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Electroclinical features of epileptic encephalopathy caused by SCN8A mutation Satoru Takahashi,1 Shiho Yamamoto,1 Akie Okayama,1 Akiko Araki,1 Hirotomo Saitsu,2 Naomichi Matsumoto,2 and Hiroshi Azuma1 1 Department of Pediatrics, Asahikawa Medical University, Asahikawa and 2Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan Abstract
Voltage-gated sodium channel Nav1.6, encoded by the gene SCN8A, plays a crucial role in controlling neuronal excitability. SCN8A mutations that cause increased channel activity are associated with seizures. We describe a patient with epileptic encephalopathy caused by de novo SCN8A mutation (c.5614C>T, p.Arg1872Trp). Seizures began 10 days after birth at which time brain magnetic resonance imaging (MRI) and electroencephalography (EEG) were normal. Seizure recurrence increased with age, leading to the development of frequent status epilepticus from 1 year of age. Seizure type included generalized tonic seizures and focal motor seizures. EEG first showed focal epileptic activity at the age of 4 months, and thereafter showed multifocal spikes. Serial MRI demonstrated brain atrophy, which appeared to progress with seizure aggravation. Clinical features that may give a clue to the diagnosis include normal EEG despite frequent seizures in early infancy and an increase in epileptic activity that occurs with aging.
Key words epilepsy, encephalopathy, mutation, SCN8A, sodium channel. Epileptic encephalopathy is an electroclinical entity in which epileptic activity is thought to be responsible for any disturbance of psychomotor function. Patients with early onset epileptic encephalopathy (EOEE) frequently have refractory seizures and psychomotor developmental delay. To ensure appropriate treatment, etiologic diagnosis is mandatory. Whole genome or exome sequencing in patients with EOEE makes it possible to detect de novo mutations in the causal genes.1,2 The first de novo mutation in SCN8A, the gene that encodes the voltage-gated sodium channel (VGSC) Nav1.6, was discovered on whole genome sequencing of a child with EOEE.3 SCN8A is located on chromosome 12q13.13. It contains 27 exons and encodes a protein of 1980 residues. Nav1.6 is localized at the nodes of Ranvier and at the axon initial segment, where it regulates neuronal firing.4 The mutant channel in the patient with EOEE exhibited a dominant gain of function, which resulted in an increase in persistent sodium current and incomplete channel inactivation.3 To date, more than 10 mutations of SCN8A have been identified in patients with EOEE.1–6 Here, we describe a new patient presenting with EOEE caused by de novo SCN8A mutation, for the first time showing temporal changes on electroencephalography (EEG) and magnetic resonance imaging (MRI).
Case report A Japanese girl aged 1 year 10 months, was the first child of healthy non-consanguineous parents. She was born at full term Correspondence: Satoru Takahashi, MD PhD, Department of Pediatrics, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa 078-8510, Japan. Email: [email protected] Received 24 July 2014; revised 9 December 2014; accepted 10 December 2014. doi: 10.1111/ped.12622
© 2015 Japan Pediatric Society
after an uneventful pregnancy. Birthweight was 2898 g with normal head circumference (33.0 cm). Generalized tonic seizures developed 10 days after birth. At that time, brain MRI and EEG were normal. Her seizures spontaneously improved, but generalized tonic and myoclonic seizures recurred at the age of 2 months. Seizures temporarily disappeared following treatment with carbamazepine (CBZ) and levetiracetam (LEV). Interictal EEG at the age of 4 months showed frontal dominant focal spikes (Fig. 1a). At the age of 5 months, she had a cluster of tonic seizures despite treatment with valproic acid (VPA). Her seizures decreased in frequency on phenytoin (PHT) treatment. She was referred to hospital when 10 months old due to intractable epilepsy. Neurological examination indicated muscle hypotonia and delayed psychomotor development. She continued to have frequent brief generalized tonic seizures and focal motor seizures. Focal motor seizures occurred in one or both limbs on the right or left side of her body and often marched to the contralateral side. At the ages of 14 and 17 months, she was hospitalized for status epilepticus. Interictal EEG showed multifocal high-amplitude spikes (Fig. 1). Serial brain MRI demonstrated diffuse progressive cerebral atrophy (Fig. 2). Seizures were refractory to anti-epileptic drugs (AED) including CBZ, LEV, VPA, and zonisamide. High-dose phenobarbital, PHT, and lamotrigine were partially effective. Psychomotor development was severely delayed without head control, eye pursuit, or the use of meaningful words. Because of feeding problems resulting from swallowing difficulties, she was fed via a gastrostomy tube. These clinical features including refractory seizures and arrest of psychomotor development were compatible with those of epileptic encephalopathy. Whole exome sequencing was performed. After obtaining parental written informed consent, genomic DNA was extracted from the peripheral blood leukocytes of the patient and her
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Fig. 1 Interictal electroencephalography at different ages showing multifocal epileptic activity. (a) Frontal dominant focal spikes at 4 months old; (b) multifocal spikes over the bilateral temporal areas at 6 months old; (c) spikes and polyspikes over the right hemisphere at 8 months old; (d) multifocal spikes at 15 months old. EEG, electroencephalogram; EOG, electro-oculogram.
parents. Genomic DNA was captured using the SureSelect Human All Exon v5 Kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) with 101 bp paired-end reads. Exome data
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processing, variant calling, and variant annotation were performed as previously described.7 The patient had a heterozygous missense mutation in SCN8A (c.5614C>T, numbered according to Genebank accession no. NM_014191.3, p.Arg1872Trp).
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Fig. 2 Serial brain magnetic resonance imaging showing diffuse progressive cerebral atrophy: (a) 14 days; (b) 3 months; (c) 6 months; (d) 15 months. © 2015 Japan Pediatric Society
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NavCTD
CaM
CaM
(c) Arg1872
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NavCTD Ser1922 (C-term)
CaM Fig. 3 Protein structure analysis for predicting the effect of mutation (p.Arg1872Trp) on Nav1.6 function. (a) Structure of the C-terminal cytoplasmic domain (CTD) of Nav1.6 (green) in complex with calmodulin (CaM, magenta). (b,c) Electrostatic potential maps in the (b) lateral view and (c) from the top, showing the charge distributions of the Nav1.6 CTD (red, lowest electrostatic potential energy; blue, highest).
Testing of the patient’s parents confirmed that the SCN8A mutation occurred de novo. The mutation, confirmed on Sanger sequencing, was predicted to be damaging on both PolyPhen-2 and SIFT. To predict the effect of the mutation on Nav1.6 function, structural modeling was performed as previously described.8 The model was built using the C-terminus of VGSC (PDB ID: 4DCK) as a template.9 The residue Arg1872 is located on the surface of the C-terminal cytoplasmic domain (CTD) with its hydrophilic side chain that may be required for interaction with other proteins (Fig. 3). The mutation that causes the substitution of arginine with tryptophan at 1872 will disturb the interaction with other proteins because tryptophan is non-polar and hydrophobic.
