- Email: [email protected]
Prevalence of SCN1A mutations in children with suspected Dravet syndrome and intractable childhood epilepsy
Epilepsy Research (2012) 102, 195—200
journal homepage: www.elsevier.com/locate/epilepsyres
Prevalence of SCN1A mutations in children with suspected Dravet syndrome and intractable childhood epilepsy Ji-wen Wang a,b,1, Xiu-yu Shi a,c,1, Hirokazu Kurahashi a,d, Su-Kyeong Hwang a, Atsushi Ishii a, Norimichi Higurashi a,e, Sunao Kaneko f, Shinichi Hirose a,∗ , The Epilepsy Genetic Study Group Japan (Chairperson, SK) a
Department of Pediatrics, School of Medicine, Central Research Institute for the Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan b Department of Pediatrics, Qilu Hospital, Shandong University, Jinan, China c Department of Pediatrics, Chinese PLA General Hospital, Beijing, China d Department of Pediatric Neurology, Central Hospital of Aichi Welfare Center for Persons with Developmental Disabilities, Kasugai, Japan e Department of Pediatrics, Jikei University School of Medicine, Japan f Department of Neuropsyschiatry, School of Medicine, Hirosaki University, Hirosaki, Japan Received 5 April 2012; received in revised form 12 June 2012; accepted 16 June 2012 Available online 20 July 2012
KEYWORDS Channelopathy; Dravet syndrome; Genetic tests; Molecular diagnosis; Oversight
∗ 1
Summary Mutations of the gene encoding the ␣1 subunit of neuronal sodium channel, SCN1A, are reported to cause Dravet syndrome (DS). The prevalence of mutations reported in such studies (mainly in clinically confirmed DS) seems high enough to make genetic diagnosis feasible. In fact, commercially operating genetic diagnostic laboratories offering genetic analyses of SCN1A are available. Still, the exact prevalence of mutations of SCN1A remains elusive. Fukuoka University has been serving as a genetic diagnostic laboratory for DS for the last 10 years. In this study, we determined the prevalence of SCN1A mutations (SCN1A, SCN2A, SCN1B and SCN2B) in 448 patients with suspected DS and intractable childhood epilepsy. A total of 192 SCN1A mutations were identified in 188 of 448 patients (42.0%). The frequencies of SCN1A mutations in suspected severe myoclonic epilepsy of infancy (SMEI), its borderline phenotype (SMEB) and intractable epilepsy were 56.2%, 41.9% and 28.9% respectively. In addition, four SCN2A mutations were identified in 4 of 325 patients. No mutations of SCN1B and SCN2B were identified. These results are potentially helpful for the diagnosis of DS at early stage. © 2012 Elsevier B.V. All rights reserved.
Corresponding author at: 45-1, 7-chome, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan. Tel.: +81 92 801 1011; fax: +81 92 863 1970. E-mail address: [email protected] (S. Hirose). These authors contributed equally to this work.
0920-1211/$ — see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2012.06.006
196
Introduction Voltage gated sodium channels are one of the mediators of normal neuronal firing. Disruption of the gene encoding the ␣1 subunit of neuronal sodium channels (SCN1A) is associated with a variety of epilepsies, such as Dravet syndrome (DS), intractable childhood epilepsy with generalized tonic—clonic seizures (ICEGTC), genetic epilepsy with febrile seizures plus (GEFS+) and other rare early onset epileptic encephalopathies (Claes et al., 2003; Fujiwara et al., 2003; Harkin et al., 2007; Mulley et al., 2005). DS is a rare and malignant epilepsy syndrome, which usually develops in the first year of life (Dravet et al., 1992; Engel, 2001) and is currently classified as a disease entity that includes severe myoclonic epilepsy in infancy (SMEI) and its borderline phenotype (SMEB) (Mullen and Scheffer, 2009). Patients initially present with recurrent and prolonged seizures, usually febrile, hemiclonic or generalized tonic clonic, which frequently progress into status epilepticus. With advancement of age, the clinical features evolve into various afebrile or fever induced seizure types. Cognitive outcome is poor, with high risk of psychomotor retardation. However, the diagnosis of DS cannot be made at an early stage of the disease according to current diagnostic criteria. Heterozygous SCN1A mutations are a major cause of DS. To date, more than 700 of these have been reported in patients with DS (Lossin, 2009). Various types of SCN1A mutations, e.g., missense and nonsense mutations, have been observed in patients with DS (Fukuma et al., 2004). Moreover, microchromosomal deletions involving SCN1A (Wang et al., 2008), which cannot be detected by direct sequencing, are related to DS. Thus, the prevalence of SCN1A mutations may be higher than that considered previously and should be reevaluated. In fact, several studies have reported the prevalence of SCN1A mutations in patients with DS, although with wide variation, ranging from 33% (Nabbout et al., 2003; Wallace et al., 2003) to 80—100% (Claes et al., 2001; Fujiwara, 2006; Fukuma et al., 2004; Mulley et al., 2005). This variability could be due to the inclusion criteria used for the recruitment of the subjects. Thus, it is plausible that a high prevalence may be found in a cohort of clinically confirmed DS and low prevalence in young children with a provisional diagnosis of DS. Genetic analysis of SCN1A has become available in recent years, at least in commercially operating diagnostic genetic laboratories. Still, the exact prevalence of SCN1A mutations remains elusive. For the last 10 years, Fukuoka University has served as a genetic diagnostic laboratory for DS and other types of intractable childhood epilepsies and has conducted analysis of SCN1A mutations including microchromosomal deletions. In this study, we analyzed the genetic prevalence of SCN1A mutations diagnosed by commercially operating genetic diagnostic laboratories in patients with suspected DS (SMEI and SMEB) and those with other types of intractable childhood epilepsy.
J.-w. Wang et al.
Methods Patients This study included 448 patients with firm or provisional diagnosis of DS (SMEI n = 185; SMEB n = 62) or intractable childhood epilepsy (n = 201, including myoclonic-astatic epilepsy, multifocal epilepsy, complex partial epilepsy, fever triggered epilepsy, West syndrome and progressive myoclonic epilepsy) at the Departments of Pediatric Neurology at various regional tertiary hospitals. The diagnoses of SMEI and SMEB were made by the method described previously (Fukuma et al., 2004). Briefly, SMEI diagnosis was based on fulfillment of all the accepted diagnostic criteria for SMEI (Dravet et al., 1992), whereas the diagnosis of SMEB was based on the presence of clinical features that were almost identical to those of SMEI, excluding myoclonic and atypical absence seizures. We also recruited 96 healthy volunteers as the control group. Each participant or the parent/guardian signed an informed consent form approved by the Ethics Review Committee of Fukuoka University or similar committees of the participating institutions.
Genetic analysis Genomic DNAs were prepared from ethylenediaminetetraacetic acid (EDTA)-treated whole blood samples by using QIAamp DNA Blood Kit (Qiagen, Hilden, Germany). SCN1A, SCN2A, SCN1B and SCN2B were screened for genetic abnormalities using direct sequencing method with an automatic sequencer. Primers were designed to amplify all exons and the flanking intronic splice sites of the genes. The purified products of polymerase chain reaction (PCR) were directly sequenced and analyzed with the automatic sequencer. Details of the PCR conditions and the primers used are available upon request. Reference sequences of messenger RNA (mRNA) were based on information available from RefSeq (accession numbers: Human SCN1A AB093548; Human SCN2A NM 021007; Human SCN1B NM 001037; Human SCN2B NM 004588).
