© 2013 Nature America, Inc. All rights reserved. Heather C Mefford Correspondence should be addressed to H.C.M. ( Neurology, University of Toronto, Toronto Western Hospital, Krembil Program, Neurosciences Toronto, Ontario, Canada. 19 Tel-Aviv, Israel. 15 University of Melbourne, The Royal Children’s Hospital, Melbourne, Victoria, Australia. Petach Tikvah, Israel. 10 Children’s Hospital, Sydney, New South Wales, Australia. School of Medicine and Health Sciences, University of Otago, Wellington, New Zealand. Washington, USA. 4 University of Melbourne, Austin Health, Melbourne, Victoria, Australia. 1 refractory by characterized ongoing with epilepsies associated are regression, or arrest of cognitive and seizures encephalopathies group Epileptic 3%. devastating of a rate incidence with lifetime disorders, a neurological common most the of one is Epilepsy molecular method resequencing. cases mutations. phenotypic for are cohort. pathogenic of genes. phenotypic to epileptic resequencing unknown epilepsies Epileptic Jeremy L Freeman Tally Lerman-Sagie Victoria Rodriguez-Casero Thorsten Stanley Adiba Khan L Gemma Carvill identifies Targeted resequencing in epileptic encephalopathies Nature Ge Nature Received 28 December 2012; accepted 1 May 2013; published online 26 May 2013; Department Department of Psychiatry & Behavioral Sciences, University of Washington, Seattle, Washington, USA. Department of Pediatrics, Division of Genetic Medicine, University of Washington, Seattle, Washington, USA. Metabolic-Neurogenetic Service, WolfsonMetabolic-Neurogenetic Medical Center, Holon, Israel. TY Nelson Department of Neurology, The Children’s Hospital at Westmead, Sydney, New South Wales, Australia. Department of Neurology, The Royal Children’s Hospital, Parkville, Melbourne, Victoria, Australia.
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3 , , Jacinta M McMahon , , Samuel F Berkovic ( syndromes epilepsy other or disorders neurodevelopmental caused ( CNVs epilepsy-associated from selected epileptic encephalopathy phenotypes ( ( genes) related disease- candidate 46 and known (19 genes 65 of analysis sequence to throughput needed approachtargeted sequencing to comprehensiveperform is encephalopathies Here we diagnosis. of advantage take a high- epileptic genetics-based facilitate addi cause of that discovery genes the that tional clear is it Furthermore, unexplained. remaining cases of majority vast the with setting, carried clinical is the in out process, expensive and inefficient an testing, gene serial As a result, syndromes. encephalopathy of epileptic spectrum a wide known. across gene not given any of role is the investigated have studies encephalopathy few Very epileptic in involved be to known gene. specific a in mutations with ciated heterogeneity genetic within a given epilepsy syndrome and both phenotypic heterogeneity asso is there as challenging, be can patients cases of rare, encephalopathies tic epilep some for responsible be to known are genes several in tions epileptic activity, that typically carry a poor prognosis n doi:10.1038/ng.264 14 = involved Usingnew genes in 13). approach, we this (i) identified The full phenotypic spectrum associated with mutations in genes genes in mutations with associated spectrum phenotypic full The 8 Paediatric Paediatric Neurology, Monash Medical Centre, Melbourne, Victoria, Australia. Department Department of Paediatric Neurology, University of New South Wales, Sydney CHD2 de novo de 3 16 , 4 . Despite this recent progress, genetics-based diagnoses of diagnoses genetics-based progress, recent this Despite . , Zaid , Afawi Zaid 15 11 ). copy number variants (CNVs) account for up to ~8% ~8% to up for account (CNVs) variants number copy , Danielle , M Danielle Andrade Epilepsy Epilepsy Unit, Schneider Children’s Medical Center of Israel, 5 Supplementary Fig. 1 Fig. Supplementary Veteran Affairs Puget Sound Health Care System, Seattle, 6 10 , , Stephen Malone 6 and , , Sara Kivity 2 2 . Furthermore, we and others have shown that that shown have others and we Furthermore, . Department Department of Medicine, Epilepsy Research Centre, 2 , , Ingrid E Scheffer 2 16 , , Brian J O’Roak 17 Department Department of Neurology, Tel-Aviv University, 22 , , Nathanel Zelnick Florey Institute, Melbourne, Victoria, Australia. SYNGAP1 21 Department Department of Medicine, Division of 11 , , Mark T Mackay Table ) in 500 cases with a range of of range a with cases 500 in ) 6 13 , Geoffrey Wallace , Geoffrey 21 n Department Department of Paediatrics, The , = 33) and mutations that and mutations = 33) 1
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© 2013 Nature America, Inc. All rights reserved. retardation of growth and/or development, genital and/or urinary abnormalities urinary and ear abnormalities and/or and deafness) syndrome, genital resulted development, and/or growth of retardation which cause CHARGE (coloboma, heart defects, atresia of the choanae, human Furthermore, in process gene target regulation. gral inte an remodeling, nucleosome ATP-dependent for responsible is of another CHD protein, CHD7, have shown that the helicase domain ers remodel as chromatin function proteins these that suggest domains These domain. helicase/ATPase SNF2-related an and modifier) tion remodeling domain termed the chromodomain (chromatin organiza proteins. of All CHD family members are by characterized a family chromatin- (CHD) DNA-binding helicase chromodomain the of ber to be damaging by both PolyPhen-2 and SIFT. ATPase and were predicted (p.Trp548Arg domain and