BRIEF COMMUNICATION
Exome sequencing identifies a de novo SCN2A mutation in a patient with intractable seizures, severe intellectual disability, optic atrophy, muscular hypotonia, and brain abnormalities *Anna-Lena Baasch, †Irina H€ uning, ‡Christian Gilissen, §Joerg Klepper, ‡Joris A. Veltman, †Gabriele Gillessen-Kaesbach, ‡Alexander Hoischen, and *Katja Lohmann Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
SUMMARY
Anna-Lena Baasch is a medical student and presents the data of her doctoral thesis in this paper.
Epilepsy is a phenotypically and genetically highly heterogeneous disorder with >200 genes linked to inherited forms of the disease. To identify the underlying genetic cause in a patient with intractable seizures, optic atrophy, severe intellectual disability (ID), brain abnormalities, and muscular hypotonia, we performed exome sequencing in a 5year-old girl and her unaffected parents. In the patient, we detected a novel, de novo missense mutation in the SCN2A (c.5645G>T; p.R1882L) gene encoding the aII-subunit of the voltage-gated sodium channel Nav1.2. A literature review revealed 33 different SCN2A mutations in 14 families with benign forms of epilepsy and in 21 cases with severe phenotypes. Although almost all benign mutations were inherited, the majority of severe mutations occurred de novo. Of interest, de novo SCN2A mutations have also been reported in five patients without seizures but with ID (n = 3) and/or autism (n = 3). In the present study, we successfully used exome sequencing to detect a de novo mutation in a genetically heterogeneous disorder with epilepsy and ID. Using this approach, we expand the phenotypic spectrum of SCN2A mutations. Our own and literature data indicate that SCN2A-linked severe phenotypes are more likely to be caused by de novo mutations. KEY WORDS: Epilepsy, Intellectual disability, Sodium channel, Epileptic encephalopathy, Rett-like syndrome.
Epilepsy is a highly heterogeneous disorder and part of the phenotypic spectrum of numerous syndromes. The development of epileptic seizures can be explained by either hyperexcitability in neuronal cells or by abnormal simultaneous electrical activity within neuronal cell complexes.1 Accepted January 6, 2014; Early View publication March 1, 2014. *Institute of Neurogenetics, University of L€ubeck, L€ubeck, Germany; †Institut f€ ur Humangenetik, Universit€at zu L€ubeck, L€ubeck, Germany; ‡Department of Human Genetics, Nijmegen Center for Molecular Life Sciences, Institute for Genetic and Metabolic Disease, Radboud University Medical Center, Nijmegen, The Netherlands; and §Children’s Hospital Aschaffenburg, Aschaffenburg, Germany Address correspondence to Katja Lohmann, Institute of Neurogenetics, University of L€ ubeck, Ratzeburger Allee 160, 23538 L€ubeck, Germany. E-mail: [email protected] Wiley Periodicals, Inc. © 2014 International League Against Epilepsy
Dysfunction of different ion channels has been shown to be frequently involved in the development and dissemination of seizures.2 The SCN2A gene encodes the aII-subunit of the Nav1.2 that represents one of four voltage-gated sodium channels highly expressed in brain. SCN2A mutations have been found in different forms of benign or severe epilepsy as well as in patients with intellectual disability (ID) and/or autism (Table 1). De novo germline base substitutions occur with a frequency of 10 8 per base pair per generation3 and can be of clinical relevance in offspring of unaffected parents.4 Exome sequencing has emerged as a cost-effective strategy to identify disease-causing mutations including de novo mutations, especially in highly heterogeneous disorders such as epilepsy and ID.5–7
e25
e26 A.-L. Baasch et al. Table 1. Mutations in SCN2A and the associated phenotype Seizures Mutation
Benign
Severe
Intellectual impairment
Autism
Positive family history
Arg102X Met136Ile
No No
Intractable epilepsy Epileptic encephalopathy
Yes Yes
Yes Unknown
No (de novo) Unknown
Arg188Trp Val208Glu Arg223Gln Met252Val Val261Met Ala263Val
No Yes Yes Yes Yes No
No No No No No No
No No No No No No
Yes Yes Yes Yes No (de novo) No (de novo in both families)
Asp322Asn
No
Yes
No
Phe328Val
No
Yes
Glu430Gln Asn503Lysfs*19
Yes No
Febrile and afebrile seizures No No No No Intractable neonatal-onset seizures (one patient), Ohtahara syndrome in monozygotic twins Severe myoclonic epilepsy in infancy Severe myoclonic epilepsy borderline No No
Ala575Val
No
Leu611Valfs*35
No
Asp649Asn
No
Val892Ile Lys905Asn Phe928Cys Arg937Cys
Other features
References 12 11
Yes
None Not specifically reported None None None None None Ataxia myoclonia (in one patient), dentate-olivary dysplasia (in one of the twins) None
No
Yes
None
19
No Yes
No No
Yes No (de novo)
20 6
Severe myoclonic epilepsy borderline No
Yes
No
Yes
None Autoaggressive behavior, typical face None
Yes
No
No (de novo)
6
Yes
No
Unknown
Yes No No No
Severe myoclonic epilepsy in infancy No Epileptic encephalopathy Epileptic encephalopathy No
Autoaggressive behavior; typical face None
No Yes Yes Yes
No Unk. Unk. Yes
Yes No (de novo) Unknown No (de novo)
15 11 11 6
Cys959X Asn1001Lys
No Yes
No No
No No
Yes No
No (de novo) Yes
Leu1003Ile Gly1013X Ile1021Tyrfs*16 Met1128Thr
Yes No No No
No No Yes Yes
No Yes Unk. Yes
Yes No (de novo) No (de novo) Unknown
Glu1211Lys
No
Yes
No
No (de novo)
Developmental delay
21
Arg1312Thr
No
Yes
No
No (de novo)
None
19
Arg1319Gln Leu1330Phe Trp1398X
Yes Yes No
No No Lennox-Gastaut syndrome Acute encephalitis with refractory, repetitive partial seizures Sporadic refractory infantile spasms, evolved to symptomatic generalized epilepsy Severe myoclonic epilepsy in infancy No No Early infantile epileptic encephalopathy
None Not specifically reported Not specifically reported Autoaggressive behavior, typical face None No neonatal or febrile seizures None None Not specifically reported None
No No Yes
No No No
Yes (2 families) Yes No (de novo)
15 25 26
Ile1473Met
No
Sporadic neonatal epileptic encephalopathy
Yes?