Multiplex ligation-dependent probe amplification (MLPA) MLPA was conducted in patients found to have no mutations and those with only missense mutations, using a commercially available kit for SCN1A (SALSA MLPA KIT P137 SCN1A, Lot 0107 or Lot 0805, MRC-Holland, Amsterdam, the Netherlands). MLPA tests were conducted according to the protocol supplied by the manufacturer. Fragment analysis of the PCR product was carried out on ABI model 310 capillary sequencer (Applied Biosystems, Foster City, CA) using GeneScanTM -500LIZ as size standards (Applied Biosystems) and deionized formamide (HiDi Formamide, Applied Biosystems). Data were analyzed using the Genescan software (Applied Biosystems). The thresholds were set at <0.65 for deletions and >1.35 for duplications. Details are available upon request.
Statistical analysis Data were analyzed by The Statistical Package for Social Sciences (version 17.0, SPSS Inc., Chicago, IL). Differences in the frequency distribution of mutations between SMEI, SMEB and Intractable Childhood Epilepsy were examined by the 2 test or Fisher’s exact test. All p values are two tailed, and significance was set at 5%.
Results A total of 192 SCN1A mutations were identified in 188 of 448 patients (42.0%). These mutations included 91 missense, 35 nonsense, 32 frameshift, 21 splice site, 4 deletion
Prevalence of SCN1A mutations Table 1
197
Exonic deletions of SCN1A identified by MLPA.
No
Diagnosis
Sex
Exonic deletion
Transmission
5 9 24 33 54 83 108 146 221
SMEI SMEI SMEI SMEI SMEI SMEI SMEI SMEI PME
F F M F M M F M M
Ex1∼Ex26 deletion Ex1∼Ex26 deletion Ex1∼Ex26 deletion Ex17∼Ex26 deletion Ex8∼Ex26 deletion Ex1∼Ex26 deletion Ex20∼Ex26 deletion Ex17∼Ex26 deletion Ex1∼Ex26 deletion
Unknown De novo De novo De novo De novo De novo De novo De novo Unknown
PME: progressive myoclonus epilepsy.
and 9 microchromosomal deletional mutations (E-Table A, Table 1). Four SCN2A mutations were identified in 4 of 325 patients. No SCN1B or SCN2B mutations were detected within the regions examined.
SCN1A point mutations 183 SCN1A point mutations were identified in 179 patients. Missense mutation of SCN1A was the most frequent mutation type (47.4%, 91/192), encountered in 89 patients. The frequencies of other mutation types were: nonsense mutation (18.2%, 35/192), frameshift mutation (16.7%, 32/192), and splice site mutation (10.9%, 21/192). Deletion mutations were identified in 4 patients (2.1%, 4/192). There were significant differences in the mutation and distribution frequencies among SMEI, SMEB and intractable epilepsy (Table 2), SCN1A mutations were most frequent in SMEI (56.2%, P = 0.001), whereas missense mutations were most frequent in SMEB 16/23 (69.2%, P = 0.027). Four patients harbored double SCN1A mutations. The first carried the de novo c.251A>G: Y84C mutation, and the c.4723C>T: R1575C variant, inherited from her apparent healthy mother. The second had the c.680T>G: I227S and the c.4723C>T: R1575C variants (samples not available from the parents). The third carried the IVS12-6 del T and the c.5674C>T: R1892X mutations (samples not available from the parents). The fourth carried the de novo c.14981538 del and the c.1497—1539 ins GAGGATGAATTCCAAAAA (R500fsX509) mutations. Biological samples from both parents of 69 patients with point mutations were available for genetic analysis. Direct sequencing showed none of the parents had any mutations
Table 2
in 64 cases out of 69 (92.8%), indicating de novo mutations. However, in one case, a boy and his sister had the same mutation but could not be detected in the blood samples from the parents, suggesting possible somatic and/or germline mosaicism in either parent. The mutation was inherited from the mother in 5 cases. In one of these, a girl and her brother with SMEI had the same SCN1A mutation inherited from the mother, but the mother had simple febrile seizures only during childhood. The other three mothers didn’t have any phenotype.
Microdeletions detected with MLPA MLPA analysis of 313 patients without mutation or with missense mutations only identified SCN1A microdeletions in 9 patients (2.9%, 9/313; Table 1). Samples from both parents were available for MLPA in 7 of these patients. The results of MLPA indicated that all 7 deletions were de novo. Deletion of the whole gene was detected in 5 patients (55.6%, 5/9). Deletion of 8—26th exons was found in one patient and 20—26th exons in another, whereas 17—26th exons were deleted in two patients. Among the 9 patients, 8 had SMEI and 1 had intractable epilepsy. No exonic deletion was found in patients with SMEB.
SCN2A mutations SCN2A polymorphism (c.56G>A: R19K) resulting in an amino acid exchange was identified, which had already been reported by our team (Ito et al., 2004). Four novel SCN2A missense mutations were identified in 4 patients. Two of the
Frequency distribution of different patients with SCN1A mutation.
Mutation
SMEI (n = 185)
SMEB (n = 62)
Intractable epilepsy (n = 201)
P value
With mutation Missense Nonsense Frameshift Splice site Deletion Exonic deletion
104(56.2%) 42(40.4%) 23(22.1%) 19(18.3%) 12(11.5%) 0 8(7.7%)
26(41.9%) 18(69.2%) 2(7.7%) 2(7.7%) 1(3.8%) 3(11.5%) 0
58(28.9%) 29(50.0%) 9(15.5%) 10(17.2%) 8(13.8%) 1(1.7%) 1(1.7%)
P = 0.001 P = 0.027 P = 0.193 P = 0.422 P = 0.402 P = 0.001 P = 0.109
198 4 patients harbored both SCN1A and SCN2A variants: one with SMEI carried de novo splice site mutation of SCN1A (IVS4 + 1G>A), and SCN2A missense mutation (c.964G>A: D322N), inherited from her mother; the other with SMEB had missense mutations of SCN1A (c.4507G>A: E1503K) and SCN2A (c.982T>G: F328V), her mother had same SCN2A mutation. The third patient with SMEI had de novo missense mutation of SCN2A (c.3935G>C: R1312T) but no SCN1A mutations (Shi et al., 2009). The fourth patient with SMEI had SCN2A missense mutation (c.1945G>A: D649N) without SCN1A mutations (samples not available from the parents).
