De Novo Loss-of-Function Mutations in CHD2 Cause a Fever-Sensitive Myoclonic Epileptic Encephalopathy Sharing Features with Dravet Syndrome
2013; Elsevier BV; Volume: 93; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2013.09.017
ISSN1537-6605
AutoresArvid Suls, Johanna A. Jaehn, Angéla Kecskés, Yvonne G. Weber, Sarah Weckhuysen, Dana Craiu, Aleksandra Siekierska, Tania Djémié, Tatiana Afrikanova, Padhraig Gormley, Sarah von Spiczak, Gerhard Kluger, Catrinel Iliescu, Tiina Talvik, Inga Talvik, Cihan Meral, Hande Çağlayan, Beatriz G. Giráldez, José M. Serratosa, Johannes R. Lemke, Dorota Hoffman‐Zacharska, Elżbieta Szczepanik, Nina Barišić, Vladimı́r Komárek, Helle Hjalgrim, Rikke S. Møller, Tarja Linnankivi, Petia Dimova, Pasquale Striano, Federico Zara, Carla Marini, Renzo Guerrini, Christel Depienne, Stéphanie Baulac, Gregor Kuhlenbäumer, Alexander D. Crawford, Anna‐Elina Lehesjoki, Peter de Witte, Aarno Palotie, Holger Lerche, Camila V. Esguerra, Peter De Jonghe, Ingo Helbig, Rik Hendrickx, Philip Holmgren, Ulrich Stephani, Hiltrud Muhle, Manuela Pendiziwiat, Silke Appenzeller, Kaja Kristine Selmer, Eva H. Brilstra, Bobby P.C. Koeleman, Felix Rosenow, Eric LeGuern, Katalin Štěrbová, Budisteanu Magdalena, Gherghiceanu Rodica, Oana Tarta Arsene, Barca Diana, Rosa Guerrero, Laura Ortega, Албена Тодорова, Andrey Kirov, Angela Robbiano, Mutluay Arslan, Uluç Yiş, Vanja Ivanović,
Tópico(s)Metabolism and Genetic Disorders
ResumoDravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resistant, fever-sensitive seizures followed by cognitive decline. Mutations in SCN1A explain about 75% of cases with Dravet syndrome; 90% of these mutations arise de novo. We studied a cohort of nine Dravet-syndrome-affected individuals without an SCN1A mutation (these included some atypical cases with onset at up to 2 years of age) by using whole-exome sequencing in proband-parent trios. In two individuals, we identified a de novo loss-of-function mutation in CHD2 (encoding chromodomain helicase DNA binding protein 2). A third CHD2 mutation was identified in an epileptic proband of a second (stage 2) cohort. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To explore the functional relevance of CHD2 haploinsufficiency in an in vivo model system, we knocked down chd2 in zebrafish by using targeted morpholino antisense oligomers. chd2-knockdown larvae exhibited altered locomotor activity, and the epileptic nature of this seizure-like behavior was confirmed by field-potential recordings that revealed epileptiform discharges similar to seizures in affected persons. Both altered locomotor activity and epileptiform discharges were absent in appropriate control larvae. Our study provides evidence that de novo loss-of-function mutations in CHD2 are a cause of epileptic encephalopathy with generalized seizures. Dravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resistant, fever-sensitive seizures followed by cognitive decline. Mutations in SCN1A explain about 75% of cases with Dravet syndrome; 90% of these mutations arise de novo. We studied a cohort of nine Dravet-syndrome-affected individuals without an SCN1A mutation (these included some atypical cases with onset at up to 2 years of age) by using whole-exome sequencing in proband-parent trios. In two individuals, we identified a de novo loss-of-function mutation in CHD2 (encoding chromodomain helicase DNA binding protein 2). A third CHD2 mutation was identified in an epileptic proband of a second (stage 2) cohort. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To explore the functional relevance of CHD2 haploinsufficiency in an in vivo model system, we knocked down chd2 in zebrafish by using targeted morpholino antisense oligomers. chd2-knockdown larvae exhibited altered locomotor activity, and the epileptic nature of this seizure-like behavior was confirmed by field-potential recordings that revealed epileptiform discharges similar to seizures in affected persons. Both altered locomotor activity and epileptiform discharges were absent in appropriate control larvae. Our study provides evidence that de novo loss-of-function mutations in CHD2 are a cause of epileptic encephalopathy with generalized seizures. Epileptic encephalopathies (EEs) are severe, intractable childhood epilepsies with concomitant cognitive impairment and other associated comorbidities. Although a broad range of exogenous factors can lead to EE, in a significant subset of affected individuals the etiology remains unidentified and might be endogenous. Consequently, a genetic cause is assumed.1Cross J.H. Guerrini R. The epileptic encephalopathies.Handb. Clin. Neurol. 2013; 111: 619-626Crossref PubMed Scopus (21) Google Scholar Among the various EEs of genetic origin, Dravet syndrome (MIM 607208) has emerged as one of the best-defined phenotypes and the one with the highest mutation detection yield. Children with Dravet syndrome are prone to repetitive and prolonged epileptic seizures in the setting of fever.2Dravet C. The core Dravet syndrome phenotype.Epilepsia. 2011; 52: 3-9Crossref PubMed Scopus (357) Google Scholar Although these fever-induced seizures start around the age of 6 months (range = 3–16 months),3Kearney J.A. Wiste A.K. Stephani U. Trudeau M.M. Siegel A. RamachandranNair R. Elterman R.D. Muhle H. Reinsdorf J. Shields W.D. et al.Recurrent de novo mutations of SCN1A in severe myoclonic epilepsy of infancy.Pediatr. Neurol. 2006; 34: 116-120Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar other seizure types occur during the course of the disease, and developmental plateauing is typical in the second year of life. Taking into account SCN1A abnormalities due to single or multiple exons deletions, de novo mutations in SCN1A (MIM 182389) are known to cause Dravet syndrome in ∼75% of affected probands.4Depienne C. Trouillard O. Saint-Martin C. Gourfinkel-An I. Bouteiller D. Carpentier W. Keren B. Abert B. Gautier A. Baulac S. et al.Spectrum of SCN1A gene mutations associated with Dravet syndrome: analysis of 333 patients.J. Med. Genet. 2009; 46: 183-191Crossref PubMed Scopus (275) Google Scholar Despite this high probability of identifying a mutation in an individual with a typical phenotype, up to 25% of affected persons do not carry SCN1A mutations, suggesting the involvement of other genes. Mutations in PCDH19 (MIM 300460) can result in a Dravet-syndrome-like phenotype in females and explain some of the cases without an SCN1A mutation.5Depienne C. Trouillard O. Bouteiller D. Gourfinkel-An I. Poirier K. Rivier F. Berquin P. Nabbout R. Chaigne D. Steschenko D. et al.Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females.Hum. Mutat. 2011; 32: E1959-E1975Crossref PubMed Scopus (99) Google Scholar For a relevant subset of probands, however, a causative mutation cannot be identified in either gene. We therefore explored the involvement of mutations in additional genes in these probands by using whole-exome sequencing in proband-parent trios. This approach provides the possibility of querying the entire exome for de novo mutations in simplex cases, which has been proven to be successful for intellectual disability, schizophrenia, autism, and some forms of epilepsy or epilepsy-related disorders.6Vissers L.E. de Ligt J. Gilissen C. Janssen I. Steehouwer M. de Vries P. van Lier B. Arts P. Wieskamp N. del Rosario M. et al.A de novo paradigm for mental retardation.Nat. Genet. 