Expanding the genetic and phenotypic relevance of KCNB1 variants in developmental and epileptic encephalopathies: 27 new patients and overview of the literature Claire Bar, Giulia Barcia, Mélanie Jennesson, Gwenaël Le Guyader, Amy Schneider, Cyril Mignot, Gaetan Lesca, Delphine Breuillard, Martino Montomoli, Boris Keren, et al.

To cite this version:

Claire Bar, Giulia Barcia, Mélanie Jennesson, Gwenaël Le Guyader, Amy Schneider, et al.. Ex- panding the genetic and phenotypic relevance of KCNB1 variants in developmental and epileptic encephalopathies: 27 new patients and overview of the literature. Human , Wiley, 2020, 41 (1), pp.69-80. ￿10.1002/humu.23915￿. ￿hal-02302579￿

HAL Id: hal-02302579 https://hal.archives-ouvertes.fr/hal-02302579 Submitted on 28 Nov 2019

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Article 1163, ImagineFrance Institute, Paris, Necker Mefford Gu Genevieve Poisson Montomoli Schneider Claire Bar genetic the Expanding 0000 iD: Bar ORCID Claire doi: 10.1002/humu.23915. as this article cite which may todifferences lead between thi through been copyediting,not pagination typesetting, the and proofreadingprocess, article acceptedThis been forhas publicationand undergonepeer has full review but 6 5 Paris,France -Hôpitauxde Publique 4 3 2 1 Affiliations: Kabashi Edor G. Lynette Alexandra Afenjar Nica variants new new Service de Cedex de BP Service 577,86021 Poitiers Génétique, CHU Poitiers, de Department of Pediatrics, American Memorial Hospital, Reims, France UniversitéDescartes Paris Reference for centre , rare Neurology, Hôpital of Pediatric Department Re Laboratory of Translational Service de Génétique, Groupe Hospitalier Necker Enfants Malades, Assistance ët 34 27 patients , , - 18 Gaetano Terrone Enfants Malades, Paris, France 31 Emmanuel Scalais , Sophie Dupont , Sophie , Katherine 8

1,2,3 23,24 , 13 in in Sa , Cyril Mignot Cyril 2,3 , Giulia Barcia , Anne De Saint Martin dleir developmental Boris Keren , Rima Nabbout

and overview of the literature 40 15 , , Isabelle Marey Arnold Munnich B. B. 35 Howell , 28 9,10 10 Tayeb SekharaTayeb -Sorbonne Paris Cité, -Sorbonne Paris Imagine Paris, Institute, France , Nathalie Dorison 2,3,4 , Diane Doum Diane , 9,19

-0003-1489-0211 -0003-1489-0211 , and phenotypic and

1,2,3 , Patrick , Mélanie Jennesson Gae 32,3 search and epileptic encephalopathies

3 tan Lesca , 25 Carla Marini 3,4 10 , Salima El Chehadeh , f , or Neurological Dis Neurological or s versions of Please the and Record. Version Berquin 36 Renzo Guerrini Marion Gerard , Anne- , m ar 11,12 29 14 , ,

, relevance of of relevance Candace 13 Sophie LebreSophie Thierry Billette 5 20 , Delphine Breuillard , Gwena , , Jeremy

Pierre Meyer 13 16 Igi E. Ingrid , , 26 T. T. orders, orders, , Jamel Chelly ë Hervé Isnard L. L. l LeGuyader l Myer 37 Freeman , Sylvie Odent

KCNB1 de Villemeur INSERM UMR UMR INSERM s 30 Scheffer C. , HeatherC. 21,2 1 32,33 , : 27 : 27 2 , David , David 17 26 6,7 Martino , Alice , Agn

, , Amy Amy , 8,32,41 Anca Anca 38, 14 39 è s , , ,

Accepted Article Montpellier, France Rec Disorders, Centre déficiences intellectuelles de causes AP rares, Congénitales duCervelet n°19, pathologies France Florence, Italy Neuroscience Department, A Meyer Children's Hospital, University of Florence, 11 10 9 of Heidelberg, Melbourne, Victoria, Australia 8 7 23 22 21 Jules Verne, Amiens France 20 France Paris, F-75013, Foix, 19 18 17 16 75012 Paris,France 15 14 13 Lyon Claude Bernard 5292, Université 1,69675BronCedex,France. 12 «Déficience de ReferenceCentre duMoelle et la épinièrede Cerveau Institut UMR S 1127, Poitiers, Pôle Biologie Santé développement, CHU Montpellier Rares et Médecine Personnalisée, Centre de référence maladies rares anomalies du - EA3808 INSERM, UUniversités 1127,CNRSSorbonne UMR 7225, Research Centre, Department ofMedicine, Austin Health, The University Département de Neuropédiatrie, CHU Montpellier Montpellier, de 28rue république 69002Lyonla Clinical genetics, CHU Côte de Nacre, Caen, France Lyon, deCivils Service Génétique, Hospices 69002Lyon, France

Service de génétique clinique du et Département de Génétique Médicale, Maladies GénoPsy, Reference Center for Diagnosis and Management Psychiatric ofGenetic APHP, Hôpital Pitié Centre de Recherche en Neurosciences de Lyon, INSERM U1028, UMR CNRS Dé Epileptology Unit and Rehabilitation Unit Epileptology and Unit Unit Rehabilitation AP Service de Neurologie Pédiatrie Department of Pediatric Neurology,Department of Pediatric AP Ped PhyMedExp, U1046 INSERM, UMR9214 CNRS, Université CNRS,PhyMedExp, UMR9214 Université Montpellier,de INSERM, U1046 herche Scientifique herche Bernard LyonUniversity) 1Claude & partement de génétique et génétique embryologiemédicale de partement

iatric Neurobiology and Neurology, Neurogenetics and Unit Laboratories, Intellectuelle et Autisme», Paris, France. NEUVACOD Unité neurovasculaire et

Hospitalier le Vinatier and EDR

- Déficience Salpêtrière

, , CHU Amiens , CHU Département Intellectuelle de Causes Rares; GRC UPMC - - LeucoDystrophies, Centre de Référence HP, Hôpital Armand Trousseau, Paris, Armand Paris, Hôpital HP, Trousseau, - HP, Hôpital Armand Trousseau, F ArmandHP, Trousseau, Hôpital de Génétique de - - HP, Pitie GH - Psy Team (Centre National de la

Picardie, Université de Picardie

troubles de cognitifs, Université

, , ICM, Paris, France Sorbonne Université, GRCSorbonne Université, , UPMC Uni UPMC , et deet Cytogénétique , France - Salpêtrière v Paris 06 v Paris -