Discussion We have described a Japanese patient with EOEE caused by de novo SCN8A mutation. The clinical features are summarized in Table 1 and were collated with those of 10 previously reported patients.2,3,5,6 In most of the patients the first seizures occurred by the age of 6 months. Initial seizure type was variable, including generalized and focal motor seizures. In 11 patients with SCN8A mutations including the present patient, interictal EEG was normal in five patients at the onset of epilepsy. In the remaining six patients, initial EEG showed focal spikes or slowing in three and diffuse or multifocal spikes in three. Brain atrophy on MRI was observed in three patients on initial imaging and in © 2015 Japan Pediatric Society
eight patients on later imaging, suggesting that brain atrophy progressed with seizure aggravation. Additional clinical characteristics included severe hypotonia and profound delay in psychomotor development. This indicates that genetic testing of SCN8A should be considered in neurologically impaired patients with early onset epilepsy of unknown etiology. Distinct clinical features that may give a clue to the diagnosis include a normal EEG despite frequent and refractory seizures in early infancy and an increase in epileptic activity that occurs with aging. The mutation c.5614C>T has been identified in an Israeli patient who presented with similar clinical features to those observed in the present Japanese patient, including early-onset seizures refractory to AED, profound delay in psychomotor development, and multifocal spikes on EEG.2 This recurrent mutation caused an arginine-to-tryptophan substitution at amino acid position 1872 in the CTD, where Nav1.6 interacts with accessory proteins such as β-subunits, fibroblast growth factors, calmodulin, and E3 ubiquitin ligase Nedd4.4 The interaction between Nav1.6 and accessory proteins plays a crucial role in the channel functions. Protein structural modeling predicted that the substitution of arginine with tryptophan at 1872 would not significantly alter the molecular structure of the Nav1.6 protein, but should change the surface electrostatics. This change should disturb the interaction between Nav1.6 and accessory proteins, resulting in functional impairment of the channel. The p.Asn1768Asp mutation has been found in the patient who had
3 months, normal
MRI
Yes Severe
Hypotonia Intellectual disability
Yes Severe
Bedridden
6 months, mild BA
Intractable
Yes Severe
wheelchair
6 years 4 months, mild BA
Yes Severe
Bedridden
4 years, mild BA
9 months, mild BA
Intractable, eventually controlled 6 months, 2 months, mild BA normal
4 months, focal or diffuse spike and waves Intractable
2 months, focal slowing
At birth, normal
Apnea, FS
No Severe
sitting/ crawling
2 years 6 months, normal
6 months, normal
Intractable
2 years 6 months, normal
3 months, normal
GTC
No Severe
walking
1 year, BA
8 months, normal
unable to sit, but able to roll over Yes Severe
Intractable, eventually controlled 4 months, normal 3 months, normal
14 months, multifocal spikes with diffuse slowing Intractable
5 months, focal sharp waves
FS with GTC generalization 4 months, normal 3 months, multifocal spikes
No Severe
Bedridden
Intractable, eventually controlled 3 months, asymmetric ventricles 1 year, BA
1y, multifocal spikes
Vaher et al., 20145
p.Thr767Ile 2 weeks
Estacion et al., 20146
Yes Severe
walking, died at 15 years
15 years, normal
6 months, normal
Intractable
2 years, delayed myelination
2 weeks, normal
Intractable
11 months, BA, delayed myelination Bedridden, Bedridden died at 17 months Yes Yes Severe Severe
5 months, BA
1 months, normal
Intractable
GTS, myoclonic 1–2 months, 2 weeks, focal sp multifocal spikes, normal background 5 years, 11 months, 2 years, multifocal burstmultifocal spikes with suppression spikes, diffuse background slowing slowing
GTS, FS
p.Asn1768Asp p.Ile132Val 6 months 0 days
Veeramah et al., 20123
Loss of tone and GTS consciousness 5 months, normal 6 months, focal spikes
Patient 6 Patient 7 p.Arg1872Trp p.Ala1650Thr 3 months 3 months
AED, anti-epileptic drug; BA, brain atrophy; EEG, electroencephalogram; FS, focal seizure; GTC, generalized tonic–clonic seizure; GTS, generalized tonic seizure; MRI, magnetic resonance imaging.