No
No (de novo)
Leu1563Val Ile1596Ser Asp1598Gly
Yes Yes No
No No Seizures
No No Yes
No No No
Yes Yes No (de novo)
Migraine in one patient None Behavior problems, sleep disturbances, stereotype behavior Hyponatremia, megalencephaly, deceased at 7 years of age None None
13 14 15 16 16 17,18
19
21
22
10 23 15 10 11 24
21
25 20 27 Continued
Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
e27 De novo SCN2A Mutation Table 1. Continued. Seizures Mutation
Benign
Severe
Intellectual impairment
Autism
Positive family history
Lys1641Asn Arg1882Gln Arg1882Leu
Yes No No
No Epileptic encephalopathy Intractable epilepsy and encephalopathy
No Yes Yes
No Unk. No
Yes No (de novo) No (de novo)
Arg1918His
No
Febrile seizures, childhood absence epilepsy
No
No
Unknown
Herein we report a de novo missense mutation in SCN2A detected by exome sequencing expanding the associated phenotype.
Methods Genetic analyses The study was approved by the ethics committee at the University of L€ ubeck (Germany) and all participants gave informed consent. We performed exome sequencing (enrichment with SureSelect Human Exome kit v2; Agilent Technologies, Santa Clara, CA, U.S.A) on a SOLiD 5500xl instrument in the 5-year-old proband with a complex phenotype and her unaffected parents. Detected variants were filtered by a systematic algorithm to identify potential de novo variants as described,8 with the exception that we kept variants with a known frequency 60 candidate genes (Table S1). These genes were selected by a search of the Online Mendelian Inheritance in Man (OMIM) database (http://omim.org/) using the search terms “temperature homeostasis,” “temperature regulation,” and “optic atrophy,” since these features have not previously been reported in carriers of SCN2A mutations. Literature review We first searched for known SCN2A mutations in the public version of Human Gene Mutation Database (HGMD at
Other features Developmental delay, hypotonia, infantile spasms and minor dysmorphisms None Not specifically reported Microcephaly, optic atrophy, muscle hypotonia, psychomotor retardation None
References
28 11 Current study
29
http://www.hgmd.cf.ac.uk/ac/index.php), which contained variants published until December 2009. After inspection of all the listed references and relevant references therein, we searched PubMed for most recent publications (from January 2010 to May 2013) using the search term “SCN2A.”
Results Identification of a de novo SCN2A mutation Exome sequencing identified about 35,000 variants in each of the three investigated individuals. Of these, 14 protein-changing variants were called only in the patient but not in either of the parents. By resequencing we confirmed two variants, but one of them was also detected in a parent, leaving one de novo variant representing a missense mutation in exon 26 of the SCN2A gene (c.5645G>T: p.R1882L). This mutation was neither observed in 500 ethnically matched controls, nor reported in dbSNP or in 6,500 exomes of the Exome Variant Server (http://evs.gs.washington.edu/EVS/). The mutation is highly conserved and predicted to be pathogenic by SIFT (http://sift.jcvi.org/www/SIFT_seq_submit2.html), Polyphen (http://genetics.bwh.harvard.edu/pph2/), MutPred (http:// mutpred.mutdb.org/), SNPs&GO (http://snps-and-go.bio comp.unibo.it/snps-and-go/), and MutationTaster (http:// www.mutationtaster.org/). The amino acid change is located in the intracellular C terminal region of the encoded voltage-gated sodium channel Nav1.2. Among another 54 patients with seizures and ID, we did not detect additional novel SCN2A mutations, but we found two known rare (minor allele frequency 60 candidate genes that had been previously linked to epilepsy, optic atrophy, or temperature regulation (Table S1). Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
e28 A.-L. Baasch et al. Case report The patient was born to healthy, nonconsanguineous parents after an uneventful pregnancy. A cesarean section was performed at 36 + 6 weeks of gestation due to silent cardiotocography. Birth weight was 2,880 g (+0.3 standard deviation [SD]), length 52 cm (+0.6 SD), occipital frontal circumference (OFC) 34.5 cm (+0.5 SD). The nondysmorphic newborn was hypotonic and required cardiorespiratory support. Neonatal sepsis was suspected on clinical grounds and antibiotic treatment was initiated. On day 1 she developed focal and generalized tonic–clonic seizures with opisthotonus, bradycardia, and cyanosis. Electroencephalography (EEG) showed generalized and irregular spike wave and polyspike wave activity. Vitamin B6, pyridoxal phosphate, and folic acid were ineffective. Seizure character changed constantly during the newborn period and was resistant to phenobarbitone, sulthiame, valproate, topiramate, levetiracetam, ketogenic diet, vigabatrin, and steroids. Intensive metabolic workups including nuclear magnetic resonance (NMR) spectroscopy of cerebrospinal fluid (CSF) were uninformative. Magnetic resonance imaging (MRI) at the