Charles Charles - ; Accepted Article Reference centre for rare epilepsies Rima Nabbout Address correspondence to Australia Neurology FedericoUniversity,Unit, 80131 II ItalyNaples, Hospital University Victoria, of Melbourne, Australia Melbourne, Seattle, WA. 30 29 28 27 Hautepierre, Strasbourg 26 France 25 24 41 Zealand 40 Renn 39 France 38 Reims, F 37 36 35 34 33 32 31 Neurology, Department ofC.H.I.R.E.C, Belgium Brussels, Pediatric Murdoch Children's Research Institute, Melbourne, Victoria, Australia UniversityWashington,WA. Departmentof ofSeattle, Pediatrics, Pediatric FoundationParis Rothschild Hospital, Neurosurgery, Université Unité MontpellierInserm et Montpellier, France U1183, Pediatric Neurology Unit, Centre Hospitalier de Luxembo de Hospitalier Centre Unit, Pediatric Neurology Service de pédiatrie Department of and Paediatrics Health, Child Uni Department of Division Pediatrics, of ofUniversity Genetic Medicine, Washington, Reference centre for rare developmental abnormalities CLAD CNRS de 6290Institut Développement UMR Génétiqueet Rennes de IGDR, Univ Service de génétique médicale, Hôpitaux Universitaires de Strasbourg, Hôpital de Hôpital Maison Blanche, Pôle de Biologie, Service de Génétique, Service Pôle Biologie, Blanche, de Maison Hôpital CHU Reims, Depart The Florey Institute ofNeurosciences and Mental Health, Heidelberg, Victoria, Hospital, Neurology,University Strasbourg, Strasbourg Department of Pediatric Department of Translational Medical Sciences, Section of Pediatrics Neurology Department, Center for Clinical Research (CIC 1414), Rennes es, Rennes, France

-51092, France -51092,

ments ofments Neurology Paediatrics, and Royal Children's University Hospital,

, Hôpital Colombes, Louis-Mourier, France

:

ersity ofversity Wellington,New Otago, urg, Luxembourg urg, - Ouest, CHU Rennes, CHURennes, Ouest,

- Child Child Accepted Article on the voltage sensor and the pore domain and voltagepore ofon the sensor K delayed of devastatingneurodevelopmental Variantsdisorders. Developmental and epileptic encephalopathies Abstract agreement no.602531,RG). DESIRE the FrameworkProgrammeproject Commission under (grant Seventh the National Health and Research Medical Council of Australia(IES), the European Curekids New Zealand and theHealth Research Council of New Zealand (LS, IES) , BettencourtFondation Schueller (CB ERC RN), and the grant consolidator the(EK), Recherche under “Investissements d’avenir” program (ANR Agence byStatefunding Nationale from de the workwassupported This la InformationFunding Necker Enfants Malades Hospital Department of Pediatric Neurology to associated with severe aless epileptic phenotype. Overall, this report provides an up variants Truncating prognosis. and syndromes - impairment. Eighty neurodevelopmental disorders with difficulties predominant language and behavioral variant (p. occur patients. Most variants theexpand mutational spectrum describing 18 novel variants reported patientspreviously carrying 29distinctKCNB1 reported in patients with early Email: [email protected] 144381536 +33 Phone: Sèvres, de 75015France149 rue Paris, - date review ofmutational the and clinical spectrum of - rectifier voltage

Arg 583*). 583*). five percent of patients developed epilepsies with variable KCNB1 - dependent K - de novode related encephalopathies - onset DEE. KCNB1

and mainly consistof missense variants (DEE) refer to a heterogeneous group group heterogeneous a to (DEE) refer v . We2.1. also report the first inherited

in the C the in encodes the alpha of the thesubunit encodes encompass awide spectrumof in in KCNB1

KCNB1 variants v 2.1. We the 2.1. 37 review -10- - terminal d

from 27unreported

- IAHU have been recently , strengthening its strengthening , and significantly 01) and the the and 01) omain omain located located are are - Accepted Article and report 18 novel variants. variants. 18novel and report review of the mutational and clinical an up- provide we update, mutation this In Kv2.1. channel potassium - the delayed of subunit alpha the encodes KCNB1 Graphical Abstract unravel underlyingto studies the mechanisms. place as a causal inDEEs and emphasizing theneed for further function channel E and “Developmental behavioral and language motor, cognitive, persist neurodevelopmental of group encephalopathies constituteDevelopmental KCNB1 Keywords developmental impairmentdevelopmental associated epileptic activity have highlighted DNA methods have sequencing 1. ing ing BACKGROUND , developmental and epileptic encephalopathy, epileptic epilepsy, and potassium, developmental channel the pathogenesis of the in s throughout lifethroughout

pile .

The clinical spectrum and regression ptic Encephalopathy” (seizures

DEE disorders s s (Wang 2017) al., et and and spectrum of the KCNB1 encephalopathy encephalopathy the KCNB1 of spectrum

(Scheffer et al., 2017)(Scheffer al., et diagnosed during childhood early and

the important role of EEG abnormalities) that contribute a broad and genetically heterogeneous includes includes

impairments (DEE) rectifier voltage .

variable degrees of social, Ionchannelscrucial are in refer . s s .

ion ion encoding Recent advances in The concept of The concept to the frequent theto - dependent dependent to - date date s s to to ly al al

Accepted Article the generationthe modulation and of excitability in nervous the system Samanta, 2017; 2018) al., al., 2017;etet al., Marini 2017; Miao 2017, et al., Parrini 2018; et al., Zhu et2017; al., Allen 2016;2016, 2015; et Kovel de al., Calhoun al.,2017; 2017; et Latypova al., al.,Thiffaultet 2015; et 2015; al., Saitsu et 2014; al., Fitzgerald et Torkamani delay and/or developmental next- new 29 2014), 2014) al., et variants onset channel subfamily B member 1( 2014, In 2017; al., 2019).et Wangal., Lehman 2017; et al., et Soldovieri 2014; et al., Pena Tabarkiet2014; Coimbra,2015; al.,and Gururaj 2016; al., 2012;Weckhuysen et Veeramah al., 2013; et Ambrosini et al., 2014; Lee al., et KCNQ5, (eg, ( spectrum of “C 2017). 2017). the in mightgenes result since KCNT1 hannelopathies” are generation- high CA, KCND2 KCNA2, one geneone often is DEE In particular, g ) p.Ser347Arg, p.Thr374Ile

, early Torkamani et al. identif KCNT2, KCNT2, . .

