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Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis
Brain & Development xxx (2012) xxx–xxx www.elsevier.com/locate/braindev
Original article
Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis Atsushi Ishii a,b,c, Sawa Yasumoto a, Yukiko Ihara a,b, Takahito Inoue a, Takako Fujita a, Noriko Nakamura a, Masaharu Ohfu a, Yushiro Yamashita d, Hideo Takatsuka e, Toshiaki Taga f, Rie Miyata g, Masahiro Ito h, Hiroshi Tsuchiya i, Taro Matsuoka j, Tetsuya Kitao k, Kiyotaka Murakami k, Wang-Tso Lee l, Sunao Kaneko m, Shinichi Hirose a,b,⇑ a Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan Central Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan c Research Fellow of the Japan Society for the promotion of Science, Fukuoka University, Fukuoka, Japan d Department of Pediatrics and Child Health, School of Medicine, Kurume University, Kurume, Japan e Division of Pediatrics, Mimuro Hospital, Ikoma, Japan f Department of Pediatrics, Nagahama City Hospital, Nagahama, Japan g Department of Pediatrics, Tokyo-kita Social Insurance Hospital, Tokyo, Japan h Department of Pediatrics, Tokyo Metropolitan Bokuto Hospital, Tokyo, Japan i Department of Neonatology, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan j Department of Pediatrics, Toyonaka Municipal Hospital, Toyonaka, Japan k Nakano Children’s Hospital, Osaka, Japan l Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan m Department of Neuropsychiatry, School of Medicine, Hirosaki University, Hirosaki, Japan b
Received 5 June 2012; received in revised form 5 September 2012; accepted 11 September 2012
Abstract Purpose: PRRT2 mutations were recently identified in benign familial infantile epilepsy (BFIE) and infantile convulsions with paroxysmal choreoathetosis (ICCA) but no abnormalities have so far been identified in their phenotypically similar seizure disorder of benign convulsions with mild gastroenteritis (CwG), while mutations in KCNQ2 and KCNQ3 have been recognized in benign familial neonatal epilepsy (BFNE). The aim of this study was to identify PRRT2 mutations in infantile convulsions in Asian families with BFIE and ICCA, CwG and BFNE. Methods: We recruited 26 unrelated Japanese affected with either BFIE or non-familial benign infantile seizures and their families, including three families with ICCA. A total of 17 Japanese and Taiwanese with CwG, 50 Japanese with BFNE and 96 healthy volunteers were also recruited. Mutations of PRRT2 were sought using direct sequencing. Results: Heterozygous truncation mutation (c.649dupC) was identified in 15 of 26 individuals with benign infantile epilepsy (52.1%). All three families of ICCA harbored the same mutation (100%). Another novel mutation (c.1012+2dupT) was found in the proband of a family with BFIE. However, no PRRT2 mutation was found in either CwG or BFNE. Conclusions: The results confirm that c.649dupC, a truncating mutation of PRRT2, is a hotspot mutation resulting in BFIE or ICCA regardless of the ethnic background. In contrast, PRRT2 mutations do not seem to be associated with CwG or BFNE. Screening for PRRT2 mutation might be useful in early-stage differentiation of BFIE from CwG. Ó 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. ⇑ Corresponding author. Address: 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). 0387-7604/$ - see front matter Ó 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.braindev.2012.09.006
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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Keywords: PRRT2; Benign familial infantile epilepsy (BFIE); Benign convulsions with mild gastroenteritis (CwG); Benign familial neonatal epilepsy (BFNE); Paroxysmal kinesigenic choreoathetosis (PKC); Infantile convulsion with paroxysmal choreoathetosis (ICCA)
1. Introduction Benign familial infantile epilepsy (BFIE: OMIM 605751) is an autosomal dominant familial epilepsy syndrome with a high penetrance, characterized by frequent seizures in infancy with onset at 3–24 months of age. The seizures tend to occur in clusters and remit spontaneously in late infancy. In general, the prognosis is favorable and psychomotor development is normal. The clinical subtype of BFIE, known as infantile convulsions with paroxysmal choreoathetosis syndrome (ICCA: OMIM 602066) shares common clinical features with BFIE, with a susceptibility locus on 16p12-q12 [1,2]. Thus, ICCA and BFIE are considered allelic variants. Until recently, the underlying genetic abnormality in BFIE and ICCA remained elusive. Using wholeexome sequencing, Chen et al. [3] identified three heterozygous truncating mutations within PRRT2 (NM_145239.2) in eight Chinese families with histories of paroxysmal kinesigenic dyskinesia, an involuntary movement disorder that includes the paroxysmal kinesigenic choreoathetosis (PKC: OMIM 128200) seen in ICCA. In addition, PKD shares its susceptible locus on 16p12-q12 [1] with BFIE and ICCA. Subsequently, an Australian group identified heterozygous mutations in PRRT2 in 14 of 17 families affected by BFIE, and PRRT2 mutations in five of six families affected by ICCA [4]. The results of these studies provide compelling evidence that mutations in PRRT2 cause BFIE, PKD, or ICCA. There are several other convulsive disorders similar to BFIE, namely benign convulsions with mild gastroenteritis (CwG), benign familial neonatal epilepsy (BFNE: OMIM 121200, 121201) and benign familial neonatal-infantile epilepsy (BFNIE: OMIM 607745). CwG is a well recognized and relatively common seizure disorder in infancy associated with mild diarrhea including rotavirus gastroenteritis. In fact, the incidence of CwG is higher in Asian than Western countries [5–7]. CwG and BFIE have common onset features and seizure semiology as well as response to certain anti-epileptic drugs, such as carbamazepine. However, the underlying genetic abnormality of CwG has not yet been identified. BFNE presents with a seizure semiology reminiscent of that of BFIE although the seizures exclusively afflict neonates [8–15]. BFNE is caused by mutation of either KCNQ2 or KCNQ3 genes, which encode the voltagegated potassium channels. However, no mutations are identified in more than 15% of BFNE.
