ings, record single-channel by observed was kinetics receptor NMDA in COS-7 to cells in form a NMDA mutant receptor. heteromeric A shift NR1 resultant wild-type with NR2A mutant coexpressing by tion func on NR2A alteration p.Thr531Met of the effect Wethe assessed showing duration of the open state for the single-channel patches in in patches single-channel the for state open the of duration showing ( mutation the by affected significantly not were amplitudes single-channel that 50 ( ( wild-type COS-7 transfected transiently from ( Thr531Met NR1–NR2A ( type) (wild WT NR1–NR2A through currents unitary state ( state. open the in receptors NMDA of Figure 2 hybridization genomic comparative array-based we performed EAS, encepha epileptic ( phenotypes lopathy remaining the in detected were variants genic patho additional No disorders. encephalopathy epileptic of group this with rate in mutation individuals a 9% (4/44) yielding EAS, with families. these that conclude we EAS, with tion segrega their and variants these of effect pathogenic the Given ics. the that show we Furthermore, p.Thr531Met variant has a profound on effect NMDA brain. receptor kinet the in composi distribution or receptor tion NMDA aberrant of because possibly receptor, NMDA the of subunit NR2 the for haploinsufficiency of result a as colleagues and by Lesca study parallel a in domain same the affecting mutations missense for reported those consequences to similar functional and clinical had variant identified newly to receptors NMDA of known properties are kinetic and gating the domain influence this within sites Specific NR2A. of domain ( SIFT and PolyPhen-2 by damaging probably be to predicted is that residue (as by predicted conserved high GERP and Grantham scores) unavailable. was proband the from RNA as possibilities, these test to We unable were codon. start truncated protein stemming from translation initiation at an alternate or a codon start at the initiation of translation failure to due product GRIN2A Nature Ge Nature exponentials. single with fitted well were Histograms A Family T epilepsy aphasiadisorder;NA,notavailable. epilepsy withspeechdyspraxia;LKS,Landau-Kleffnersyndrome;ESCWS,epilepticencephalopathycontinuousspikeandwaveinslow-wavesleep;IEAD,intermediate relative toisoform1( Chromosomal coordinatesaregivenrelativetothehg19genomebuild,cDNApositionstranscriptvariant2( D C B n Table able able 2

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). This variant is located in the extracellular ligand-binding ligand-binding extracellular the in located is variant This ). The NR2A p.Thr531Met alteration increases the mean time time mean the increases alteration p.Thr531Met NR2A The protein synthesis, resulting in either complete absence of of absence complete either in resulting synthesis, protein Pathogenic Pathogenic P P Chr. < 0.0001, Mann-Whitney test, two-tailed) ( two-tailed) test, Mann-Whitney 0.0001, < 16 16 16 16 value = 0.953, Mann-Whitney, two-tailed). Right, histograms histograms Right, two-tailed). Mann-Whitney, = 0.953, value n etics N P

Position (bp) _ 10031815 10031815 10274267 mM glutamate. O, open state; C, closed state. Note Note state. closed C, state; open O, glutamate. mM 0 ADVANCE ONLINE PUBLICATION ONLINE ADVANCE 9934563 GRIN2A 0 Table Table 0 b 8 ) channels, recorded in cell-attached patches patches cell-attached in recorded ) channels, 2 4 . 1 1 ). Chr., chromosome;GERP, genomicevolutionaryrateprofiling;SIFT, sortingintolerantfromtolerant;ADRESD,autosomaldominantrolandic ). In the 40 remaining individuals with with individuals remaining 40 In the ). mutations in four families with ± c.1007+1G>A cDNA change c.1592C>T c.1007+1G>A c.2T>C 2.5 ms; ms; 2.5 GRIN2A a , b ) Left, representative steady- representative ) Left, GRIN2A ± n n 0.2 ms; ms; 0.2 = 2,299 channel events) events) channel 2,299 = mutations are causal in in causal are mutations = 7) and Thr531Met Thr531Met and = 7) mutations presented GERP n 5.1 5.2 4.5 5.2 1 8 = 6,715 channel channel = 6,715