2010; 42: 1109-1112Crossref PubMed Scopus (624) Google Scholar, 7de Ligt J. Veltman J.A. Vissers L.E. Point mutations as a source of de novo genetic disease.Curr. Opin. Genet. Dev. 2013; 23: 257-263Crossref PubMed Scopus (38) Google Scholar We first studied nine Dravet-syndrome-affected individuals in whom SCN1A sequence mutations or copy-number variations had been excluded. These probands were selected on the basis of broad inclusion criteria, extending the onset age of seizures to an age between 3 months and 2 years of life. Further inclusion criteria consisted of (1) the presence of both febrile and afebrile seizures; (2) multiple seizure types including tonic-clonic, hemiclonic, myoclonic, absence, and/or focal seizures; (3) pharmacoresistant epilepsy at least during childhood or frequent status epilepticus; (4) normal development prior to epilepsy onset, although some minor degree of developmental delay could be present; and (5) slowing or stagnation of development after onset of seizures and absence of epileptogenic lesions on brain MRI. Signed informed consent was obtained from all study participants or their legal representatives. The local ethical committees of the University of Antwerp and Antwerp University Hospital, University of Kiel, and other collaborating centers approved this study. Genomic DNA was extracted from peripheral blood according to standard procedures. We performed whole-exome sequencing on genomic DNA of the nine selected individuals with Dravet syndrome and both unaffected parents at the Wellcome Trust Sanger Institute (Hinxton, Cambridgeshire). In brief, genomic DNA (∼3 μg) was fragmented by sonication, and fragments with a length of 150–200 bp were purified. After a paired-end DNA library was prepared from the DNA fragments (with the TruSeq DNA Sample Preparation Kit from Illumina), targeted enrichment was performed with the SureSelect Human All Exon 50Mb Kit (Agilent Technologies). Captured DNA was then sequenced on a HiSeq2000 (Illumina) as paired-end 75 bp reads according to the manufacturer’s protocol. Sequencing reads passing quality filtering were aligned to the human reference genome (hg19, UCSC Genome Browser) with the Burrows-Wheeler Aligner.8Li H. Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform.Bioinformatics. 2010; 26: 589-595Crossref PubMed Scopus (7016) Google Scholar The Genome Analysis Toolkit (GATK)9McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. Garimella K. Altshuler D. Gabriel S. Daly M. DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14776) Google Scholar was used to recalibrate base quality scores, realign around indels, and mark duplicate reads. Independent variant calling was performed on the mapped reads with SAMtools10Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31559) Google Scholar mpileup, GATK UnifiedGenotyper, and Dindel.11Albers C.A. Lunter G. MacArthur D.G. McVean G. Ouwehand W.H. Durbin R. Dindel: accurate indel calls from short-read data.Genome Res. 2011; 21: 961-973Crossref PubMed Scopus (327) Google Scholar The GenomeComb12Reumers J. De Rijk P. Zhao H. Liekens A. Smeets D. Cleary J. Van Loo P. Van Den Bossche M. Catthoor K. Sabbe B. et al.Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing.Nat. Biotechnol. 2012; 30: 61-68Crossref Scopus (167) Google Scholar program was used for annotating, comparing, and filtering the data. For de novo variant calling, the DeNovoGear13Conrad D.F. Keebler J.E.M. DePristo M.A. Lindsay S.J. Zhang Y. Casals F. Idaghdour Y. Hartl C.L. Torroja C. Garimella K.V. et al.1000 Genomes ProjectVariation in genome-wide mutation rates within and between human families.Nat. Genet. 