p.Leu823Pro) ants disrupted highly conserved residues in the SNF2-related helicase/ premature truncation of CHD2 ( ( encephalopathies with a in individuals range detected deletions of the 15q26.1 epileptic CHD2 NP_006763. CHD2 encephalopathy: epileptic for genes carrying new 2 the of either in mutations individuals multiple with genes, candidate 46 our of 6 in cohort. discovery the in included not were cases these cases; tested in previously for variants 24 known sensitivity and 100% sequencing ants (161 loci) from 12 samples that had previously undergone exome calling ( variant accurate for required depth sequencing a coverage, encephalopathies. for and in known genes new epileptic both and frequencies mutation spectra phenotypic the delineated (ii) and encephalopathy epileptic a MAE, myoclonicatonicepilepsy. syndrome; LKS,Landau-KleffnerLGS,Lennox-Gastaut EOEE, early-onsetepilepticencephalopathy;FIRES,febrileinfection-relatedepilepsy continuous spikeandwaveduringsleep;EME,earlymyoclonicencephalopathy, ABPE, atypicalbenignpartialepilepsy;ECSWS,epilepticencephalopathywith Total Ohtahara syndrome MAE LGS LKS Infantile spasms FIRES Epilepsy aphasia EOEE EME Epileptic encephalopathy ECSWS Dravet ABPE Syndrome 65 new and known genes disease-relevant T s r e t t e l ability remodeling decreased in In fouroftheDravetsyndromecasesincluded, Supplementary Fig. Supplementary 2 able able 1 not otherwisespecified Notably, we detected six six detected we Notably, mutations pathogenic likely or pathogenic more or 1 We detected 91% of Overall, the target genes (65 genes) were at sequenced >25× 7 . Although functional studies of studies . CHD2 have functional studies Although limited, been ( (
NM_001 CHD7 Figs. 1 1 Figs. E pileptic encephalopathy pileptic cohort encephalopathy screened for mutations in 2 ) ( ) showed that mutations affecting the helicase domain, domain, helicase the affecting mutations that showed and Fig. Fig. 271. Supplementary Fig. 3 Fig. Supplementary
2 1 3 ), selected from within the interval shared by shared interval the within from selected ), and and ; ; ). ). We achieved 91% sensitivity across 685 vari NP_001262. Tables 1 Number ofcases de novo de 500 173 8 Table . These results suggest that the two two the that suggest results These . 81 40 81 12 27 39 10 19 4 3 5 6 SCN1A 3 and variants in the candidate gene gene candidate the in variants ) ) and 2
). ). Two testinghadnotyetbeenundertaken. 2 ). ) SYNGAP1 5 , 6 . Four mutations led to to led mutations Four . CHD2 de de novo pathogenic variants(%) Pathogenic orlikely in in vivo encodes a mem ( 52 (10) 22 (13) NM_006772. missense vari 2 (50) 3 (4) 5 (13) 0 4 (5) 0 3 (11) 8 (21) 1 (20) 0 4 (21) 0 studies of studies a
2 ------;
of this chromatin-remodeling family in epileptogenesis. in family chromatin-remodeling this of disorders and will provide new avenues of research focused on the role alterations in each protein across the spectrum of neurodevelopmental the role determine family will of of studies Further this nine-member system. neurodevelopmental the affect that disorders in implicated ( family chromodomain in mutations Notably, cohorts. diverse have cally recent studies implicated localization ( intragenic phenotype-specific or mutation of type the either to respect with correlation genotype-phenotype clear no was There disorders. of neurodevelopmental to a spectrum tribute broad (ASD) order dis spectrum autism with individual an in identified was mutation seizures absence and disability six). intellectual (all severe to moderate A from ranging disability intellectual years; 1–3 of Table (range months 18 of onset seizure at age median ing probably mutations haploinsufficiency. in result truncating whereas manner, similar a in tion de novo disease-related genes. disease-related (blue) known and (red) new in encephalopathies epileptic of cases 1 Figure testing. diagnostic with ated associ progressiveand encephalopathies static core featurea ofboth with geneity. Our study identifies the first cases of epileptic encephalopathies conditions and mechanisms, genetic or otherwise, underlie this hetero heterogeneity. Rather, we hypothesize that alternative neurobiological suggesting that the nature of the mutation does not underlie phenotypic genotype-phenotype correlation with respect to controlled well typically are epilepsy,seizures have also cases most although disability, and, intellectual with ciated disability. intellectual severe to delay and subsequent regression. Outcomes were poor, with moderate 3 years) ( onset ofseizure 14 months (mean of 14 months, range of 6 months to SYNGAP1 epilepsy-associated genes ( Fig. Fig. de de novo CHD2 The six cases with Number of pathogenic mutations We identified 9 pathogenic or likely pathogenic variants in 4 of the 13
SCN1A 0 1 2 3 4 5 6 7 8 SYNGAP1 2 2 ), motivating further further ), motivating missense mutations described here may func CHD2 disrupt mutations described missense
CDKL5 and (three) photosensitivity six), (all seizures myoclonic ), Table Pathogenic and likely pathogenic mutations identified in 500 500 in identified mutations pathogenic likely and Pathogenic SYNGAP1 (
STXBP1 Fig. 1 0 CHD8 2 . These results suggest that mutations in in mutations that suggest results These . mutations. These observations suggest that epilepsy is ). They had multiple seizure types, early developmental 2 a frameshift mutation frameshift was in reported a proband with
CHD2 ). Cases with DVANCE ONLINE PUBLICATION ONLINE DVANCE SYNGAP1 mutations and carry important implications for implications important carry and mutations in individuals with ASD with individuals in CHD2