Bedridden
Motor function
17 months, moderate BA
6 months, mild BA
Intractable
5y, focal or diffuse spike and waves
17 months, multifocal spikes
Absence seizures 5 months, normal
7 months, focal spikes
6 months, focal or diffuse spike and waves
GTC
4 months, focal spikes
3 months, normal
Response to AED
EEG
Mutation Age at onset of epilepsy Seizure type GTS, FS
Present study
Ohba et al. 20142 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 p.Arg1872Trp p.Asn1466Lys p.Val216Asp p.Phe846Ser p.Arg1617Gln p.Asn1466Thr 10 days 3 days 7 months 2 months 3 months 4 months
Table 1 Clinical patient features in reported SCN8A mutations
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severe epileptic encephalopathy consisting of early onset seizures, intellectual disability, and sudden unexplained death in epilepsy (Table 1).3 Analysis of the biophysical properties of the mutant channel demonstrated a dramatic increase in persistent sodium current, incomplete channel inactivation, and a depolarizing shift in the voltage dependence of steady-state fast inactivation, consistent with a gain-of-function phenotype in the patient.3 The residue Asn1768 is located on the same side as the residue Arg1872 in the N-terminus of the Nav1.6 CTD (Fig. 3a). The mutation p.Asn1768Asp will not significantly alter the molecular structure. This suggests that the N-terminal surface of Nav1.6 CTD is important for channel function through the molecular interactions. Nav1.6 encoded by the gene SCN8A is one of the major VGSC in the brain, and plays an important role in controlling neuronal excitability.4 Scn8a null heterozygotes were resistant to kainic acid-induced seizures.10 Furthermore, heterozygosity for a null mutation of Scn8a ameliorated the seizure phenotype of Scn1a null heterozygotes.10 This suggests that reduced expression of Scn8a protects against seizures. Thus, loss of Nav1.6 activity results in reduced neuronal excitability, whereas gain-of-function mutations identified in EOEE patients can increase neuronal excitability.3 This is consistent with the increase in epileptic activity that occurred with aging in the present patient. The Scn8a mRNA level increases 5.5-fold from postnatal day (P) 8 to P30 in the mouse brain.11 The developmental change in SCN8A expression might explain how gain-of-function mutations in SCN8A led to the age-dependent seizure phenotype. Kindling, an animal model of epilepsy produced by focal electrical stimulation of the brain, has been associated with increased expression of Nav1.6 in neurons.12 Scn8a null heterozygotes are resistant to the initiation and development of kindling.12 This suggests that Nav1.6 may participate in a mechanism that contributes to the development of epilepsy. Cellular alterations, induced by seizures, such as neuronal loss and gliosis, occur in the human brain with epilepsy. Serial MRI in the present patient demonstrated brain atrophy, which appeared to progress with seizure aggravation. The progressive decline of neurological function requires therapeutic strategies aimed at attenuating seizure-related neuronal loss. Identifying the etiology of epileptic encephalopathy may stimulate the development of such neuroprotective therapies. Conclusion
Epileptic activity in a patient with a missense mutation in SCN8A increased with age during early infancy, and caused severe disturbance of psychomotor development. This case provides addi-
© 2015 Japan Pediatric Society
tional support for delineating the clinical features associated with gain-of-function mutation in SCN8A.
Acknowledgments We thank the family members of the present patient, whose help and participation made this work possible. This work was supported in part by Grant-in-Aid for Scientific Research C from the Japan Society for the Promotion of Science (#22591118). The authors have no conflicts of interest to disclose.
References 1 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: 825–30. 2 Ohba C, Kato M, Takahashi S et al. Early onset epileptic encephalopathy caused by de novo SCN8A mutations. Epilepsia 2014; 55: 994–1000. 3 Veeramah KR, O’Brien JE, Meisler MH et al. De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP. Am. J. Hum. Genet. 2012; 90: 502–10. 4 O’Brien JE, Meisler MH. Sodium channel SCN8A (Nav1.6): Properties and de novo mutations in epileptic encephalopathy and intellectual disability. Front. Genet. 2013; 4: 213. 5 Vaher U, Nõukas M, Nikopensius T et al. De novo SCN8A mutation identified by whole-exome sequencing in a boy with neonatal epileptic encephalopathy, multiple congenital anomalies, and movement disorders. J. Child Neurol. 2014; 29: NP202–6. 6 Estacion M, O’Brien JE, Conravey A et al. A novel de novo mutation of SCN8A (Nav1.6) with enhanced channel activation in a child with epileptic encephalopathy. Neurobiol. Dis. 2014; 69C: 117–23. 7 Saitsu H, Nishimura T, Muramatsu K et al. De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood. Nat. Genet. 2013; 45: 445–9. 8 Ishii A, Shioda M, Okumura A et al. A recurrent KCNT1 mutation in two sporadic cases with malignant migrating partial seizures in infancy. Gene 2013; 531: 467–71. 9 Wang C, Chung BC, Yan H, Lee SY, Pitt GS. Crystal structure of the ternary complex of a NaV C-terminal domain, a fibroblast growth factor homologous factor, and calmodulin. Structure 2012; 20: 1167–76. 10 Martin MS, Tang B, Papale LA, Yu FH, Catterall WA, Escayg A. The voltage-gated sodium channel Scn8a is a genetic modifier of severe myoclonic epilepsy of infancy. Hum. Mol. Genet. 2007; 16: 2892–9. 11 Liao Y, Deprez L, Maljevic S et al. Molecular correlates of agedependent seizures in an inherited neonatal-infantile epilepsy. Brain 2010; 133 (Pt 5): 1403–14. 12 Blumenfeld H, Lampert A, Klein JP et al. Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis. Epilepsia 2009; 50: 44–55.
