BRIEF COMMUNICATION
DEPDC5 Mutations in Genetic Focal Epilepsies of Childhood Dennis Lal, PhD,1,2,3 Eva M. Reinthaler, MS,4 Julian Schubert, MSc,5,6 Hiltrud Muhle, MD,7 Erik Riesch, MD,8 Gerhard Kluger, MD,8 Kamel Jabbari, PhD,1 Amit Kawalia, MSc,1 Christine B€ aumel, MD,9 Hans Holthausen, MD,9 Andreas Hahn, MD,2 Martha Feucht, MD,10 Birgit Neophytou, MD,11 Edda Haberlandt, MD,12 Felicitas Becker, MD,5 Janine Altm€ uller, MD,1 Holger Thiele, MD,1,13 Johannes R. Lemke, MD,14 Holger Lerche, MD,5 Peter N€ urnberg, PhD,1,3,15 Thomas Sander, MD,1 Yvonne Weber, MD,5 Fritz Zimprich, MD, PhD,4 and Bernd A. Neubauer, MD2 Recent studies reported DEPDC5 loss-of-function mutations in different focal epilepsy syndromes. Here we identified 1 predicted truncation and 2 missense mutations in 3 children with rolandic epilepsy (3 of 207). In addition, we identified 3 families with unclassified focal childhood epilepsies carrying predicted truncating DEPDC5 mutations (3 of 82). The detected variants were all novel, inherited, and present in all tested affected (n 5 11) and in 7 unaffected family members, indicating low penetrance. Our findings extend the phenotypic spectrum associated with mutations in DEPDC5 and suggest that rolandic epilepsy, albeit rarely, and other nonlesional childhood epilepsies are among the associated syndromes.
(ADNFLE), familial temporal lobe epilepsy (FTLE), and other small families with genetic focal epilepsies.1–3 DEPDC5 is a member of the GATOR1 complex that interacts with the mammalian target of rapamycin (mTOR) complex1, which is potentially accessible by mTOR inhibitors.4 Rolandic epilepsy (RE), or benign epilepsy with centrotemporal spikes (BECTS), is a common focal epilepsy syndrome of childhood.5,6 RE is considered related to rare, less benign epilepsy syndromes, often referred to as atypical RE (ARE).7 Notably, “atypical benign partial epilepsy” (ABPE) with astatic seizures and atypical absences,8–10 Landau–Kleffner syndrome (LKS) with acquired speech dyspraxia, epileptic encephalopathies with continuous spike-and-wave during sleep (CSWS), and “epilepsy aphasia spectrum” with language delay and autistic features11,12 might belong to a spectrum with RE on its benign and CSWS/LKS on its severe end.7,10,12 Among those, only BECTS, CSWS, and LKS are currently recognized by the international classification.5 Recently, mutations and copy number variations in GRIN2A and RBFOX genes have been identified in some patients with typical and atypical RE.13,14 From the 1Cologne Center for Genomics, University of Cologne, Cologne, Germany; 2Department of Neuropediatrics, University Medical Center Giessen and Marburg, Giessen, Germany; 3Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany; 4Department of Neurology, Medical University of Vienna, Vienna, Austria; 5Department of Neurology and Epileptology, Hertie Institute of Clinical Brain Research, University of T€ ubingen, T€ ubingen, Germany; 6Graduate School of Cellular and Molecular Neuroscience, University of T€ ubingen, T€ ubingen, Germany; 7 Department of Neuropediatrics, University Medical Center Schleswig8 ubingen, GerHolstein (Kiel Campus), Kiel, Germany; CeGaT GmbH, T€ many; 9Department of Neuropediatrics and Neurorehabilitation, Epilepsy Center for Children and Adolescents, Schoen Clinic Vogtareuth, Vogtareuth, Germany; 10Department of Pediatrics and Neonatology, Medical University of Vienna, Vienna, Austria; 11St Anna Children’s Hospital, Department of Neuropediatrics, Vienna, Austria; 12Department of Pediatrics, Medical University of Innsbruck, Innsbruck, Austria; 13EuroEPINOMICS Consortium; 14Division of Human Genetics, University Children’s Hospital Inselspital, Bern, Switzerland; and 15Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. Address correspondence to Dr Bernd A. Neubauer, Department of Neuropediatrics, University Medical Faculty Giessen, Feulgenstrasse 12, 35385 Giessen, Germany. E-mail: [email protected]
ANN NEUROL 2014;75:788–792 Additional Supporting Information may be found in the online version of this article.