SCN2A CHD2 Fig. MEF2C mutations had distinctive features, includ CHD2 SYNGAP1 1 SYNGAP1 KCNQ2 , ). We found five truncating variants in CHD7
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© 2013 Nature America, Inc. All rights reserved. not available for analysis. A recent report identified identified report recent A analysis. for available not was DNA paternal and inherited, maternally was mutation Neither 4 Fig. changes, and c.471T>C single-nucleotide ( c.472A>T gene NP_114 candidate CNV-identified the GABRG2 ants in found we Furthermore, (p.*464SerExt*?). NP_002388. two were There genes. father unavailable. a spike wave;MH,modifiedhypsarrhythmia. fast activity;GPSW, generalizedpolyspikewave;GSW, generalizedspikewave;MFD,multifocaldischarges;PPR,photoparoxysmalresponse;MA,myoclonicabsence;SSW, slow epilepticus; SE,statusT, tonic;TC,tonic-clonic;EEG,electroencephalography;DS,diffuseslowing;ED,epileptiformdischarge;GPFA, generalizedparoxysmal M, male;F, female;A,atonic;FDS,focaldyscognitiveseizures;FS,febrileH,hemiclonic; IS,infantilespasms;MJ,myoclonicjerks;NCS,non-convulsivestatus T162 T2528 T15924 T19988 Likely pathogenic T18044 T23549 T20719 T1898 T22387 T15923 T18431 T17756 T20240 T2608 T18697 T38 Pathogenic variants( Proband T Nature Ge Nature that haploinsufficiency for for haploinsufficiency that mental phenotype including epilepsy site variant was identified in a proband with a complex neurodevelop of microdeletions phenotypes 1q44 with probands seizure and disability intellectual the for candidate PolyPhen-2 andSIFTscoresaregivenonlyformissensevariants. able able 2 We detected a premature truncation mutation (p.Tyr805*) in in (p.Tyr805*) mutation truncation premature a detected We epilepsy-associated additional three in identified were Variants d d ), that occurred two amino acids upstream of the stop codon. codon. stop the of upstream acids amino two occurred that ), d c MBD5
032. Pathogenic Pathogenic and likely pathogenic variants in new genes for epileptic encephalopathy ( age (years) Sex, study NM_000816. n M, 33 M, 26 M, 11 M, 18 M, 4 F, 3.5 M, 2.5 F, 20 F, 7 F, 26 M, 2.5 M, 15 M, 12 F, 29 F, 12 M, 17 2 2 etics ), a missense variant (p.Cys39Arg) and a stop-loss variant ). ). The p.Tyr805* change arose as a result of two adjacent ( NM_018328. de novo
ADVANCE ONLINE PUBLICATION ONLINE ADVANCE SYNGAP1 SYNGAP1 SYNGAP1 MEF2C MEF2C GABRG2 MBD5 SYNGAP1 SYNGAP1 CHD2 CHD2 CHD2 CHD2 CHD2 CHD2 HNRNPU Gene de novo de 3 ) ; ; NP_000807. 4 HNRNPU ; ; NP_060798. p.Tyr805* p.Gln702* Unknown p.Lys108Valfs*25 p.*464SerExt*? p.Cys39Arg p.Arg323Gln p.Thr157Glnfs*4 p.Arg143* p.Trp267* p.Leu823Pro p.Trp548Arg p.Arg1644Lysfs*22 p.Gly491Valfs*13 p.Arg121* p.Glu1412Glyfs*64 variants in in variants (Polyphen, SIFT) (c.389–2A>T) (0.998, 0) (0.998, 0) (0.999, 0) (1, 0) Protein change 2 1 0 9 . Collectively these data suggest . In addition, a a addition, In . 2 s soitd with associated is ) (p.Arg323Gln) ( ) (p.Arg323Gln)
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b Initial seizurelistedfirst(ageofonset)followedbysubsequenttypes. Diagnosis ( pathogenic vari pathogenic LGS EE EE EE EE EE MAE EE EE EE EE EE MAE LGS EE MAE (
Supplementary Supplementary NM_031844. NM_0 de novo de HNRNPU Table Atypical absence,MJ, A (2years) Absence, aura,FDS,MJ, FS (18months) TC Absence (6months) FDS Unknown (infostercare) Absence, A,focal,IS, H (4months) Absence, SE,TC FS (13months) Absence. A,MJ,TC FS (8months) Absence, FDS,focal,T TC (6months) MJ Absence (10months) A, aura,FDS,MJ Atypical absence(3years) MA, T FDS, MJ(2.5years) FDS, H,MJ TC (3years) MJ, SE,TC A (2years) A, MJ,NCS,SE,T, TC Atypical absence(1year) NCS, T, TC,MA MJ (1year) FS, absence,MJ-A, A (1year)
epileptic epileptic MJ, TC NCS, T, TC NCS, TC MJ, TC 02397. splice- 2 (age ofonset) as a as ). Seizures 2 4 - - ; ;
We identified multiple cases with mutations in in mutations with cases multiple identified We mutations are with associated early infantile epileptic encephalopathy encephalopathy. epileptic unclassified with proband single a in mutation example, missense recessive a homozygous For we identified spectrum. phenotypic known the expanded have we ( listed are disease in involved be to ( pathogenic also are variants of these a that number it is probable analysis; segregation to conduct an additional 16 rare detected variants We in 6 of the cohort. 19 known genes butour were unable in individuals mutation-positive all of SCN1A genes involved in epileptic encephalopathy ( known 19 of 10 in pathogenic likely are that variants 5 additional an to corresponding spectrum phenotypic the of understanding improve will correlation genotype-phenotype encephalopathy as well as with intellectual disability, although further b
The phenotypes identified in with mutations cases identified in The genes phenotypes known We identified 31 variants fulfilling our criteria for pathogenicity and
, ,
SCN2A Normal GPSW, MFD,DS GSW SSW, MFD GSW, GPSW, MFD GSW, MFD,DS DS, GPSW, 2.5-HzGSW SSW, MFD,DS,GPSW, GPSW, MFD,GPFA, SSW 3.8-Hz GSW GSW, GPSW, DS,SSW, SSW, bioccipitalED,DS GSW, GPSW, MFD MFD, DS MH, MFD MFD, DS PPR-triggered MJ GPFA , , PCDH19 EEG and and Supplementary Table 1 Supplementary KCNQ2 before seizure Development c Normal Delayed Delayed Delayed Delayed Delayed Normal Delayed Normal Mild delay, Delayed Delayed Delayed Unknown Normal Delayed DNA fromparentsunavailable. problems behavioral onset Table , accounting for 69% (36/52) (36/52) 69% for accounting , HNRNPU 3 Fig.