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Electroclinical features of epileptic encephalopathy caused by SCN8A mutation Satoru Takahashi,1 Shiho Yamamoto,1 Akie Okayama,1 Akiko Araki,1 Hirotomo Saitsu,2 Naomichi Matsumoto,2 and Hiroshi Azuma1 1 Department of Pediatrics, Asahikawa Medical University, Asahikawa and 2Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan Abstract
Voltage-gated sodium channel Nav1.6, encoded by the gene SCN8A, plays a crucial role in controlling neuronal excitability. SCN8A mutations that cause increased channel activity are associated with seizures. We describe a patient with epileptic encephalopathy caused by de novo SCN8A mutation (c.5614C>T, p.Arg1872Trp). Seizures began 10 days after birth at which time brain magnetic resonance imaging (MRI) and electroencephalography (EEG) were normal. Seizure recurrence increased with age, leading to the development of frequent status epilepticus from 1 year of age. Seizure type included generalized tonic seizures and focal motor seizures. EEG first showed focal epileptic activity at the age of 4 months, and thereafter showed multifocal spikes. Serial MRI demonstrated brain atrophy, which appeared to progress with seizure aggravation. Clinical features that may give a clue to the diagnosis include normal EEG despite frequent seizures in early infancy and an increase in epileptic activity that occurs with aging.
Key words epilepsy, encephalopathy, mutation, SCN8A, sodium channel. Epileptic encephalopathy is an electroclinical entity in which epileptic activity is thought to be responsible for any disturbance of psychomotor function. Patients with early onset epileptic encephalopathy (EOEE) frequently have refractory seizures and psychomotor developmental delay. To ensure appropriate treatment, etiologic diagnosis is mandatory. Whole genome or exome sequencing in patients with EOEE makes it possible to detect de novo mutations in the causal genes.1,2 The first de novo mutation in SCN8A, the gene that encodes the voltage-gated sodium channel (VGSC) Nav1.6, was discovered on whole genome sequencing of a child with EOEE.3 SCN8A is located on chromosome 12q13.13. It contains 27 exons and encodes a protein of 1980 residues. Nav1.6 is localized at the nodes of Ranvier and at the axon initial segment, where it regulates neuronal firing.4 The mutant channel in the patient with EOEE exhibited a dominant gain of function, which resulted in an increase in persistent sodium current and incomplete channel inactivation.3 To date, more than 10 mutations of SCN8A have been identified in patients with EOEE.1–6 Here, we describe a new patient presenting with EOEE caused by de novo SCN8A mutation, for the first time showing temporal changes on electroencephalography (EEG) and magnetic resonance imaging (MRI).