This Following the initialFollowing DEE

- KCNB1 onset study provided provided study KCNV2 icuig epilepsy including s DEE , sequencing throughput ene

associa variants associated CD, KCNH5 KCND3, same ) ) . - s with suppression related p (Jorge (Jorge et al., 2011; Sicca et al., Barcia2011; et al., 2012; The m The

DEE ted with epilepsy phenotype epilepsy have been reported 37patients in ied identif and p.Gly379Arg and KCNB1 ajority of these

the initial evidence of evidence the initial with

otassium channel dysfunction cause dysfunction s otassium channel (Soden et al., 2014; Srivastava et al., 2014; 2014; 2014; al., al., Srivastava et et (Soden novo de

ication of KCNB1 a different phenotypes and variants phenotypesand different of with infancy seizures focal migrating ) in in ) wide , KCNJ2, KCNJ10,KCNMA1,KCNQ3 variants - burst (KCNQ2 3

phenotypic and genotypic spectrum genotypic and phenotypic sporadic in cohorts of individuals with with of cohorts in individuals

(McTague et al., 2016; Wei al., et 2016; al., (McTague et patients on KCNB1 (Torkamani function in in the the

patients affectedby

variants , epilepsy intellectual had the deleterious effectof potassium voltage ) and and )

(Wei et al., 2017) et (Wei nonspecific DEE

(Torkamani et al., detected in several a clinical through through

-gated early s s - . . , ,

Accepted Article these region that modulate region 27 new of unrelateddescription patients carrying 1 KCNB1 to mutation update, we aim this In 26).infantile, disability familiesof channel K addition, -P- domain(S5 pore transmembranehelices (S1 surrounding subunits alpha al., 2014)et soma, axonal and proximal dendrites segment initial of neurons (Trimmer, King 1991; expressedare acrossthe , especially clustersin large on the subunit of the voltage alpha forming the 858 aminoacids 96of a is kDa core KCNB1 protein full a has and KCNB1 K TVGYG aminoacids motif located in the re openingchannel and The closing. filterselectivity of the formed pore is bythe charged amino acidsthat senses the change in themembrane potential leading to v 8 ( 2. KCNV variants STRUCTURE OF K OF STRUCTURE

(MIM# 600397) (MIM# variants

and v . ) 2.1 voltage Like other eairl problems behavioral n K and variant we. Furthermore, expandvariant the - length ofcontaining 2exons. length transcript 11.879kb(NM_004975) and and - forming such as subunits, the silent alpha subunits K v S6). Thevoltage s s 9 ( homotetramerization as well as heterotetramerization with other - discuss gated potassium chann KCNS

- voltage- is a potassium channel gene located on 20q13.3 located potassium a gene onchromosome 20q13.3 is channel gated potassium channels have a N - S6) thatS6) voltage a include v the the .1 CHANNEL 2.1 ) the evidence for evidence pathophysiologicalthe relevance of the (Xu et al., 1995; Hugnot et al., 1996; Salinas et 1995; al., 1996; al., Hugnotet al., Salinas et (Xu gated potassium channels ion conduction Each pore. alpha subunithas 6

present an exhaustive re (MIM# - sensor S4 helix contains sensor S4 helix

- 616056; Epileptic encephalopathy, early entrant pore loop between S5 and S6. In loopS5S6. and In pore between entrant el subfamily 2 (K 8 novel variants8 novel - spectrum of of spectrum sensing domain- (S1 , of four four they are composed of view of patients carrying ofview patients

- terminal cytoplasmic a series of positively v 2.1). K . KCNB1 v 2.1 channels 2.1 channels v 6 ( S4) and a a and S4)

with the KCNG ), ),

Accepted Article of Medical Genetics (ACMG) Laboratory Practice Committee Working Group class 1) byWhole (WES) (n=16)or (n=1 disability Sequencing intellectual Exome - by targeted detected ( essential for membrane repolarization during high mediate adelayed channel termin proximal localization of K terminal al.,et Bocksteins Althoughfunctionalroleofintracellular1997; 2014). C the ( French We collected 27 new unrelated patients with KCNB1 pathogeni variant the novo confirmation of de Database HGMD, Biobase, Qiagen). crossed with KCNB1 the The database Pubmed used searchto was compositionet 2002). al.,Sano 2002; al., subunit (Ottschytsch et moreon channel ofare depending complex, the properties channels heterotetrameric dependent activation gating,and Belgium, Italy, Luxembourg,Belgium, New Italy, Zealand Richards 2015; Richards et al., 3. terms “KCNB1” “variants” and or“”. ified variants according to the variants accordingified the to international College guidelines of American the al IDENTIFICATION OF KCNB1 OF IDENTIFICATION

nentoa collaboration reference network for rare epilepsies and international (Ju et al., 2003; al., al., Mohapatra(Ju 2008) et et domain understood, yet fully not is it mediates the restricted clustered and domain to regulate intracellular domaintrafficking, voltage surface regulate expression, to - rectifier voltage city

Next Generationfor either Sequencingepilepsy (NGS) panels or variants listed in professional database inheri (Zhu et al., et (Zhu 2017). Supp. Table S1) Supp. Table v 2.1 channel(Lim 2000) al., et tance - and and dependent outward potassium current which is is outwardwhich potassiumdependent current

one patient patient We one excluded insufficient VARIANTS . Single nucleotide variants (SNVs) were KCNB1

and Australia) - dependent modulation of dependent modulation

. Homotetrameric K pathogenic variants by combining

clinical data

pathogenic variants t pathogenic - A frequency firing. Functional rticles were reviewed and

and interacts with the N s . (Human Gene Mutation (Human Mutation Gene KCNB1 available without Sanger

variants werevariants v 2.1 channels channels 2.1 hrough to validate the K the v We We 2.1 the s s - - -

Accepted Article (http://varnomen.hgvs.org/ (https://www.ncbi.nlm.nih.gov/clinvar/ RefSeq variants were described according toHG variants family. sequencingconfirmed Sanger and by segregation analysis was completed ineach order to confirm the Segregation the in Study epilepsy. Arg (p. variants novel 18 I 2017, al., al., Parrini 2017; Miao 2018; et et 2017; Samanta, 2018). al., et Marini 2017; et al., Latypova 2017; et Calhoun al., al., 2016,2017; et Kovel de 2016; et al., al.,Allen 2015; et et al., Thiffault et2015; Saitsu 2015; Fitzgerald al., literature We research participation gaveguardians for written procedures informeddiagnosis consent genetic data when available. electroencephalogram imaging (EEG)and epilepsy behavioral, neurological, and including developmental,patients, n our cohortn our 4. identified identified 8* i patient 64 in 583*)

VARIANT SPECTRUM VARIANT arising , proband’s mother In silico re NM_ 004975.2.NM_ variants (Table 1). All patients carried heterozygousmissense or truncating (Soden al., et Srivastava et 2014; al., Torkamani 2014; 2014; et al., ported in ourported in 27new could not be completed be not could

of 27 unrelated patients unrelated of 27 29 pathogenic variants from 37 unrelated patients reported in in variants from29 pathogenic reported unrelated patients 37 of of predictions predictions de novode the the ir according according thecommitteeto ethics of each institution. maternal grandparents showed thatthe novo de and his his and ; Dunnen; 2016), et al., using referencethe sequence

4/27 24/27 in Variant who carried the at heterozygousvariation state. the occurrence supporting evidence of pathogenicity for of evidence supporting pathogenicity

patients s s mother affected by intellectual disability without , patients in both parents both in we found have been submitted to ). ). C ( linical and EEG data were obtainedfor all patients 51 are detailed in Supp. detailed in S2. KCNB1 are Table . VS VS We report the first 25 distinct25 KCNB1 variant

for for , 58) the remaining 2 , but were both considered nomenclature guidelines nomenclature variant variant All parents or legal