M
utations in DEPDC5 encoding the disheveled, Egl-10, and pleckstrin domain-containing protein 5, have recently been identified as a major cause of inherited focal epilepsies including familial focal epilepsy with variable foci (FFEVF), autosomal dominant nocturnal frontal epilepsy
C 2014 American Neurological Association 788 V
Members of the EuroEPINOMICS Consortium are listed in the Appendix on page 5. Received Oct 20, 2013, and in revised form Feb 18, 2014. Accepted for publication Feb 26, 2014. View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana. 24127
Lal et al: DEPDC5 in Focal Childhood Epilepsies
by all respective local institutional review boards. The investigated cohort consisted of index patients selected from 87 multiplex families with at least 2 affected siblings. In 87 families, at least 1 of the affected probands suffered from RE or ARE; the second affected sibling presented either with RE, ARE, or the trait only detected by electroencephalogram (EEG). Of 87 familial index patients tested (male 5 53, female 5 34), 72 presented with RE, 12 with ARE, and 3 with electrical status epilepticus during sleep (ESES). In addition, we included 120 nonfamilial patients (69 male, 51 female). Of these sporadic patients, 111 suffered from RE, and 9 from ARE.
Sequencing: RE Cohort For all 207 patients, exome data were generated that featured an average coverage >303 for 77% of the target sequences (for details, see Lal et al14). Variants were filtered for high-quality rare variants in DEPDC5 using a comparison with an in-house variation database, dbSNP build 137 (www.ncbi.nlm.nih.gov/ projects/SNP), 1000 Genomes database (www.1000genomes. org), and the Exome Variant Server (http://evs.gs.washington. edu/EVS). Variant validation and segregation analyses were performed using Sanger sequencing following standard protocols.
Screening for DEPDC5 Mutations in Nonrolandic Genetic Focal Epilepsy Syndromes (n 5 82)
FIGURE 1: Segregation of DEPDC5-affecting variants. Segregation analyses could be performed for which DNA samples of family members were available. The respective DEPDC5 mutations (NM_001136029.2, NP_001129501.1) cosegregated with a variable phenotype of either seizures or pathologic electroencephalographic (EEG) patterns in most family members. Only a few individuals carried the respective familial mutation but did not present any clinical features, indicating incomplete penetrance of the mutations. However, subclinical phenotypes (eg, EEG patterns) have not been investigated in these individuals (indicated by question mark). CTS 5 centrotemporal spikes; n.a. 5 DNA was not available for testing; RE 5 rolandic epilepsy.
RE, like FFEVF, ADNFLE, and FTLE, is a nonlesional genetic focal epilepsy, rendering DEPDC5 a plausible candidate. In the present study, we examined whether DEPDC5 mutations are associated with RE (n 5 207) and other less specifiable genetic focal childhood epilepsies (n 5 82).
Patients and Methods DEPDC5 Screening Cohorts To investigate an involvement of DEPDC5 in the etiology of focal epilepsies in childhood, 2 cohorts comprised of 289 European patients were included in the DEPDC5 mutation screen.
Patients: RE Cohort (n 5 207) Patients of European ancestry were recruited at several centers from Germany, Austria, and Canada. The study was approved May 2014
Mutations in DEPDC5 were also screened in a cohort of 82 families with a broad spectrum of genetic focal epilepsy phenotypes, with variable degrees of intellectual disability. This cohort was analyzed using a targeted next generation sequencing panel approach covering 285 epilepsy-associated genes as described previously.15
Results Detection and Inheritance Testing of Rare Variants in DEPDC5 Mutational screening of DEPDC5 in 207 unrelated RE and ARE patients, who underwent whole exome sequencing, identified a total of 2 novel, heterozygous truncating mutations (Fig 1, Fig 2, Supplementary Table), including a frameshift variant p.I1139Mfs*24 (c.3417delA; according to reference transcript NM_001136029.2, reference protein NP_001129501.1) and a splice site variant c.59-1G>C. However, the epilepsy classification of the splice site variant carriers (Family 2) is not consistent with typical RE, because the epilepsy persisted beyond adolescence. We use the term focal epilepsy with centrotemporal spikes in the Supplementary Table and text. In the same cohort, we identified 3 missense mutations (p.V272L, c.814G>T; p.V90I, c.268G>A; p.S1153G, c.3457A>G) of uncertain significance (see Fig 1). The p.V272L (Family 5) and the p.S1153G (Family 7) mutations are predicted to be diseasecausing, whereas the p.V90I (Family 6) exchange is 789
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FIGURE 2: Overview of DEPDC5 variants. Top: Genomic localization and structure of the identified DEPDC5 variants (NM_001136029.2, hg19). Black dashes highlight the variant position. Bottom: Schematic diagram of the DEPDC5 protein (NP_001129501.1); alterations are indicated.