Discussion DS is one of the catastrophic pediatric epilepsy syndromes and it is mainly due to SCN1A mutations. The first major clinical sign of DS is prolonged febrile seizure occurring 2—10 months of age; however, without genetic information, it is difficult to distinguish febrile seizure plus from those that will evolve into DS before they present other seizures. Therefore, many pediatric neurologists send blood samples of patients with suspected DS to genetic diagnostic centers and are often interested in information on the prevalence of SCN1A mutations in highly suspected babies. To our knowledge, however, there is no such information at present. Our study indicates that the prevalence of SCN1A mutation in patients with suspected SMEI, SMEB and intractable epilepsy is 56.2%, 41.9% and 28.9% respectively. The prevalence in our cohort was lower than previously reported data (70—80%) (Ottman et al., 2010). The difference could be due to the inclusion criteria used for recruitment of subjects. Previous studies recruited mainly patients with clinically proven DS, while in our study; we also included patients suspected with DS. For pediatric neurologists, it is important to know the genetic status of young children suspected to develop DS. Early diagnosis of DS and management with appropriate anticonvulsants and provision of a treatment plan may reduce the seizure burden and improve long-term developmental outcome (Millichap et al., 2009). All types of SCN1A mutations were identified in this study, however, the frequency distribution of missense mutations was significantly different among SMEI, SMEB and intractable childhood epilepsy [missense mutation 42 (40.4%), 18 (69.2%), 29 (50.0%), P = 0.027]. Missense mutation was most frequent in SMEB. Although there is no significant difference, nonsense and frameshift mutations were more common in SMEI. This finding is similar to our study reported previously (Fukuma et al., 2004). Fukuma et al. (2004) reported that the frequency of molecular truncation mutations, including nonsense and frameshift mutations, was high only in SMEI. Such information should help in establishing the diagnosis. In addition to mutations detected by direct sequencing, we identified partial (n = 4) and complete (n = 5) gene deletions by MLPA in 9 patients. Microchromosomal deletions affect not only SCN1A but also often involve the adjacent genes (Wang et al., 2008). The frequency of MLPA-detected chromosomal deletions was 2.9% (9/313) among the different types of epilepsy, mostly SMEI, and no exonic deletions were found in patients with SMEB. The prevalence in our cohort of 313 patients is similar to that reported by Suls et al.
J.-w. Wang et al. (2.7%) (Suls et al., 2006) but lower than that of Madia et al. (7.7%) (Madia et al., 2006) and Marini et al. (12.5%) (Marini et al., 2009). The difference from the latter two studies could be due to differences in phenotyping or to the larger number of patients analyzed. Our investigation forms the largest study of MLPA, encompassing 313 patients with various types of epilepsies. Moreover, previous studies included only SCN1A negative patients, whereas this study included also patients with SCN1A missense mutations. Although the overall frequency of microchromosomal deletions is 2—3%, these abnormalities are important pathogenic factors of DS. Several groups of researchers have stressed the importance of conducting MLPA in patients with sequencing negative mutations (Mulley et al., 2006; Wang et al., 2008). Parental samples were available in 40.4% (76/188) of cases with SCN1A abnormalities. Among them, 93.4% (71/76) were de novo cases whereas 6.6% (5/76) inherited from a healthy parent. Similar to our findings, familial SCN1A mutations have been reported previously in around 5% of patients with DS (Mulley et al., 2005). The mechanisms involved in these inherited mutations remain unclear. Gennaro et al. (2006) found that parental somatic and germline mosaicism can cause recurrent transmission of SCN1A mutations in DS. This partly explains the phenotypic variability and complex inheritance characteristics of DS patients whose SCN1A mutations appear to occur de novo. In our study, we identified four novel SCN2A missense mutations in four DS patients. All four mutations affected highly conserved amino acids in many species. Two of the SCN2A mutations coexisted with de novo SCN1A mutations. These two mutations were also inherited from one of the parents and accordingly were not likely to be pathogenic although they could have had modifying effect on the phenotypes of DS. In contrast, the third patient with SMEI had a de novo missense mutation of SCN2A (c.3935G>C: R1312T) without any SCN1A mutations. Arginine at the 1312 position is a crucial charged amino acid in the fourth transmembrane segment of domain III, which functions as a voltage sensor of the NaV 1.2 channel. This mutation in such an important position may downgrade the function of the channel and lead to DS. The fourth patient with SMEI only had SCN2A mutation, and since blood samples were not available from the parents, we were unable to determine whether or not the mutation was de novo. Ogiwara et al. (2009) reported de novo SCN2A mutations in intractable epilepsies, similar to our results, further confirming the potential role NaV 1.2 channel dysfunctions and its involvement in the molecular etiology of epilepsy. Recently, Patino et al. (2009) presented the first case of DS attributable to a homozygous mutation in SCN1B. Nevertheless, we are unable to identify any SCN1B or SCN2B mutations in our cohort of 448 patients. In summary, genetic information in young patients especially those with prolonged seizures aged less than 6 months, provides valuable information that does not only help in establishing the etiology but also early diagnosis, aggressive prevention and proper treatment.
Conflict of interest None of the authors has any conflict of interest to disclose.
Prevalence of SCN1A mutations
Acknowledgments We are indebted to all members of the family for their helpful cooperation in this study. We thank Ms. Takako Umemoto and Hideko Takeda for formatting and typing the manuscript and Ms. Minako Yonetani and Akiyo Hamachi for the technical assistance. This study was supported in part by Grantsin-Aid for Scientific Research (S) 16109006, (A) 18209035, 21249062 and 24249060, Exploratory Research 1659272 and 23659529, and ‘‘High-Tech Research Center’’ Project for Private Universities-matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology, 2006—2010- ‘‘The Research Center for the Molecular Pathomechanisms of Epilepsy, Fukuoka University’’, Research Grants (19A-6) and (21B-5) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare and the Central Research Institute of Fukuoka University.Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.eplepsyres.2012.06.006.