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Segregation was perfect in the seven affected members of the origi the of members affected seven the in perfect was Segregation identified. were variants pathogenic No additional sequencing. Sanger using variants for BECTS with probands 81 screened next we spectrum, detected. were alterations number copy No bp. ~350 every probe one of density average an at spanning probes with microarray custom a using (aCGH) Intellectual disability occurred in 6 of 16 mutation carriers, and a ( and intellect borderline of were 2 carriers, further mutation 16 of 6 in occurred (16/16). disability difficulties Intellectual language and speech and (14/16) epilepsy ing includ phenotype, complex a had members family Affected CSWS. to detect performed EEG a sleep not had had IEAD with Individuals studies. CSWS EEG in showed ECSWS and LKS with individuals All ( IEAD and ECSWS LKS, including phenotypes, spectrum Notably, C). (family ECSWS GRIN2A with pair father-son a in found was A; (family ADRESD with family nal Although two cases had EEG patterns suggestive of CSWS, only only CSWS, of suggestive patterns EEG had cases two Although disability intellectual features and dysmorphic moderate-to-severe including phenotypes, complex more of setting the in seizures had LKS had which of one reported, been including microdeletions Four 16p13 with cases cases. fied a consistent epilepsy phenotype but have shared features with our b a o o o o o o c c c c c c In total, we identified 16 subjects with with subjects 16 identified we total, In epilepsy-aphasia the of end mild the at lies BECTS that Given Previous cases implicating mutations in in mutations implicating cases Previous damaging) 1.000 (probably NA 0.213 (benign) NA PolyPhen score mutations were associated with a range of epilepsy-aphasia of epilepsy-aphasia a range with associated were mutations 100 ms 100 ms 5 pA 5 pA 0 (damaging) NA 0 (damaging) NA SIFT N NR1–NR2A T531M M NR1–NR2A WT _ 0 0 0

Count Count 8 0.04 0.08 0.04 0.08 3 1.2 p.Thr531Met (predicted) p.Phe139Ilefs*15 p.Met1Thr (predicted) p.Phe139Ilefs*15 3 0 0 Supplementary Table 2 Supplementary 1 Protein change Fig. Fig. . 0 3 –1 –1 . The remaining three cases cases three remaining The . ), andproteinpositionsaregiven GRIN2A 1 Open-state duration(log,ms Open-state duration(log, ms ) 5 1 0 1 0 . The same mutation mutation same The . GRIN2A s r e t t e l have not identi not have GRIN2A ECSWS, IEAD ECSWS LKS ADRESD mutations. mutations. Phenotype 3 2 3 2 Table GRIN2A GRIN2A ) ) )

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© 2013 Nature America, Inc. All rights reserved. Methods and any associated references are available in the the in available are references associated any and Methods M g grant to L.G.S. P.S. is supported by ANR (Agence Nationale de la Recherche) Recherche) la de P.S.Nationale (Agence by L.G.S. to ANR grant supported is project of Zealand New Council Research by and a Health M.S.H.) to (546493) Fellowship Martin CJ and I.E.S. to 1006110 Fellowship Practitioner I.E.S., and S.F.B.to 628952 Grant (Program of Australia Council Research Medical and Health National by the supported was work This AwardScientists. for Medical Wellcome Career Fund of a Burroughs a recipient is and 1R01NS069605) NINDS (NIH; of Health Institutes National US the from by a grant supported is H.C.M. research. our in for participating families their and We subjects the thank o Note: Any Supplementary Information and Source Data files are available in the v w e h 2012), October (accessed (ESP) Project Sequencing Exome GO (NHLBI) Institute URLs. families. for counseling and genetic prognostic enhance and will disorders spectrum aphasia that suggest strongly results GRIN2A These 9%. of rate mutational a with EAS for cause monogenic a detect to knowledge, our to first, study the This is research. future of area important an be will sleep slow during network corticothalamic the disrupting in role potential its and aberration receptor NMDA cascade. signaling downstream the of disruption and flux ion aberrant in results mutations missense or to due activity receptor NMDA altered ping for disorders. Weetiology neurodevelopmental that hypothesize an toward overlap trend current the balks that an observation tinct, of may dis EAS be well etiology genetic the that demonstrate results autism probands ( of in series large a or in Furthermore, phenotypes BECTS. with probands 81 encephalopathy epileptic other any with detect probands not did we note, Of EAS. to epilepsy rolandic impairment atypical speech with and CSWSS) syndrome, CSWS called report colleagues and Lesca study, separate a in Furthermore, IEAD. and ECSWS LKS, include that disorders spectrum epilepsy-aphasia the syndrome determined. was epilepsy no and family, this of studies EEG in CSWS of or disability.intellectual difficulties learning There was no suggestion of setting the in convulsions adolescent-onset and childhood- with a had family epilepsy partial atypical of one fell clearly along the epilepsy-aphasia spectrum with the syndrome s r e t t e l  A n d e s t cknowledgments w l t . e r u We conclude that that conclude We i w p n s thod w . i e : s a o /