The aim of this study was to characterize PRRT2 mutations and their phenotypes in Asians and whether they are associated with seizure disorders similar to BFIE, such as CwG and BFNE. For this purpose, mutation analysis of PRRT2 was conducted in an Asian cohort with BFIE, ICCA, CwG and BFNE. 2. Materials and methods 2.1. Subjects and families The study subjects were 26 Japanese families with individuals affected by infantile seizures with seizure phenotypes compatible with BFIE (19 families), ICCA (3 families) and benign infantile epilepsy [(BIE), i.e., without apparent family history (3 families)] (Table 1). The diagnoses of BIE and BFIE were established based on clinical manifestations such as afebrile seizures occurring at the age of 3–24 months with spontaneous remission within infancy, with and without family history, respectively. ICCA was diagnosed when family members had a history of PKC. We also recruited 50 Japanese families with BFNE. BFNE was diagnosed based on the accepted diagnostic criteria of seizures appearing at the age of 1–7 days in otherwise healthy neonates, followed by spontaneous remission, and evidence of autosomal inheritance. The study also included 2 Japanese and 15 Taiwanese patients diagnosed with CwG. The diagnosis of CwG was established based on the clinical manifestation of a cluster of afebrile seizures appearing at 4–30 months of age with mild gastroenteritis. Any other etiology of seizures was excluded by blood tests, cerebrospinal fluid examination, electroencephalography and neuroimaging studies. Linkage analysis was not performed in the present cohort because of the small number of samples. The control group comprised 96 unrelated healthy Japanese volunteers free of seizures and with negative history for epilepsy. The parent or guardian of each participant signed an informed consent form before the study. Furthermore, the study was approved by the Ethics Review Committee of Fukuoka University and similar committees of the participating institutions. 2.2. Polymerase chain reaction Genetic analysis of PRRT2 was performed using the direct sequence method described in detail previously [13,14,16–18]. Briefly, genomic DNA was prepared from
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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Table 1 Clinical features and PRRT2 mutations of 26 probands. Proband
Family
Gender
Mutation
Mean age at onset, months (range)
Seizure type
Seizure status
Effective medicine
Family history
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
F F M M M F M M M M M F F M F M F F M M M F M M M F
c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.1012+2dupT ND ND ND ND ND ND ND ND ND ND
10.5 (5–16) 16.5 (5–28) 4 4 5 5 (4–6) 5 3.3 (3–3.5) 3.5 (3–4) 4 6.5 (4–9) 6.5 (5–8) 4.5 (4–5) 5 3.4 (3–4) 4 7 8 16 5 (4–6) Unknown 5 5.5 (3–8) 6 1.5 (1–2) 4
GTC GTC Unknown Unknown GT Partial Partial Partial Partial GTC Partial Unknown GTC GTC Partial GTC Unknown Unknown Unknown Partial Unknown Unknown GTC Partial Partial GTC
Cluster SE Unknown Unknown Cluster Cluster Cluster cluster Cluster Unknown Cluster SE Cluster Cluster Cluster Cluster Unknown Unknown Unknown Cluster Unknown Unknown Unknown Unknown SE Cluster
CBZ CBZ Unknown CBZ CBZ CBZ CBZ VPA VPA, PHT Lidocaine Lidocaine VPA, PHT CBZ VPA CBZ CBZ, VPA Unknown Unknown Unknown None CBZ VPA, PHT PB – CBZ CBZ
BFIE BFIE BFIE BFIE BFIE BFIE BFIE BFIE BFIE BFIE BFIE BIE ICCA ICCA ICCA BFIE Unknown BFIE BFIE BIE BFIE BFIE BFIE BFIE BFIE BIE
M, male; F, female; ND, not detected; GTC, generalized tonic clonic seizure; SE, status epilepticus; CBZ, carbamazepine; VPA, valproic acid; PHT, phenitoin; PB, phenobarbital.
blood samples containing EDTA-Na2 using the QIAamp DNA Blood Maxi Kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer. The entire PRRT2 coding sequence (accession number; RefSeq: NM_145239.2), including adjacent short intronic sequences of the gene (accession number; NC_000016.9), was amplified by PCR with genomic DNA obtained from one affected individual of each family. Once PRRT2 variations were detected, the variations were sought in the families and controls. The primer sequences and corresponding PCR protocols are available on request. After amplification and purification on a 96-well PCR Clean-up Kit (Whatman, Piscataway, NJ), the PCR products were sequenced directly by the dye terminator sequencing method using the amplification primers on an ABI PRISMÒ 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). 3. Results Two genetic variations were identified within the investigated region of PRRT2 (Fig. 1A–C). One was a heterozygous single nucleotide duplicational variant, c.649dupC (Fig. 1A and C) and the other was a novel heterozygous variant, c.1012+2dupT (Fig. 1B and C).