References Claes, L., Ceulemans, B., Audenaert, D., Smets, K., Lofgren, A., Del-Favero, J., Ala-Mello, S., Basel-Vanagaite, L., Plecko, B., Raskin, S., Thiry, P., Wolf, N.I., Van Broeckhoven, C., De Jonghe, P., 2003. De novo SCN1A mutations are a major cause of severe myoclonic epilepsy of infancy. Hum. Mutat. 21, 615—621. Claes, L., Del-Favero, J., Ceulemans, B., Lagae, L., Van Broeckhoven, C., De Jonghe, P., 2001. De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am. J. Hum. Genet. 68, 1327—1332. Dravet, C., Bureau, M., Guerrini, R., Giraud, N., Roger, J., 1992. Severe myoclonic epilepsy in infants. In: Roger, J., Dravet, C., Bureau, M., Dreifuss, F.E., Perret, A., Wolf, P. (Eds.), Epileptic Syndromes in Infancy, Childhood and Adolescence. John Libbey, London, pp. 75—88. Engel Jr., J., 2001. A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE Task Force on Classification and Terminology. Epilepsia 42, 796—803. Fujiwara, T., 2006. Clinical spectrum of mutations in SCN1A gene: severe myoclonic epilepsy in infancy and related epilepsies. Epilepsy Res. 70 (Suppl 1), S223—S230. Fujiwara, T., Sugawara, T., Mazaki-Miyazaki, E., Takahashi, Y., Fukushima, K., Watanabe, M., Hara, K., Morikawa, T., Yagi, K., Yamakawa, K., Inoue, Y., 2003. Mutations of sodium channel alpha subunit type 1 (SCN1A) in intractable childhood. Epilepsies with frequent generalized tonic—clonic seizures. Brain 126, 531—546. Fukuma, G., Oguni, H., Shirasaka, Y., Watanabe, K., Miyajima, T., Yasumoto, S., Ohfu, M., Inoue, T., Watanachai, A., Kira, R., Matsuo, M., Muranaka, H., Sofue, F., Zhang, B., Kaneko, S., Mitsudome, A., Hirose, S., 2004. Mutations of neuronal voltage-gated Na+ channel ␣1 subunit gene SCN1A in core severe myoclonic epilepsy in infancy (SMEI) and in borderline SMEI (SMEB). Epilepsia 45, 140—148. Gennaro, E., Santorelli, F.M., Bertini, E., Buti, D., Gaggero, R., Gobbi, G., Lini, M., Granata, T., Freri, E., Parmeggiani, A., Striano, P., Veggiotti, P., Cardinali, S., Bricarelli, F.D., Minetti, C., Zara, F., 2006. Somatic and germline mosaicisms in severe myoclonic epilepsy of infancy. Biochem. Biophys. Res. Commun. 341, 489—493. Harkin, L.A., McMahon, J.M., Iona, X., Dibbens, L., Pelekanos, J.T., Zuberi, S.M., Sadleir, L.G., Andermann, E., Gill, D., Farrell, K.,
199 Connolly, M., Stanley, T., Harbord, M., Andermann, F., Wang, J., Batish, S.D., Jones, J.G., Seltzer, W.K., Gardner, A., Sutherland, G., Berkovic, S.F., Mulley, J.C., Scheffer, I.E., 2007. The spectrum of SCN1A-related infantile epileptic encephalopathies. Brain 130, 843—852. Ito, M., Shirasaka, Y., Hirose, S., Sugawara, T., Yamakawa, K., 2004. Seizure phenotypes of a family with missense mutations in SCN2A. Pediatr Neurol 31, 150—152. Lossin, C., 2009. A catalog of SCN1A variants. Brain Dev 31, 114—130. Madia, F., Striano, P., Gennaro, E., Malacarne, M., Paravidino, R., Biancheri, R., Budetta, M., Cilio, M.R., Gaggero, R., Pierluigi, M., Minetti, C., Zara, F., 2006. Cryptic chromosome deletions involving SCN1A in severe myoclonic epilepsy of infancy. Neurology 67, 1230—1235. Marini, C., Scheffer, I.E., Nabbout, R., Mei, D., Cox, K., Dibbens, L.M., McMahon, J.M., Iona, X., Carpintero, R.S., Elia, M., Cilio, M.R., Specchio, N., Giordano, L., Striano, P., Gennaro, E., Cross, J.H., Kivity, S., Neufeld, M.Y., Afawi, Z., Andermann, E., Keene, D., Dulac, O., Zara, F., Berkovic, S.F., Guerrini, R., Mulley, J.C., 2009. SCN1A duplications and deletions detected in Dravet syndrome: implications for molecular diagnosis. Epilepsia 50, 1670—1678. Millichap, J.J., Koh, S., Laux, L.C., Nordli Jr., D.R., 2009. Child Neurology: Dravet syndrome: when to suspect the diagnosis. Neurology 73, e59—e62. Mullen, S.A., Scheffer, I.E., 2009. Translational research in epilepsy genetics: sodium channels in man to interneuronopathy in mouse. Arch Neurol 66, 21—26. Mulley, J.C., Nelson, P., Guerrero, S., Dibbens, L., Iona, X., McMahon, J.M., Harkin, L., Schouten, J., Yu, S., Berkovic, S.F., Scheffer, I.E., 2006. A new molecular mechanism for severe myoclonic epilepsy of infancy: exonic deletions in SCN1A. Neurology 67, 1094—1095. Mulley, J.C., Scheffer, I.E., Petrou, S., Dibbens, L.M., Berkovic, S.F., Harkin, L.A., 2005. SCN1A mutations and epilepsy. Hum Mutat 25, 535—542. Nabbout, R., Gennaro, E., Dalla Bernardina, B., Dulac, O., Madia, F., Bertini, E., Capovilla, G., Chiron, C., Cristofori, G., Elia, M., Fontana, E., Gaggero, R., Granata, T., Guerrini, R., Loi, M., La Selva, L., Lispi, M.L., Matricardi, A., Romeo, A., Tzolas, V., Valseriati, D., Veggiotti, P., Vigevano, F., Vallee, L., Dagna Bricarelli, F., Bianchi, A., Zara, F., 2003. Spectrum of SCN1A mutations in severe myoclonic epilepsy of infancy. Neurology 60, 1961—1967. Ogiwara, I., Ito, K., Sawaishi, Y., Osaka, H., Mazaki, E., Inoue, I., Montal, M., Hashikawa, T., Shike, T., Fujiwara, T., Inoue, Y., Kaneda, M., Yamakawa, K., 2009. De novo mutations of voltagegated sodium channel ␣II gene SCN2A in intractable epilepsies. Neurology 73, 1046—1053. Ottman, R., Hirose, S., Jain, S., Lerche, H., Lopes-Cendes, I., Noebels, J.L., Serratosa, J., Zara, F., Scheffer, I.E., 2010. Genetic testing in the epilepsies-report of the ILAE Genetics Commission. Epilepsia 51, 655—670. Patino, G.A., Claes, L.R., Lopez-Santiago, L.F., Slat, E.A., Dondeti, R.S., Chen, C., O’Malley, H.A., Gray, C.B., Miyazaki, H., Nukina, N., Oyama, F., De Jonghe, P., Isom, L.L., 2009. A functional null mutation of SCN1B in a patient with Dravet syndrome. J. Neurosci. 29, 10764—10778. Shi, X., Yasumoto, S., Nakagawa, E., Fukasawa, T., Uchiya, S., Hirose, S., 2009. Missense mutation of the sodium channel gene SCN2A causes Dravet syndrome. Brain Dev. 31, 758—762. Suls, A., Claeys, K.G., Goossens, D., Harding, B., Van Luijk, R., Scheers, S., Deprez, L., Audenaert, D., Van Dyck, T., Beeckmans, S., Smouts, I., Ceulemans, B., Lagae, L., Buyse, G., Barisic, N., Misson, J.P., Wauters, J., Del-Favero, J., De Jonghe, P., Claes, L.R., 2006. Microdeletions involving the SCN1A gene may
200 be common in SCN1A-mutation-negative SMEI patients. Hum. Mutat. 27, 914—920. Wallace, R.H., Hodgson, B.L., Grinton, B.E., Gardiner, R.M., Robinson, R., Rodriguez-Casero, V., Sadleir, L., Morgan, J., Harkin, L.A., Dibbens, L.M., Yamamoto, T., Andermann, E., Mulley, J.C., Berkovic, S.F., Scheffer, I.E., 2003. Sodium channel alpha1subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms. Neurology 61, 765—769.