ClinVar database variants variants occurred occurred inherited variant

variants variants including missense de novo history,

and and the the in in

Accepted Article variants 6 in identified variants pathogenic includingOverall, data and our those of liter the 2016; al., twopatient in pathogenic likely as reported 50 patient in pathogenic. 53,54).and p.Gly401Arg p.Gly395Arg and p.Phe416Leu The 2017) al., p.Val349Phe two patients (p.Ser347Arg, protein patient different ( affectedvariants potentialvariant p.Val378Leu),a representing p.Val378Phe; variants had 381) 377to segments acid (amino the protein Most were recurrent. nonsense 8 Patients

variants (4 (

(S5S6). to Patient 16 Patient except one were except one ) and the p.Arg312His p.Arg312His the and 15) 12 to in change at this p.Arg312Cys position, . ID . variants Three recurrent variants

The The variants The K+ selectivity filter located between theS5 and S6 transmembrane

-2010D05815). -2010D05815). our series. Three were in this series.this in variant

2/4 p.Gly395Arg the voltage sensor domain. ). ). variant 7, 89%) were Two were in the in were Two /4 (8 Six recurrent variants found patient in in in 7; 1 The patients p.Val349Phe, patients 27; 26 and was found in 3patients in was found exon 2. exon 7%) and 2 variant p.Phe416Leu variant variant p.Phe416Leu variants

located 4 They includ were located in theS6 transmembrane segment

S5 transmembrane unrelated patients (Table 1 and Fig

in 29 8 distinct missense variants, including 3 novel includingmissense 3novel variants, 8 distinct at amino acid position 378 (p.Val378Ala; 378(p.Val378Ala; amino position at acid

w was patient 51patient frameshift

resulted from s in the S1 ere s ( s The p.Arg306Cys The were were found in 5 patients ( 5patients in found Allen et al., 2016; patient 8; de Kovel et Kovel de 8; patient 2016; al., Allen et found 2patients in ed ature, localized in the pore domain of the domain inpore the of the localized 37

was identified to to variants variants in ( missense variantsmissense S6 transmembrane segments of we

Patients while 58) 56to the patient

omatic mosaicismomatic

segment reviewed /4 (2 , was , patients 28 The patients 29). and hotspot. T hotspot. was 58 ha 58

7 each Patients

, each one found in found in one each , ; as a as

found in 4patientsin found also %). 4%). data of 4 s been previously been ( variant variant novo de Patients 50,51 (3 found one in wo recurrent Ten Ten 17 to 21) 17 to 7/4 ure ure

(Marini et 7 7; variants distinct 1) . %), 9%), All . A . A .

Accepted Article provided functional functional Butler et al. provided subunit alpha5 novo de variant self determinant for subunit (p.Glu43Gly). region 1hadPatient a voltage- The studies. 2017) al., et Calhoun 2015; different cellular models et 2014;al.,Thiffault 2015; al., al., Saitsu et (Torkamani et To date, seven KCNB1 Thr (p.Ser363 (p.Val286Glyfs*58), truncating were variants (Patients The truncating. All phenotype. both Therefore, GABRA5 5. variants KCNB1 OF AND CHARACTERIZATION RELEVANCE FUNCTIONAL was thus sensing domain, channel reduces availability due to shift in the voltage variant in GABRA5 in variant

62 64; to variant

variants p.Ile199Phe p.Ile199Phe located w fs*13). in the in of of S VARIANT non- KCNB1 predicted d be to (p.Val294Leu) the the variants were

pore helix between pore helix inherited from the motherthe from inherited gamma es variant sense ithin S4 to S5S4 to linker (p.Tyr328 *)

missense variants missense The The

variant in the in variant the the were considered to be to contributing -

. Distinct functional effects have been described among and and association intotetrameric channels - gene# (MIM aminobutyric acid type acid aminobutyric highly conserved N- conserved highly linkers:

intracellular C in the 1, exon , located in the S1 transmembrane segment of the segment the the of in S1 transmembrane , located amaging in

evidence of evidence a patient with patient severea p.Arg583* S5 S5 S1 to S2 linker S2S1 to (p.Gln228 S4 S3linker linker to *), (Xu et al., 1995) et (Xu have been functionally characterized using 137142, p.Thr301Met), which encodes the whichthe 137142, encodes p.Thr301Met), and and - *) p.Trp370 *; (p.Trp369 S6 in in termin the the affecting was Patient 64 terminal

- A (GABA pathogenic effect of found in 3 unrelated patients found 3unrelated in al domainal protein of the

the cytoplasmic N DEE . and S5and to S6 linker Th . T ). is patientis alsocarrie d domain

A (Butler al., 2018) et (Butler ) receptor ) wo te nonsense other the p.Glu43Gly is is the . T .

patient a critical Recently, a - 4 4 he last terminal nearby nearby were ’s a .

Accepted Article sufficiently deep interspike voltages (Saitsu e 2015) effectand onWTnegative (Saitsuchannels currents abolishes endogenous et al., while p.Gly401Argsensor the variant similar 2017) dependence of compared activation to the wild disorders associated with su K lacking cultured from delayed hippocampal is rectifierpotassiumdiminishedin Finally, neurons current gain rather should consideredloss-of-function than be they as T of pathophysiology the to contribute 2014) membrane repolarization leading to increased cellular excitability were predicted to result in depolarized restingmembrane potential and impaired these mutants alsoinduce reduced current density conductance of K affecting herefore, given the biophysical properties of these pathogenic variants on K pathogenic biophysicalof properties the these given herefore, sceptibility in response to proconvulsant drugs to response sceptibility in . Bothvariants Avariant. . Changes in channelexpression and localization to those in the the disruptsof those in WTbut sensitivityto cooperativity the and channel the pore domain result in loss ofselectivity ion and gain of inward cation v 2.1 have no spontaneous seizures but display increased seizure increased display seizure but seizures nospontaneous 2.1 have Kcnb1−/− v in in the sensordomain voltage currents S4, p.Arg306Cys, induces 2.1 channel inhibit repetitive neuronal firing by preventing the productioninhibit repetitivethe of firing bypreventing neuronal marked hyperactivity (Speca et al.,marked (Speca et hyperactivity 2014).