The c.649dupC, which has been reported in BFIE and ICCA [4], is assumed to result in a frame shift followed by a premature stop codon (p.Arg217ProfsX8). The c.1012+2dupT locates at the splice donor site. In silico, c.1012+2dupT was considered to affect mRNA splicing (see URL). The predicted splice donor site of exon3 caused by c.1012+2dupT showed different exon/intron boundary with normal splice site and lower acceptor score (maximum 0.75) than normal (real site: 0.99) (Table 2). Therefore, the c.1012+2dupT was predicted to produce aberrant protein. Neither of the variants was found in the controls. Based on the deduced consequences of both mutations and the fact that they were not found in controls, both were considered disease causing mutations. The c.649dupC was identified in 12 probands of families with BFIE (Families 1–11) and BIE without family history (Family 12) ( Fig. 2). The same mutation was found in three families with ICCA. In turn, all families with ICCA in this study harbored the same c.649dupC mutation although they were unrelated. The c.1012 +2dupT was found in a family with BFIE (Family 16). No PRRT2 variation was detected within the investigated region in the remaining families (10 families either with BFIE or BIE). Likewise, no PRRT2 variation was
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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A. Ishii et al. / Brain & Development xxx (2012) xxx–xxx
A
B
C
Fig. 1. Nucleotide sequence of the relevant region of PRRT2 mutations of the patients. Genomic DNA from the patient was amplified for short fragments segmented exons 2, 3 and 4 of PRRT2 by PCR. The nucleotide sequences of the products were determined by the dye terminator cycle sequencing method in an ABI auto-sequencer. The nucleotide sequence was determined in 50 –30 direction. (A): DNA sequence of the wild type allele (top) and the c.649dupC mutation (bottom). Arrow indicates nucleotide 650 of the coding sequence, where a single nucleotide was inserted. (B): DNA sequence of the wild type allele (top) and the c.1012+2dupT mutation (bottom). Capital letters indicate nucleotides in exon, and lower cases indicate those in intron. Arrow indicates nucleotide 3 of intron 3, where a single nucleotide was inserted. (C): Positions of the identified mutations of PRRT2. The c.649dupC mutation locates at exon 2, whereas the c.1012+2dupT mutation locates at canonical donor splice site of intron 3.
Table 2 Prediction of splice donor site of mutation. Normal
c.1012+3_4insT
PRRT2 transcript variant1 (NM_145239.2)
Start
End
Score
Exon/Intron
Start
End
Score
Exon/Intron
Exon1
5230 1109 1729 2111 2269 2801 3715 3786 3851
1002 1123 1743 2125 2283 2815 3729 3800 3865
0.99 0.47 0.77 0.52 0.99 0.99 0.70 0.74 0.75
cgtcgagGTgagacc caaaaggGTtaatct accagaaGTgagcaa ccccgagGTagcctg tgtcatgGTgagccc ttaggcgGTgagtgg gactcggGTgagggt cttccagGTcagctg cttgaagGTgtgggg
5230 1109 1729 2111 2269 3716 3787 3852
1002 1123 1743 2125 2283 3730 3801 3866
0.99 0.47 0.77 0.52 0.99 0.70 0.74 0.75
cgtcgagGTgagacc caaaaggGTtaatct accagaaGTgagcaa ccccgagGTagcctg tgtcatgGTgagccc gactcggGTgagggt cttccagGTcagctg cttgaaggtGTgggg
Exon2 Exon3
Start and End position means genomic number of chromosome 16. Large letters “GT” represent boundary of exon and intron. The score represents donor score.
found in CwG or BFNE. Comparison of the clinical characteristics of carriers of PRRT2 mutations and non-carriers showed no difference (Table 1). 4. Discussion Mutations of PRRT2 have been reported recently as the cause of PKC, BFIE and ICCA [3,4,19–23]. To date, mutations in PKC have been reported exclusively in Asians with c.649dupC mutation being a hot spot mutation. In contrast, mutations of PRRT2 in BFIE
and ICCA were reported in both Asians and nonAsians. Intriguingly, however, the hot spot mutation underlying BFIE and ICCA was also c.649dupC mutation. In this study, we showed that c.649dupC mutation is associated with the majority of BFIE, BIE and ICCA Japanese families (15/26) and described a novel c.1012+2dupT mutation as the underlying cause of BFIE. These findings suggest that c.649dupC mutation is a recurrent mutation among PKC, BFIE and ICCA regardless of ethnicities. Therefore, the c.649dupC
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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Fig. 2. Pedigrees of 16 families with PRRT2 mutations. Arrow: proband, E+: individuals with heterozygous PRRT2 mutations, E: individuals negative for PRRT2 mutations. The c.649dupC mutation was identified in Family 1 to 15. The c.1012+2dupT mutation was identified only in Family 16.