predicted to affect splicing using in silico analysis, using the Mutation T@ster Tool (http://www.mutationtaster.org). In a gene panel approach15 performed on 82 unrelated patients with genetic focal or cryptogenic epilepsy referred for diagnostic purposes, we detected 2 novel nonsense variants p.R865* (c.2593C>T) and p.R243* (c.727C>T). The 7 mutations were distributed throughout the whole gene and protein (see Fig 2). None of these variants were present in dbSNP137, the 1000 Genomes Project database, or in the 6,503 exomes for which variants are currently listed in the National Heart, Lung, and Blood Institute (NHLBI) exome variant server (EVS; accessed June 2013) database. One stop codongenerating variant and 1 frameshift variant in DEPDC5 have been annotated in the 6,503 exomes listed in the EVS database, and no deleterious mutation was observed in >450 exomes of our in-house database fulfilling the same exome sequencing quality filters (various nonepilepsy projects; approximately 80% Caucasian ancestry). Familial Segregation Analysis The segregation of DEPDC5 mutations identified in the RE index patients could be tracked in 6 of 7 families (see Fig 1). In Family 3, the mutation was introduced by the affected grandfather and transmitted to the index patient by the mother. In the remaining 5 families, the variants were inherited from an unaffected parent, once maternally and 4 times paternally. One family (Family 4 in Fig 1) denied DNA sampling in affected relatives, but the family history with many affected individuals suggests an inherited mutation. Four of 18 variant carriers were affected by RE, 1 by centrotemporal spikes without seizures, 2 by a focal genetic epilepsy with centrotemporal spikes (Family 2, termed RE persisting to adulthood by the treating physicians). Four patients were affected by unclassified 790
focal childhood epilepsies (Family 3, Family 4), 1 with a relatively benign course (F3.I-2). The unaffected mutation carriers might best be explained by incomplete penetrance, which has been reported previously for DEPDC5, even for truncation mutations.11–13 In Family 6, the mutation is homozygous in the unaffected father, questioning its pathogenicity. All tested individuals exhibiting seizures carried mutations in DEPDC5. Clinical Features of Individuals with Deleterious DEPDC5 Mutations FAMILY 1 (P.I1139MFS*24; C.3417DELA). The index patient (F1-II.1) presented with 2 nocturnal perioral sensorimotor focal seizures (“twitching of the right side of the mouth and face”) at age 4.8 years. The EEG showed the characteristic left-sided centrotemporal spike focus with sleep activation. Physical examination and developmental history were entirely normal. The child was diagnosed with (classic) RE and was successfully treated with sulthiame. FAMILY 2 (C.59-1G>C, SPLICE SITE). The male index patient (F2-II.1; c.59-1G>C) suffered from sensorimotor seizures of his right arm and revealed a characteristically shaped frontocentral sharp wave focus at age 8 years. He was diagnosed with RE and treated successfully with carbamazepine. After weaning the medication in adolescence, seizures reoccurred. The epilepsy was tentatively labeled “RE persisting to adulthood” by the treating physicians because of the characteristic EEG trait. Focal epilepsy with centrotemporal spikes might be a less equivocal term (FE/CTS in Fig 1). His younger sister (F2-II-2; c.59-1G>C) presented with atypical absences (staring attacks) at age 3.5 years. Characteristically shaped centrotemporal spikes were recorded bilaterally at age 3.5 Volume 75, No. 5
Lal et al: DEPDC5 in Focal Childhood Epilepsies
years, and left-sided only at age 5.5 years. At age 8 years, she suffered from repeated exercise-induced atypical absences preceded by an unspecific aura. Sequence analysis of the SLC2A1 gene was unsuspicious. She is seizurefree with ongoing oxcarbazepine therapy. Both patients are still receiving therapy. High-resolution magnetic resonance imaging scans were negative in both children. FAMILY 3 (P.R865*; C.2593C>T). The mother (F3-II.1;
p.R865*) presented with atonic seizures, starting at age 3 years, without any intellectual deficits. Her first documented EEG at 9 years and later at 9.5 years revealed temporoposterior sharp waves bilaterally. Since age 10 years she has remained seizurefree without medication. Repeated EEG recordings were normal. The benign epilepsy is possibly consistent with ABPE (RE with astatic seizures). We prefer to label it “unclassified benign epilepsy” in Figure 1. Her son (F3-III.1, p.R865*) expressed myoclonic, atonic, and generalized tonic–clonic seizures that started at age 12 weeks. The epilepsy proved therapyresistant, and the child’s development remained severely impaired. The epilepsy was labeled unclassified focal epilepsy (unknown etiology). The grandfather in this family (F3-I.2) was reported to have experienced epileptic seizures in childhood, but no details were available to classify the syndrome. FAMILY 4 (P.R243*; C.727C>T). The father (F4-II.2, DNA not available for testing) of our index patient reportedly had a focal epilepsy that resolved spontaneously during adolescence, possibly consistent with RE. The available index patient (F4-III.3, p.Arg243*), however, did not present with a benign course. The patient’s seizures started at age 8 months. At age 2 years, he showed myoclonic seizures with centrotemporal spikes, and later during adulthood focal tonic seizures and multifocal spikes were documented. The epilepsy could not be attributed to a specific epilepsy syndrome.
Clinical Features of Individuals with DEPDC5 Missense Mutations FAMILY 5 (PV272L; C.814G>T). Family 6 (p.V90I; c.268G>A), Family 7 (p. S1153G; c.3457A>G). For all index patients in these families (F5-II.1, F6-II.1, F7-II.1) the epilepsy was diagnosed as RE based on seizure semiology and EEG findings with characteristically shaped centrotemporal spikes with sleep activation. In Family 5, the mutation-carrying healthy brother (F5-II.2; pV272L) also displayed a characteristic EEG focus at age 4.9 years. The index child (F5-II.1) has been therapy- and seizure-free for many years, with several normal follow-up EEG recordings. In Family 6, the index case (F6-II.1), now 15 years old, has been seizure- and therapy-free since the age of 12. In Family 7, the affected child (F7-II.1) does show very pronounced sleep activation with multifocal spikes and sharp May 2014
waves, at times labeled “transient ESES” by treating physicians. Therapy was initiated with levetiracetam, and was changed to valproate since nocturnal seizures recurred. The patient is seizure-free but still using therapy at age 9 years.