(K v s s 2.1-/- (Torkamani et al., 2014; Thiffault et al., (Torkamani al., Since 2015). Thiffaultet 2014; al., et ) mice 2014) et al., (Speca , located , located in the pore domainS6, has a dominant K v 2.1 pore variants 2.1 pore t al., 2015) al., t - (Calhoun et al., et (Calhoun channel (WT)type

at moreat they depolarized voltages, and exhibit exhibit and rangea of behavioral . Other w -of-function. (Thiffault et al., 2015) al., et (Thiffault ere . Interestingly, mice also suggestedto (Torkamani al., et KCNB1 variants variants v 2.1, 2.1, - . Accepted Article (Patients 16, 34,35, 55,59,64). We here report 6 new patients aged who from 23years 8to (median agestudy at 8 years, data available for n= 6 available for n= 6 The patients ( ambulation was verbal while theremaining were able to speak someor shortsentences. words Data on whom in expressive language severe disorder was found all in A data (n=62/64). available patients with all in delay wasreported Developmental 12 and in hyperactivity (24%). 20 (41%) available data (76%), including spasm 5- monthsmean 12(range of age seizurespatientsonly clonic (n=5).Nine (18%) and epileptic developed seizures (n=14), myoclonic (n=14), clonic seizures (n=28), focal seizures (n=24), epileptic e %) (70 patientsAll developmental had delay to prior onset. seizure 10days months (range: of 12 53/63Overall, patientsdeveloped (85%) epilepsy with mediana age at 6. cohort of 64 of cohort s s VARIANTS OF SPECTRUM PHENOTYPIC THE EXPANDING xhibited xhibited typesseveral seizure during follow (Pavone et al.,(Pavone 2014)

72 some %) %) achieved independent walking.

language datawas available. Twenty available for 4 patients included included patients 2/6 ) with pathogenic pathogenic with 4) - 5 years, meanmonths 5 years, 15 . 3 autism 21 months) Behavioral issues 1 of 61 out 34

atypical males spectrum disorder in 26(53%),spectrum aggressi disorder KCNB1 patients aged 2 years or older or 2 years aged patients , consistent with the syndrome of infantile absences (n= 13), atonic seizures (n=9) and 28females(sexand ratio M/F 1 Two patients

variant

2/6 occurred in - 42 patients aged 3or 42 patients aged years up, including generalizedup, including tonic - four patients (57%) were non 4). 4).

spasms tonicseizures (n=21), , data available for). n=52/53 s , aged from 1. did not develop not did (5%) 37/ Thirty 49 patients with 49 patients aged 11and 17 -s

even 2 to 33years 2 to seizure onset . Thirty spasms a at

KCNB1 seizures patients patients 2; data .2;

on in in on older older - one - -

Accepted Article Electroencephalographic (EEG) datawere available for 51 patients choreiform 15%) hyperlaxity(n=8,and movements15 (n=8, %). ataxia19%), extra (n=10, 21%), followed (n=11,2, 48%), mostfrequentlybyspasticity signwas the (n=25/5 reported months). median years walked with assistance heterotopias carbamazepine ( associated with focal seizures, multifocal spikes on EEG and patients 54). patients normal a 7 had computedin atrophywas reported tomography scan. (CT) Mild MRI onbrain Data spasms became- seizure infantile who developed only particular,ofpatients antiepileptic In 6out 9nine drugs. patients (72%)had photosensitivityin and 13patients in 4 wasfound patients.abnormalities Thirty Hypsarrhythmia(n=23). wasreported 8 in focalwaves spikesand multifocal (n=12) (n=30), of spikes and/or generalized or EEG s recording

Non was normal in 42 of the 42 in was normal

and documented and (patients age of wal specific Neurological examination datawere available for 5 (Patient 34). magnetic resonance imaging (MRI) w imagingmagnetic resonance (MRI) were characterized by slow P 37 atient atient periventricular pharmacoresistant king king and and free with treatment. 52) 52) as prog reported for 22/31 patients forwas 22/31 reported 4). 4). s s twoarea had One patient ().

Another patient patient Another patients carrying KCNB1 and 10 and ressive w pyramidal hite matter abnormalities epilepsy while 13 patients (26%) by serial MRIs in 3 of them ( patients (2

background activity,with a combination children symptoms had small bilateral periventricular 3%) were non as . Sleep activation of EEG . Sleep activation of EEG variants variants available for 5 including dystonia dystonia including

of of 24 months (range 18 months24 (range focal cortical focal

2

were reported in 2 2 in reported were patients. Hypotonia (78%). One patient -ambulatory. P with epilepsy atients 47,53,atients responded to to responded 4/6 responded responded 4 dysplasia patients. - eight eight The The and and -54 -54 to to . Accepted Article evolving into severe cognitive impairment and intractable focal and generalized a have variant pathogenic We reported the first localization disability Patients with KCNB1 most the phenotype to severe domains in these mutations delay a de and S4 tono epilepsy patientsmoderate the with S6in in domains language acquisition However,the test). variant with a 2017). a more severephenotype to sensorporewere correlate regionofprotein the thought domainthe voltage or with I hit mechanism damaging, double a et(Butler al., 2018) seizure patientthat earlier had reported receptor in this patient, located in the pore- seizures n a previousn a review of 26patients KCNB1 a with 7. (Patients 16,34, 35, 55) variant

Our Our - GENOTYPE in other regions ofprotein other in the (p.Thr301Met). DEE (Patient 1). Interestingly, a in a patient w , behavioral disorders and or the type type the or results with early developmental delay early seizurewith a and onset developmental at 16months, in the S4 to S6 crucial domains the S6in S4crucial to show that re re . or behavioral issues.

As variants both variants PHENOTYPE CORRELATION PHENOTYPE ith ith was no variant correlated variant of of of A severe epilepsy and global and epilepsy nearby pathogenic pathogenic nearby non-

have

. infcn difference significant This s finding developmental delay who delay developmental onset our patient our for may responsible be forming transmembranedomain M2 ofGABA the pathogenic ambulatory patients a frequent epilepsy. epilepsy. frequent wide phenotypic wide ( , , (n=0/10 KCNB1 autistic behavior andautistic behavior in the N the in In variant particular, 4new here we report with the phenotype the with does supporta not variant ) (n=10/31 GABRA5 developmental delay developmental and and 48, 0,048, p = - ter

variant GABRA5 minal patient a domainin

in terms of in in spectrum intellectual including

weremore frequent

GABRA5

We examined whether the variant (de Kovel et al.,(de 2017) et Kovel differ compare , missense variants in the unilateral ) were predicted to be . spastic quadriparesisspastic s s (p.Val294Leu) was clear

from our was epilepsy severity, epilepsy d ’s (de Kovel to those with a a with those to phenotype.

also identif Fisher's exact correlation of velopmental velopmental patient

in those those in

et al., .T who who i ed he he as as s

Accepted Article had a had change the at same position ( generalized seizures (Patient (Patient was associated with infantile spasm variant epilepsy et 2016) Puckerin al., functional onchannelpropertiesconsequences thus channel voltage- terminal truncation of K dominant- potential ain nonsense these truncating variants occur within the last exon, they are not anticipated to result hadepilepsy spasms pharmacoresponsiveinfantile 61). syndrome (Patients of seizures not (Patients 6did develop 59,60,63, both and 64) patients that developed While these Interestingly heterogeneity. studies localization proteinof missense variants onthe domains the more and experimental genotype no Therefore, impact traffickingof tetrameric K

developmental encephalopathy developmental . are understand to theare needed under - dependent gating phosphorylation and function For For ), 19), (Mohapatra et al., 2008; Jensen et al., (Mohapatra al., Jensen2017) et 2008; et al., phenotype phenotype instance, t , all 4 all , patients patients pharmaco - mediat (Aizawa et al., 2004; Duarri et al., 2015; Mezghrani et al., 2008; al., 2015;et 2004; al., Mezghrani al., Duarri et (Aizawa et variants negative effect on channel function (Khajavi et al., 2006) (Khajavi function effect onchannel negative was was ed mRNAdecay had . he he responsive responsive Other truncating variants v -

