These authors contributed equally to this work.
A homozygous mutation of voltage-gated sodium channel βI gene SCN1B in a patient with Dravet syndrome
Article first published online: 13 NOV 2012
Wiley Periodicals, Inc. © 2012 International League Against Epilepsy
Volume 53, Issue 12, pages e200–e203, December 2012
How to Cite
Ogiwara, I., Nakayama, T., Yamagata, T., Ohtani, H., Mazaki, E., Tsuchiya, S., Inoue, Y. and Yamakawa, K. (2012), A homozygous mutation of voltage-gated sodium channel βI gene SCN1B in a patient with Dravet syndrome. Epilepsia, 53: e200–e203. doi: 10.1111/epi.12040
- Issue published online: 3 DEC 2012
- Article first published online: 13 NOV 2012
- Accepted September 27, 2012; Early View publication November 13, 2012.
- Dravet syndrome;
- SCN1B ;
- Voltage-gated sodium channel βI
Dravet syndrome is a severe form of epileptic encephalopathy characterized by early onset epileptic seizures followed by ataxia and cognitive decline. Approximately 80% of patients with Dravet syndrome have been associated with heterozygous mutations in SCN1A gene encoding voltage-gated sodium channel (VGSC) αI subunit, whereas a homozygous mutation (p.Arg125Cys) of SCN1B gene encoding VGSC βI subunit was recently described in a patient with Dravet syndrome. To further examine the involvement of homozygous SCN1B mutations in the etiology of Dravet syndrome, we performed mutational analyses on SCN1B in 286 patients with epileptic disorders, including 67 patients with Dravet syndrome who have been negative for SCN1A and SCN2A mutations. In the cohort, we found one additional homozygous mutation (p.Ile106Phe) in a patient with Dravet syndrome. The identified homozygous SCN1B mutations indicate that SCN1B is an etiologic candidate underlying Dravet syndrome.
Voltage-gated sodium channels (VGSCs) are essential for the generation and propagation of action potentials in brain, muscle, and heart. VGSCs consist of one α pore–forming main subunit and one or two β accessory subunits that modulate the voltage dependence, gating, and cellular localization of the α subunit (Brackenbury & Isom, 2011).
Mutations in VGSC αI gene, SCN1A, encoding Nav1.1 have been associated with a broad spectrum of childhood epilepsies including generalized epilepsy with febrile seizure plus (GEFS+; OMIM#604233) and Dravet syndrome (OMIM#607208) (Escayg et al., 2000; Claes et al., 2001). GEFS+ is a dominantly inherited epilepsy characterized by febrile seizures in early childhood that progress to afebrile seizures in late childhood and is responsive to antiepileptic drugs (Scheffer & Berkovic, 1997). Dravet syndrome is one of the most major forms of SCN1A-related epilepsies, and is also an intractable epileptic encephalopathy. Typically, the first seizure in Dravet syndrome is a unilateral or generalized tonic–clonic or clonic seizure often associated with fever, which is progressively followed by additional generalized and focal seizures, ataxia, and cognitive decline (Dravet et al., 2005). Although SCN1A is the major and thus far the best-characterized responsible gene for childhood epilepsies, other candidate genes underlying SCN1A-negative cases remain largely unknown.
Voltage-gated sodium channel βI subunit, encoded by SCN1B gene, is an immunoglobulin-like protein that is noncovalently linked to a VGSC α subunit (Brackenbury & Isom, 2011). Heterozygous mutations in SCN1B gene have been associated with families with temporal lobe epilepsy (TLE) and GEFS+ phenotypes (Wallace et al., 1998; Scheffer et al., 2007). Recently, a homozygous SCN1B mutation was described in a patient with Dravet syndrome (Patino et al., 2009). Here, we screened SCN1B for mutations in Japanese patients with epilepsies including Dravet syndrome, GEFS+, TLE, and other phenotypes. We report a novel homozygous SCN1B mutation in a patient with Dravet syndrome.
Materials and Methods
Ethics committees in the Shizuoka Institute of Epilepsy and Neurological Disorders, the Tohoku University School of Medicine, and the RIKEN Institute approved this study. Each adult participant or, where necessary, responsible guardians of adult subjects, as well as the parents and/or legal guardians of subjects who were minors at the time the study began, signed an informed consent form as approved by the ethics committees.