), and, for some of these, these, of some for and, ),
Normal (yes) Severe intellectual Severe intellectual Severe intellectual Severe intellectual Moderate intellectual Severe intellectual Severe intellectual Severe intellectual Moderate intellectual Severe intellectual Moderate intellectual Severe intellectual Moderate intellectual Intellectual disability(no) Severe intellectual disability, ASD(no) disability, ASD(yes) disability, ASD(yes) disability, ASD(yes) disability (yes) disability (yes) disability (yes) disability (yes) disability, ASD(no) disability (yes) disability (yes) disability, ASD(yes) disability, ASD(unknown) disability, ASD(yes) 1 Cognitive outcome and mutations. STXBP1 (regression) s r e t t e l Tables 1 ). d DNA from , , PNKP CDKL5
and PNKP
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© 2013 Nature America, Inc. All rights reserved. numbers of cases with mutations of specific genes are identified, identified, are genes specific of mutations with cases larger of as numbers Furthermore, pathogenic. be to likely are which of some identified, we that variants of subset a for analysis segregation duct to con and we unable were mutations, identified previously with als Our individu cohort excluded burden of encephalopathies. epileptic overall the to gene each of contribution true the of underestimation an probably is rate mutation this However, genes. 16 in found tions genic or likely pathogenic mutations in 10% of our cohort, with muta overall the patho We identified frame disorders. of group this of to architecture genetic begin to us allow encephalopathies epileptic mosaicism parental with encephalopathies genetic other in as seen as pathogenic variant of this interpretation the supporting 13% of (with the with mutant to cells have allele), mosaicism somatic shown was father the although inherited, paternally was variant fied identi the Notably, age. of months 18 at beginning encephalopathy in epilepsy death unexplained sudden and encephalopathy epileptic severe with mutation p.Asn1768Asp) (encoding in had a been described proband in p.Leu1290Val) for accounting encephalopathies, cases. of 1% epileptic of burden overall the intellectual that Weconclude probands) disability. (three severe to probands) (two mild from ranged which outcome, intellectual with not did correlate of seizures stopped seizures nature at early and 5 cases, 7 weeks relatively of months age. The refractory two In age). of months 13 and weeks 6 (at in infancy (11 h beginning to and 2 two seizures having d birth) after period neonatal the in beginning seizures having three with BFNIS, in seen as onset at age of range the in variability similar showed bly encephalopathies epileptic with individuals seizures (BFNIS) autosomal neonatal-infantile familial self-limited benign of the syndrome with dominant associated been have mutations of a channel. Untilsodium voltage-gated now, the majority of exome and approach. association, effective an represent could cases this these of sequencing clarify to warranted is work Further ( encephalopathies epileptic of group this to the drome, individual but predisposing it be could a modifier genetic the syndromes epilepsy-aphasia in reported been previously not have but (GEFS+) plus seizures febrile with epilepsy (FS+). epilepsy-aphasia phenotype, with two also having febrile seizures plus zure types. Also, three cases with sei multiple with epilepsy refractory despite cognition, normal had was in circumference or the 50th delay percentile) developmental but mental delay comprising microcephaly, early-onset intractable seizures and develop CHD2 either in mutations for exist correlations genotype-phenotype evident No epilepsy. with individuals in found variants pathogenic indicate entries autism and/or disability intellectual in SYNGAP1 of and (Thr604Leufs*19) disability intellectual in CHD2 of variants reported previously denote entries black study; ( SYNGAP1 and ( encephalopathies. epileptic for 2 Figure s r e t t e l are to likely phenotypes emerge. encephalopathy epileptic distinctive The findings in this large series of cases with hitherto unsolved unsolved hitherto with cases of series large this in findings The (encoding mutation missense pathogenic a identified also We We five variants in detected SCN1A or or SCN1A