Discussion Our DEPDC5 screen revealed in total 7 rare mutations, 4 of which are predicted to be severely damaging due to the insertion of a stop codon or consensus splice site alteration, whereas the 3 missense variations are considered of uncertain significance. In patients with typical RE, 1 truncating mutation (Family 1) and 3 missense mutations were detected (Families 5, 6, and 7). In silico analysis predicted 2 of 3 missense variants as disease-causing (Families 5 and 7) and 1 modifying splicing (Family 6). However, in Family 6 the unaffected father is homozygous for this variant, which makes it unlikely to be a disease-causing mutation of major effect. The observed frequency of approximately 1.5% (3 in 207) is less than in previous reports on familial focal epilepsy with variable foci, autosomal dominant nocturnal frontal epilepsy, and familial temporal lobe epilepsy.1–3 Five families were part of our initial RE/ARE screening cohort (Families 1, 2, 5, 6, and 7). Family 2 is exceptional, because the epilepsy did not resolve in adolescence. This family was reclassified post hoc as focal epilepsy with centrotemporal spikes (and not RE). In today’s view, this family might also receive the diagnosis of “familial focal epilepsy with variable foci.” Two additional families (Families 3 and 4) of our second cohort, comprised of 82 families with unclassified genetic focal childhood epilepsies, revealed truncating mutations. Both families presented with a more or less private epilepsy phenotype that differs greatly between affected individuals ranging from benign to intractable epilepsy and cognitive impairment. In summary, our results strengthen the association of mutations in DEPDC5 with focal epilepsies and extend the DEPDC5-related phenotypic spectrum to RE and other genetic focal childhood epilepsies with variable symptomatology and course. However, RE does not seem to be a major phenotype in DEPDC5-associated epilepsies.
Acknowledgment This study was supported by the EuroEPINOMICS programme (DFG grants SA434/5-1, T.S.; BN416/5-1, B.N.; LE1030/11-1, H.L.; and NU50/8-1, P.N.; and FWF grant 1643-B09, F.Z.) within the EUROCORES framework of the European Science Foundation. We thank all participants and their families for participating in this study, and the NHLBI GO Exome Sequencing Project and its ongoing studies, which produced and provided 791
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exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL102925), the Seattle GO Sequencing Project (HL-102926), and the Heart GO Sequencing Project (HL-103010).
2.
Ishida S, Picard F, Rudolf G, et al. Mutations of DEPDC5 cause autosomal dominant focal epilepsies. Nat Genet 2013;45:552– 555.
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Martin C, Meloche C, Rioux MF, et al. A recurrent mutation in DEPDC5 predisposes to focal epilepsies in the French-Canadian population. Clin Genet 2013.
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Bar-Peled L, Chantranupong L, Cherniack AD, et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 2013;340: 1100–1106.
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Berg AT, Berkovic SF, Brodie MJ, et al. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005–2009. Epilepsia 2010;51:676–685.
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Sidenvall R, Forsgren L, Heijbel J. Prevalence and characteristics of epilepsy in children in northern Sweden. Seizure 1996;5:139–146.
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Fejerman N. Atypical rolandic epilepsy. Epilepsia 2009;50(suppl 7):9–12.
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Aicardi J, Chevrie JJ. Atypical benign partial epilepsy of childhood. Dev Med Child Neurol 1982;24:281–292.
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Hahn A, Pistohl J, Neubauer BA, Stephani U. Atypical “benign” partial epilepsy or pseudo-Lennox syndrome. Part I: Symptomatology and long-term prognosis. Neuropediatrics 2001;32:1–8.
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Doose H, Hahn A, Neubauer BA, et al. Atypical “benign” partial epilepsy of childhood or pseudo-lennox syndrome. Part II: Family study. Neuropediatrics 2001;32:9–13.
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Pal DK. Epilepsy and neurodevelopmental disorders of language. Curr Opin Neurol 2011;24:126–131.
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Vears DF, Tsai MH, Sadleir LG, et al. Clinical genetic studies in benign childhood epilepsy with centrotemporal spikes. Epilepsia 2012;53:319–324.
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Lemke JR, Lal D, Reinthaler EM, et al. Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nat Genet 2013;45: 1067–1072.
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Lal D, Reinthaler EM, Altm€ uller J, et al. RBFOX1 and RBFOX3 mutations in rolandic epilepsy. PLoS One 2013;8:e73323.
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Lemke JR, Riesch E, Scheurenbrand T, et al. Targeted next generation sequencing as a diagnostic tool in epileptic disorders. Epilepsia 2012;53:1387–1398.
Authorship D.L., E.M.R., and J.S. contributed equally to this work.