age of 11 months showed supratentorial atrophy, hypoplastic corpus callosum (Fig. 1A). Calcifications were detected by computerized tomography (CT) of the brain. Reexamination at the age of 21 months showed global motor, intellectual, and language impairment. She was severely hypotonic, and unable to turn, sit, crawl, or stand unsupported. No development of speech or purposeful movements was observed. Communication and eye contact was extremely limited in the presence of bilateral optic atrophy and marked microcephaly (OFC 44 cm, 3.4 SD). She currently remains severely impaired with prominent truncal hypotonia, no purposeful hand movements, no speech, and inability to sit or stand (Fig. 1B). The neurodevelopmental decline eventually stabilized. Family history was unremarkable for genetic and neurologic diseases.
A
B
Figure 1. (A) Cranial MRI at the age of 1 year displaying marked global atrophy of supratentorial gray and white matter and hydrocephalus e vacuo. (B) The nondysmorphic patient at the age of 5 years illustrating marked facial hypotonia. Epilepsia ILAE Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
Genetic studies including chromosome analysis, array comparative genomic hybridization (aCGH), X-inactivation studies and sequence analysis of candidate genes such as ARX, CDKL5, FOXG, MCF2C, MECP2, PCDH19, PNPO, RAB3GAP-1/-2, RNASEH2-A/-B/-C, SCN1, SLC9A6, TCF4, TREX1, and TUBA were all negative. Reported SCN2A mutations Literature review revealed 16 SCN2A missense mutations reported in HGMD and 17 additional mutations in PubMed. Most of the mutations (n = 33) were described in patients with epilepsy including mutations in 14 families with benign and 21 patients with severe phenotypes. Although almost all variants reported to cause benign forms of epilepsy were inherited (13/14), the majority of severe mutations occurred de novo (12/16, no information for five patients; Table 1). A total of 15 patients presented with ID, 3 of whom did not report any seizures, and another 5 patients had autism, 2 of whom also had severe seizures (Table 1).
Discussion In clinical practice, pediatric neurologists may encounter patients with complex and severe but unspecific neurologic impairment and epileptic encephalopathy. Extensive laboratory, metabolic, imaging, and molecular workup may not yield diagnostic clues to the etiology of the disease. In our patient, single gene analysis of 17 candidate genes was costand labor-intensive and remained uninformative. However, using exome sequencing in the patient and her unaffected parents, we detected a novel de novo mutation in SCN2A in the proband expanding the phenotypic spectrum of SCN2A mutations. Based on our own data and the literature review, de novo SCN2A mutations appear to cause a more severe phenotype than inherited mutations (Table 1). De novo SCN2A mutations are also related to autism, especially nonsense variants.10 The phenotype in our patient with a novel SNC2A mutation is more severe than previously described for any SCN2A mutation, and included optic atrophy and temperature regulation problems. Using the exome data, we did not detect any mutation in >60 candidate genes that would explain these additional clinical features. Therefore, the question remains whether these features are related to a primary genetic cause (SCN2A or other) or are secondary and acquired due to complications during neonatal phase (sepsis), or severe seizures leading to encephalopathy. The phenomenon of phenotypic heterogeneity is well known for many diseases and refers to differences in the clinical presentation in carriers of mutations in the same gene or even among carriers of the same mutation. For instance, carriers of the same Arg583Gln mutation in CACNA1A can present with spinocerebellar ataxia or migraine with hemiplegia with or without cerebellar ataxia (OMIM #601011). Environmental or genetic modifiers are currently discussed to
e29 De novo SCN2A Mutation explain this phenomenon but are difficult to elucidate, especially in single cases. Notably, a different substitution of the very same amino acid (R1882Q) has recently been reported in another patient with epileptic encephalopathy, but no additional clinical features have been reported.11 It remains to be investigated if the altered nucleotide represents a mutational hot spot. This study provides another example that exome sequencing is a powerful tool to detect de novo mutations, especially when applied for heterogeneous disorders such as epilepsy and ID. We identified a mutation in SCN2A that was not detected by candidate gene approaches due to the atypical phenotype.
Acknowledgments The authors would like to thank the patients for their participation and support of this study. This work was supported by a grant from the Renate Maass foundation (to KL) and intramural funding of the University of L€ ubeck “Schwerpunktprogramm: Medizinische Genetik – Von seltenen Varianten zur Krankheitsentstehung” (to GGK and KL).