recurrent mutation does not suggest a founder effect but may result from proneness of the nucleotide repeat in the relevant position of PRRT2, since another novel mutation, c.1012+2dupT, occurred in the GC-rich region of the gene. In our cohort, the available clinical reports could not explain the phenotypic difference between BFIE with and without PRRT2 mutation. This was in part due to unavailability of the clinical records of BFIE without PRRT2 mutation. In general, however, in most patients with PRRT2 mutations, seizures tended to start as partial seizures and formed clusters. These partial seizures showed no seizure status, whereas the partial seizures in patients without PRRT2 mutation showed sei-
zure status. All ICCA families harbored the c.649dupC mutation. This implies genetic heterogeneity in BFIE and BIE. The pathological changes resulting from PRRT2 in PKC, BFIE and ICCA remain unclear. In fact, the physiological functions of PRRT2 per se have yet to be clarified, although the expression of PRRT2 mRNA has been reported in the brain and spinal cord [3]. Furthermore, PRRT2 mRNA levels in the whole brain reach peak levels at postnatal day 14 then decline in the adult brain of mice [3]. This profile is in agreement with the clinical course of BFIE, which is characterized by infantile onset followed by spontaneous remission. PRRT2 is known to interact with SNAP25 (NM_003081.3) both in vitro and in vivo [24]. SNAP25,
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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synaptosomal-associated protein 25, is a presynaptic plasma membrane protein engaged in synaptic vesicle membrane docking and fusion pathway [25] and plays a role in the regulation of intracellular calcium dynamics as well as in the exocytosis [26–28]. The SNARE complex, which includes SNAP25, is associated with the N or P/Q types of voltage-dependent calcium channels, which open in response to an action potential generated by voltage-gated sodium channels. Carbamazepine has little pharmacological effect on the calcium channels. The association between PRRT2 and SNAP25 is intriguing because PRRT2 includes two transmembrane domains while SNAP25 has no membrane-binding regions. PRRT2 may serve as an anchor to connect SNAP25 to the presynaptic membrane. Other players in the genetic heterogeneity of BFIE and BIE may reside in putative molecules in such interactions whereas PRRT2 mutations are required for ICCA. The present study also demonstrated that PRRT2 is unlikely to be associated with the pathogenesis of CwG, a common infantile seizure disorder in the Asian population. Also, the lack of mutation in BFNE negates the role of PRRT2 in this phenotypically similar seizure disorder in early infancy. Both CwG and BFNE are sometimes difficult to differentiate from BFIE and BIE at clinical settings. Since c.649dupC is a hotspot mutation in BFIE and BIE, screening for PRRT2 mutation might be useful to distinguish between BFIE and CwG or BFNE at an early stage. In summary, the identification of PRRT2 mutations in PKC and BFIE opens a new avenue toward our understanding of so called “benign” paroxysmal, such as movements, which respond well to medications, and also of epilepsy that shows spontaneous remission. Further studies on PRRT2 should shed light on the development of novel antiepileptic drugs for not only BFIE but also other epilepsies. 5. URL Splice Site Prediction by Neural Network http:// www.fruitfly.org/seq_tools/splice.html. Conflict of interest The authors have no financial or personal relations that could pose a conflict of interest. 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.
Acknowledgments The authors are indebted to all members of the family for their helpful cooperation in this study. The authors
also thank Akiyo Hamachi and Minako Yonetani for the excellent technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research (A) 21249062, Grant-in-Aid for Challenging Exploratory Research (23659529), Japan Society for the Promotion of Science (JSPS); Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) Exploratory Research, Japan Science and Technology Agency (JSP); Research Grants for Nervous and Mental Disorder (21B-5) and Health and Labor Science Research Grant KB230019, KB230004 from the Ministry of Health, Labor and Welfare; 2013–2017 “Central Research Institute for the Molecular Pathomechanisms of Epilepsy of Fukuoka University” Recommended Projects of Fukuoka University (117016); and Research foundation for Clinical Medical Promotion. References [1] Caraballo R, Pavek S, Lemainque A, Gastaldi M, Echenne B, Motte J, et al. Linkage of benign familial infantile convulsions to chromosome 16p12-q12 suggests allelism to the infantile convulsions and choreoathetosis syndrome. Am J Hum Genet (in eng) 2001;68:788–94. [2] Szepetowski P, Rochette J, Berquin P, Piussan C, Lathrop GM, Monaco AP. Familial infantile convulsions and paroxysmal choreoathetosis: a new neurological syndrome linked to the pericentromeric region of human chromosome 16. Am J Hum Genet (in Eng) 1997;61:889–98. [3] Chen WJ, Lin Y, Xiong ZQ, Wei W, Ni W, Tan GH, et al. Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia. Nat Genet (in Eng) 2011;43:1252–5. [4] Heron SE, Grinton BE, Kivity S, Afawi Z, Zuberi SM, Hughes JN, et al. PRRT2 mutations cause benign familial infantile epilepsy and infantile convulsions with choreoathetosis syndrome. Am J Hum Genet (in Eng) 2012;90:152–60. [5] Contino MF, Lebby T, Arcinue EL. Rotaviral gastrointestinal infection causing afebrile seizures in infancy and childhood. Am J Emerg Med (in Eng) 1994;12:94–5. [6] Kawano G, Oshige K, Syutou S, Koteda Y, Yokoyama T, Kim BG, et al. Benign infantile convulsions associated with mild gastroenteritis: a retrospective study of 39 cases including virological tests and efficacy of anticonvulsants. Brain Dev (in Eng) 2007;29:617–22. [7] Lin SC, Hsu HY, Wang PJ, Lee CN, Chang MH, Shen YZ, et al. Rotavirus gastroenteritis associated with afebrile seizure in childhood. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi (in Eng) 1996;37:204–7. [8] Biervert C, Schroeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ, et al. A potassium channel mutation in neonatal human epilepsy. Science (in Eng) 1998;279:403–6. [9] Charlier C, Singh NA, Ryan SG, Lewis TB, Reus BE, Leach RJ, et al. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat Genet (in Eng) 1998;18:53–5. [10] Claes LR, Ceulemans B, Audenaert D, Deprez L, Jansen A, Hasaerts D, et al. De novo KCNQ2 mutations in patients with benign neonatal seizures. Neurology (in Eng) 2004;63:2155–8. [11] de Haan GJ, Pinto D, Carton D, Bader A, Witte J, Peters E, et al. A novel splicing mutation in KCNQ2 in a multigenerational
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
A. Ishii et al. / Brain & Development xxx (2012) xxx–xxx
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
family with BFNC followed for 25 years. Epilepsia (in Eng) 2006;47:851–9. Hirose S, Okada M, Kaneko S, Mitsudome A. Are some idiopathic epilepsies disorders of ion channels?: a working hypothesis. Epilepsy Res (in Eng) 2000;41:191–204. Hirose S, Zenri F, Akiyoshi H, Fukuma G, Iwata H, Inoue T, et al. A novel mutation of KCNQ3 (c.925T –>C) in a Japanese family with benign familial neonatal convulsions. Ann Neurol (in Eng) 2000;47:822–6. Ishii A, Fukuma G, Uehara A, Miyajima T, Makita Y, Hamachi A, et al. A de novo KCNQ2 mutation detected in non-familial benign neonatal convulsions. Brain Dev (in Eng) 2009;31:27–33. Singh NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R, et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat Genet (in Eng) 1998;18:25–9. Fukuma G, Oguni H, Shirasaka Y, Watanabe K, Miyajima T, Yasumoto S, et al. Mutations of neuronal voltage-gated Na+ channel alpha 1 subunit gene SCN1A in core severe myoclonic epilepsy in infancy (SMEI) and in borderline SMEI (SMEB). Epilepsia (in Eng) 2004;45:140–8. Hirose S, Iwata H, Akiyoshi H, Kobayashi K, Ito M, Wada K, et al. A novel mutation of CHRNA4 responsible for autosomal dominant nocturnal frontal lobe epilepsy. Neurology (in Eng) 1999;53:1749–53. Ishii A, Zhang B, Kaneko S, Hirose S. Positive association between benign familial infantile convulsions and LGI4. Brain Dev (in Eng) 2010;32:538–43. Cao L, Huang XJ, Zheng L, Xiao Q, Wang XJ, Chen SD. Identification of a novel PRRT2 mutation in patients with paroxysmal kinesigenic dyskinesias and c.649dupC as a mutation hot-spot. Parkinsonism Relat Disord (in Eng) 2012.
7
[20] Li J, Zhu X, Wang X, Sun W, Feng B, Du T, et al. Targeted genomic sequencing identifies PRRT2 mutations as a cause of paroxysmal kinesigenic choreoathetosis. J Med Genet (in Eng) 2012;49:76–8. [21] Liu Q, Qi Z, Wan XH, Li JY, Shi L, Lu Q, et al. Mutations in PRRT2 result in paroxysmal dyskinesias with marked variability in clinical expression. J Med Genet (in Eng) 2012;49:79–82. [22] Wang JL, Cao L, Li XH, Hu ZM, Li JD, Zhang JG, et al. Identification of PRRT2 as the causative gene of paroxysmal kinesigenic dyskinesias. Brain (in Eng) 2011;134:3493–501. [23] Ono S, Yoshiura K, Kinoshita A, Kikuchi T, Nakane Y, Kato N, et al. Mutations in PRRT2 responsible for paroxysmal kinesigenic dyskinesias also cause benign familial infantile convulsions. J Hum Genet (in Eng) 2012;57:338–41. [24] Lee H-Y, Huang Y, Bruneau N, Roll P, Roberson Elisha DO, Hermann M, et al. Mutations in the gene PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions. Cell Rep 2012;1:2–12. [25] Zhao N, Hashida H, Takahashi N, Sakaki Y. Cloning and sequence analysis of the human SNAP25 cDNA. Gene (in Eng) 1994;145:313–4. [26] Seagar M, Takahashi M. Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis. J Bioenerg Biomembr (in Eng) 1998;30:347–56. [27] Sheng ZH, Rettig J, Cook T, Catterall WA. Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature (in Eng) 1996;379:451–4. [28] Verderio C, Pozzi D, Pravettoni E, Inverardi F, Schenk U, Coco S, et al. SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization. Neuron (in Eng) 2004;41:599–610.
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
Original article
Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis Atsushi Ishii a,b,c, Sawa Yasumoto a, Yukiko Ihara a,b, Takahito Inoue a, Takako Fujita a, Noriko Nakamura a, Masaharu Ohfu a, Yushiro Yamashita d, Hideo Takatsuka e, Toshiaki Taga f, Rie Miyata g, Masahiro Ito h, Hiroshi Tsuchiya i, Taro Matsuoka j, Tetsuya Kitao k, Kiyotaka Murakami k, Wang-Tso Lee l, Sunao Kaneko m, Shinichi Hirose a,b,⇑ a Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan Central Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan c Research Fellow of the Japan Society for the promotion of Science, Fukuoka University, Fukuoka, Japan d Department of Pediatrics and Child Health, School of Medicine, Kurume University, Kurume, Japan e Division of Pediatrics, Mimuro Hospital, Ikoma, Japan f Department of Pediatrics, Nagahama City Hospital, Nagahama, Japan g Department of Pediatrics, Tokyo-kita Social Insurance Hospital, Tokyo, Japan h Department of Pediatrics, Tokyo Metropolitan Bokuto Hospital, Tokyo, Japan i Department of Neonatology, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Japan j Department of Pediatrics, Toyonaka Municipal Hospital, Toyonaka, Japan k Nakano Children’s Hospital, Osaka, Japan l Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan m Department of Neuropsychiatry, School of Medicine, Hirosaki University, Hirosaki, Japan b
Received 5 June 2012; received in revised form 5 September 2012; accepted 11 September 2012