Case report A Japanese girl aged 1 year 10 months, was the first child of healthy non-consanguineous parents. She was born at full term Correspondence: Satoru Takahashi, MD PhD, Department of Pediatrics, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa 078-8510, Japan. Email: [email protected] Received 24 July 2014; revised 9 December 2014; accepted 10 December 2014. doi: 10.1111/ped.12622
© 2015 Japan Pediatric Society
after an uneventful pregnancy. Birthweight was 2898 g with normal head circumference (33.0 cm). Generalized tonic seizures developed 10 days after birth. At that time, brain MRI and EEG were normal. Her seizures spontaneously improved, but generalized tonic and myoclonic seizures recurred at the age of 2 months. Seizures temporarily disappeared following treatment with carbamazepine (CBZ) and levetiracetam (LEV). Interictal EEG at the age of 4 months showed frontal dominant focal spikes (Fig. 1a). At the age of 5 months, she had a cluster of tonic seizures despite treatment with valproic acid (VPA). Her seizures decreased in frequency on phenytoin (PHT) treatment. She was referred to hospital when 10 months old due to intractable epilepsy. Neurological examination indicated muscle hypotonia and delayed psychomotor development. She continued to have frequent brief generalized tonic seizures and focal motor seizures. Focal motor seizures occurred in one or both limbs on the right or left side of her body and often marched to the contralateral side. At the ages of 14 and 17 months, she was hospitalized for status epilepticus. Interictal EEG showed multifocal high-amplitude spikes (Fig. 1). Serial brain MRI demonstrated diffuse progressive cerebral atrophy (Fig. 2). Seizures were refractory to anti-epileptic drugs (AED) including CBZ, LEV, VPA, and zonisamide. High-dose phenobarbital, PHT, and lamotrigine were partially effective. Psychomotor development was severely delayed without head control, eye pursuit, or the use of meaningful words. Because of feeding problems resulting from swallowing difficulties, she was fed via a gastrostomy tube. These clinical features including refractory seizures and arrest of psychomotor development were compatible with those of epileptic encephalopathy. Whole exome sequencing was performed. After obtaining parental written informed consent, genomic DNA was extracted from the peripheral blood leukocytes of the patient and her
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Fig. 1 Interictal electroencephalography at different ages showing multifocal epileptic activity. (a) Frontal dominant focal spikes at 4 months old; (b) multifocal spikes over the bilateral temporal areas at 6 months old; (c) spikes and polyspikes over the right hemisphere at 8 months old; (d) multifocal spikes at 15 months old. EEG, electroencephalogram; EOG, electro-oculogram.
parents. Genomic DNA was captured using the SureSelect Human All Exon v5 Kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) with 101 bp paired-end reads. Exome data
a
R
b
processing, variant calling, and variant annotation were performed as previously described.7 The patient had a heterozygous missense mutation in SCN8A (c.5614C>T, numbered according to Genebank accession no. NM_014191.3, p.Arg1872Trp).
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Fig. 2 Serial brain magnetic resonance imaging showing diffuse progressive cerebral atrophy: (a) 14 days; (b) 3 months; (c) 6 months; (d) 15 months. © 2015 Japan Pediatric Society
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CaM Fig. 3 Protein structure analysis for predicting the effect of mutation (p.Arg1872Trp) on Nav1.6 function. (a) Structure of the C-terminal cytoplasmic domain (CTD) of Nav1.6 (green) in complex with calmodulin (CaM, magenta). (b,c) Electrostatic potential maps in the (b) lateral view and (c) from the top, showing the charge distributions of the Nav1.6 CTD (red, lowest electrostatic potential energy; blue, highest).
Testing of the patient’s parents confirmed that the SCN8A mutation occurred de novo. The mutation, confirmed on Sanger sequencing, was predicted to be damaging on both PolyPhen-2 and SIFT. To predict the effect of the mutation on Nav1.6 function, structural modeling was performed as previously described.8 The model was built using the C-terminus of VGSC (PDB ID: 4DCK) as a template.9 The residue Arg1872 is located on the surface of the C-terminal cytoplasmic domain (CTD) with its hydrophilic side chain that may be required for interaction with other proteins (Fig. 3). The mutation that causes the substitution of arginine with tryptophan at 1872 will disturb the interaction with other proteins because tryptophan is non-polar and hydrophobic.