A total of 286 unrelated probands consisting of 67 individuals diagnosed with Dravet syndrome, 8 with GEFS+, 30 with TLE, 65 with juvenile myoclonic epilepsy (JME), and 116 with other phenotypes were screened. The patients with Dravet syndrome and GEFS+ have been negative for mutations of SCN1A and SCN2A (Ogiwara et al., 2009; Nakayama et al., 2010). Epileptic seizures and epilepsy syndrome diagnoses were performed according to the International League Against Epilepsy classification of epileptic syndromes (ILAE, 1989; Engel, 2001). Dravet syndrome was defined following the criteria described by Dravet et al. (2005).
Mutational analysis of SCN1B in the probands was performed on all of the five coding exons and exon–intron boundary using a direct sequencing. Genomic DNA was extracted from heparin-treated blood samples using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). All genomic DNA samples were amplified by polymerase chain reaction (PCR) using PrimeSTAR (Takara Bio, Shiga, Japan) DNA polymerase system according to the manufacturer’s instructions. The PCR products were then analyzed by the ABI 3700 Genetic Analyzer (Life Technologies, Carlsbad, CA, U.S.A.). Primer sequences are available upon request.
Microsatellite haplotype analysis
The microsatellite markers flanking the SCN1B locus, D19S216, D19S221, D19S226, D19S414, and D19S220, were genotyped using ABI PRISM Linkage Mapping Set v2.5 (Life Technologies) according to the manufacturer’s instructions.
The proband (Fig. 1A; II-1) is a 24-year old Japanese man born to unrelated, healthy parents after an uneventful pregnancy. Neither of his parents had intellectual impairment or epilepsy. He began having recurrent hemiclonic seizures lasting for 15 min and myoclonic seizures at 6 months. He subsequently developed fever-provoked myoclonic seizures and generalized tonic–clonic seizures, which often led to status epilepticus (SE). First SE appeared at 13 months by fever, starting with hemiconvulsion and then generalized, lasting for 1 h. Myoclonic seizures occurred more than weekly and clustered with elevation of body temperature. He also experienced atypical absence seizures at 12 months, which gradually appeared frequently, eventually daily. Myoclonic atonic seizures and focal dyscognitive seizures with cyanosis were observed at 20 months and 3 years, respectively, for a short period. Myoclonic seizures and atypical absence seizures became gradually less frequent and disappeared at 4 years. GTCS was the only seizure type since then and repeated more than weekly throughout childhood. Presently GTCS is occurring weekly. These seizures were refractory to multiple medications. His development was normal until the seizures began at 6 months, but then the development stagnated. On examination at 4 years, he showed global developmental delays. The developmental quotient was assessed as 35 on the Mother Child Counseling test (Tokyo, Japan, 1967). A physical examination revealed ataxia of the extremities. He showed slight pyramidal signs. No distinctive deformities were noted. Interictal sleep electroencephalograpy (EEG) showed no paroxysmal abnormality at around 1 year, but then generalized or multifocal spike and slow waves appeared in isolation or in bursts. His EEG still shows infrequent polyspikes, spikes, and slow waves, predominantly over the frontal area. Brain magnetic resonance imaging (MRI) showed mild nonspecific atrophy with enlargement of the lateral ventricles. The clinical evolution, seizure presentation, and EEG findings in this patient is the typical phenotype of Dravet syndrome and can be categorized as core Dravet syndrome.
Mutational analysis of SCN1B
A nucleotide change resulting in an amino acid substitution (c.316A>T in exon 3, p.Ile106Phe) was identified homozygously in the proband and heterozygously in both of his asymptomatic parents (Fig. 1A,B). The p.Ile106Phe was not listed in the NCBI single nucleotide polymorphisms (SNPs) (http://www.ncbi.nlm.nih.gov/), JSNPs (http://snp.ims.u-tokyo.ac.jp/), Exome Variant Server (http://evs.gs.washington.edu/EVS/), and 1000 Genomes (http://browser.1000genomes.org/index.html) databases, or observed in our pool of 312 healthy control subjects. Analysis of alleles of the microsatellite markers flanking the SCN1B locus (between D19S216 and D19S220) showed the putative maternal and paternal haplotypes in the patient, which were different from each other (Fig. 1A). The isoleucine of the p.Ile106Phe was putatively located on the extracellular immunoglobulin loop domain (Fig. 1C) and was highly conserved among vertebrate VGSC βI and βIII subunits (Fig. 1D). The p.Ile106Phe was predicted to be probably damaging by PolyPhen-2 analysis (HumDiv score = 0.997, Humvar score = 0.992) (http://genetics.bwh.harvard.edu/pph2/). SIFT analysis also predicted the p.Ile106Phe to be damaging (SIFT score = 0.03, Median Information Content = 2.05) (http://sift.jcvi.org/).