Potential Conflicts of Interest H.L.: consultancy, GSK, Valeant, UCB, Eisai; grants, UCB, Sanofi-Aventis; speaking fees, GSK, Valeant, UCB, Eisai, Desitin; paid educational presentations, Virepa/ ILAE, UCB; travel/accommodations/meeting expenses, Pfizer, Desitin. P.N.: grant, Federal Ministry of Education and research (Germany).
Appendix EuroEPINOMICS Consortium contributing members: Dennis Lal, Eva M. Reinthaler, Julian Schubert, Hiltrud Muhle, Gerhard Kluger, Christine B€aumel, Hans Holthausen, Andreas Hahn, Martha Feucht, Birgit Neophytou, Edda Haberlandt, Felicitas Becker, Janine Altm€ uller, Holger Thiele, Johannes R. Lemke, Holger Lerche, Peter N€ urnberg, Thomas Sander, Yvonne Weber, Fritz Zimprich, Bernd A. Neubauer, H. Steinb€ ock, G. M. Ronen, U. Gruber-Sedlmayr, and J. Geldner.
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Volume 75, No. 5
DEPDC5 Mutations in Genetic Focal Epilepsies of Childhood Dennis Lal, PhD,1,2,3 Eva M. Reinthaler, MS,4 Julian Schubert, MSc,5,6 Hiltrud Muhle, MD,7 Erik Riesch, MD,8 Gerhard Kluger, MD,8 Kamel Jabbari, PhD,1 Amit Kawalia, MSc,1 Christine B€ aumel, MD,9 Hans Holthausen, MD,9 Andreas Hahn, MD,2 Martha Feucht, MD,10 Birgit Neophytou, MD,11 Edda Haberlandt, MD,12 Felicitas Becker, MD,5 Janine Altm€ uller, MD,1 Holger Thiele, MD,1,13 Johannes R. Lemke, MD,14 Holger Lerche, MD,5 Peter N€ urnberg, PhD,1,3,15 Thomas Sander, MD,1 Yvonne Weber, MD,5 Fritz Zimprich, MD, PhD,4 and Bernd A. Neubauer, MD2 Recent studies reported DEPDC5 loss-of-function mutations in different focal epilepsy syndromes. Here we identified 1 predicted truncation and 2 missense mutations in 3 children with rolandic epilepsy (3 of 207). In addition, we identified 3 families with unclassified focal childhood epilepsies carrying predicted truncating DEPDC5 mutations (3 of 82). The detected variants were all novel, inherited, and present in all tested affected (n 5 11) and in 7 unaffected family members, indicating low penetrance. Our findings extend the phenotypic spectrum associated with mutations in DEPDC5 and suggest that rolandic epilepsy, albeit rarely, and other nonlesional childhood epilepsies are among the associated syndromes.
(ADNFLE), familial temporal lobe epilepsy (FTLE), and other small families with genetic focal epilepsies.1–3 DEPDC5 is a member of the GATOR1 complex that interacts with the mammalian target of rapamycin (mTOR) complex1, which is potentially accessible by mTOR inhibitors.4 Rolandic epilepsy (RE), or benign epilepsy with centrotemporal spikes (BECTS), is a common focal epilepsy syndrome of childhood.5,6 RE is considered related to rare, less benign epilepsy syndromes, often referred to as atypical RE (ARE).7 Notably, “atypical benign partial epilepsy” (ABPE) with astatic seizures and atypical absences,8–10 Landau–Kleffner syndrome (LKS) with acquired speech dyspraxia, epileptic encephalopathies with continuous spike-and-wave during sleep (CSWS), and “epilepsy aphasia spectrum” with language delay and autistic features11,12 might belong to a spectrum with RE on its benign and CSWS/LKS on its severe end.7,10,12 Among those, only BECTS, CSWS, and LKS are currently recognized by the international classification.5 Recently, mutations and copy number variations in GRIN2A and RBFOX genes have been identified in some patients with typical and atypical RE.13,14 From the 1Cologne Center for Genomics, University of Cologne, Cologne, Germany; 2Department of Neuropediatrics, University Medical Center Giessen and Marburg, Giessen, Germany; 3Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany; 4Department of Neurology, Medical University of Vienna, Vienna, Austria; 5Department of Neurology and Epileptology, Hertie Institute of Clinical Brain Research, University of T€ ubingen, T€ ubingen, Germany; 6Graduate School of Cellular and Molecular Neuroscience, University of T€ ubingen, T€ ubingen, Germany; 7 Department of Neuropediatrics, University Medical Center Schleswig8 ubingen, GerHolstein (Kiel Campus), Kiel, Germany; CeGaT GmbH, T€ many; 9Department of Neuropediatrics and Neurorehabilitation, Epilepsy Center for Children and Adolescents, Schoen Clinic Vogtareuth, Vogtareuth, Germany; 10Department of Pediatrics and Neonatology, Medical University of Vienna, Vienna, Austria; 11St Anna Children’s Hospital, Department of Neuropediatrics, Vienna, Austria; 12Department of Pediatrics, Medical University of Innsbruck, Innsbruck, Austria; 13EuroEPINOMICS Consortium; 14Division of Human Genetics, University Children’s Hospital Inselspital, Bern, Switzerland; and 15Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. Address correspondence to Dr Bernd A. Neubauer, Department of Neuropediatrics, University Medical Faculty Giessen, Feulgenstrasse 12, 35385 Giessen, Germany. E-mail: [email protected]
ANN NEUROL 2014;75:788–792 Additional Supporting Information may be found in the online version of this article.