highly variable, as illustrated variable, highly in 2.1 channelhave been shown to expression, impact surface severe phenotype be correlations can established recurrent

found in the C 20). p.Arg312Cys developmental delay In addition, late

s s variant

(Patient 1 with intellectual disability behavioral and issues intellectual with -

onset focal 21) seizures (Patient but rather to produce a truncated protein with a v 2.1 channel to to cellthe membrane2.1 channel to leading - terminal part of thewere protein truncating. p.Arg312His p.Arg312His , patient 16) , patient lying lying a patient carry 7 and 18), 18), 7 and were pathophysiology ofpathophysiology thisphenotype demonstrated in few in demonstrated as , . Truncated K Truncated . also inthe and behavioral disorders behavioral and but but epilepsy develop not did dependent modulation of the dependent modulation of the in the voltage sensor the domainin voltage infantile p ing atients a a last exon of the gene exonlast ofgene the different -onset -onset v carrying the 2.1 channel

and based on the based onthe focal

amino

intractable Since all

seizures other , could

4 out same same - C- . acid . .

Accepted Article DEE helix, pore located but p.Arg583* inform parents about prognos though Even confirm pathogenic the phenotype. that with interact developmentfactors the could the mutation during shaping phenotype The mechanism 2017) al., et Calhoun 2015; dominant-negative domains. non a either speculate that they may alsoescape nonsense- delayed language skills language delayed All the KCNB1 K diagnosis to is important practice clinical in not developnot epilepsy behavioral disorder neurodevelopmental disorder with nolanguage acquisition, autismspectrum disorder and intrafamilialvariable This expressivity. that observation v 2.1 8.

(Jorge et al., 2011; Orhan et al., 2014; Smets et al., 2015; Masnada et al., 2017) 2015;al., al., Masnada et et al.,Smets 2011; 2014; et al., Orhan et (Jorge channel impairment CLINICAL AND DIAGNOSTIC RELEVANCE DIAGNOSTIC AND CLINICAL Indeed, m variant with noobviouswith phenotypic More e of

upstream theC al deleterious-function even or targeted therapy is not yet available for KCNB1 for available yet not is therapy targeted

KCNB1 variants severity from s . gicallectrophysiolo studies effects . Her 12- Her . ost ost We We , noreading

identified occurred de novo variants, asfor some

mechanism her her might be functionally studied missense variants displayed of range a thus report the first inherited KCNB1

, in addition other to (Torkamani et al., 2014; Saitsuet al., Thiffault 2015; et al., is affected mother. H mother. affected - that

and allow terminal domain,terminal either in extracellular loops year and might

-old related to of truncatingKCNB1

writing abilitieswriting but

daughter accurate geneticaccurate counselling. underlying pathogenic pathogenic main representthe underlying thus difference stop

the extent of mutation truncated protein missing other potassium channelopathies other

er

and animal models did not have (genetic or environmental)modulating unnecessary unnecessary

mediated mRNA decay and result mother except in one patient one in except compared could live independently. She did individuals harboring a KCNB1 had variants diagnostic

a severe severe a had but seizures intellectual disability to missense , genetic genetic , encephalopathies

variant, associated with with associated variant, .

- are are induced functional

who tests now

vital channelvital variants

, to to , inherited or or needed to correctly correctly - related related in the

. with

We We her in in .

Accepted Article out epilepsy with or with associated domain C-terminal the pore domain of the protein. Few truncating variants and mainly consist of the heterogenous phenotype neurodevelopmental outcome data fully fully genes syndrome difficulties disorders, including early-onset global developmental delay with In a In interpretation of inherited variants, prenatal diagnosis and genetic counselling. variants literature the of review exhaustive description of 18unreported pathogenic variants in 27 variant with patients of spectrum molecular and clinical the review We DEEs. further strengthening the importanceof KCNB1 AEDs disappears and from exome from withdrawal, DEEof social with variants deleterious two ddition, ddition, 9. will delineate the phenotyp of spectrum ic do not inform further is likely to be to another likely is modifier of the phenotype . et al.,(Marini 2017)

with CONCLUSIONS AND PERSPECTIVES AND CONCLUSIONS to

helpin genetic type and prognosis are

47. over

and p a and genomeand studies will enable us to better understand such double hit carrying atient carrying 1 disease severe a transmit can phenotype -severe” “less KCNB1

time behavioral impairment

with variants missense

encephalopathies encompass widea spectrum of neurodevelopmental in the same patient. patient. same the in

counselling. prolonged periods of remission and . Potentialmodulation coexisting by pathogenic variants In this study, we also reported 6new patients without epilepsy, .

The variety of reported func KCNB1 genotype no language

variable. variable. increasing the number of patients to patients of number the increasing

and In some patients, seizure frequency attenuates and and attenuates frequency seizure some patients, In with somehotspot pilepsy . Epilepsy phenotype correlations, especially in term in especially correlations, -phenotype

KCNB1 dentification of new patients is is patients new of Identification GABRA This

acquisition

30- in neurodevelopmental disorders, beyond

dysfunction. 5 is frequent year

variants a are reported with one hotspot in the . Beyond the variant the Beyond .

-old and mild s might effectstional located in the in located voltage sensor and an an and patients unrelated new

patient had a severe phenotype daily seizures. A seizures. daily suggests

epilepsy , the possibility of withdrawn of the possibility KCNB1 variants Most including the DEEs, is important for accurate accurate for important is predominant language

64 available available However, . the co the

variants and o and thus

-occurrence effects on ion ion on effects occur occur dditional data dditional data contribute f pathogenic important to important to through

genotypes de novo de in other s

but the the

of of of of to

Accepted Article their participation in this study. DISCLOSURE STATEMENT DISCLOSURE The authors thank the ACKNOWLEDGMENTS p neurodevelopmental disrupted of mechanisms pathophysiological theassociated accurately currents, new animalmodels reproducing 2012.De novo al. A, et P, Kaminska R, A,Nitschke Abhyankar Cilio J, Kronengold Barcia G, FlemingDeligniere MR, A, Gazula V autism dysfunctions QT3 of channels: syndromeinduced Kir2.1 implications for short and S, A, 2014. G,GuglielmiF, L, Lanciotti al. RuanPieroni Servillo Y, Genetically et Ambrosini F, E, MS, Sicca Brignone MC, C, D’Adamo Napolitano Servettini MoroI, 57:e12-17. andexpansion.Epilepsia encephalopathy: screening phenotype Exomeepileptic Magalhães TR, Ennis S, LynchSA, KingMD. 2016.Unexplained onsetearly D, RG, SDJ, Pena McCreary Lynch B, NM,ShahwanCorrea ConroyJ, A,Allen dominant- - A178fs/105,mutant, forms hetero KCNQ1 al. A, 2004.Truncated et Kimura Y,Aizawa S, Yasunami Hirano M, Wu L K, Ueda Y, Aizawa REFERENCES interest. of conflict no declare authors The athways and athways -epilepsy phenotype. Hum Mol Genet 23:4875–4886. Genet phenotype. Hum-epilepsy Mol ppression due due ppression su tonegative FEBS trafficking574:145–150. defect. Lett