Abstract Purpose: PRRT2 mutations were recently identified in benign familial infantile epilepsy (BFIE) and infantile convulsions with paroxysmal choreoathetosis (ICCA) but no abnormalities have so far been identified in their phenotypically similar seizure disorder of benign convulsions with mild gastroenteritis (CwG), while mutations in KCNQ2 and KCNQ3 have been recognized in benign familial neonatal epilepsy (BFNE). The aim of this study was to identify PRRT2 mutations in infantile convulsions in Asian families with BFIE and ICCA, CwG and BFNE. Methods: We recruited 26 unrelated Japanese affected with either BFIE or non-familial benign infantile seizures and their families, including three families with ICCA. A total of 17 Japanese and Taiwanese with CwG, 50 Japanese with BFNE and 96 healthy volunteers were also recruited. Mutations of PRRT2 were sought using direct sequencing. Results: Heterozygous truncation mutation (c.649dupC) was identified in 15 of 26 individuals with benign infantile epilepsy (52.1%). All three families of ICCA harbored the same mutation (100%). Another novel mutation (c.1012+2dupT) was found in the proband of a family with BFIE. However, no PRRT2 mutation was found in either CwG or BFNE. Conclusions: The results confirm that c.649dupC, a truncating mutation of PRRT2, is a hotspot mutation resulting in BFIE or ICCA regardless of the ethnic background. In contrast, PRRT2 mutations do not seem to be associated with CwG or BFNE. Screening for PRRT2 mutation might be useful in early-stage differentiation of BFIE from CwG. Ó 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. ⇑ Corresponding author. Address: 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). 0387-7604/$ - see front matter Ó 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.braindev.2012.09.006
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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Keywords: PRRT2; Benign familial infantile epilepsy (BFIE); Benign convulsions with mild gastroenteritis (CwG); Benign familial neonatal epilepsy (BFNE); Paroxysmal kinesigenic choreoathetosis (PKC); Infantile convulsion with paroxysmal choreoathetosis (ICCA)
1. Introduction Benign familial infantile epilepsy (BFIE: OMIM 605751) is an autosomal dominant familial epilepsy syndrome with a high penetrance, characterized by frequent seizures in infancy with onset at 3–24 months of age. The seizures tend to occur in clusters and remit spontaneously in late infancy. In general, the prognosis is favorable and psychomotor development is normal. The clinical subtype of BFIE, known as infantile convulsions with paroxysmal choreoathetosis syndrome (ICCA: OMIM 602066) shares common clinical features with BFIE, with a susceptibility locus on 16p12-q12 [1,2]. Thus, ICCA and BFIE are considered allelic variants. Until recently, the underlying genetic abnormality in BFIE and ICCA remained elusive. Using wholeexome sequencing, Chen et al. [3] identified three heterozygous truncating mutations within PRRT2 (NM_145239.2) in eight Chinese families with histories of paroxysmal kinesigenic dyskinesia, an involuntary movement disorder that includes the paroxysmal kinesigenic choreoathetosis (PKC: OMIM 128200) seen in ICCA. In addition, PKD shares its susceptible locus on 16p12-q12 [1] with BFIE and ICCA. Subsequently, an Australian group identified heterozygous mutations in PRRT2 in 14 of 17 families affected by BFIE, and PRRT2 mutations in five of six families affected by ICCA [4]. The results of these studies provide compelling evidence that mutations in PRRT2 cause BFIE, PKD, or ICCA. There are several other convulsive disorders similar to BFIE, namely benign convulsions with mild gastroenteritis (CwG), benign familial neonatal epilepsy (BFNE: OMIM 121200, 121201) and benign familial neonatal-infantile epilepsy (BFNIE: OMIM 607745). CwG is a well recognized and relatively common seizure disorder in infancy associated with mild diarrhea including rotavirus gastroenteritis. In fact, the incidence of CwG is higher in Asian than Western countries [5–7]. CwG and BFIE have common onset features and seizure semiology as well as response to certain anti-epileptic drugs, such as carbamazepine. However, the underlying genetic abnormality of CwG has not yet been identified. BFNE presents with a seizure semiology reminiscent of that of BFIE although the seizures exclusively afflict neonates [8–15]. BFNE is caused by mutation of either KCNQ2 or KCNQ3 genes, which encode the voltagegated potassium channels. However, no mutations are identified in more than 15% of BFNE.
The aim of this study was to characterize PRRT2 mutations and their phenotypes in Asians and whether they are associated with seizure disorders similar to BFIE, such as CwG and BFNE. For this purpose, mutation analysis of PRRT2 was conducted in an Asian cohort with BFIE, ICCA, CwG and BFNE. 2. Materials and methods 2.1. Subjects and families The study subjects were 26 Japanese families with individuals affected by infantile seizures with seizure phenotypes compatible with BFIE (19 families), ICCA (3 families) and benign infantile epilepsy [(BIE), i.e., without apparent family history (3 families)] (Table 1). The diagnoses of BIE and BFIE were established based on clinical manifestations such as afebrile seizures occurring at the age of 3–24 months with spontaneous remission within infancy, with and without family history, respectively. ICCA was diagnosed when family members had a history of PKC. We also recruited 50 Japanese families with BFNE. BFNE was diagnosed based on the accepted diagnostic criteria of seizures appearing at the age of 1–7 days in otherwise healthy neonates, followed by spontaneous remission, and evidence of autosomal inheritance. The study also included 2 Japanese and 15 Taiwanese patients diagnosed with CwG. The diagnosis of CwG was established based on the clinical manifestation of a cluster of afebrile seizures appearing at 4–30 months of age with mild gastroenteritis. Any other etiology of seizures was excluded by blood tests, cerebrospinal fluid examination, electroencephalography and neuroimaging studies. Linkage analysis was not performed in the present cohort because of the small number of samples. The control group comprised 96 unrelated healthy Japanese volunteers free of seizures and with negative history for epilepsy. The parent or guardian of each participant signed an informed consent form before the study. Furthermore, the study was approved by the Ethics Review Committee of Fukuoka University and similar committees of the participating institutions. 2.2. Polymerase chain reaction Genetic analysis of PRRT2 was performed using the direct sequence method described in detail previously [13,14,16–18]. Briefly, genomic DNA was prepared from