Discussion We have described a Japanese patient with EOEE caused by de novo SCN8A mutation. The clinical features are summarized in Table 1 and were collated with those of 10 previously reported patients.2,3,5,6 In most of the patients the first seizures occurred by the age of 6 months. Initial seizure type was variable, including generalized and focal motor seizures. In 11 patients with SCN8A mutations including the present patient, interictal EEG was normal in five patients at the onset of epilepsy. In the remaining six patients, initial EEG showed focal spikes or slowing in three and diffuse or multifocal spikes in three. Brain atrophy on MRI was observed in three patients on initial imaging and in © 2015 Japan Pediatric Society
eight patients on later imaging, suggesting that brain atrophy progressed with seizure aggravation. Additional clinical characteristics included severe hypotonia and profound delay in psychomotor development. This indicates that genetic testing of SCN8A should be considered in neurologically impaired patients with early onset epilepsy of unknown etiology. Distinct clinical features that may give a clue to the diagnosis include a normal EEG despite frequent and refractory seizures in early infancy and an increase in epileptic activity that occurs with aging. The mutation c.5614C>T has been identified in an Israeli patient who presented with similar clinical features to those observed in the present Japanese patient, including early-onset seizures refractory to AED, profound delay in psychomotor development, and multifocal spikes on EEG.2 This recurrent mutation caused an arginine-to-tryptophan substitution at amino acid position 1872 in the CTD, where Nav1.6 interacts with accessory proteins such as β-subunits, fibroblast growth factors, calmodulin, and E3 ubiquitin ligase Nedd4.4 The interaction between Nav1.6 and accessory proteins plays a crucial role in the channel functions. Protein structural modeling predicted that the substitution of arginine with tryptophan at 1872 would not significantly alter the molecular structure of the Nav1.6 protein, but should change the surface electrostatics. This change should disturb the interaction between Nav1.6 and accessory proteins, resulting in functional impairment of the channel. The p.Asn1768Asp mutation has been found in the patient who had
3 months, normal
MRI
Yes Severe
Hypotonia Intellectual disability
Yes Severe
Bedridden
6 months, mild BA
Intractable
Yes Severe
wheelchair
6 years 4 months, mild BA
Yes Severe
Bedridden
4 years, mild BA
9 months, mild BA
Intractable, eventually controlled 6 months, 2 months, mild BA normal
4 months, focal or diffuse spike and waves Intractable
2 months, focal slowing
At birth, normal
Apnea, FS
No Severe
sitting/ crawling
2 years 6 months, normal
6 months, normal
Intractable
2 years 6 months, normal
3 months, normal
GTC
No Severe
walking
1 year, BA
8 months, normal
unable to sit, but able to roll over Yes Severe
Intractable, eventually controlled 4 months, normal 3 months, normal
14 months, multifocal spikes with diffuse slowing Intractable
5 months, focal sharp waves
FS with GTC generalization 4 months, normal 3 months, multifocal spikes
No Severe
Bedridden
Intractable, eventually controlled 3 months, asymmetric ventricles 1 year, BA
1y, multifocal spikes
Vaher et al., 20145
p.Thr767Ile 2 weeks
Estacion et al., 20146
Yes Severe
walking, died at 15 years
15 years, normal
6 months, normal
Intractable
2 years, delayed myelination
2 weeks, normal
Intractable
11 months, BA, delayed myelination Bedridden, Bedridden died at 17 months Yes Yes Severe Severe
5 months, BA
1 months, normal
Intractable
GTS, myoclonic 1–2 months, 2 weeks, focal sp multifocal spikes, normal background 5 years, 11 months, 2 years, multifocal burstmultifocal spikes with suppression spikes, diffuse background slowing slowing
GTS, FS
p.Asn1768Asp p.Ile132Val 6 months 0 days
Veeramah et al., 20123
Loss of tone and GTS consciousness 5 months, normal 6 months, focal spikes
Patient 6 Patient 7 p.Arg1872Trp p.Ala1650Thr 3 months 3 months
AED, anti-epileptic drug; BA, brain atrophy; EEG, electroencephalogram; FS, focal seizure; GTC, generalized tonic–clonic seizure; GTS, generalized tonic seizure; MRI, magnetic resonance imaging.