J.-w. Wang et al. Wang, J.W., Kurahashi, H., Ishii, A., Kojima, T., Ohfu, M., Inoue, T., Ogawa, A., Yasumoto, S., Oguni, H., Kure, S., Fujii, T., Ito, M., Okuno, T., Shirasaka, Y., Natsume, J., Hasegawa, A., Konagaya, A., Kaneko, S., Hirose, S., 2008. Microchromosomal deletions involving SCN1A and adjacent genes in severe myoclonic epilepsy in infancy. Epilepsia 49, 1528—1534.
journal homepage: www.elsevier.com/locate/epilepsyres
Prevalence of SCN1A mutations in children with suspected Dravet syndrome and intractable childhood epilepsy Ji-wen Wang a,b,1, Xiu-yu Shi a,c,1, Hirokazu Kurahashi a,d, Su-Kyeong Hwang a, Atsushi Ishii a, Norimichi Higurashi a,e, Sunao Kaneko f, Shinichi Hirose a,∗ , The Epilepsy Genetic Study Group Japan (Chairperson, SK) a
Department of Pediatrics, School of Medicine, Central Research Institute for the Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan b Department of Pediatrics, Qilu Hospital, Shandong University, Jinan, China c Department of Pediatrics, Chinese PLA General Hospital, Beijing, China d Department of Pediatric Neurology, Central Hospital of Aichi Welfare Center for Persons with Developmental Disabilities, Kasugai, Japan e Department of Pediatrics, Jikei University School of Medicine, Japan f Department of Neuropsyschiatry, School of Medicine, Hirosaki University, Hirosaki, Japan Received 5 April 2012; received in revised form 12 June 2012; accepted 16 June 2012 Available online 20 July 2012
KEYWORDS Channelopathy; Dravet syndrome; Genetic tests; Molecular diagnosis; Oversight
∗ 1
Summary Mutations of the gene encoding the ␣1 subunit of neuronal sodium channel, SCN1A, are reported to cause Dravet syndrome (DS). The prevalence of mutations reported in such studies (mainly in clinically confirmed DS) seems high enough to make genetic diagnosis feasible. In fact, commercially operating genetic diagnostic laboratories offering genetic analyses of SCN1A are available. Still, the exact prevalence of mutations of SCN1A remains elusive. Fukuoka University has been serving as a genetic diagnostic laboratory for DS for the last 10 years. In this study, we determined the prevalence of SCN1A mutations (SCN1A, SCN2A, SCN1B and SCN2B) in 448 patients with suspected DS and intractable childhood epilepsy. A total of 192 SCN1A mutations were identified in 188 of 448 patients (42.0%). The frequencies of SCN1A mutations in suspected severe myoclonic epilepsy of infancy (SMEI), its borderline phenotype (SMEB) and intractable epilepsy were 56.2%, 41.9% and 28.9% respectively. In addition, four SCN2A mutations were identified in 4 of 325 patients. No mutations of SCN1B and SCN2B were identified. These results are potentially helpful for the diagnosis of DS at early stage. © 2012 Elsevier B.V. All rights reserved.
Corresponding author at: 45-1, 7-chome, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan. Tel.: +81 92 801 1011; fax: +81 92 863 1970. E-mail address: [email protected] (S. Hirose). These authors contributed equally to this work.
0920-1211/$ — see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2012.06.006
196
Introduction Voltage gated sodium channels are one of the mediators of normal neuronal firing. Disruption of the gene encoding the ␣1 subunit of neuronal sodium channels (SCN1A) is associated with a variety of epilepsies, such as Dravet syndrome (DS), intractable childhood epilepsy with generalized tonic—clonic seizures (ICEGTC), genetic epilepsy with febrile seizures plus (GEFS+) and other rare early onset epileptic encephalopathies (Claes et al., 2003; Fujiwara et al., 2003; Harkin et al., 2007; Mulley et al., 2005). DS is a rare and malignant epilepsy syndrome, which usually develops in the first year of life (Dravet et al., 1992; Engel, 2001) and is currently classified as a disease entity that includes severe myoclonic epilepsy in infancy (SMEI) and its borderline phenotype (SMEB) (Mullen and Scheffer, 2009). Patients initially present with recurrent and prolonged seizures, usually febrile, hemiclonic or generalized tonic clonic, which frequently progress into status epilepticus. With advancement of age, the clinical features evolve into various afebrile or fever induced seizure types. Cognitive outcome is poor, with high risk of psychomotor retardation. However, the diagnosis of DS cannot be made at an early stage of the disease according to current diagnostic criteria. Heterozygous SCN1A mutations are a major cause of DS. To date, more than 700 of these have been reported in patients with DS (Lossin, 2009). Various types of SCN1A mutations, e.g., missense and nonsense mutations, have been observed in patients with DS (Fukuma et al., 2004). Moreover, microchromosomal deletions involving SCN1A (Wang et al., 2008), which cannot be detected by direct sequencing, are related to DS. Thus, the prevalence of SCN1A mutations may be higher than that considered previously and should be reevaluated. In fact, several studies have reported the prevalence of SCN1A mutations in patients with DS, although with wide variation, ranging from 33% (Nabbout et al., 2003; Wallace et al., 2003) to 80—100% (Claes et al., 2001; Fujiwara, 2006; Fukuma et al., 2004; Mulley et al., 2005). This variability could be due to the inclusion criteria used for the recruitment of the subjects. Thus, it is plausible that a high prevalence may be found in a cohort of clinically confirmed DS and low prevalence in young children with a provisional diagnosis of DS. Genetic analysis of SCN1A has become available in recent years, at least in commercially operating diagnostic genetic laboratories. Still, the exact prevalence of SCN1A mutations remains elusive. For the last 10 years, Fukuoka University has served as a genetic diagnostic laboratory for DS and other types of intractable childhood epilepsies and has conducted analysis of SCN1A mutations including microchromosomal deletions. In this study, we analyzed the genetic prevalence of SCN1A mutations diagnosed by commercially operating genetic diagnostic laboratories in patients with suspected DS (SMEI and SMEB) and those with other types of intractable childhood epilepsy.
J.-w. Wang et al.
Methods Patients This study included 448 patients with firm or provisional diagnosis of DS (SMEI n = 185; SMEB n = 62) or intractable childhood epilepsy (n = 201, including myoclonic-astatic epilepsy, multifocal epilepsy, complex partial epilepsy, fever triggered epilepsy, West syndrome and progressive myoclonic epilepsy) at the Departments of Pediatric Neurology at various regional tertiary hospitals. The diagnoses of SMEI and SMEB were made by the method described previously (Fukuma et al., 2004). Briefly, SMEI diagnosis was based on fulfillment of all the accepted diagnostic criteria for SMEI (Dravet et al., 1992), whereas the diagnosis of SMEB was based on the presence of clinical features that were almost identical to those of SMEI, excluding myoclonic and atypical absence seizures. We also recruited 96 healthy volunteers as the control group. Each participant or the parent/guardian signed an informed consent form approved by the Ethics Review Committee of Fukuoka University or similar committees of the participating institutions.
Genetic analysis Genomic DNAs were prepared from ethylenediaminetetraacetic acid (EDTA)-treated whole blood samples by using QIAamp DNA Blood Kit (Qiagen, Hilden, Germany). SCN1A, SCN2A, SCN1B and SCN2B were screened for genetic abnormalities using direct sequencing method with an automatic sequencer. Primers were designed to amplify all exons and the flanking intronic splice sites of the genes. The purified products of polymerase chain reaction (PCR) were directly sequenced and analyzed with the automatic sequencer. Details of the PCR conditions and the primers used are available upon request. Reference sequences of messenger RNA (mRNA) were based on information available from RefSeq (accession numbers: Human SCN1A AB093548; Human SCN2A NM 021007; Human SCN1B NM 001037; Human SCN2B NM 004588).