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control collapsing analysis identifies epilepsy genes implicated in trio –30. - M, SuM, T, He N, Z Lin PHILIC MOTIFS DETERMINE SUBFAMILY DETERMINE MOTIFS PHILIC - J, WangJ, Y Shi J, - -W SUBUNITS. J Biol Chem Biol J SUBUNITS. , Yi Y Yi , - H, Liao W Liao H, - gated Potassium

al. 2017. al. A - P. 2017. - - Accepted Article between parenthesesbetween next to variant the in this report. Underlined level (p). and variants. described novel previously S1 Functional bold. V indicate missense variants Schematic representation of K 1 Figure Figure S3 S6: SegmentS6S6: ( 331-351), ariants : Segment S1 Segment : ( residues residues

where P: pore helix 260-280) For

functional (residues domains represented are by residues 392-420),

recurren variants , S4: Segment S4 , Segment S4: ( residues 187-208)

and truncatingvariants studies have been previously conducted t

variants, correspond to novel variantscorrespond novel to v 2.1 protein structure protein 2.1 365 , S2 Segment S2: C: C- C: -376) ( the the residues residues Variant terminal domain Tinls and Triangles . number ofpatients reported ( , S: Selectivity filter N: N- N: s 295-316) are are ( terminal domain frameshift frameshift ( residues displayed aschanges at

(residues 421-858). (residues , S5: Segment S5 , Segment S5: and location of KCNB1

black circle reported for the first time 229-250) or or nonsense variants) nonsense residues ( residues are indic s , S3: Segment Segment , S3:

respectively is indicatedis 377-381) ( residues residues protein protein 1-186), 1-186), ated in in ated , .

Accepted Article literature. 1. Table restrictions. corresponding author. The data are not publicly available due to privacy or ethical the from request on available are study this of thefindings support that data The STATEMENT AVAILABILITY DATA 7 6 5 4 3 2 1 ID t ien Pat

T 9C> c.62 T 9C> c.62 G 9C> c.62 T 5C> c.60 T 5A> c.59 T 6A> c.58 G 8A> c.12 ge ge chan e eotid nucl KCNB1

10Met p.Thr2 10Met p.Thr2 g 210Ar p.Thr 02Phe p.Ser2 9Phe p.Ile19 6Phe p.Ile19 43Gly p.Glu nge cha d aci no ami -

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Sex / (y) e Ag F 8 / F 4 / M 11 / M 7 / / M NA F 12 / M 29 /

D D s s e Y A N s e Y s e Y s e Y s e Y s e Y k al W A N A N es Y es Y A N A N es Y

ls skil l rba Ve bal ver n- No NA rds Wo ces ten Sen NA bal ver n- No bal ver n- No

rs de or dis ral vio ha Be s s Ye A N s Ye s Ye s Ye s Ye No

(m) et ons sz at e Ag / sy lep Epi / 12 / Yes No 48 / Yes 24 / Yes NA / Yes 18 / Yes 16 / Yes

References (ID Marini et al. 2017 24)(patient 2017 De et Kovel al. study This 26)(patient Kovel De al. 2017 et Calhoun (ID Marini et al. 2017 study This -5) -4)

et al. 2017 al. et

Accepted Article 1 0 1 9 8 0 2 9 1 8 1 7 1 6 1 5 1 4 1 3 1 2 1 1

5G> c.93 A 5G> c.93 A 5G> c.93 A 5G> c.93 T 4C> c.93 T 6C> c.91 T 6C> c.91 T 6C> c.91 T 6C> c.91 7del c.85 T 2C> c.68 C 2T> c.63 A 9C> c.62

12His p.Arg3 12His p.Arg3 12His p.Arg3 12His p.Arg3 s 312Cy p.Arg 06Cys p.Arg3 06Cys p.Arg3 06Cys p.Arg3 06Cys p.Arg3 fs*58 86Gly p.Val2 28* p.Gln2 11Pro p.Leu2 10Lys p.Thr2 sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi ft shi me fra se en ns no e ns sse mi e ns sse mi

nov de NA o nov de o nov de vo no de o nov de o nov de o nov de o nov de vo no de o nov de o nov de o nov de

F 22 / F 8 / M 2 / / F 3,5 F 7 / M 7 / M 33 / M 11 / / F 1,2 M 9 / M 23 / M 9 / M 7 /

s s e Y s e Y s e Y s e Y s e Y s e Y e Y s e Y s e Y s e Y s e Y s e Y s e Y es Y es Y es Y es Y A N es Y es Y o N o N es Y es Y A N es Y

ces ten Sen ces ten Sen ces ten Sen NA NA ces ten Sen n No bal ver n- No bal ver n- No bal ver n- No rds Wo NA rds Wo -

No s Ye No s Ye No s Ye s Ye s Ye A N s Ye s Ye s Ye s Ye

/ 6 Yes 48 / Yes / 5 Yes 10 / Yes 11 / Yes / 4 Yes / 18 / Yes 14 / Yes 14 / Yes 10 / Yes No 12 / Yes 12 / Yes

(ID Marini et al. 2017 study This study This 22)(patient 2017 De et Kovel al. 23)(patient Kovel De 25)(patient 2017 De et Kovel al. This study This 18)(patient al. et Kovel 2017 (ID 2016 De et Kovel al. al. 2018 et Samanta 19)(patient 2017 De et Kovel al. study This 21)(patient 2017 De et Kovel al. 20)(patient 2017 De et Kovel al. (patient 2) Saitsu -3) -KIEL20), De -KIEL20),

et al. 2015 al. et

et al. 2017 al. et

Accepted Article 3 2 2 2 1 2 2 3 1 3 0 3 9 2 8 2 7 2 6 2 5 2 4 2 >A 07G c.11 >C 05T c.11 lG 88de c.10 >T 45G c.10 >T 45G c.10 >A 41C c.10 >G 41C c.10 >C 01T c.10 C 0G> c.99 G 4C> c.98 T 8C> c.96 A 5G> c.93 A

69* p.Trp3 g 369Ar p.Trp s*13 63Thrf p.Ser3 49Phe p.Val3 49Phe p.Val3 47Arg p.Ser3 47Arg p.Ser3 34Pro p.leu3 30Asp p.Glu3 328* p.Tyr 323Ile p.Thr 12His p.Arg3 se en ns no e ns sse mi ft shi me fra e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi se en ns no e ns sse mi e ns sse mi e ns

o nov de vo no de o nov de o nov de o nov de o nov de o nov de vo no de o nov de vo no de vo no de o nov de o