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3. 2. 1. r at online available is information permissions and Reprints The authors declare no competing interests.financial and performed single-channel recordings and analysis. manuscript. N.L., N. Bruneau, N. Burnashev and P.S. generated mutant transcripts screening in the BECTS cohort. B.M.R., S.F.B. and I.E.S. critically reviewed the R.W. performed phenotypic analysis. R.O., J.A.D. and M.S.H. conducted mutation MIP methodology and analysis pipeline. B.M.R., S.C.Y., L.G.S., S.J.T., M.-H.T. and aCGH. A.K. performed mutation segregation analysis. B.J.O. and J.S. developed the analysis (assisted by E.G.) and performed haplotyping. J.C. and G.L.C. performed calling pipeline (assisted by J.C.), analyzed sequence data, conducted RNA transcript and I.E.S. supervised the study. G.L.C. constructed libraries, developed the variant G.L.C., H.C.M. and I.E.S. designed the study and wrote the manuscript. H.C.M. by INSERM. are supported P.S., Bruneau and N. and label, EuroBiomed Burnashev N. with EPILAND grant 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING AUTHOR CONTRIBUTIONS e p

r

Tsai,M.H. Tassinari,C.A. A.T.Berg, er, D.F. Vears, Roll, P. R. Michelucci, S.L. Kugler, I.E. Scheffer, deficits oromotor and J.P.Speech Marcoz, & D. Fontan, T.W.,E., Deonna, Roulet, 54 (2000). S94–S102 2), (suppl. aphasia. acquired the including syndrome ESES or sleep Terminology, and Classification on Commission ILAE 2005–2009. the of report epilepsies: centrotemporal spikes. centrotemporal Genet. Mol. of screening and inheritance dominant autosomal loci. with candidate pedigree Italian an of disorder. speech and epilepsy rolandic anticipation. with syndrome new a spikes rolandic 24 with childhood of epilepsy syndrome. aphasia-epilepsy acquired the to partial Relationship (BPERS). benign in origin epileptic of O’Roak, B.J. O’Roak, Lesca, G. S.F.Traynelis, the ligand-binding within sites Specific L.P. Wollmuth, & P. Borker, I., Talukder, B.J. O’Roak, G.L. Carvill, J. Ligt, de S. Endele, C. Reutlinger, G. Lesca, network of network dysfunction. language and speech withGenet. Nat. encephalopathies and epilepsies focal function. (2010). 11792–11804 gating. receptor NMDA modulate linkers channel ion and domain disorders. spectrum autism in genes novo de disability. phenotypes. 42 neurodevelopmental variable cause receptors NMDA of the of disorders region. rolandic seizure and features, dysmorphic various disability, intellectual autism. with link the makes dissection genomic types: sleep slow-wave during waves and spike i n t , 280–287 (2013). 280–287 , , 83–87 (1993). 83–87 , s , 1021–1026 (2010). 1021–1026 , / i n d et al. e mutations in mutations x et al. Pharmacol. Rev. Pharmacol. . et al. et N. Engl. J. Med. J. Engl. N. et al. et h et al. et et al. et et al. et publishedonline; de novo de