M
utations in DEPDC5 encoding the disheveled, Egl-10, and pleckstrin domain-containing protein 5, have recently been identified as a major cause of inherited focal epilepsies including familial focal epilepsy with variable foci (FFEVF), autosomal dominant nocturnal frontal epilepsy
C 2014 American Neurological Association 788 V
Members of the EuroEPINOMICS Consortium are listed in the Appendix on page 5. Received Oct 20, 2013, and in revised form Feb 18, 2014. Accepted for publication Feb 26, 2014. View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana. 24127
Lal et al: DEPDC5 in Focal Childhood Epilepsies
by all respective local institutional review boards. The investigated cohort consisted of index patients selected from 87 multiplex families with at least 2 affected siblings. In 87 families, at least 1 of the affected probands suffered from RE or ARE; the second affected sibling presented either with RE, ARE, or the trait only detected by electroencephalogram (EEG). Of 87 familial index patients tested (male 5 53, female 5 34), 72 presented with RE, 12 with ARE, and 3 with electrical status epilepticus during sleep (ESES). In addition, we included 120 nonfamilial patients (69 male, 51 female). Of these sporadic patients, 111 suffered from RE, and 9 from ARE.
Sequencing: RE Cohort For all 207 patients, exome data were generated that featured an average coverage >303 for 77% of the target sequences (for details, see Lal et al14). Variants were filtered for high-quality rare variants in DEPDC5 using a comparison with an in-house variation database, dbSNP build 137 (www.ncbi.nlm.nih.gov/ projects/SNP), 1000 Genomes database (www.1000genomes. org), and the Exome Variant Server (http://evs.gs.washington. edu/EVS). Variant validation and segregation analyses were performed using Sanger sequencing following standard protocols.
Screening for DEPDC5 Mutations in Nonrolandic Genetic Focal Epilepsy Syndromes (n 5 82)
FIGURE 1: Segregation of DEPDC5-affecting variants. Segregation analyses could be performed for which DNA samples of family members were available. The respective DEPDC5 mutations (NM_001136029.2, NP_001129501.1) cosegregated with a variable phenotype of either seizures or pathologic electroencephalographic (EEG) patterns in most family members. Only a few individuals carried the respective familial mutation but did not present any clinical features, indicating incomplete penetrance of the mutations. However, subclinical phenotypes (eg, EEG patterns) have not been investigated in these individuals (indicated by question mark). CTS 5 centrotemporal spikes; n.a. 5 DNA was not available for testing; RE 5 rolandic epilepsy.
RE, like FFEVF, ADNFLE, and FTLE, is a nonlesional genetic focal epilepsy, rendering DEPDC5 a plausible candidate. In the present study, we examined whether DEPDC5 mutations are associated with RE (n 5 207) and other less specifiable genetic focal childhood epilepsies (n 5 82).
Patients and Methods DEPDC5 Screening Cohorts To investigate an involvement of DEPDC5 in the etiology of focal epilepsies in childhood, 2 cohorts comprised of 289 European patients were included in the DEPDC5 mutation screen.
Patients: RE Cohort (n 5 207) Patients of European ancestry were recruited at several centers from Germany, Austria, and Canada. The study was approved May 2014
Mutations in DEPDC5 were also screened in a cohort of 82 families with a broad spectrum of genetic focal epilepsy phenotypes, with variable degrees of intellectual disability. This cohort was analyzed using a targeted next generation sequencing panel approach covering 285 epilepsy-associated genes as described previously.15
Results Detection and Inheritance Testing of Rare Variants in DEPDC5 Mutational screening of DEPDC5 in 207 unrelated RE and ARE patients, who underwent whole exome sequencing, identified a total of 2 novel, heterozygous truncating mutations (Fig 1, Fig 2, Supplementary Table), including a frameshift variant p.I1139Mfs*24 (c.3417delA; according to reference transcript NM_001136029.2, reference protein NP_001129501.1) and a splice site variant c.59-1G>C. However, the epilepsy classification of the splice site variant carriers (Family 2) is not consistent with typical RE, because the epilepsy persisted beyond adolescence. We use the term focal epilepsy with centrotemporal spikes in the Supplementary Table and text. In the same cohort, we identified 3 missense mutations (p.V272L, c.814G>T; p.V90I, c.268G>A; p.S1153G, c.3457A>G) of uncertain significance (see Fig 1). The p.V272L (Family 5) and the p.S1153G (Family 7) mutations are predicted to be diseasecausing, whereas the p.V90I (Family 6) exchange is 789
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FIGURE 2: Overview of DEPDC5 variants. Top: Genomic localization and structure of the identified DEPDC5 variants (NM_001136029.2, hg19). Black dashes highlight the variant position. Bottom: Schematic diagram of the DEPDC5 protein (NP_001129501.1); alterations are indicated.