Multiplex ligation-dependent probe amplification (MLPA) MLPA was conducted in patients found to have no mutations and those with only missense mutations, using a commercially available kit for SCN1A (SALSA MLPA KIT P137 SCN1A, Lot 0107 or Lot 0805, MRC-Holland, Amsterdam, the Netherlands). MLPA tests were conducted according to the protocol supplied by the manufacturer. Fragment analysis of the PCR product was carried out on ABI model 310 capillary sequencer (Applied Biosystems, Foster City, CA) using GeneScanTM -500LIZ as size standards (Applied Biosystems) and deionized formamide (HiDi Formamide, Applied Biosystems). Data were analyzed using the Genescan software (Applied Biosystems). The thresholds were set at <0.65 for deletions and >1.35 for duplications. Details are available upon request.
Statistical analysis Data were analyzed by The Statistical Package for Social Sciences (version 17.0, SPSS Inc., Chicago, IL). Differences in the frequency distribution of mutations between SMEI, SMEB and Intractable Childhood Epilepsy were examined by the 2 test or Fisher’s exact test. All p values are two tailed, and significance was set at 5%.
Results A total of 192 SCN1A mutations were identified in 188 of 448 patients (42.0%). These mutations included 91 missense, 35 nonsense, 32 frameshift, 21 splice site, 4 deletion
Prevalence of SCN1A mutations Table 1
197
Exonic deletions of SCN1A identified by MLPA.
No
Diagnosis
Sex
Exonic deletion
Transmission
5 9 24 33 54 83 108 146 221
SMEI SMEI SMEI SMEI SMEI SMEI SMEI SMEI PME
F F M F M M F M M
Ex1∼Ex26 deletion Ex1∼Ex26 deletion Ex1∼Ex26 deletion Ex17∼Ex26 deletion Ex8∼Ex26 deletion Ex1∼Ex26 deletion Ex20∼Ex26 deletion Ex17∼Ex26 deletion Ex1∼Ex26 deletion
Unknown De novo De novo De novo De novo De novo De novo De novo Unknown
PME: progressive myoclonus epilepsy.
and 9 microchromosomal deletional mutations (E-Table A, Table 1). Four SCN2A mutations were identified in 4 of 325 patients. No SCN1B or SCN2B mutations were detected within the regions examined.
SCN1A point mutations 183 SCN1A point mutations were identified in 179 patients. Missense mutation of SCN1A was the most frequent mutation type (47.4%, 91/192), encountered in 89 patients. The frequencies of other mutation types were: nonsense mutation (18.2%, 35/192), frameshift mutation (16.7%, 32/192), and splice site mutation (10.9%, 21/192). Deletion mutations were identified in 4 patients (2.1%, 4/192). There were significant differences in the mutation and distribution frequencies among SMEI, SMEB and intractable epilepsy (Table 2), SCN1A mutations were most frequent in SMEI (56.2%, P = 0.001), whereas missense mutations were most frequent in SMEB 16/23 (69.2%, P = 0.027). Four patients harbored double SCN1A mutations. The first carried the de novo c.251A>G: Y84C mutation, and the c.4723C>T: R1575C variant, inherited from her apparent healthy mother. The second had the c.680T>G: I227S and the c.4723C>T: R1575C variants (samples not available from the parents). The third carried the IVS12-6 del T and the c.5674C>T: R1892X mutations (samples not available from the parents). The fourth carried the de novo c.14981538 del and the c.1497—1539 ins GAGGATGAATTCCAAAAA (R500fsX509) mutations. Biological samples from both parents of 69 patients with point mutations were available for genetic analysis. Direct sequencing showed none of the parents had any mutations
Table 2
in 64 cases out of 69 (92.8%), indicating de novo mutations. However, in one case, a boy and his sister had the same mutation but could not be detected in the blood samples from the parents, suggesting possible somatic and/or germline mosaicism in either parent. The mutation was inherited from the mother in 5 cases. In one of these, a girl and her brother with SMEI had the same SCN1A mutation inherited from the mother, but the mother had simple febrile seizures only during childhood. The other three mothers didn’t have any phenotype.
Microdeletions detected with MLPA MLPA analysis of 313 patients without mutation or with missense mutations only identified SCN1A microdeletions in 9 patients (2.9%, 9/313; Table 1). Samples from both parents were available for MLPA in 7 of these patients. The results of MLPA indicated that all 7 deletions were de novo. Deletion of the whole gene was detected in 5 patients (55.6%, 5/9). Deletion of 8—26th exons was found in one patient and 20—26th exons in another, whereas 17—26th exons were deleted in two patients. Among the 9 patients, 8 had SMEI and 1 had intractable epilepsy. No exonic deletion was found in patients with SMEB.
SCN2A mutations SCN2A polymorphism (c.56G>A: R19K) resulting in an amino acid exchange was identified, which had already been reported by our team (Ito et al., 2004). Four novel SCN2A missense mutations were identified in 4 patients. Two of the
Frequency distribution of different patients with SCN1A mutation.
Mutation
SMEI (n = 185)
SMEB (n = 62)
Intractable epilepsy (n = 201)
P value
With mutation Missense Nonsense Frameshift Splice site Deletion Exonic deletion
104(56.2%) 42(40.4%) 23(22.1%) 19(18.3%) 12(11.5%) 0 8(7.7%)
26(41.9%) 18(69.2%) 2(7.7%) 2(7.7%) 1(3.8%) 3(11.5%) 0
58(28.9%) 29(50.0%) 9(15.5%) 10(17.2%) 8(13.8%) 1(1.7%) 1(1.7%)
P = 0.001 P = 0.027 P = 0.193 P = 0.422 P = 0.402 P = 0.001 P = 0.109
198 4 patients harbored both SCN1A and SCN2A variants: one with SMEI carried de novo splice site mutation of SCN1A (IVS4 + 1G>A), and SCN2A missense mutation (c.964G>A: D322N), inherited from her mother; the other with SMEB had missense mutations of SCN1A (c.4507G>A: E1503K) and SCN2A (c.982T>G: F328V), her mother had same SCN2A mutation. The third patient with SMEI had de novo missense mutation of SCN2A (c.3935G>C: R1312T) but no SCN1A mutations (Shi et al., 2009). The fourth patient with SMEI had SCN2A missense mutation (c.1945G>A: D649N) without SCN1A mutations (samples not available from the parents).