Disclosure None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
References 1. Oliva M, Berkovic SF, Petrou S. Sodium channels and the neurobiology of epilepsy. Epilepsia 2012;53:1849–1859. 2. Mizielinska SM. Ion channels in epilepsy. Biochem Soc Trans 2007;35:1077–1079. 3. Abecasis GR, Altshuler D, Auton A, et al. A map of human genome variation from population-scale sequencing. Nature 2010;467:1061– 1073. 4. Ku CS, Tan EK, Cooper DN. From the periphery to centre stage: de novo single nucleotide variants play a key role in human genetic disease. J Med Genet 2013;50:203–211. 5. Veltman JA, Brunner HG. De novo mutations in human genetic disease. Nat Rev Genet 2012;13:565–575. 6. Rauch A, Wieczorek D, Graf E, et al. Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study. Lancet 2012;380:1674–1682. 7. Veeramah KR, Johnstone L, Karafet TM, et al. Exome sequencing reveals new causal mutations in children with epileptic encephalopathies. Epilepsia 2013;54:1270–1281. 8. Vissers LE, de Ligt J, Gilissen C, et al. A de novo paradigm for mental retardation. Nat Genet 2010;42:1109–1112. 9. Kasten M, Hagenah J, Graf J, et al. Cohort Profile: a population-based cohort to study non-motor symptoms in parkinsonism (EPIPARK). Int J Epidemiol 2013;42:128–128k. 10. Sanders SJ, Murtha MT, Gupta AR, et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature 2012;485:237–241. 11. Carvill GL, Heavin SB, Yendle SC, et al. Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1. Nat Genet 2013;45:825–830.
12. Kamiya K, Kaneda M, Sugawara T, et al. A nonsense mutation of the sodium channel gene SCN2A in a patient with intractable epilepsy and mental decline. J Neurosci 2004;24:2690–2698. 13. Sugawara T, Tsurubuchi Y, Agarwala KL, et al. A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction. Proc Natl Acad Sci U S A 2001;98:6384–6389. 14. Lemke JR, Riesch E, Scheurenbrand T, et al. Targeted next generation sequencing as a diagnostic tool in epileptic disorders. Epilepsia 2012;53:1387–1398. 15. Berkovic SF, Heron SE, Giordano L, et al. Benign familial neonatalinfantile seizures: characterization of a new sodium channelopathy. Ann Neurol 2004;55:550–557. 16. Liao Y, Deprez L, Maljevic S, et al. Molecular correlates of agedependent seizures in an inherited neonatal-infantile epilepsy. Brain 2010;133:1403–1414. 17. Liao Y, Anttonen AK, Liukkonen E, et al. SCN2A mutation associated with neonatal epilepsy, late-onset episodic ataxia, myoclonus, and pain. Neurology 2010;75:1454–1458. 18. Touma M, Joshi M, Connolly MC, et al. Whole genome sequencing identifies SCN2A mutation in monozygotic twins with Ohtahara syndrome and unique neuropathologic findings. Epilepsia 2013;54: e81–85. 19. Shi X, Yasumoto S, Nakagawa E, et al. Missense mutation of the sodium channel gene SCN2A causes Dravet syndrome. Brain Dev 2009;31:758–762. 20. Herlenius E, Heron SE, Grinton BE, et al. SCN2A mutations and benign familial neonatal-infantile seizures: the phenotypic spectrum. Epilepsia 2007;48:1138–1142. 21. Ogiwara I, Ito K, Sawaishi Y, et al. De novo mutations of voltagegated sodium channel alphaII gene SCN2A in intractable epilepsies. Neurology 2009;73:1046–1053. 22. Wang JW, Shi XY, Kurahashi H, et al. Prevalence of SCN1A mutations in children with suspected Dravet syndrome and intractable childhood epilepsy. Epilepsy Res 2012;102:195–200. 23. Striano P, Bordo L, Lispi ML, et al. A novel SCN2A mutation in family with benign familial infantile seizures. Epilepsia 2006;47:218– 220. 24. Kobayashi K, Ohzono H, Shinohara M, et al. Acute encephalopathy with a novel point mutation in the SCN2A gene. Epilepsy Res 2012;102:109–112. 25. Heron SE, Crossland KM, Andermann E, et al. Sodium-channel defects in benign familial neonatal-infantile seizures. Lancet 2002;360:851–852. 26. de Ligt J, Willemsen MH, van Bon BW, et al. Diagnostic exome sequencing in persons with severe intellectual disability. N Engl J Med 2012;367:1921–1929. 27. Need AC, Shashi V, Hitomi Y, et al. Clinical application of exome sequencing in undiagnosed genetic conditions. J Med Genet 2012;49:353–361. 28. Zara F, Specchio N, Striano P, et al. Genetic testing in benign familial epilepsies of the first year of life: clinical and diagnostic significance. Epilepsia 2013;54:425–436. 29. Haug K, Hallmann K, Rebstock J, et al. The voltage-gated sodium channel gene SCN2A and idiopathic generalized epilepsy. Epilepsy Res 2001;47:243–246.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Candidate genes for disturbed temperature regulation and optic atrophy.
Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
Exome sequencing identifies a de novo SCN2A mutation in a patient with intractable seizures, severe intellectual disability, optic atrophy, muscular hypotonia, and brain abnormalities *Anna-Lena Baasch, †Irina H€ uning, ‡Christian Gilissen, §Joerg Klepper, ‡Joris A. Veltman, †Gabriele Gillessen-Kaesbach, ‡Alexander Hoischen, and *Katja Lohmann Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
SUMMARY
Anna-Lena Baasch is a medical student and presents the data of her doctoral thesis in this paper.
Epilepsy is a phenotypically and genetically highly heterogeneous disorder with >200 genes linked to inherited forms of the disease. To identify the underlying genetic cause in a patient with intractable seizures, optic atrophy, severe intellectual disability (ID), brain abnormalities, and muscular hypotonia, we performed exome sequencing in a 5year-old girl and her unaffected parents. In the patient, we detected a novel, de novo missense mutation in the SCN2A (c.5645G>T; p.R1882L) gene encoding the aII-subunit of the voltage-gated sodium channel Nav1.2. A literature review revealed 33 different SCN2A mutations in 14 families with benign forms of epilepsy and in 21 cases with severe phenotypes. Although almost all benign mutations were inherited, the majority of severe mutations occurred de novo. Of interest, de novo SCN2A mutations have also been reported in five patients without seizures but with ID (n = 3) and/or autism (n = 3). In the present study, we successfully used exome sequencing to detect a de novo mutation in a genetically heterogeneous disorder with epilepsy and ID. Using this approach, we expand the phenotypic spectrum of SCN2A mutations. Our own and literature data indicate that SCN2A-linked severe phenotypes are more likely to be caused by de novo mutations. KEY WORDS: Epilepsy, Intellectual disability, Sodium channel, Epileptic encephalopathy, Rett-like syndrome.
Epilepsy is a highly heterogeneous disorder and part of the phenotypic spectrum of numerous syndromes. The development of epileptic seizures can be explained by either hyperexcitability in neuronal cells or by abnormal simultaneous electrical activity within neuronal cell complexes.1 Accepted January 6, 2014; Early View publication March 1, 2014. *Institute of Neurogenetics, University of L€ubeck, L€ubeck, Germany; †Institut f€ ur Humangenetik, Universit€at zu L€ubeck, L€ubeck, Germany; ‡Department of Human Genetics, Nijmegen Center for Molecular Life Sciences, Institute for Genetic and Metabolic Disease, Radboud University Medical Center, Nijmegen, The Netherlands; and §Children’s Hospital Aschaffenburg, Aschaffenburg, Germany Address correspondence to Katja Lohmann, Institute of Neurogenetics, University of L€ ubeck, Ratzeburger Allee 160, 23538 L€ubeck, Germany. E-mail: [email protected] Wiley Periodicals, Inc. © 2014 International League Against Epilepsy
Dysfunction of different ion channels has been shown to be frequently involved in the development and dissemination of seizures.2 The SCN2A gene encodes the aII-subunit of the Nav1.2 that represents one of four voltage-gated sodium channels highly expressed in brain. SCN2A mutations have been found in different forms of benign or severe epilepsy as well as in patients with intellectual disability (ID) and/or autism (Table 1). De novo germline base substitutions occur with a frequency of 10 8 per base pair per generation3 and can be of clinical relevance in offspring of unaffected parents.4 Exome sequencing has emerged as a cost-effective strategy to identify disease-causing mutations including de novo mutations, especially in highly heterogeneous disorders such as epilepsy and ID.5–7
e25
e26 A.-L. Baasch et al. Table 1. Mutations in SCN2A and the associated phenotype Seizures Mutation
Benign
Severe
Intellectual impairment
Autism
Positive family history
Arg102X Met136Ile
No No
Intractable epilepsy Epileptic encephalopathy
Yes Yes
Yes Unknown
No (de novo) Unknown
Arg188Trp Val208Glu Arg223Gln Met252Val Val261Met Ala263Val
No Yes Yes Yes Yes No
No No No No No No
No No No No No No
Yes Yes Yes Yes No (de novo) No (de novo in both families)
Asp322Asn
No
Yes
No
Phe328Val
No
Yes
Glu430Gln Asn503Lysfs*19
Yes No
Febrile and afebrile seizures No No No No Intractable neonatal-onset seizures (one patient), Ohtahara syndrome in monozygotic twins Severe myoclonic epilepsy in infancy Severe myoclonic epilepsy borderline No No
Ala575Val
No
Leu611Valfs*35
No
Asp649Asn
No
Val892Ile Lys905Asn Phe928Cys Arg937Cys
Other features
References 12 11
Yes
None Not specifically reported None None None None None Ataxia myoclonia (in one patient), dentate-olivary dysplasia (in one of the twins) None
No
Yes
None
19
No Yes
No No
Yes No (de novo)
20 6
Severe myoclonic epilepsy borderline No
Yes
No
Yes
None Autoaggressive behavior, typical face None
Yes
No
No (de novo)
6
Yes
No
Unknown
Yes No No No
Severe myoclonic epilepsy in infancy No Epileptic encephalopathy Epileptic encephalopathy No
Autoaggressive behavior; typical face None
No Yes Yes Yes
No Unk. Unk. Yes
Yes No (de novo) Unknown No (de novo)
15 11 11 6
Cys959X Asn1001Lys
No Yes
No No
No No
Yes No
No (de novo) Yes
Leu1003Ile Gly1013X Ile1021Tyrfs*16 Met1128Thr
Yes No No No
No No Yes Yes
No Yes Unk. Yes
Yes No (de novo) No (de novo) Unknown
Glu1211Lys
No
Yes
No
No (de novo)
Developmental delay
21
Arg1312Thr
No
Yes
No
No (de novo)
None
19
Arg1319Gln Leu1330Phe Trp1398X
Yes Yes No
No No Lennox-Gastaut syndrome Acute encephalitis with refractory, repetitive partial seizures Sporadic refractory infantile spasms, evolved to symptomatic generalized epilepsy Severe myoclonic epilepsy in infancy No No Early infantile epileptic encephalopathy
None Not specifically reported Not specifically reported Autoaggressive behavior, typical face None No neonatal or febrile seizures None None Not specifically reported None
No No Yes
No No No
Yes (2 families) Yes No (de novo)
15 25 26
Ile1473Met
No
Sporadic neonatal epileptic encephalopathy
Yes?