2011; 43: 712-714Crossref PubMed Scopus (394) Google Scholar program by Conrad and colleagues was used and double-checked by GenomeComb analysis. Our analysis revealed a heterozygous de novo mutation in CHD2 (MIM 302119; RefSeq accession number NM_001271.3), encoding chromodomain helicase DNA binding protein 2 (CHD2; RefSeq NP_001262.3), in two out of nine probands. One individual carried a nonsense mutation, c.4971G>A (p.Trp1657∗), in exon 38; the second person carried a splice-site mutation, c.1810−2A>C, affecting the splice acceptor site of exon 16. Presence or absence of the mutations was confirmed on genomic DNA of the probands or parents, respectively, by bidirectional Sanger sequencing using the ABI BigDye Terminator v.3.1 cycle sequencing kit on an ABI 3730xl automated DNA Analyzer (Applied Biosystems). To obtain further genetic evidence of pathogenicity, we performed a mutation analysis of all 39 coding exons and intron-exon boundaries of CHD2 by using bidirectional sequencing (primer sequences are available upon request) in a cohort of 150 EE probands similar to the two individuals carrying a CHD2 mutation: all selected probands had infantile- or childhood-onset epilepsy with subsequent developmental delay. All individuals had normal brain MRI and at least one of the following seizure types: tonic-clonic seizures, myoclonic seizures, (atypical) absence seizures, or atonic seizures. In this cohort, we identified a third person carrying a de novo mutation in CHD2 (c.1396C>T [p.Arg466∗]) in exon 13. The two identified premature stop codons are predicted to result in degradation of the mutant transcript by means of nonsense-mediated mRNA decay (NMD). The effect of the splice-site mutation on transcript level is unclear, given that both exon skipping and (partial) intron retention are possible. Most likely, the splice acceptor mutation results in skipping of exon 16 and a subsequent out-of-frame deletion of exon 16, leading to a premature stop codon in exon 17 (p.Thr604Valfs2∗). Therefore, NMD could also be active in this case. To test the NMD hypothesis, we extracted RNA from fresh blood (QIAGEN RNeasy Micro Kit) of the three probands and a parent as a control individual, removed contaminated DNA by Ambion DNA-free DNase treatment (Life Technologies), and synthesized cDNA by Superscript III reverse transcriptase (Life Technologies). For the two nonsense mutations, we performed bidirectional sequencing on cDNA with primers flanking the mutation. With these experiments, we were able to detect the mutation with a 50/50 ratio, proving that the aberrant allele was not degraded by NMD. These results were confirmed by Sybr-Green-based quantitative PCR (qPCR) experiments (Sigma-Aldrich) with six primer pairs complementary to CHD2 cDNA (data not shown). For the splice-site mutation, we performed qPCR and developed sequencing primers flanking exon 16 on cDNA. Also here, qPCR showed the presence of both alleles. Additionally, the sequencing experiment showed an abundance of alternative splicing events in the probands compared to the control individual, but the consequence of the alternative splicing remained unknown. Although skipping of exon 16 appeared to be a logical consequence, additional investigations revealed more complex alternative splicing events. Whereas definite identification of all alternatively spliced transcripts was not possible, we were able to rule out NMD. We assumed that either the aberrant proteins were degraded by the proteasome or the shorter proteins were not fully functional; both of these cases would result in loss of function. In support of the pathogenicity of these mutations, none of the identified mutations have been observed in the 1000 Genomes Project, National Heart, Lung, and Blood Institute Exome Sequencing Project Exome Variant Server (EVS), or dbSNP (build 137). Furthermore, splice-site, frameshift, or nonsense variations are absent in the EVS and 1000 Genomes Project cohorts. Between the age of 14 months and 3.5 years, the three persons carrying a