SYNGAP1 2 Protein alterations encoded by by encoded alterations Protein 4 . Previously, only three three only Previously, . 2 mutation is not responsible for the epilepsy-aphasia syn epilepsy-aphasia for the is not responsible mutation 7 . . Here we describe a second case presenting with epileptic 2 1 mutations are well known to be associated with genetic . . By contrast, our case did not have microcephaly (head b ). Alterations shown in red were identified in this this in identified were red in shown Alterations ). . SCN8A . Before now, only a single single a now, only Before . SCN2A a SCN2A , b SCN1A ) Alterations are shown for CHD2 ( CHD2 for shown are ) Alterations de novo de is an important contributor to to contributor important an is 9 de novo de , which encodes , the which encodes and autism (Asp856Gly) autism and mutations presented with an variants were reported in in reported were variants 25 mutations in new genes genes new in mutations Supplementary Fig. 5 Fig. Supplementary , 2 22 6 . Our five cases . nota Our five cases , 2 3 . It is possible that that possible Itis . 9 , 14 de novo SCN8A novo de – 1 8 2 . Bold . Bold α 8 . 2 2 subunit 1 SCN2A
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approach across various neurodevelopmental disorders will disorders neurodevelopmental various across approach Applying this correlations. genotype-phenotype and type to describe approaches, to determine overall mutation frequency in a given pheno single a where genes in mutations with individuals additional identify to critical is approach This manner. cost-effective and high-throughput a in als of targeted resequencing to screen large numbers of affected individu novo de with cases more or two identify to required be will cohorts large but discovery, gene to avenue an provide sequencing whole-genome or exome as such approaches Unbiased disorders. neurodevelopmental of basis genetic shared the highlighting ASD phenotypes, and ability in described probands with epileptic encephalopathy, dis intellectual a in gene causative the individual. priori a given diffi determine large remain to will employs it expensive as and that cult sequencing, diagnosis whole-exome or genetic panels to notion gene the approach support clinical findings a were These of that cohort. syndromes our in distinct prevalent very not only cause or each <0.2% cohort) for our in (accounting encephalopathies epileptic of causes rare are important, although genes, these in mutations pathogenic SPTAN1 ( genes disease-related known 9 other in seen were mutations no However, that to similar in frequency mutations for a seen cases, of 1–1.2% for accounting each with in mutated encephalopathies, commonly are epileptic cases. that of genes 1.6% new to up identified only we for Notably, accounted each study our in genes ( genes disease-associated new or known nine in found were variants pathogenic likely erable; previ cause. of unknown ously encephalopathies epileptic all of ~18% to variants causal series current of the of a subset analysis earlier in an identified encephalopathy epileptic with in cases of CNVs rare rate up the to 8% prevalence with together results these Considering b a Notably, mutations in in mutations Notably, is consid encephalopathies of epileptic heterogeneity The genetic K114Sfs*20 H95Pfs*5 SY CHD2 R121* ARX mutations in the same gene same the in mutations NGAP1 K108Vfs*25 and and Chromodomain homology Pleckstrin K138fs* c.389–2A>T R143* c.510–1G>A W267* , , FOXG1, KCNT1 FOXG1, V348Afs*70 ARHGEF9 K418Rfs*54 de novo de a G491Vfs*13 W362R DVANCE ONLINE PUBLICATION ONLINE DVANCE T604Lfs*19 P562L conserved Protein kinase W548R SCN1A mutation is identified by exome sequencing sequencing exome by identified is mutation Arg579* DEXDc helicase ) in 500 cases. These results suggest that that suggest results These cases. 500 in ) c.2294+1G>A Q702* Fig. Fig. SYNGAP1 N729Tfs*31 S738* , , L823P , , 2 MECP2 D856G L813Rfs*22 STXBP1 ). Even the most commonly mutated mutated ). commonly Even most the C T878Dfs*60 Q893Rfs*184 CHD2 9 , 1 and and 8 , , RASGAP . Our results show the power power the show results Our . PLCB1 and and ATP helicase , , SYNGAP1 CHD2 CDKL5 , , 3 E1412Gfs*64
SLC25A22 , we can now ascribe ascribe now , can we Nature Ge Nature have now been been now have Coiled-coil motif in our cohort. cohort. our in R1644Kfs*22 and and Coiled-coil motif , , SLC2A1
identify identify SCN2A n etics - - - - - ,
© 2013 Nature America, Inc. All rights reserved. (GATK, version 2.2, 2.2, version (GATK, October 2012), (accessed (ESP) Project Sequencing Exome GO (NHLBI) URLs. mechanisms. disease of understanding enhance and gene specific a for cases mutation-positive additional d a EE, epilepticencephalopathynototherwisespecified;ICEGTC,intractablechildhood epilepsywithgeneralizedtonicclonicseizures. PNKP PNPO KCNQ2 SCN8A UBE3A STXBP1 CDKL5 PCDH19 SCN2A SCN1A Gene T Nature Ge Nature (version 134, Variant segregateswiththedisorder(pedigreesshownin Father issomaticmosaic,with13%ofcellscarryinganalternate,pathogenicallele. able able 3 NP_009185. ( NP_06 ( NP_004509. ( NP_001171455. ( NP_00 ( NP_001027392. ( NP_001032420. ( NP_001171809. ( NP_066287. ( NP_001159435. ( NM_007254. NM_018129. NM_004518. NM_001177 NM_000462. NM_001032221. NM_001037343. NM_001184880. NM_021007. NM_001165963.