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l GRIN2A et al. et , 1195–1207 (2006). 1195–1207 , t al. et . Epileptic encephalopathies of the Landau-Kleffner and continuous and Landau-Kleffner the of encephalopathies Epileptic Epilepsy Res. Epilepsy t al. et Revised terminology and concepts for organization of seizures and seizures of organization for concepts and terminology Revised t al. et Clinical genetic study of the epilepsy-aphasia spectrum. epilepsy-aphasia the of study genetic Clinical a Epilepsia Mutations in Mutations Diagnostic exome sequencing in persons with severe intellectual severe with persons in sequencing exome Diagnostic DVANCE ONLINE PUBLICATION ONLINE DVANCE agtd eeunig n plpi ecpaoahe identifies encephalopathies epileptic in resequencing Targeted lncl eei suis n ein hlho eies with epilepsy childhood benign in studies genetic Clinical Sporadic autism exomes reveal a highly interconnected protein interconnected highly a reveal exomes autism Sporadic n uooa dmnn gntcly eeoeeu vrat of variant heterogeneous genetically dominant autosomal An mutations. Multiplex targeted sequencing identifies recurrently mutated recurrently identifies sequencing targeted Multiplex uooa dmnn rlni eies ad peh dyspraxia: speech and epilepsy rolandic dominant Autosomal Encephalopathy with electrical status epilepticus during slow during epilepticus status electrical with Encephalopathy mutationsindisordersoflanguagecortexandcognition. ltmt rcpo in hnes srcue rglto, and regulation, structure, channels: ion receptor Glutamate aiil plpy n dvlpetl ypai: description dysphasia: developmental and epilepsy Familial eein i 1p3 including 16p13 in Deletions CHD2

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45 h Clin. Neurophysiol. Clin. Nature Ge Nature t t , 825–830 (2013). 825–830 , p : / / n ains with patients in w w J. Neurosci. J. Neuropediatrics w . n a. Genet. Nat. a t n u Epilepsia r etics e . Hum. c

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© 2013 Nature America, Inc. All rights reserved. Sanger DNA sequencing. Sanger exonic rare the for members family all PeakScanner V2.0 software (Applied Biosystems). Furthermore, we genotyped ranges size allele and were with determined the analyzer, GS500LIZ size standard (Applied Biosystems) using genetic ABI3100 an on analyzed were ucts spanning a across 0.56-Mb interval D16S407, and D16S3126 D16S404, markers, microsatellite three Wevariant. selected c.1007+1G>A the carried who B and A families of members fected Genotyping. Workbench Technologies). Genomic (Agilent using conducted was analysis data and manufacturer’s instructions, the to according performed were experiments All bp. ~350 epilepsy-related genes. known in alterations copy number to detect designed Technologies) (Agilent aCGH. previously. described as methods population. PCR and Sanger sequencing were conducted according to standard the and URLS), (see ESP6500 data set (see URLs) SeattleSeq was used to with assess variant frequency in annotated the control were Variants bp). (5 runs in homopolymer of and size 10) variants (window variants clustered of <25×, from further analysis: allele balance of >0.70, QUAL of <30, QD of <5, coverage see URLs). Variants that did not adhere to the following were criteria excluded Tool Analysis Kit Genome (GATK; the using performed was ing and filtering performed as previously calling. variant and analysis Data International League Against Epilepsy Commission on Classification 1. studies. Epilepsy syndromes were classified according to the Organization of the were obtained together with the results of investigations including EEG and MRI quent, ongoing epileptiform activityepilepsy 1. Detailed and histories medical fre with associated regressionor slowing cognitive and seizures refractory as epileptic encephalopathy phenotypes. An epileptic encephalopathy was defined informed consent. The cohort consisted of 519 individuals withafter internationallyand Australia around from researcha genetics diverseepilepsy for range of epilepsy clinic at Austin Health, the practices of the investigators and by referral Washington. Probands with epileptic encephalopathies were of Universityrecruited from the the and Health Austin of Committees Ethics Research Human diagnosis. and recruitment subject Human O doi: NLINE 10.1038/ng.2727 We performed aCGH using a custom-designed 8-plex microarray microarray 8-plex custom-designed a using aCGH performed We