In addition to the p.Ile106Phe, a novel nucleotide change, c.448+8 G>T in intron 3, was found in a proband with JME. The c.448+8 G>T was not observed in her mother with a history of epileptic seizures. Genomic DNA of her father was not available for the analysis. Although the c.448+8 G>T was not listed in the NCBI SNPs, JSNPs, Exome Variant Server, and 1000 Genomes databases, the G to C nucleotide substitution at the same site, c.448+8 G>C, was described in one of 2,184 alleles in the 1000 Genomes database. The c.448+8 G>T seemed not to cause aberrant splicing of SCN1B messenger RNA (mRNA). The c.448+8 G>T is therefore very likely a benign variant.
We have described an SCN1B missense mutation that is, to our knowledge, the second homozygous mutation in the sodium channel accessory subunit gene in Dravet syndrome.
The homozygous mutation identified in our patient with Dravet syndrome results in an isoleucine to phenylalanine substitution at amino acid position 106 of VGSC βI. The conservation of the isoleucine residue through vertebrate VGSC βI and βIII subunits implicates a pathogenic potential for the p.Ile106Phe. Pathogenicity of the p.Ile106Phe was also supported by PolyPhen-2 and SIFT analyses. The isoleucine residue at position 106 of VGSC βI was putatively located on the immunoglobulin loop domain that mediates interaction of VGSC βI with cellular adhesion molecules (Brackenbury & Isom, 2011). The substation of the isoleucine residue by the more bulky phenylalanine residue with a sizeable benzene ring may perturb interaction between VGSC βI and those cellular adhesion molecules. It is interesting to note that all the previous SCN1B mutations associated with epilepsies also converge at this protein domain (Scheffer et al., 2007; Patino et al., 2009). Although the in vivo roles of the immunoglobulin loop domain of VGSC βI in neural excitability remain largely unknown, given that a GEFS+ mutation resulted in increased excitability in mouse hippocampal pyramidal neurons (Wimmer et al., 2010), altered function of VGSC βI–mediated cell adhesion seems to contribute to neuronal hyperexcitability, thereby leading to epileptic seizures.
The parents of our proband both harbor the heterozygous p.Ile106Phe and have no history of seizures, similarly to the previous report describing the first homozygous SCN1B mutation in the patient with Dravet syndrome (Patino et al., 2009), suggesting that the p.Ile106Phe seems to be nonpathogenic in the heterozygous state. Nonetheless, given that the heterozygous SCN1B mutations identified in GEFS+ patients were also observed in their asymptomatic family members (Scheffer et al., 2007), we cannot completely exclude the possibility that the p.Ile106Phe has potential to contribute to the susceptibility to develop seizures in heterozygous state.
SCN1B has a splice variant that retains intron 3 and results in a VGSC βI isoform, designated βIB (Brackenbury & Isom, 2011). A nucleotide change in intron 3 leading to amino acid substitution in the VGSC βIB isoform was recently identified in unrelated patients with epilepsy and Tourette syndrome (Patino et al., 2011). The present study also identified a novel nucleotide change in intron 3, c.448+8 G>T, in a patient with JME. However, the c.448+8 G>T is predicted not to change the corresponding amino acid sequence in the VGSC βIB isoform. In addition, the nucleotide change was not observed in the patients symptomatic mother. Therefore, the c.448+8 G>T is likely a rare nonpathogenic variant.
In conclusion, we identified a homozygous SCN1B mutation, p.Ile106Phe, in 1 of 67 patients with Dravet syndrome who have been negative for SCN1A and SCN2A mutations. Together with the homozygous mutation, p.Arg125Cys, which was previously discovered in a patient with Dravet syndrome (Patino et al., 2009), these mutations may underlie a recessive form of Dravet syndrome.
We thank all the patients and families for their contributions and cooperation. We also thank Drs. S. Kaneko (Hirosaki University) and S. Hirose (Fukuoka University) for providing materials, and the Research Resource Center of the RIKEN Brain Science Institute for DNA sequencing. This work was supported in part by grants from the RIKEN Brain Science Institute.
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.
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