Exome Variant Server, National Heart, Lung, and Blood Institute
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ADVANCE ONLINE PUBLICATION ONLINE ADVANCE http://www.broa Proband (sex) T23451 (M) T23919 (F) T24158 (M) T3929 (M) T23859 (F) T23289 (M) T22856 (M) T23122 (M) T23553 (F) T23151 (F) T1266 (M) T22595 (M) T24139 (M) T23234 (F) T23951 (M) T23057 (M) T897 (F) T22954 (F) T22724 (M) T20819 (M) T23305 (F) T23579 (F) T24127 (F) T20340 (M) T22816 (F) T21005 (F) T20632 (F) T19963 (M) T18997 (M) T18775 (M) T19875 (F) T18594 (M) T18466 (M) T1639 (M) T23445 (F) T23141 (M) ngton.edu/SeattleSeq EVS dinstitute.org/gatk Homozygous recessive Homozygous recessive De novo De novo Inherited fromsomaticmosaic Inherited fromunaffected De novo De novo De novo De novo De novo Unknown, motherunavailable De novo Unknown, parentsunavailable De novo De novo De novo De novo De novo Inherited fromunaffected De novo X linked,femalerestricted X linked,femalerestricted De novo De novo Unknown, fatherunavailable De novo Unknown, parentsunavailable De novo Unknown, fatherunavailable Segregates De novo Segregates De novo Segregates De novo / father had mutation mother, affectedsiblingalso mother, Xlinked ; Genome Analysis Toolkit Analysis Genome ; S upplementary Fig.5 d Inheritance a a Annotation134
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Known pathogenicvariantindbSNP, rs104894629(ref.
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Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Likely pathogenic Pathogenic Likely pathogenic Pathogenic Likely pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Likely pathogenic Pathogenic Likely pathogenic Pathogenic Pathogenic Pathogenic Inferred effect Methods and any associated references are available in the the in vers available are references associated any and Methods M Intolerant from Tolerant), PolyPhen-2, for Burrows-Wheeler (BWA,Aligner version 0.5.9, et ge.net ion of the pape the of ion h o / d ; Picard tools (version 1.82), 1.82), (version tools Picard ; s h cDNA change c.2564C>G c.545T>C c.433C>T c.464-2A>G c.2873C>T c.1681G>A c.5645G>A c.3057delA c.2783T>G c.2715G>C c.408G>T c.4453T>C c.1209delA c.1076T>G c.3977G>A c.133C>T c.4033G>A c.5962G>A c.4836delC c.58G>A c.686G>A c.602C>T c.587G>A c.3868C>G c.1585G>A c.1708A>G c.1060T>C c.568C>T c.238T>C c.125C>T c.1630G>T c.1154delC c.1926delT c.620G>A c.533G>A c.1926delT ttp://genetics.bwh.h r . htt p.Ser855* p.Leu182Pro p.His145Tyr Unknown p.Arg958Gln p.Pro561Ser p.Arg1882Gln p.Ile1021Tyrfs*16 p.Phe928Cys p.Lys905Asn p.Met136Ile p.Asn1485Asp p.Phe403Leufs*12 p.Asn359Thr p.Ala1326Val p.Asp45Asn p.Pro1345Ser p.Arg1988Trp p.Ile1613Phefs*5 p.Pro20Ser p.Arg229Gln p.Arg201His p.Ala196Val p.Leu1290Val p.Arg506Cys p.Thr570Ala p.Cys354Arg p.Arg190Trp p.Ser80Pro p.Ser42Phe p.Gly544Cys p.Met387Tyrfs*17 p.Leu642Argfs*16 p.Gly207Glu p.Arg178Gln p.Leu642Argfs*16 p://sift.bii.a-star. Protein change 3 1 ). arvard.edu/pph2 c Known pathogenicvariant http://picar b b c e b edu.sg Infantile spasms EOEE EE EOEE LGS EE EE LGS EE EE EOEE evolvingto Dravet Dravet Dravet (ICEGTC) Epilepsy- aphasia Epilepsy aphasia,FS+ EOEE Epilepsy aphasia,FS+ Dravet EE EME EOEE, IS EOEE EE EE (featuressuggestiveof EE Ohtahara syndrome EOEE EE EOEE LGS Ohtahara syndrome EOEE EE EOEE Infantile spasms http: infantile spasms Angelman syndrome) d.sourceforge.net / s r e t t e l / //bio-bwa.source . ; SIFT (Sorting (Sorting SIFT ; Diagnosis 3 0 .