de novo CHD2 mutation presented with febrile seizures followed by therapy-resistant generalized seizures (Table 1). Frequent myoclonic seizures, generalized tonic-clonic seizures (GTCSs), and absences were seen in all three individuals. Proband 1 developed normally during the first year of life. The first febrile seizure occurred when he was 14 months old and was soon followed by afebrile head drops occurring several times a day. When he was 2 years old, therapy-resistant myoclonic seizures, atypical absences, and GTCSs rarely associated with fever developed. On one occasion, he had a status epilepticus. Electroencephalography (EEG) showed generalized polyspike wave discharges and, later, focal epileptic discharges. He has been treated with valproic acid, levetiracetam, phenytoin, topiramate, bromide, phenobarbital, ethosuximide, topiramate, vitamine B6, and prednisolon. He now has moderate intellectual disability (ID), dysarthria, and ataxia. Proband 2 had a normal early development. At the age of 2 years, she had a cluster of febrile seizures. At the age of 2.5 years, she developed therapy-resistant absence seizures accompanied by eyelid myoclonias, myoclonic seizures, and febrile and afebrile GTCSs. EEG showed frequent generalized spike-wave complexes and polyspikes. She has been treated with vigabatrin, valproate, bromide, ethosuximide, lamotrigine, and levetiracetam and is currently taking a combination of topiramate and valproic acid. She still has afebrile GTCSs and moderate ID. Proband 3 had slightly delayed early motor and speech development, given that he walked at the age of 1.5 years and produced his first words at the age of 2 years. At the age of 3.5 years, he had two GTCSs during an episode of high fever. During the following 2 years, he had pharmacoresistant febrile and afebrile GTCSs, hemiclonic, atonic, and myoclonic seizures, and atypical absences. He was treated with valproic acid, topiramate, and levetiracetam. After the start of clobazam at the age of 5.5 years, he became seizure free. EEG showed generalized (poly)spike-wave complexes. After seizure onset, a cognitive decline was seen but partially improved when seizures were controlled. He now has mild ID, autism spectrum disorder (ASD), attention deficit hyperactivity disorder, and mild ataxia. Brain MRI was normal in probands 1 and 2 and showed atrophic changes in proband 3.Table 1Clinical and Genetic Characteristics of Persons with a CHD2 MutationProbandSexAge at InclusionMutationDevelopment Prior to EpilepsyAge of Seizure OnsetFirst Seizure TypeFurther Seizure TypesFever SensitivityEEGImagingClinical ExamCognitive Outcome1male6 yearsc.1810−2A>C (p.?)normal14 monthssimple FSshead drops, GTCSs, myoclonic seizures, atypical absences, status epilepticus+frequent generalized (poly)SWsnormalataxia, dysarthriamild ID2female24 yearsc.4971G>A (p.Trp1657∗)normal2 yearscluster of FSsmyoclonic absences, myoclonic seizures, GTCSs+frequent generalized SWs and polyspikesnormalnormalmild ID3male6 yearsc.1396C>T (p.Arg466∗)subtle delay in motor and speech development3 years, 6 monthstwo FSs during single fever episodeGTCSs and hemiclonic, atonic, myoclonic, atypical absences+generalized (poly)SWsnonspecific atrophymild ataxiamild ID, ASD, ADHDAbbreviations are as follows: ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder; FS, febrile seizure; GTCS, generalized tonic-clonic seizure; ID, intellectual disability; and SW, spike-wave complex. Open table in a new tab Abbreviations are as follows: ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder; FS, febrile seizure; GTCS, generalized tonic-clonic seizure; ID, intellectual disability; and SW, spike-wave complex. In addition to our three individuals with a CHD2 mutation, one simplex case with ID and absence epilepsy and one simplex ASD case both carrying a mutation in CHD2 have been reported in the literature.14Rauch A. Wieczorek D. Graf E. Wieland T. Endele S. Schwarzmayr T. Albrecht B. Bartholdi D. Beygo J. Di Donato N. et al.Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study.Lancet. 2012; 380: 1674-1682Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar, 15Neale B.M. Kou Y. Liu L. Ma’ayan A. Samocha K.E. Sabo A. Lin C.-F. Stevens C. Wang L.-S. Makarov V. et al.Patterns and rates of exonic de novo mutations in autism spectrum disorders.Nature. 2012; 485: 242-245Crossref PubMed Scopus (1275) Google Scholar Recently, two other research groups have also shown the involvement of CHD2 mutations in EEs.16Carvill G.L. Heavin S.B. Yendle S.C. McMahon J.M. O’Roak B.J. Cook J. Khan A. Dorschner M.O. Weaver M. Calvert S. et al.Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1.Nat. Genet. 2013; 45: 825-830Crossref PubMed Scopus (473) Google Scholar, 17Allen A.S. Berkovic S.F. Cossette P. Delanty N. Dlugos D. Eichler E.E. Epstein M.P. Glauser T. Goldstein D.B. Han Y. et al.Epi4K ConsortiumEpilepsy Phenome/Genome ProjectDe novo mutations in epileptic encephalopathies.Nature. 2013; 501: 217-221Crossref PubMed Scopus (1086) Google Scholar Furthermore, several individuals affected by ID and generalized epilepsy and carrying a multigenic chromosomal deletion of 15q26.2, including CHD2, have been described (Table 2).18Capelli L.P. Krepischi A.C. Gurgel-Giannetti J. Mendes M.F. Rodrigues T. Varela M.C. Koiffmann C.P. Rosenberg C. Deletion of the RMGA and CHD2 genes in a child with epilepsy and mental deficiency.Eur. J. Med. Genet. 2012; 55: 132-134Crossref PubMed Scopus (37) Google Scholar, 19Dhamija R. Patterson M.C. Wirrell E.C. Epilepsy in children—when should we think neurometabolic disease?.J. Child Neurol. 2012; 27: 663-671Crossref PubMed Scopus (10) Google Scholar, 20Veredice C. Bianco F. Contaldo I. Orteschi D. Stefanini M.C. Battaglia D. Lettori D. Guzzetta F. Zollino M. Early onset myoclonic epilepsy and 15q26 microdeletion: observation of the first case.Epilepsia. 2009; 50: 1810-1815Crossref PubMed Scopus (27) Google Scholar, 21Li M.M. Nimmakayalu M.A. Mercer D. Andersson H.C. Emanuel B.S. Characterization of a cryptic 3.3 Mb deletion in a patient with a “balanced t(15;22) translocation” using high density oligo array CGH and gene expression arrays.Am. J. Med. Genet. A. 2008; 146: 368-375Crossref PubMed Scopus (21) Google Scholar, 22Lund C. Brodtkorb E. Røsby O. Rødningen O.K. Selmer K.K. Copy number variants in adult patients with Lennox-Gastaut syndrome features.Epilepsy Res. 2013; 105: 110-117Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar The presumed haploinsufficiency of CHD2 in these probands prompted us to screen all individuals without a previous detected mutation from both studied cohorts for copy-number variants in CHD2 with the multiplex amplicon quantification (MAQ) technique, an in-house-developed technique based on a semiquantified multiplex PCR. The multiplex PCR reaction consisted of six target amplicons located in the genomic region of CHD2 and four reference amplicons randomly located on different chromosomes. The MAQ analysis was performed as described previously.23Suls A. Claeys K.G. Goossens D. Harding B. Van Luijk R. Scheers S. Deprez L. Audenaert D. Van Dyck T. Beeckmans S. et al.Microdeletions involving the SCN1A gene may be common in SCN1A-mutation-negative SMEI patients.Hum. Mutat. 2006; 27: 914-920Crossref PubMed Scopus (109) Google Scholar A partial or full deletion or duplication of the gene was not identified in any of the remaining studied persons (7 of the initial cohort and 149 of the second-stage cohort).Table 2Clinical Phenotype of Probands Reported in Literature with CHD2 Deletions or MutationsReferenceCapelli et al.18Capelli L.P. Krepischi A.C. Gurgel-Giannetti J. Mendes M.F. Rodrigues T. Varela M.C. Koiffmann C.P. Rosenberg C. Deletion of the RMGA and CHD2 genes in a child with epilepsy and mental deficiency.Eur. J. Med. Genet. 