predicted to affect splicing using in silico analysis, using the Mutation T@ster Tool (http://www.mutationtaster.org). In a gene panel approach15 performed on 82 unrelated patients with genetic focal or cryptogenic epilepsy referred for diagnostic purposes, we detected 2 novel nonsense variants p.R865* (c.2593C>T) and p.R243* (c.727C>T). The 7 mutations were distributed throughout the whole gene and protein (see Fig 2). None of these variants were present in dbSNP137, the 1000 Genomes Project database, or in the 6,503 exomes for which variants are currently listed in the National Heart, Lung, and Blood Institute (NHLBI) exome variant server (EVS; accessed June 2013) database. One stop codongenerating variant and 1 frameshift variant in DEPDC5 have been annotated in the 6,503 exomes listed in the EVS database, and no deleterious mutation was observed in >450 exomes of our in-house database fulfilling the same exome sequencing quality filters (various nonepilepsy projects; approximately 80% Caucasian ancestry). Familial Segregation Analysis The segregation of DEPDC5 mutations identified in the RE index patients could be tracked in 6 of 7 families (see Fig 1). In Family 3, the mutation was introduced by the affected grandfather and transmitted to the index patient by the mother. In the remaining 5 families, the variants were inherited from an unaffected parent, once maternally and 4 times paternally. One family (Family 4 in Fig 1) denied DNA sampling in affected relatives, but the family history with many affected individuals suggests an inherited mutation. Four of 18 variant carriers were affected by RE, 1 by centrotemporal spikes without seizures, 2 by a focal genetic epilepsy with centrotemporal spikes (Family 2, termed RE persisting to adulthood by the treating physicians). Four patients were affected by unclassified 790
focal childhood epilepsies (Family 3, Family 4), 1 with a relatively benign course (F3.I-2). The unaffected mutation carriers might best be explained by incomplete penetrance, which has been reported previously for DEPDC5, even for truncation mutations.11–13 In Family 6, the mutation is homozygous in the unaffected father, questioning its pathogenicity. All tested individuals exhibiting seizures carried mutations in DEPDC5. Clinical Features of Individuals with Deleterious DEPDC5 Mutations FAMILY 1 (P.I1139MFS*24; C.3417DELA). The index patient (F1-II.1) presented with 2 nocturnal perioral sensorimotor focal seizures (“twitching of the right side of the mouth and face”) at age 4.8 years. The EEG showed the characteristic left-sided centrotemporal spike focus with sleep activation. Physical examination and developmental history were entirely normal. The child was diagnosed with (classic) RE and was successfully treated with sulthiame. FAMILY 2 (C.59-1G>C, SPLICE SITE). The male index patient (F2-II.1; c.59-1G>C) suffered from sensorimotor seizures of his right arm and revealed a characteristically shaped frontocentral sharp wave focus at age 8 years. He was diagnosed with RE and treated successfully with carbamazepine. After weaning the medication in adolescence, seizures reoccurred. The epilepsy was tentatively labeled “RE persisting to adulthood” by the treating physicians because of the characteristic EEG trait. Focal epilepsy with centrotemporal spikes might be a less equivocal term (FE/CTS in Fig 1). His younger sister (F2-II-2; c.59-1G>C) presented with atypical absences (staring attacks) at age 3.5 years. Characteristically shaped centrotemporal spikes were recorded bilaterally at age 3.5 Volume 75, No. 5
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years, and left-sided only at age 5.5 years. At age 8 years, she suffered from repeated exercise-induced atypical absences preceded by an unspecific aura. Sequence analysis of the SLC2A1 gene was unsuspicious. She is seizurefree with ongoing oxcarbazepine therapy. Both patients are still receiving therapy. High-resolution magnetic resonance imaging scans were negative in both children. FAMILY 3 (P.R865*; C.2593C>T). The mother (F3-II.1;
p.R865*) presented with atonic seizures, starting at age 3 years, without any intellectual deficits. Her first documented EEG at 9 years and later at 9.5 years revealed temporoposterior sharp waves bilaterally. Since age 10 years she has remained seizurefree without medication. Repeated EEG recordings were normal. The benign epilepsy is possibly consistent with ABPE (RE with astatic seizures). We prefer to label it “unclassified benign epilepsy” in Figure 1. Her son (F3-III.1, p.R865*) expressed myoclonic, atonic, and generalized tonic–clonic seizures that started at age 12 weeks. The epilepsy proved therapyresistant, and the child’s development remained severely impaired. The epilepsy was labeled unclassified focal epilepsy (unknown etiology). The grandfather in this family (F3-I.2) was reported to have experienced epileptic seizures in childhood, but no details were available to classify the syndrome. FAMILY 4 (P.R243*; C.727C>T). The father (F4-II.2, DNA not available for testing) of our index patient reportedly had a focal epilepsy that resolved spontaneously during adolescence, possibly consistent with RE. The available index patient (F4-III.3, p.Arg243*), however, did not present with a benign course. The patient’s seizures started at age 8 months. At age 2 years, he showed myoclonic seizures with centrotemporal spikes, and later during adulthood focal tonic seizures and multifocal spikes were documented. The epilepsy could not be attributed to a specific epilepsy syndrome.