F 5 / / F 3,6 / F 4,5 M 3 / M 13 / M M 5 / M 14 / M 2 / M 17 / / F 1,6 F 9 M 10 /

/

s s e Y s e Y s e Y s e Y s e Y s s s e Y s e Y s e Y s e Y s e Y s e Y s e Y

o N A N es Y es Y es Y A N o N A N A N o N es Y es Y bal ver n- No NA rds Wo bal ver n- No bal ver n- No bal ver NA bal ver n- No bal ver n- No rds Wo bal ver n- No NA ces ten Sen

s s Ye A N s Ye s Ye s Ye s s Ye s Ye No s Ye s Ye A N s Ye

/ 24 / Yes 18 / Yes / 7 Yes 21 / Yes 60 / Yes / 18 / Yes 10 / Yes 14 / Yes 11 / Yes / 9 Yes 48 / Yes 18 / Yes

This study This 2018 et al. Miao study This study This study This (patient 15)(patient 2017 De et Kovel al. study This 16)(patient 2017 De et Kovel al. (ID Marini et al. 2017 study This 17) (Patient al. et Kovel 2017 2014 (ID- Torkamani al. et study This -6) 9), De De 9),

Accepted Article 3 5 3 4 3 3 3 4 4 3 4 2 4 1 4 0 4 9 3 8 3 7 3 6 >G 39A c.11 >T 36G c.11 >A 35G c.11 >C 33T c.11 >T 32G c.11 >C 32G c.11 >A 30C c.11 >T 21C c.11 >T 21C c.11 >A 15C c.11 >T 15C c.11 >A 09G c.11

s 380Cy p.Tyr 79Val p.Gly3 79Arg p.Gly3 78Ala p.Val3 78Phe p.Val3 78Leu p.Val3 n 377As p.Thr 74Ile p.Thr3 74Ile p.Thr3 n 372As p.Thr 372Ile p.Thr 70* p.Trp3

ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi se en ns no

o nov de o nov de o nov de o nov de vo no de o nov de vo no de o nov de o nov de vo no de vo no de o nov de

F 11 / F 5 / F 12 / M 8 / M 7 / / F 5,5 ND 2 / M 7 / F 3 / M 8 / F 10 / M 2 /

s s e Y s e Y s e Y s e Y s e Y s e Y s e Y s e Y s e Y s e Y s e Y s e Y

ds ai ith W A N es Y es Y A N es Y A N es Y o N o N es Y o N

bal ver n- No NA ces ten Sen ces ten Sen rds Wo rds Wo NA bal ver n- No bal ver n- No bal ver n- No rds Wo bal ver n- No

s s Ye A N s Ye No No No A N s Ye A N A N s Ye No

/13 /13 Yes / 6 Yes No No / 9 Yes / 6 Yes 24 / Yes / 8 Yes 13 / Yes 14 / Yes No 6 / Yes

(patient 14)(patient 2017 De et Kovel al. 13)(patient al. et Kovel 2017 De 2014 (patient3), Torkamani al. et (ND27062), 2013 al. et Allen study This study This (ID Marini et al. 2017 Parrini et al. 2017 This study This 2017 et al. Miao 2017 (patient11) al. et 2014, De Kovel Srivastava et al. 2), 2014 (individual Torkamani al. et 12)(patient 2017 De et Kovel al. 2015, Thiffault et al. al. 2014, Soden et study This 2016 et al. Latypova study This -1)

Accepted Article 7 4 6 4 5 4 6 5 5 5 4 5 3 5 2 5 1 5 0 5 9 4 8 4 >G 48C c.12 >C 26T c.12 >A 01G c.12 >A 01G c.12 >T 90G c.11 >A 83G c.11 >A 83G c.11 >A 80G c.11 >C 73A c.11 >A 53C c.11 >A 44G c.11 >A 41G c.11

16Leu p.Phe4 09Thr p.Ile4 01Arg p.Gly4 01Arg p.Gly4 97Phe p.Cys3 95Arg p.Gly3 95Arg p.Gly3 94Arg p.Gly3 91Asn p.Lys3 85Thr p.Pro3 n 382As p.Asp 81Arg p.Gly3 e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e ns sse mi e

o nov de vo no de o nov de o nov de o nov de NA vo no de vo no de o nov de o nov de vo no de o nov de

9.5 F 9 / NA M 17 / F 6 / M 7 / F 15 / M 8 / M 9 / M 4 / M 22 / M 14 / / F

Y s e Y A N s e Y s e Y s e Y s s e Y s e Y s e Y s e Y s e Y s e Y s e Y es Y A N ds ai ith W es Y o N A N es Y o N o N A N o N es

No bal ver n- No NA bal ver n- No bal ver n- No NA NA ces ten Sen bal ver n- No bal ver n- No NA bal ver n- No bal ver n-

Ye s Ye A N s Ye No A N s s Ye s Ye A N A N A N s Ye s

Yes 3,5 / Yes NA 13 / Yes / 8 Yes / 3 Yes / 14 / Yes No / 8 Yes 17 / Yes 10 / Yes / 8 Yes 12 /

This study This study This 2017 (patient8) et al. 2015, De Kovel Fitzgerald et al. (patient 9) 2017 De et Kovel al. study This 10) 2017 al. (patient et 7), (patient De Kovel 2016 al. et Allen et al. 2017 al. (patient 5)et 8), (patient De Kovel 2016 al. et Allen study This This 2017 al. (patient 6)et 1), (patient De Kovel et al. 2015 Saitsu (patient 7) Kovel De study This

study

et al. 2017 al. et

Accepted Article 6 9 5 8 5 7 5 p.Thr301Met). † m: delay, sz: developmental seizures, years, months,DD: y: NA: Notavailable mutationsare bold.Novel indicated in Numbering is according to thecDNA sequence (RefSeq NM_ 4 6 3 6 2 6 1 6 0

This patient also carries variant novo ade in GABRA5 >T 47C c.17 >T 47C c.17 >T 47C c.17 >A 99C c.15 p 03du c.15 >T 89G c.14 >A 48C c.12 >G 48C c.12

83* p.Arg5 83* p.Arg5 83* p.Arg5 33* p.Tyr5 02* p.Lys5 497* p.Glu 16Leu p.Phe4 16Leu p.Phe4 se en ns no se en ns no se en ns no se en ns no se en ns no se en ns no e ns sse mi e ns sse mi

nal ter ma o nov de o nov de o nov de o nov de vo no de NA o nov de

F 8 / M 32 / M 10 / M 20 / F 14 / F 18 / F 12 / F 11 / s s e Y s e Y s e Y s e Y s e Y s e Y s s e Y s e Y A N es Y A N es Y es Y A N es Y es Y ver n- No NA NA ces ten Sen bal ver n- No NA bal ver n- No ces ten Sen bal

No A N s Ye s Ye No A N s s Ye s Ye (NM_000810:c.902C>T,

/ 5 Yes / 5 Yes No No s day 10 / Yes 42 / Yes No No

004975.2 (ID Marini et al. 2017 (patient 2) al. et Kovel 2017 (ID Kovel De 2017 (patient3) et al. 2015, De Kovel Fitzgerald et al. study This study This (patient 4) 2017 De et Kovel al. (ID 2016 De et Kovel al. This study This (patient 1) Kovel De -2) De -EP1852), -2010D05815), ).

et al. 2016 al. et et al. 2017 al. et