2012; 55: 132-134Crossref PubMed Scopus (37) Google ScholarDhamija et al.19Dhamija R. Patterson M.C. Wirrell E.C. Epilepsy in children—when should we think neurometabolic disease?.J. Child Neurol. 2012; 27: 663-671Crossref PubMed Scopus (10) Google ScholarVeredice et al.20Veredice C. Bianco F. Contaldo I. Orteschi D. Stefanini M.C. Battaglia D. Lettori D. Guzzetta F. Zollino M. Early onset myoclonic epilepsy and 15q26 microdeletion: observation of the first case.Epilepsia. 2009; 50: 1810-1815Crossref PubMed Scopus (27) Google ScholarLi et al.21Li M.M. Nimmakayalu M.A. Mercer D. Andersson H.C. Emanuel B.S. Characterization of a cryptic 3.3 Mb deletion in a patient with a “balanced t(15;22) translocation” using high density oligo array CGH and gene expression arrays.Am. J. Med. Genet. A. 2008; 146: 368-375Crossref PubMed Scopus (21) Google ScholarLund et al.22Lund C. Brodtkorb E. Røsby O. Rødningen O.K. Selmer K.K. Copy number variants in adult patients with Lennox-Gastaut syndrome features.Epilepsy Res. 2013; 105: 110-117Abstract Full Text Full Text PDF PubMed Scopus (28) Google ScholarRauch et al.14Rauch A. Wieczorek D. Graf E. Wieland T. Endele S. Schwarzmayr T. Albrecht B. Bartholdi D. Beygo J. Di Donato N. et al.Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study.Lancet. 2012; 380: 1674-1682Abstract Full Text Full Text PDF PubMed Scopus (764) Google ScholarCarvill et al.16Carvill G.L. Heavin S.B. Yendle S.C. McMahon J.M. O’Roak B.J. Cook J. Khan A. Dorschner M.O. Weaver M. Calvert S. et al.Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1.Nat. Genet. 2013; 45: 825-830Crossref PubMed Scopus (473) Google ScholarAllen et al.17Allen A.S. Berkovic S.F. Cossette P. Delanty N. Dlugos D. Eichler E.E. Epstein M.P. Glauser T. Goldstein D.B. Han Y. et al.Epi4K ConsortiumEpilepsy Phenome/Genome ProjectDe novo mutations in epileptic encephalopathies.Nature. 2013; 501: 217-221Crossref PubMed Scopus (1086) Google ScholarNumber of individuals11111161Genetic findingsde novo 0.5 Mb deletion including CHD2 and RGMAde novo 0.9 Mb deletion including CHD2 and three other genesde novo 5 Mb deletion including CHD2 and 55 other genes3.3 Mb deletion including CHD2 and 17 other genes (no segregation analysis)2 Mb deletion including CHD2 and seven other genes. Also carried five additional deletions and duplications, including a total of 100 genes (paternal DNA not available)de novo frameshift mutation c.1809 del (p.Thr604Leufs∗19)four de novo frameshift and two de novo missense alterations: p.Glu1412Glyfs∗64, p.Arg121∗, p.Gly491Valfs∗13, p.Arg1644Lysfs∗22, p.Trp548Arg, p.Leu823Prode novo splice mutation c.1502+1G>AAge at seizure onset2 years3.5 years6 monthsnot specified4 years5 years1–3 years6 monthsSeizure type at onsetnot specifiedCPSsfebrile generalized clonic SEtwo episodes of FSsatypical ASs, MSsASsatypical ASs, AtSs, MSs, GTCSs, FSs, FDSsunknownFurther seizure typesnot specifiedtherapy-resistant ASs with eyelid flutter, TSs, MSs, GTCSstherapy-resistant massive MSs with head drop, eyelid MSs, prolonged hemiclonic FSsnoneTSs, MSs, atypical ASs, nonconvulsive SEnot specifiedFSs, AtSs, MSs, GTCSs, NCS, SE, TSs, HSs, FDSs, MAs, atypical ASsMSs, FDSs, GTCSs, atypical ASs, AtSsFever sensitivitynot specifiednoyesyesnonot specifiedone patientnoEEGgeneralized spike waves and focal dischargesgeneralized spike waves, PPRirregular generalized spike waves, PPRnot specifiedgeneralized slow spike waves and runs of fast spikesnot specifiedgeneralized (poly)spike waves, slow spike