No
No (de novo)
Leu1563Val Ile1596Ser Asp1598Gly
Yes Yes No
No No Seizures
No No Yes
No No No
Yes Yes No (de novo)
Migraine in one patient None Behavior problems, sleep disturbances, stereotype behavior Hyponatremia, megalencephaly, deceased at 7 years of age None None
13 14 15 16 16 17,18
19
21
22
10 23 15 10 11 24
21
25 20 27 Continued
Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
e27 De novo SCN2A Mutation Table 1. Continued. Seizures Mutation
Benign
Severe
Intellectual impairment
Autism
Positive family history
Lys1641Asn Arg1882Gln Arg1882Leu
Yes No No
No Epileptic encephalopathy Intractable epilepsy and encephalopathy
No Yes Yes
No Unk. No
Yes No (de novo) No (de novo)
Arg1918His
No
Febrile seizures, childhood absence epilepsy
No
No
Unknown
Herein we report a de novo missense mutation in SCN2A detected by exome sequencing expanding the associated phenotype.
Methods Genetic analyses The study was approved by the ethics committee at the University of L€ ubeck (Germany) and all participants gave informed consent. We performed exome sequencing (enrichment with SureSelect Human Exome kit v2; Agilent Technologies, Santa Clara, CA, U.S.A) on a SOLiD 5500xl instrument in the 5-year-old proband with a complex phenotype and her unaffected parents. Detected variants were filtered by a systematic algorithm to identify potential de novo variants as described,8 with the exception that we kept variants with a known frequency 60 candidate genes (Table S1). These genes were selected by a search of the Online Mendelian Inheritance in Man (OMIM) database (http://omim.org/) using the search terms “temperature homeostasis,” “temperature regulation,” and “optic atrophy,” since these features have not previously been reported in carriers of SCN2A mutations. Literature review We first searched for known SCN2A mutations in the public version of Human Gene Mutation Database (HGMD at
Other features Developmental delay, hypotonia, infantile spasms and minor dysmorphisms None Not specifically reported Microcephaly, optic atrophy, muscle hypotonia, psychomotor retardation None
References
28 11 Current study
29
http://www.hgmd.cf.ac.uk/ac/index.php), which contained variants published until December 2009. After inspection of all the listed references and relevant references therein, we searched PubMed for most recent publications (from January 2010 to May 2013) using the search term “SCN2A.”
Results Identification of a de novo SCN2A mutation Exome sequencing identified about 35,000 variants in each of the three investigated individuals. Of these, 14 protein-changing variants were called only in the patient but not in either of the parents. By resequencing we confirmed two variants, but one of them was also detected in a parent, leaving one de novo variant representing a missense mutation in exon 26 of the SCN2A gene (c.5645G>T: p.R1882L). This mutation was neither observed in 500 ethnically matched controls, nor reported in dbSNP or in 6,500 exomes of the Exome Variant Server (http://evs.gs.washington.edu/EVS/). The mutation is highly conserved and predicted to be pathogenic by SIFT (http://sift.jcvi.org/www/SIFT_seq_submit2.html), Polyphen (http://genetics.bwh.harvard.edu/pph2/), MutPred (http:// mutpred.mutdb.org/), SNPs&GO (http://snps-and-go.bio comp.unibo.it/snps-and-go/), and MutationTaster (http:// www.mutationtaster.org/). The amino acid change is located in the intracellular C terminal region of the encoded voltage-gated sodium channel Nav1.2. Among another 54 patients with seizures and ID, we did not detect additional novel SCN2A mutations, but we found two known rare (minor allele frequency 60 candidate genes that had been previously linked to epilepsy, optic atrophy, or temperature regulation (Table S1). Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
e28 A.-L. Baasch et al. Case report The patient was born to healthy, nonconsanguineous parents after an uneventful pregnancy. A cesarean section was performed at 36 + 6 weeks of gestation due to silent cardiotocography. Birth weight was 2,880 g (+0.3 standard deviation [SD]), length 52 cm (+0.6 SD), occipital frontal circumference (OFC) 34.5 cm (+0.5 SD). The nondysmorphic newborn was hypotonic and required cardiorespiratory support. Neonatal sepsis was suspected on clinical grounds and antibiotic treatment was initiated. On day 1 she developed focal and generalized tonic–clonic seizures with opisthotonus, bradycardia, and cyanosis. Electroencephalography (EEG) showed generalized and irregular spike wave and polyspike wave activity. Vitamin B6, pyridoxal phosphate, and folic acid were ineffective. Seizure character changed constantly during the newborn period and was resistant to phenobarbitone, sulthiame, valproate, topiramate, levetiracetam, ketogenic diet, vigabatrin, and steroids. Intensive metabolic workups including nuclear magnetic resonance (NMR) spectroscopy of cerebrospinal fluid (CSF) were uninformative. Magnetic resonance imaging (MRI) at the