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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Table 1 Clinical features and PRRT2 mutations of 26 probands. Proband
Family
Gender
Mutation
Mean age at onset, months (range)
Seizure type
Seizure status
Effective medicine
Family history
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
F F M M M F M M M M M F F M F M F F M M M F M M M F
c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.649dupC c.1012+2dupT ND ND ND ND ND ND ND ND ND ND
10.5 (5–16) 16.5 (5–28) 4 4 5 5 (4–6) 5 3.3 (3–3.5) 3.5 (3–4) 4 6.5 (4–9) 6.5 (5–8) 4.5 (4–5) 5 3.4 (3–4) 4 7 8 16 5 (4–6) Unknown 5 5.5 (3–8) 6 1.5 (1–2) 4
GTC GTC Unknown Unknown GT Partial Partial Partial Partial GTC Partial Unknown GTC GTC Partial GTC Unknown Unknown Unknown Partial Unknown Unknown GTC Partial Partial GTC
Cluster SE Unknown Unknown Cluster Cluster Cluster cluster Cluster Unknown Cluster SE Cluster Cluster Cluster Cluster Unknown Unknown Unknown Cluster Unknown Unknown Unknown Unknown SE Cluster
CBZ CBZ Unknown CBZ CBZ CBZ CBZ VPA VPA, PHT Lidocaine Lidocaine VPA, PHT CBZ VPA CBZ CBZ, VPA Unknown Unknown Unknown None CBZ VPA, PHT PB – CBZ CBZ
BFIE BFIE BFIE BFIE BFIE BFIE BFIE BFIE BFIE BFIE BFIE BIE ICCA ICCA ICCA BFIE Unknown BFIE BFIE BIE BFIE BFIE BFIE BFIE BFIE BIE
M, male; F, female; ND, not detected; GTC, generalized tonic clonic seizure; SE, status epilepticus; CBZ, carbamazepine; VPA, valproic acid; PHT, phenitoin; PB, phenobarbital.
blood samples containing EDTA-Na2 using the QIAamp DNA Blood Maxi Kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer. The entire PRRT2 coding sequence (accession number; RefSeq: NM_145239.2), including adjacent short intronic sequences of the gene (accession number; NC_000016.9), was amplified by PCR with genomic DNA obtained from one affected individual of each family. Once PRRT2 variations were detected, the variations were sought in the families and controls. The primer sequences and corresponding PCR protocols are available on request. After amplification and purification on a 96-well PCR Clean-up Kit (Whatman, Piscataway, NJ), the PCR products were sequenced directly by the dye terminator sequencing method using the amplification primers on an ABI PRISMÒ 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). 3. Results Two genetic variations were identified within the investigated region of PRRT2 (Fig. 1A–C). One was a heterozygous single nucleotide duplicational variant, c.649dupC (Fig. 1A and C) and the other was a novel heterozygous variant, c.1012+2dupT (Fig. 1B and C).
The c.649dupC, which has been reported in BFIE and ICCA [4], is assumed to result in a frame shift followed by a premature stop codon (p.Arg217ProfsX8). The c.1012+2dupT locates at the splice donor site. In silico, c.1012+2dupT was considered to affect mRNA splicing (see URL). The predicted splice donor site of exon3 caused by c.1012+2dupT showed different exon/intron boundary with normal splice site and lower acceptor score (maximum 0.75) than normal (real site: 0.99) (Table 2). Therefore, the c.1012+2dupT was predicted to produce aberrant protein. Neither of the variants was found in the controls. Based on the deduced consequences of both mutations and the fact that they were not found in controls, both were considered disease causing mutations. The c.649dupC was identified in 12 probands of families with BFIE (Families 1–11) and BIE without family history (Family 12) ( Fig. 2). The same mutation was found in three families with ICCA. In turn, all families with ICCA in this study harbored the same c.649dupC mutation although they were unrelated. The c.1012 +2dupT was found in a family with BFIE (Family 16). No PRRT2 variation was detected within the investigated region in the remaining families (10 families either with BFIE or BIE). Likewise, no PRRT2 variation was
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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A
B
C
Fig. 1. Nucleotide sequence of the relevant region of PRRT2 mutations of the patients. Genomic DNA from the patient was amplified for short fragments segmented exons 2, 3 and 4 of PRRT2 by PCR. The nucleotide sequences of the products were determined by the dye terminator cycle sequencing method in an ABI auto-sequencer. The nucleotide sequence was determined in 50 –30 direction. (A): DNA sequence of the wild type allele (top) and the c.649dupC mutation (bottom). Arrow indicates nucleotide 650 of the coding sequence, where a single nucleotide was inserted. (B): DNA sequence of the wild type allele (top) and the c.1012+2dupT mutation (bottom). Capital letters indicate nucleotides in exon, and lower cases indicate those in intron. Arrow indicates nucleotide 3 of intron 3, where a single nucleotide was inserted. (C): Positions of the identified mutations of PRRT2. The c.649dupC mutation locates at exon 2, whereas the c.1012+2dupT mutation locates at canonical donor splice site of intron 3.