ME We performed genotyping in all available affected and unaf and affected available all in genotyping performed We THOD GRIN2A S 1 4 . . However, variant (single nucleotide and indel) call was covered at a density of one probe for every GRIN2A Raw read processing and alignment was was alignment and processing read Raw GRIN2A . . Fluorescently labeled PCR prod This study This approvedwas by the variant (rs61753382) using using (rs61753382) variant - - - - Cells were negative for mycoplasma contamination using the MycoTrace MycoTrace the using Mycoplasma contamination PCR detection kit mycoplasma (PAA). for No authentication negative was made. Cells were in the Cells of holding potential +100 mV using an (HEKA Elektronik). amplifier EPC-10 at patches cell-attached in CRL-1651) (ATCC, cells COS-7 transfected from Single-channel recordings and analysis. shown). not (data experiments cytochemistry by immuno was verified and at membrane mutant) type subunits plasma the (wild NR2A and NR1 the of presence The Biosciences). (Oz kit transfection Magnetofectamine the using was performed ratio) (1:3 plates constructs and NR2A 6-well in seeded were 10 × (1 cells COS-7 sequencing. fibroblast–like Sanger by kidney verified Monkey were constructs mutant and wild-type All generate the mutant NR2A (Thr531Met) construct ( to used was Technologies) (Agilent mutagenesis Site-directed (Genecopeia). transfections. and Constructs (see the of presence mutation c.1007+1G>A to the linked the was allele minor the variant; C rs6173382 assessed We and mutation. III-5) and c.1007+1G>A (II-5 the A with families (III-1) from members family affected three in for performed were sequencing Sanger and PCR Nested (Bio-Rad). kit Supermix 1 Transcription using Reverse iScript the performed was synthesis kit RNA cDNA Blood PAXgene (PreAnalytiX). the using controls and members family affected analysis. transcript RNA Instruments) Instruments) and had resistances of 4 to 7 M (World capillaries glass patch were from pipettes borosilicate Precision pulled NaH NaCl, 3.5 mM KCl, 1 mM MgCl ACSF with oxygenated 126 mM containing were chamber recording perfused no means and Devices) as expressed were Average values kHz. at 1 set was filter low-pass (Molecular Bessel pole 10.2 Clampfit using Origin 8.5 (Origin-Lab) software. For data analysis by Clampfit, a digital performed eight- was Lifetime analysis kHz. 20 at digitized and dB) (−3 kHz 2 at filtered were recordings analysis, For °C). (22–24 temperature room at performed were Recordings 50 with activated were Channels mOsm). 320–330 7.5, (pH and 20 mM HEPES/NaOH glucose mM mM 140 2.8 NaCl, KCl, 2 mM CaCl composition: following the n parametric Mann-Whitney test (two-tailed). test Mann-Whitney parametric Supplementary Table 3 Table Supplementary 2 PO ± 5 cells/well) 1 d before transfection. Magnetofection of cells with NR1 NR1 with cells of Magnetofection transfection. before d 1 cells/well) s.e.m. The statistical significance of the differences was evaluated by evaluated was of differences the significance statistical The s.e.m. 4 and 26 mM NaHCO µ M glycine and 1 mM glutamate in the pipette solution. solution. pipette the in glutamate mM 1 and glycine M RNA was isolated from the whole blood of of blood whole the from isolated was RNA for the sequences of primer pairs). primer of sequences the for 3 (oxygenated with 5% CO 2 NR1 and NR2A constructs were purchased purchased were constructs NR2A and NR1 , , 2 mM CaCl Single-channel recordings were made GRIN2A Ω 2 , , 10 mM when filled with when the filled solution of RNA transcript analysis analysis transcript RNA Supplementary Table Supplementary d (+)-glucose, 1.20 (+)-glucose, mM 2 Nature Ge Nature /95% O µ g of DNA with with DNA of g 2 ). Recording 2 , , 10 n etics

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