waves, multifocal discharges, generalized paroxysmal fast activity, diffuse slowingslow background, generalized spike wavesMRInormalnormalvermis hypoplasia, cisterna magnanormalpartial agenesis of vermisunknownunknownnormalDevelopment prior to epilepsynot specifieddelayeddelayeddelayeddelayeddelayednormal or delayednormalDevelopmental outcomeglobally delayed, severe speech impairmentmild IDmild IDmild to moderate ID, speech impairmentsevere IDmild IDmoderate to severe IDunspecified delayOther clinical findingsataxia, relative microcephaly, mild facial dysmorphismsmicrocephaly, short stature, mild facial dysmorphismsmicrocephaly, congenital hypothyroidism, bicuspid aortic valve, hypotoniamicrocephaly, short stature, mild facial dysmorphismsshort stature, hypertelorism, epicantal fold, micropenis, single palmar creasesDuane anomalyAbbreviations are as follows: AS, absence seizure; AtS, atonic seizure; CPS, complex partial seizure; FDS, focal dyscognitive seizure; FS, febrile seizure; GTCS, generalized tonic-clonic seizure; HS, hemiclonic seizure; ID, intellectual disability; MA, myoclonic absence; MS, myoclonic seizure; NCS, nonconvulsive status epilepticus; PPR, photo paroxysmal response; SE, status epilepticus; and TS, tonic seizure. Open table in a new tab Abbreviations are as follows: AS, absence seizure; AtS, atonic seizure; CPS, complex partial seizure; FDS, focal dyscognitive seizure; FS, febrile seizure; GTCS, generalized tonic-clonic seizure; HS, hemiclonic seizure; ID, intellectual disability; MA, myoclonic absence; MS, myoclonic seizure; NCS, nonconvulsive status epilepticus; PPR, photo paroxysmal response; SE, status epilepticus; and TS, tonic seizure. To establish additional evidence of the implication of CHD2 in the development of epilepsy, we examined the functional consequence of CHD2 haploinsufficiency by knocking down chd2 in zebrafish by using targeted morpholino (MO) antisense oligomers.24Nasevicius A. Ekker S.C. Effective targeted gene ‘knockdown’ in zebrafish.Nat. Genet. 2000; 26: 216-220Crossref PubMed Scopus (2116) Google Scholar All zebrafish experiments carried out were approved by the ethics committee of the University of Leuven (Ethische Commissie van de KU Leuven, approval number P05090) and by the Belgian Federal Public Service of Health, Food Chain Safety, and Environment (Federale Overheidsdienst Volksgezondheid, Veiligheid van de Voedselketen en Leefmileu, approval number LA1210199). In order to mimic loss-of-function mutations, we designed a MO (E2I2 MO, 5′-GATCAGACTGGCCTTTTTGTGTACC-3′) to target the splice donor site of exon 2 and interfere with normal pre-mRNA splicing of zebrafish chd2 (ENSDART00000127730). Targeting of the exon 2-intron 2 boundary should result in abnormal exon 2 splicing, leading to its complete or partial deletion together with its flanking introns. This should result in an mRNA shorter than the wild-type transcript (Figure 1). A control MO (randomized 25 N oligomer) was used as a negative control (ctrl MO). All MOs were designed and synthesized by GeneTools. Gene knockdowns were achieved through microinjection of MOs into 1- to 2-cell-stage embryos from the AB (wild-type) strain according to the method previously described.25Summerton J. Weller D. Morpholino antisense oligomers: design, preparation, and properties.Antisense Nucleic Acid Drug Dev. 1997; 7: 187-195Crossref PubMed Scopus (879) Google Scholar In order to mimic haploinsufficiency, we titrated the amount of E2I2 MO to 9 ng per injection so as to reduce correctly spliced chd2 mRNA levels by approximately 50%. The same amount of ctrl MO was injected into sibling control embryos. To evaluate the level of knockdown in zebrafish embryos and larvae, we perfo
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