Clinical Features of Individuals with DEPDC5 Missense Mutations FAMILY 5 (PV272L; C.814G>T). Family 6 (p.V90I; c.268G>A), Family 7 (p. S1153G; c.3457A>G). For all index patients in these families (F5-II.1, F6-II.1, F7-II.1) the epilepsy was diagnosed as RE based on seizure semiology and EEG findings with characteristically shaped centrotemporal spikes with sleep activation. In Family 5, the mutation-carrying healthy brother (F5-II.2; pV272L) also displayed a characteristic EEG focus at age 4.9 years. The index child (F5-II.1) has been therapy- and seizure-free for many years, with several normal follow-up EEG recordings. In Family 6, the index case (F6-II.1), now 15 years old, has been seizure- and therapy-free since the age of 12. In Family 7, the affected child (F7-II.1) does show very pronounced sleep activation with multifocal spikes and sharp May 2014
waves, at times labeled “transient ESES” by treating physicians. Therapy was initiated with levetiracetam, and was changed to valproate since nocturnal seizures recurred. The patient is seizure-free but still using therapy at age 9 years.
Discussion Our DEPDC5 screen revealed in total 7 rare mutations, 4 of which are predicted to be severely damaging due to the insertion of a stop codon or consensus splice site alteration, whereas the 3 missense variations are considered of uncertain significance. In patients with typical RE, 1 truncating mutation (Family 1) and 3 missense mutations were detected (Families 5, 6, and 7). In silico analysis predicted 2 of 3 missense variants as disease-causing (Families 5 and 7) and 1 modifying splicing (Family 6). However, in Family 6 the unaffected father is homozygous for this variant, which makes it unlikely to be a disease-causing mutation of major effect. The observed frequency of approximately 1.5% (3 in 207) is less than in previous reports on familial focal epilepsy with variable foci, autosomal dominant nocturnal frontal epilepsy, and familial temporal lobe epilepsy.1–3 Five families were part of our initial RE/ARE screening cohort (Families 1, 2, 5, 6, and 7). Family 2 is exceptional, because the epilepsy did not resolve in adolescence. This family was reclassified post hoc as focal epilepsy with centrotemporal spikes (and not RE). In today’s view, this family might also receive the diagnosis of “familial focal epilepsy with variable foci.” Two additional families (Families 3 and 4) of our second cohort, comprised of 82 families with unclassified genetic focal childhood epilepsies, revealed truncating mutations. Both families presented with a more or less private epilepsy phenotype that differs greatly between affected individuals ranging from benign to intractable epilepsy and cognitive impairment. In summary, our results strengthen the association of mutations in DEPDC5 with focal epilepsies and extend the DEPDC5-related phenotypic spectrum to RE and other genetic focal childhood epilepsies with variable symptomatology and course. However, RE does not seem to be a major phenotype in DEPDC5-associated epilepsies.
Acknowledgment This study was supported by the EuroEPINOMICS programme (DFG grants SA434/5-1, T.S.; BN416/5-1, B.N.; LE1030/11-1, H.L.; and NU50/8-1, P.N.; and FWF grant 1643-B09, F.Z.) within the EUROCORES framework of the European Science Foundation. We thank all participants and their families for participating in this study, and the NHLBI GO Exome Sequencing Project and its ongoing studies, which produced and provided 791
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exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL102925), the Seattle GO Sequencing Project (HL-102926), and the Heart GO Sequencing Project (HL-103010).
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Lemke JR, Lal D, Reinthaler EM, et al. Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nat Genet 2013;45: 1067–1072.
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Authorship D.L., E.M.R., and J.S. contributed equally to this work.
Potential Conflicts of Interest H.L.: consultancy, GSK, Valeant, UCB, Eisai; grants, UCB, Sanofi-Aventis; speaking fees, GSK, Valeant, UCB, Eisai, Desitin; paid educational presentations, Virepa/ ILAE, UCB; travel/accommodations/meeting expenses, Pfizer, Desitin. P.N.: grant, Federal Ministry of Education and research (Germany).
Appendix EuroEPINOMICS Consortium contributing members: Dennis Lal, Eva M. Reinthaler, Julian Schubert, Hiltrud Muhle, Gerhard Kluger, Christine B€aumel, Hans Holthausen, Andreas Hahn, Martha Feucht, Birgit Neophytou, Edda Haberlandt, Felicitas Becker, Janine Altm€ uller, Holger Thiele, Johannes R. Lemke, Holger Lerche, Peter N€ urnberg, Thomas Sander, Yvonne Weber, Fritz Zimprich, Bernd A. Neubauer, H. Steinb€ ock, G. M. Ronen, U. Gruber-Sedlmayr, and J. Geldner.
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