age of 11 months showed supratentorial atrophy, hypoplastic corpus callosum (Fig. 1A). Calcifications were detected by computerized tomography (CT) of the brain. Reexamination at the age of 21 months showed global motor, intellectual, and language impairment. She was severely hypotonic, and unable to turn, sit, crawl, or stand unsupported. No development of speech or purposeful movements was observed. Communication and eye contact was extremely limited in the presence of bilateral optic atrophy and marked microcephaly (OFC 44 cm, 3.4 SD). She currently remains severely impaired with prominent truncal hypotonia, no purposeful hand movements, no speech, and inability to sit or stand (Fig. 1B). The neurodevelopmental decline eventually stabilized. Family history was unremarkable for genetic and neurologic diseases.
A
B
Figure 1. (A) Cranial MRI at the age of 1 year displaying marked global atrophy of supratentorial gray and white matter and hydrocephalus e vacuo. (B) The nondysmorphic patient at the age of 5 years illustrating marked facial hypotonia. Epilepsia ILAE Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
Genetic studies including chromosome analysis, array comparative genomic hybridization (aCGH), X-inactivation studies and sequence analysis of candidate genes such as ARX, CDKL5, FOXG, MCF2C, MECP2, PCDH19, PNPO, RAB3GAP-1/-2, RNASEH2-A/-B/-C, SCN1, SLC9A6, TCF4, TREX1, and TUBA were all negative. Reported SCN2A mutations Literature review revealed 16 SCN2A missense mutations reported in HGMD and 17 additional mutations in PubMed. Most of the mutations (n = 33) were described in patients with epilepsy including mutations in 14 families with benign and 21 patients with severe phenotypes. Although almost all variants reported to cause benign forms of epilepsy were inherited (13/14), the majority of severe mutations occurred de novo (12/16, no information for five patients; Table 1). A total of 15 patients presented with ID, 3 of whom did not report any seizures, and another 5 patients had autism, 2 of whom also had severe seizures (Table 1).
Discussion In clinical practice, pediatric neurologists may encounter patients with complex and severe but unspecific neurologic impairment and epileptic encephalopathy. Extensive laboratory, metabolic, imaging, and molecular workup may not yield diagnostic clues to the etiology of the disease. In our patient, single gene analysis of 17 candidate genes was costand labor-intensive and remained uninformative. However, using exome sequencing in the patient and her unaffected parents, we detected a novel de novo mutation in SCN2A in the proband expanding the phenotypic spectrum of SCN2A mutations. Based on our own data and the literature review, de novo SCN2A mutations appear to cause a more severe phenotype than inherited mutations (Table 1). De novo SCN2A mutations are also related to autism, especially nonsense variants.10 The phenotype in our patient with a novel SNC2A mutation is more severe than previously described for any SCN2A mutation, and included optic atrophy and temperature regulation problems. Using the exome data, we did not detect any mutation in >60 candidate genes that would explain these additional clinical features. Therefore, the question remains whether these features are related to a primary genetic cause (SCN2A or other) or are secondary and acquired due to complications during neonatal phase (sepsis), or severe seizures leading to encephalopathy. The phenomenon of phenotypic heterogeneity is well known for many diseases and refers to differences in the clinical presentation in carriers of mutations in the same gene or even among carriers of the same mutation. For instance, carriers of the same Arg583Gln mutation in CACNA1A can present with spinocerebellar ataxia or migraine with hemiplegia with or without cerebellar ataxia (OMIM #601011). Environmental or genetic modifiers are currently discussed to
e29 De novo SCN2A Mutation explain this phenomenon but are difficult to elucidate, especially in single cases. Notably, a different substitution of the very same amino acid (R1882Q) has recently been reported in another patient with epileptic encephalopathy, but no additional clinical features have been reported.11 It remains to be investigated if the altered nucleotide represents a mutational hot spot. This study provides another example that exome sequencing is a powerful tool to detect de novo mutations, especially when applied for heterogeneous disorders such as epilepsy and ID. We identified a mutation in SCN2A that was not detected by candidate gene approaches due to the atypical phenotype.
Acknowledgments The authors would like to thank the patients for their participation and support of this study. This work was supported by a grant from the Renate Maass foundation (to KL) and intramural funding of the University of L€ ubeck “Schwerpunktprogramm: Medizinische Genetik – Von seltenen Varianten zur Krankheitsentstehung” (to GGK and KL).