Bedridden
Motor function
17 months, moderate BA
6 months, mild BA
Intractable
5y, focal or diffuse spike and waves
17 months, multifocal spikes
Absence seizures 5 months, normal
7 months, focal spikes
6 months, focal or diffuse spike and waves
GTC
4 months, focal spikes
3 months, normal
Response to AED
EEG
Mutation Age at onset of epilepsy Seizure type GTS, FS
Present study
Ohba et al. 20142 Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 p.Arg1872Trp p.Asn1466Lys p.Val216Asp p.Phe846Ser p.Arg1617Gln p.Asn1466Thr 10 days 3 days 7 months 2 months 3 months 4 months
Table 1 Clinical patient features in reported SCN8A mutations
Epilepsy caused by SCN8A mutation 761
© 2015 Japan Pediatric Society
762
S Takahashi et al.
severe epileptic encephalopathy consisting of early onset seizures, intellectual disability, and sudden unexplained death in epilepsy (Table 1).3 Analysis of the biophysical properties of the mutant channel demonstrated a dramatic increase in persistent sodium current, incomplete channel inactivation, and a depolarizing shift in the voltage dependence of steady-state fast inactivation, consistent with a gain-of-function phenotype in the patient.3 The residue Asn1768 is located on the same side as the residue Arg1872 in the N-terminus of the Nav1.6 CTD (Fig. 3a). The mutation p.Asn1768Asp will not significantly alter the molecular structure. This suggests that the N-terminal surface of Nav1.6 CTD is important for channel function through the molecular interactions. Nav1.6 encoded by the gene SCN8A is one of the major VGSC in the brain, and plays an important role in controlling neuronal excitability.4 Scn8a null heterozygotes were resistant to kainic acid-induced seizures.10 Furthermore, heterozygosity for a null mutation of Scn8a ameliorated the seizure phenotype of Scn1a null heterozygotes.10 This suggests that reduced expression of Scn8a protects against seizures. Thus, loss of Nav1.6 activity results in reduced neuronal excitability, whereas gain-of-function mutations identified in EOEE patients can increase neuronal excitability.3 This is consistent with the increase in epileptic activity that occurred with aging in the present patient. The Scn8a mRNA level increases 5.5-fold from postnatal day (P) 8 to P30 in the mouse brain.11 The developmental change in SCN8A expression might explain how gain-of-function mutations in SCN8A led to the age-dependent seizure phenotype. Kindling, an animal model of epilepsy produced by focal electrical stimulation of the brain, has been associated with increased expression of Nav1.6 in neurons.12 Scn8a null heterozygotes are resistant to the initiation and development of kindling.12 This suggests that Nav1.6 may participate in a mechanism that contributes to the development of epilepsy. Cellular alterations, induced by seizures, such as neuronal loss and gliosis, occur in the human brain with epilepsy. Serial MRI in the present patient demonstrated brain atrophy, which appeared to progress with seizure aggravation. The progressive decline of neurological function requires therapeutic strategies aimed at attenuating seizure-related neuronal loss. Identifying the etiology of epileptic encephalopathy may stimulate the development of such neuroprotective therapies. Conclusion
Epileptic activity in a patient with a missense mutation in SCN8A increased with age during early infancy, and caused severe disturbance of psychomotor development. This case provides addi-
© 2015 Japan Pediatric Society
tional support for delineating the clinical features associated with gain-of-function mutation in SCN8A.
Acknowledgments We thank the family members of the present patient, whose help and participation made this work possible. This work was supported in part by Grant-in-Aid for Scientific Research C from the Japan Society for the Promotion of Science (#22591118). The authors have no conflicts of interest to disclose.
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