Discussion DS is one of the catastrophic pediatric epilepsy syndromes and it is mainly due to SCN1A mutations. The first major clinical sign of DS is prolonged febrile seizure occurring 2—10 months of age; however, without genetic information, it is difficult to distinguish febrile seizure plus from those that will evolve into DS before they present other seizures. Therefore, many pediatric neurologists send blood samples of patients with suspected DS to genetic diagnostic centers and are often interested in information on the prevalence of SCN1A mutations in highly suspected babies. To our knowledge, however, there is no such information at present. Our study indicates that the prevalence of SCN1A mutation in patients with suspected SMEI, SMEB and intractable epilepsy is 56.2%, 41.9% and 28.9% respectively. The prevalence in our cohort was lower than previously reported data (70—80%) (Ottman et al., 2010). The difference could be due to the inclusion criteria used for recruitment of subjects. Previous studies recruited mainly patients with clinically proven DS, while in our study; we also included patients suspected with DS. For pediatric neurologists, it is important to know the genetic status of young children suspected to develop DS. Early diagnosis of DS and management with appropriate anticonvulsants and provision of a treatment plan may reduce the seizure burden and improve long-term developmental outcome (Millichap et al., 2009). All types of SCN1A mutations were identified in this study, however, the frequency distribution of missense mutations was significantly different among SMEI, SMEB and intractable childhood epilepsy [missense mutation 42 (40.4%), 18 (69.2%), 29 (50.0%), P = 0.027]. Missense mutation was most frequent in SMEB. Although there is no significant difference, nonsense and frameshift mutations were more common in SMEI. This finding is similar to our study reported previously (Fukuma et al., 2004). Fukuma et al. (2004) reported that the frequency of molecular truncation mutations, including nonsense and frameshift mutations, was high only in SMEI. Such information should help in establishing the diagnosis. In addition to mutations detected by direct sequencing, we identified partial (n = 4) and complete (n = 5) gene deletions by MLPA in 9 patients. Microchromosomal deletions affect not only SCN1A but also often involve the adjacent genes (Wang et al., 2008). The frequency of MLPA-detected chromosomal deletions was 2.9% (9/313) among the different types of epilepsy, mostly SMEI, and no exonic deletions were found in patients with SMEB. The prevalence in our cohort of 313 patients is similar to that reported by Suls et al.
J.-w. Wang et al. (2.7%) (Suls et al., 2006) but lower than that of Madia et al. (7.7%) (Madia et al., 2006) and Marini et al. (12.5%) (Marini et al., 2009). The difference from the latter two studies could be due to differences in phenotyping or to the larger number of patients analyzed. Our investigation forms the largest study of MLPA, encompassing 313 patients with various types of epilepsies. Moreover, previous studies included only SCN1A negative patients, whereas this study included also patients with SCN1A missense mutations. Although the overall frequency of microchromosomal deletions is 2—3%, these abnormalities are important pathogenic factors of DS. Several groups of researchers have stressed the importance of conducting MLPA in patients with sequencing negative mutations (Mulley et al., 2006; Wang et al., 2008). Parental samples were available in 40.4% (76/188) of cases with SCN1A abnormalities. Among them, 93.4% (71/76) were de novo cases whereas 6.6% (5/76) inherited from a healthy parent. Similar to our findings, familial SCN1A mutations have been reported previously in around 5% of patients with DS (Mulley et al., 2005). The mechanisms involved in these inherited mutations remain unclear. Gennaro et al. (2006) found that parental somatic and germline mosaicism can cause recurrent transmission of SCN1A mutations in DS. This partly explains the phenotypic variability and complex inheritance characteristics of DS patients whose SCN1A mutations appear to occur de novo. In our study, we identified four novel SCN2A missense mutations in four DS patients. All four mutations affected highly conserved amino acids in many species. Two of the SCN2A mutations coexisted with de novo SCN1A mutations. These two mutations were also inherited from one of the parents and accordingly were not likely to be pathogenic although they could have had modifying effect on the phenotypes of DS. In contrast, the third patient with SMEI had a de novo missense mutation of SCN2A (c.3935G>C: R1312T) without any SCN1A mutations. Arginine at the 1312 position is a crucial charged amino acid in the fourth transmembrane segment of domain III, which functions as a voltage sensor of the NaV 1.2 channel. This mutation in such an important position may downgrade the function of the channel and lead to DS. The fourth patient with SMEI only had SCN2A mutation, and since blood samples were not available from the parents, we were unable to determine whether or not the mutation was de novo. Ogiwara et al. (2009) reported de novo SCN2A mutations in intractable epilepsies, similar to our results, further confirming the potential role NaV 1.2 channel dysfunctions and its involvement in the molecular etiology of epilepsy. Recently, Patino et al. (2009) presented the first case of DS attributable to a homozygous mutation in SCN1B. Nevertheless, we are unable to identify any SCN1B or SCN2B mutations in our cohort of 448 patients. In summary, genetic information in young patients especially those with prolonged seizures aged less than 6 months, provides valuable information that does not only help in establishing the etiology but also early diagnosis, aggressive prevention and proper treatment.
Conflict of interest None of the authors has any conflict of interest to disclose.
Prevalence of SCN1A mutations
Acknowledgments We are indebted to all members of the family for their helpful cooperation in this study. We thank Ms. Takako Umemoto and Hideko Takeda for formatting and typing the manuscript and Ms. Minako Yonetani and Akiyo Hamachi for the technical assistance. This study was supported in part by Grantsin-Aid for Scientific Research (S) 16109006, (A) 18209035, 21249062 and 24249060, Exploratory Research 1659272 and 23659529, and ‘‘High-Tech Research Center’’ Project for Private Universities-matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology, 2006—2010- ‘‘The Research Center for the Molecular Pathomechanisms of Epilepsy, Fukuoka University’’, Research Grants (19A-6) and (21B-5) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare and the Central Research Institute of Fukuoka University.Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.eplepsyres.2012.06.006.