Disclosure None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
References 1. Oliva M, Berkovic SF, Petrou S. Sodium channels and the neurobiology of epilepsy. Epilepsia 2012;53:1849–1859. 2. Mizielinska SM. Ion channels in epilepsy. Biochem Soc Trans 2007;35:1077–1079. 3. Abecasis GR, Altshuler D, Auton A, et al. A map of human genome variation from population-scale sequencing. Nature 2010;467:1061– 1073. 4. Ku CS, Tan EK, Cooper DN. From the periphery to centre stage: de novo single nucleotide variants play a key role in human genetic disease. J Med Genet 2013;50:203–211. 5. Veltman JA, Brunner HG. De novo mutations in human genetic disease. Nat Rev Genet 2012;13:565–575. 6. Rauch A, Wieczorek D, Graf E, et al. Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study. Lancet 2012;380:1674–1682. 7. Veeramah KR, Johnstone L, Karafet TM, et al. Exome sequencing reveals new causal mutations in children with epileptic encephalopathies. Epilepsia 2013;54:1270–1281. 8. Vissers LE, de Ligt J, Gilissen C, et al. A de novo paradigm for mental retardation. Nat Genet 2010;42:1109–1112. 9. Kasten M, Hagenah J, Graf J, et al. Cohort Profile: a population-based cohort to study non-motor symptoms in parkinsonism (EPIPARK). Int J Epidemiol 2013;42:128–128k. 10. Sanders SJ, Murtha MT, Gupta AR, et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature 2012;485:237–241. 11. Carvill GL, Heavin SB, Yendle SC, et al. Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1. Nat Genet 2013;45:825–830.
12. Kamiya K, Kaneda M, Sugawara T, et al. A nonsense mutation of the sodium channel gene SCN2A in a patient with intractable epilepsy and mental decline. J Neurosci 2004;24:2690–2698. 13. Sugawara T, Tsurubuchi Y, Agarwala KL, et al. A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction. Proc Natl Acad Sci U S A 2001;98:6384–6389. 14. Lemke JR, Riesch E, Scheurenbrand T, et al. Targeted next generation sequencing as a diagnostic tool in epileptic disorders. Epilepsia 2012;53:1387–1398. 15. Berkovic SF, Heron SE, Giordano L, et al. Benign familial neonatalinfantile seizures: characterization of a new sodium channelopathy. Ann Neurol 2004;55:550–557. 16. Liao Y, Deprez L, Maljevic S, et al. Molecular correlates of agedependent seizures in an inherited neonatal-infantile epilepsy. Brain 2010;133:1403–1414. 17. Liao Y, Anttonen AK, Liukkonen E, et al. SCN2A mutation associated with neonatal epilepsy, late-onset episodic ataxia, myoclonus, and pain. Neurology 2010;75:1454–1458. 18. Touma M, Joshi M, Connolly MC, et al. Whole genome sequencing identifies SCN2A mutation in monozygotic twins with Ohtahara syndrome and unique neuropathologic findings. Epilepsia 2013;54: e81–85. 19. Shi X, Yasumoto S, Nakagawa E, et al. Missense mutation of the sodium channel gene SCN2A causes Dravet syndrome. Brain Dev 2009;31:758–762. 20. Herlenius E, Heron SE, Grinton BE, et al. SCN2A mutations and benign familial neonatal-infantile seizures: the phenotypic spectrum. Epilepsia 2007;48:1138–1142. 21. Ogiwara I, Ito K, Sawaishi Y, et al. De novo mutations of voltagegated sodium channel alphaII gene SCN2A in intractable epilepsies. Neurology 2009;73:1046–1053. 22. Wang JW, Shi XY, Kurahashi H, et al. Prevalence of SCN1A mutations in children with suspected Dravet syndrome and intractable childhood epilepsy. Epilepsy Res 2012;102:195–200. 23. Striano P, Bordo L, Lispi ML, et al. A novel SCN2A mutation in family with benign familial infantile seizures. Epilepsia 2006;47:218– 220. 24. Kobayashi K, Ohzono H, Shinohara M, et al. Acute encephalopathy with a novel point mutation in the SCN2A gene. Epilepsy Res 2012;102:109–112. 25. Heron SE, Crossland KM, Andermann E, et al. Sodium-channel defects in benign familial neonatal-infantile seizures. Lancet 2002;360:851–852. 26. de Ligt J, Willemsen MH, van Bon BW, et al. Diagnostic exome sequencing in persons with severe intellectual disability. N Engl J Med 2012;367:1921–1929. 27. Need AC, Shashi V, Hitomi Y, et al. Clinical application of exome sequencing in undiagnosed genetic conditions. J Med Genet 2012;49:353–361. 28. Zara F, Specchio N, Striano P, et al. Genetic testing in benign familial epilepsies of the first year of life: clinical and diagnostic significance. Epilepsia 2013;54:425–436. 29. Haug K, Hallmann K, Rebstock J, et al. The voltage-gated sodium channel gene SCN2A and idiopathic generalized epilepsy. Epilepsy Res 2001;47:243–246.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Candidate genes for disturbed temperature regulation and optic atrophy.
Epilepsia, 55(4):e25–e29, 2014 doi: 10.1111/epi.12554