2 9
online online / ;
© 2013 Nature America, Inc. All rights reserved. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial Illumina HiSeq sequencing. A. Khan performed segregation analysis experiments. M.O.D. and M.W. performed S.B.H., J.M.M., S.F.B. and I.E.S. reviewed the critically manuscript. G.L.C. and D.G.,R.S.M., D.M.A., J.L.F., L.G.S., S.F.B. and I.E.S. performed phenotypic analysis. A.B., K.B.H., S.K., M.T.M., V.R.-C., R.W., A. Korczyn, Z.A., N.Z., T.L.-S., D.L., and analysis pipeline. S.B.H., S.C.Y., J.M.M., S.C., S.M., G.W., T.S., A.M.E.B., data. B.J.O. and J.S. developed the molecular inversion probe (MIP) methodology variant calling pipeline (with assistance from J.C.) and analyzed the sequence the and study. I.E.S. supervised G.L.C. constructed libraries, developed the G.L.C., H.C.M. and I.E.S. the designed study and wrote the manuscript. H.C.M. project Zealand grant to L.G.S. practitioner fellowship 1006110 to I.E.S.) and a Health Research Council of New Medical Research Council of Australia (program grant 628952 to S.F.B. and I.E.S., Award for Medical Scientists. This work was supported by the National Health and and(NINDS) 1R01NS069605) is a recipient of a Burroughs Wellcome Fund Career Institutes of Health (NIH; National Institute of Neurological Disorders and Stroke participating in our research. H.C.M. is supported by a grant from the US National We thank the individuals with epileptic encephalopathies and their families for Note: Supplementary information is available in the s r e t t e l COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING AUTHOR CONTRIBUTIONS A cknowledgments
oaon, . Knso, .. hoai rmdln b te H7 rti is protein CHD7 the remodelers. by remodeling chromatin Chromatin R.E. of Kingston, & family K. Bouazoune, Chd The A.N. Imbalzano, & C.G. Marfella, Dhamija, R. L.P.Capelli, E.L. Heinzen, H.C. Mefford, diagnostic genetic A R. Sachdev, & J.A. Lawson, M., Cardamone, B.A., Kamien, A.T.Berg, Sci. USA Sci. impaired by mutations that cause human developmental disorders. Res. Mutat. epilepsy. generalized deficiency. mental and (2010). syndromes. epilepsy sporadic of spectrum encephalopathies. (2012). encephalopathies. epileptic infantile to approach Terminology, and Classification on Commission ILAE 2005–2009. the of report epilepsies:
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© 2013 Nature America, Inc. All rights reserved. QUAL< 30, QD < 5, coverage < 25× and clustered variants (window size of 10). the following criteria were excluded from further analysis: allele balance > 0.70, to adhere not Variants did that URLs). Toolkit (GATK; see Analysis Genome as performed was URLs) described see (BWA; Aligner Burrows-Wheeler with ping calling. variant and analysis Data genes. 2) (batch 35 and 1) (batch 30 of total a comprising batches, 2 in sequenced and prepared were Libraries platform. HiSeq the on to protocol paired-end 101 a subjected using were sequencing parallel libraries massively Pooled primer. reverse tagged the on barcodes was 8-base unique PCR of introduction proband. the with each primers, from universal using DNA of performed ng 100 from exons target capture elsewhere described is ( build) methodology hg19 (RefSeq, genes target of to capture all exon and intron-exon (and boundaries 5-bp sequences) flanking Target and capture sequencing. approach. our validate further to gene disease-related candidate ant Wecalling. 24 included also probands with a known variant in a known or ing were included in all and analyses were used to assess the of sensitivity vari Controls. selection). gene Note Supplementary data unpublished or reports case cohorts, published from either CNVs, epilepsy-associated from primarily genes, candidate 33 selected also have mutations that are an cause established of Weencephalopathies. epileptic MBD5 cause one or more epileptic encephalopathy syndromes ( Fig. 1 selection. Gene ( pathogenic CNVs, either in the research setting ( covery cohort. Furthermore, 369 of 500 cases had previously been screened for as mutation-positive controls but were not included in the 500 cases in the dis were mutation from excluded this study. with known Some mutations subjects were included disease-causing identified previously a with Subjects ment. involve known with genes all for screened been had none encephalopathy; in involved genes epilepsy-related for specific screening mutation undergone on Classification Commission Epilepsy Against League International the of Organization the to were according syndromes classified Epilepsy studies. (MRI) imaging resonance magnetic and tories were with obtained together the results of including investigations, EEG with activity associated epileptiform ongoing regression frequent, or slowing cognitive and seizures refractory by defined was encephalopathy epileptic An phenotypes. encephalopathy leptic epi of range diverse a with interna individuals 500 of and consisted cohort The Australia tionally. around from research genetics epilepsy for referral by and investigators the of practices the and Health Austin at clinic epilepsy the from consent informed obtaining after recruited were encephalopathies epileptic with Probands Washington. of University the and Health Austin of Subjects. ONLINE doi: neurodevelopmental disorder with epilepsy as a comorbid feature ( feature comorbid a as epilepsy with disorder neurodevelopmental KCNQ3 in individuals with non-epileptic encephalopathy forms of epilepsy ( The first set included 13 epilepsy-associated genes, more encephalopathies. commonly implicatedepileptic for genes candidate of sets two selected Wealso n = 112). Probands with pathogenic CNVs were not included in this study. this in included not were CNVs pathogenic with Probands 112). = 10.1038/ng.2646 ). The known gene group included genes in which mutations are known to , , , , MEF2C This study was approved by the Human Research Ethics Committees GABRD Twelve samples that had previously been subject to exome sequenc 1