References Claes, L., Ceulemans, B., Audenaert, D., Smets, K., Lofgren, A., Del-Favero, J., Ala-Mello, S., Basel-Vanagaite, L., Plecko, B., Raskin, S., Thiry, P., Wolf, N.I., Van Broeckhoven, C., De Jonghe, P., 2003. De novo SCN1A mutations are a major cause of severe myoclonic epilepsy of infancy. Hum. Mutat. 21, 615—621. Claes, L., Del-Favero, J., Ceulemans, B., Lagae, L., Van Broeckhoven, C., De Jonghe, P., 2001. De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am. J. Hum. Genet. 68, 1327—1332. Dravet, C., Bureau, M., Guerrini, R., Giraud, N., Roger, J., 1992. Severe myoclonic epilepsy in infants. In: Roger, J., Dravet, C., Bureau, M., Dreifuss, F.E., Perret, A., Wolf, P. (Eds.), Epileptic Syndromes in Infancy, Childhood and Adolescence. John Libbey, London, pp. 75—88. Engel Jr., J., 2001. A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE Task Force on Classification and Terminology. Epilepsia 42, 796—803. Fujiwara, T., 2006. Clinical spectrum of mutations in SCN1A gene: severe myoclonic epilepsy in infancy and related epilepsies. Epilepsy Res. 70 (Suppl 1), S223—S230. Fujiwara, T., Sugawara, T., Mazaki-Miyazaki, E., Takahashi, Y., Fukushima, K., Watanabe, M., Hara, K., Morikawa, T., Yagi, K., Yamakawa, K., Inoue, Y., 2003. Mutations of sodium channel alpha subunit type 1 (SCN1A) in intractable childhood. Epilepsies with frequent generalized tonic—clonic seizures. Brain 126, 531—546. Fukuma, G., Oguni, H., Shirasaka, Y., Watanabe, K., Miyajima, T., Yasumoto, S., Ohfu, M., Inoue, T., Watanachai, A., Kira, R., Matsuo, M., Muranaka, H., Sofue, F., Zhang, B., Kaneko, S., Mitsudome, A., Hirose, S., 2004. Mutations of neuronal voltage-gated Na+ channel ␣1 subunit gene SCN1A in core severe myoclonic epilepsy in infancy (SMEI) and in borderline SMEI (SMEB). Epilepsia 45, 140—148. Gennaro, E., Santorelli, F.M., Bertini, E., Buti, D., Gaggero, R., Gobbi, G., Lini, M., Granata, T., Freri, E., Parmeggiani, A., Striano, P., Veggiotti, P., Cardinali, S., Bricarelli, F.D., Minetti, C., Zara, F., 2006. Somatic and germline mosaicisms in severe myoclonic epilepsy of infancy. Biochem. Biophys. Res. Commun. 341, 489—493. Harkin, L.A., McMahon, J.M., Iona, X., Dibbens, L., Pelekanos, J.T., Zuberi, S.M., Sadleir, L.G., Andermann, E., Gill, D., Farrell, K.,
199 Connolly, M., Stanley, T., Harbord, M., Andermann, F., Wang, J., Batish, S.D., Jones, J.G., Seltzer, W.K., Gardner, A., Sutherland, G., Berkovic, S.F., Mulley, J.C., Scheffer, I.E., 2007. The spectrum of SCN1A-related infantile epileptic encephalopathies. Brain 130, 843—852. Ito, M., Shirasaka, Y., Hirose, S., Sugawara, T., Yamakawa, K., 2004. Seizure phenotypes of a family with missense mutations in SCN2A. Pediatr Neurol 31, 150—152. Lossin, C., 2009. A catalog of SCN1A variants. Brain Dev 31, 114—130. Madia, F., Striano, P., Gennaro, E., Malacarne, M., Paravidino, R., Biancheri, R., Budetta, M., Cilio, M.R., Gaggero, R., Pierluigi, M., Minetti, C., Zara, F., 2006. Cryptic chromosome deletions involving SCN1A in severe myoclonic epilepsy of infancy. Neurology 67, 1230—1235. Marini, C., Scheffer, I.E., Nabbout, R., Mei, D., Cox, K., Dibbens, L.M., McMahon, J.M., Iona, X., Carpintero, R.S., Elia, M., Cilio, M.R., Specchio, N., Giordano, L., Striano, P., Gennaro, E., Cross, J.H., Kivity, S., Neufeld, M.Y., Afawi, Z., Andermann, E., Keene, D., Dulac, O., Zara, F., Berkovic, S.F., Guerrini, R., Mulley, J.C., 2009. SCN1A duplications and deletions detected in Dravet syndrome: implications for molecular diagnosis. Epilepsia 50, 1670—1678. Millichap, J.J., Koh, S., Laux, L.C., Nordli Jr., D.R., 2009. Child Neurology: Dravet syndrome: when to suspect the diagnosis. Neurology 73, e59—e62. Mullen, S.A., Scheffer, I.E., 2009. Translational research in epilepsy genetics: sodium channels in man to interneuronopathy in mouse. Arch Neurol 66, 21—26. Mulley, J.C., Nelson, P., Guerrero, S., Dibbens, L., Iona, X., McMahon, J.M., Harkin, L., Schouten, J., Yu, S., Berkovic, S.F., Scheffer, I.E., 2006. A new molecular mechanism for severe myoclonic epilepsy of infancy: exonic deletions in SCN1A. Neurology 67, 1094—1095. Mulley, J.C., Scheffer, I.E., Petrou, S., Dibbens, L.M., Berkovic, S.F., Harkin, L.A., 2005. SCN1A mutations and epilepsy. Hum Mutat 25, 535—542. Nabbout, R., Gennaro, E., Dalla Bernardina, B., Dulac, O., Madia, F., Bertini, E., Capovilla, G., Chiron, C., Cristofori, G., Elia, M., Fontana, E., Gaggero, R., Granata, T., Guerrini, R., Loi, M., La Selva, L., Lispi, M.L., Matricardi, A., Romeo, A., Tzolas, V., Valseriati, D., Veggiotti, P., Vigevano, F., Vallee, L., Dagna Bricarelli, F., Bianchi, A., Zara, F., 2003. Spectrum of SCN1A mutations in severe myoclonic epilepsy of infancy. Neurology 60, 1961—1967. Ogiwara, I., Ito, K., Sawaishi, Y., Osaka, H., Mazaki, E., Inoue, I., Montal, M., Hashikawa, T., Shike, T., Fujiwara, T., Inoue, Y., Kaneda, M., Yamakawa, K., 2009. De novo mutations of voltagegated sodium channel ␣II gene SCN2A in intractable epilepsies. Neurology 73, 1046—1053. Ottman, R., Hirose, S., Jain, S., Lerche, H., Lopes-Cendes, I., Noebels, J.L., Serratosa, J., Zara, F., Scheffer, I.E., 2010. Genetic testing in the epilepsies-report of the ILAE Genetics Commission. Epilepsia 51, 655—670. Patino, G.A., Claes, L.R., Lopez-Santiago, L.F., Slat, E.A., Dondeti, R.S., Chen, C., O’Malley, H.A., Gray, C.B., Miyazaki, H., Nukina, N., Oyama, F., De Jonghe, P., Isom, L.L., 2009. A functional null mutation of SCN1B in a patient with Dravet syndrome. J. Neurosci. 29, 10764—10778. Shi, X., Yasumoto, S., Nakagawa, E., Fukasawa, T., Uchiya, S., Hirose, S., 2009. Missense mutation of the sodium channel gene SCN2A causes Dravet syndrome. Brain Dev. 31, 758—762. Suls, A., Claeys, K.G., Goossens, D., Harding, B., Van Luijk, R., Scheers, S., Deprez, L., Audenaert, D., Van Dyck, T., Beeckmans, S., Smouts, I., Ceulemans, B., Lagae, L., Buyse, G., Barisic, N., Misson, J.P., Wauters, J., Del-Favero, J., De Jonghe, P., Claes, L.R., 2006. Microdeletions involving the SCN1A gene may
200 be common in SCN1A-mutation-negative SMEI patients. Hum. Mutat. 27, 914—920. Wallace, R.H., Hodgson, B.L., Grinton, B.E., Gardiner, R.M., Robinson, R., Rodriguez-Casero, V., Sadleir, L., Morgan, J., Harkin, L.A., Dibbens, L.M., Yamamoto, T., Andermann, E., Mulley, J.C., Berkovic, S.F., Scheffer, I.E., 2003. Sodium channel alpha1subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms. Neurology 61, 765—769.
J.-w. Wang et al. Wang, J.W., Kurahashi, H., Ishii, A., Kojima, T., Ohfu, M., Inoue, T., Ogawa, A., Yasumoto, S., Oguni, H., Kure, S., Fujii, T., Ito, M., Okuno, T., Shirasaka, Y., Natsume, J., Hasegawa, A., Konagaya, A., Kaneko, S., Hirose, S., 2008. Microchromosomal deletions involving SCN1A and adjacent genes in severe myoclonic epilepsy in infancy. Epilepsia 49, 1528—1534.