1 MET . SNV and indel calling and filtering were performed using the the using performed were filtering and calling indel and SNV . , , We selected 65 genes for sequence analysis ( analysis sequence for genes 65 We selected H SYNGAP1 , , GABRG2 O D and and S Supplementary Table 2 Table Supplementary , , , , SYN1 PRICKLE1 We (MIPs) probes inversion molecular used and and a ra dt poesn ad map and processing data read Raw 1 1 1 1 . Briefly, pooled MIPs were used to to used were MIPs pooled Briefly, . ( ATP1A2 , , upeetr Tbe 3 Table Supplementary . Detailed epilepsy and medical his medical and epilepsy Detailed . Table CACNB4 1 n ). Some subjects had already ). Some subjects ) = 257) 35– and and for details on candidate candidate on details for 3 9 . None of these genes genes these of None . n 3 = 19) SCN1B or by clinical testing Supplementary Supplementary 2 , 21 ) or a related related a or ) 3 , , 25 4 ). Detailed Detailed ). , 4 CHRNA7 , 27 0 GRIN2B (see the the (see , 28 , 32– 3 4 ------. , ,
40. 39. 38. 37. 36. 35. 34. 33. 32. (iii) be inherited from a parent with somatic mosaicism or (iv) adhere to a to applicable where of mode inheritance, or adhere parent-of-origin X-linked recessive, (iv) or mosaicism somatic with parent a from inherited be (iii) arise (i) to had variants Pathogenic ant to meet one of the following criteria to constitute a new pathogenic variant. we the vari required be could performed, analysis where segregation variants Criteria for of pathogenicity rare variants. parents. in mosaicism somatic detect to and depth high at very probands and family members. This approach allowed us to variants sequence and interest of variant performed target-enrichment rare PCR and sequencing the as described for all relevant harboring sequence genomic the captured per was analysis This a using MIP-pick formed strategy. We only MIPs the genes. that and pooled selected target 65 all for variants frameshift) and site in present (not rare all splice- essential (nonsynonymous, for damaging and possibly controls) ESP6500 out carried was analysis segregation available, analysis. segregation variant Rare gene. each for at >25× covered bases of samples) all for each sample with a threshold of 25×, as well as the mean percentage (across of depth GATK coverage overall of to tool was Depth used calculate Coverage nonsynonymous, splice-site and frameshift variants were assessed further. The of Only in controls <1%. a with frequency variants we considered candidates, recessive For set. sample control this in present not variants only considered confirm maternity and paternity. and maternity confirm a with probands of parents all in (Promega) system S5 DNA was available. We microsatellite analysis using performed the PowerPlex by supported the lack tion of was further the variant in the parent from whom incidence of pathogenic missense mutations in these genes, and the interpreta and ( genes known in mutations missense three Additionally, changes). prob and stop-loss ably frameshift disease, nonsense, lead to (splice-site, protein that truncation variants causes those only also pathogenic variants likely as here these report we of although subset a that probable is It parents. both or one from DNA of unavailability the to owing variant rare a for nism ( Variants were annotated with SeattleSeq (see URLs), and the Exome Exome the (or dominant and For population. control the in URLs), frequency (see variant assess to used was URLs) SeattleSeq (see set data (ESP6500) Project Sequencing with annotated were Variants Supplementary Fig. 1 Fig. Supplementary In certain instances, we were unable to determine the inheritance mecha inheritance the determine to unable were we instances, certain In
efr, H.C. Mefford, Zweier, M. M.E. Talkowski, Hoppman-Chaney,Wain,N., Seger,K., P., Identification J. Hodge, & D. Superneau, S. Endele, H. Tao, from encephalopathies: V.epileptic Leuzzi, early-onset & of M. Genes Mastrangelo, E.J. Marco, S.E. Heron, e1000962 (2010). e1000962 epilepsies. focal and generalized idiopathic in loci susceptibility CDKL5 diminish and retardation mental severe of cause frequent a are region disorder. spectrum MBD5 phenotype. syndrome that microdeletion 15q13.3 evidence further 15q13.3: at deletions gene single of phenotypes. 42 neurodevelopmental variable cause receptors NMDA of humans. phenotype. to genotype hyperarousal. sensory 2. Jul 2009 Epub (2009) and bcr06.2009.1999 retardation mental linked Genet. Nat. KCNT1 SCN2A , 1021–1026 (2010). 1021–1026 , s snl cua lcs f nelcul iaiiy eies, n autism and epilepsy, disability, intellectual of locus causal single a as expression. as svr atsml oiat otra fotl oe epilepsy. lobe frontal nocturnal dominant autosomal severe cause t al. et ) were interpreted to be likely pathogenic on the basis of the high high of the basis on the pathogenic likely be to interpreted ) were Am. J. Hum. Genet. Hum. J. Am. et al. et al. et
et al. et 44 t al. et Mutations in prickle orthologs cause seizures in flies, mice, and mice, flies, in seizures cause orthologs prickle in Mutations , 1188–1190 (2012). 1188–1190 , t al. et Mutations in Mutations in Mutations t al. et Missense mutations in the sodium-gated potassium channel gene channel potassium sodium-gated the in mutations Missense
ARHGEF9 Am. J. Hum. Genet. Hum. J. Am. Hum. Mutat. Hum. eoewd cp nme vrain n plpy novel epilepsy: in variation number copy Genome-wide seset f q31 irdlto snrm implicates syndrome microdeletion 2q23.1 of Assessment ). Pediatr. Neurol. Pediatr. irpin n fml ptet s soitd ih X with associated is patient female a in disruption
MEF2C 88 GRIN2A
, 138–149 (2011). 138–149 , 31 , 722–733 (2010). 722–733 , Where DNA from family members was was members family from DNA Where de novo de from the 5q14.3q15 microdeletion syndrome and
89 46 , 551–563 (2011). 551–563 , , 24–31 (2012). 24–31 , GRIN2B For those rare, possibly damaging , (ii) segregate with the disorder, disorder, the with segregate (ii) , Clin. Genet. Clin. encoding regulatory subunits regulatory encoding M Cs Rep. Case BMJ
83 Nature Ge Nature de novo de de novo de CHRNA7 , 345–351 (2013). 345–351 , LS Genet. PLoS ) models, we we models, ) mutation to to mutation ass the causes MECP2 a. Genet. Nat. STXBP1 n etics 2009 and
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