Table 2 Prediction of splice donor site of mutation. Normal
c.1012+3_4insT
PRRT2 transcript variant1 (NM_145239.2)
Start
End
Score
Exon/Intron
Start
End
Score
Exon/Intron
Exon1
5230 1109 1729 2111 2269 2801 3715 3786 3851
1002 1123 1743 2125 2283 2815 3729 3800 3865
0.99 0.47 0.77 0.52 0.99 0.99 0.70 0.74 0.75
cgtcgagGTgagacc caaaaggGTtaatct accagaaGTgagcaa ccccgagGTagcctg tgtcatgGTgagccc ttaggcgGTgagtgg gactcggGTgagggt cttccagGTcagctg cttgaagGTgtgggg
5230 1109 1729 2111 2269 3716 3787 3852
1002 1123 1743 2125 2283 3730 3801 3866
0.99 0.47 0.77 0.52 0.99 0.70 0.74 0.75
cgtcgagGTgagacc caaaaggGTtaatct accagaaGTgagcaa ccccgagGTagcctg tgtcatgGTgagccc gactcggGTgagggt cttccagGTcagctg cttgaaggtGTgggg
Exon2 Exon3
Start and End position means genomic number of chromosome 16. Large letters “GT” represent boundary of exon and intron. The score represents donor score.
found in CwG or BFNE. Comparison of the clinical characteristics of carriers of PRRT2 mutations and non-carriers showed no difference (Table 1). 4. Discussion Mutations of PRRT2 have been reported recently as the cause of PKC, BFIE and ICCA [3,4,19–23]. To date, mutations in PKC have been reported exclusively in Asians with c.649dupC mutation being a hot spot mutation. In contrast, mutations of PRRT2 in BFIE
and ICCA were reported in both Asians and nonAsians. Intriguingly, however, the hot spot mutation underlying BFIE and ICCA was also c.649dupC mutation. In this study, we showed that c.649dupC mutation is associated with the majority of BFIE, BIE and ICCA Japanese families (15/26) and described a novel c.1012+2dupT mutation as the underlying cause of BFIE. These findings suggest that c.649dupC mutation is a recurrent mutation among PKC, BFIE and ICCA regardless of ethnicities. Therefore, the c.649dupC
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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Fig. 2. Pedigrees of 16 families with PRRT2 mutations. Arrow: proband, E+: individuals with heterozygous PRRT2 mutations, E: individuals negative for PRRT2 mutations. The c.649dupC mutation was identified in Family 1 to 15. The c.1012+2dupT mutation was identified only in Family 16.
recurrent mutation does not suggest a founder effect but may result from proneness of the nucleotide repeat in the relevant position of PRRT2, since another novel mutation, c.1012+2dupT, occurred in the GC-rich region of the gene. In our cohort, the available clinical reports could not explain the phenotypic difference between BFIE with and without PRRT2 mutation. This was in part due to unavailability of the clinical records of BFIE without PRRT2 mutation. In general, however, in most patients with PRRT2 mutations, seizures tended to start as partial seizures and formed clusters. These partial seizures showed no seizure status, whereas the partial seizures in patients without PRRT2 mutation showed sei-
zure status. All ICCA families harbored the c.649dupC mutation. This implies genetic heterogeneity in BFIE and BIE. The pathological changes resulting from PRRT2 in PKC, BFIE and ICCA remain unclear. In fact, the physiological functions of PRRT2 per se have yet to be clarified, although the expression of PRRT2 mRNA has been reported in the brain and spinal cord [3]. Furthermore, PRRT2 mRNA levels in the whole brain reach peak levels at postnatal day 14 then decline in the adult brain of mice [3]. This profile is in agreement with the clinical course of BFIE, which is characterized by infantile onset followed by spontaneous remission. PRRT2 is known to interact with SNAP25 (NM_003081.3) both in vitro and in vivo [24]. SNAP25,
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
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synaptosomal-associated protein 25, is a presynaptic plasma membrane protein engaged in synaptic vesicle membrane docking and fusion pathway [25] and plays a role in the regulation of intracellular calcium dynamics as well as in the exocytosis [26–28]. The SNARE complex, which includes SNAP25, is associated with the N or P/Q types of voltage-dependent calcium channels, which open in response to an action potential generated by voltage-gated sodium channels. Carbamazepine has little pharmacological effect on the calcium channels. The association between PRRT2 and SNAP25 is intriguing because PRRT2 includes two transmembrane domains while SNAP25 has no membrane-binding regions. PRRT2 may serve as an anchor to connect SNAP25 to the presynaptic membrane. Other players in the genetic heterogeneity of BFIE and BIE may reside in putative molecules in such interactions whereas PRRT2 mutations are required for ICCA. The present study also demonstrated that PRRT2 is unlikely to be associated with the pathogenesis of CwG, a common infantile seizure disorder in the Asian population. Also, the lack of mutation in BFNE negates the role of PRRT2 in this phenotypically similar seizure disorder in early infancy. Both CwG and BFNE are sometimes difficult to differentiate from BFIE and BIE at clinical settings. Since c.649dupC is a hotspot mutation in BFIE and BIE, screening for PRRT2 mutation might be useful to distinguish between BFIE and CwG or BFNE at an early stage. In summary, the identification of PRRT2 mutations in PKC and BFIE opens a new avenue toward our understanding of so called “benign” paroxysmal, such as movements, which respond well to medications, and also of epilepsy that shows spontaneous remission. Further studies on PRRT2 should shed light on the development of novel antiepileptic drugs for not only BFIE but also other epilepsies. 5. URL Splice Site Prediction by Neural Network http:// www.fruitfly.org/seq_tools/splice.html. Conflict of interest The authors have no financial or personal relations that could pose a conflict of interest. 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.
Acknowledgments The authors are indebted to all members of the family for their helpful cooperation in this study. The authors
also thank Akiyo Hamachi and Minako Yonetani for the excellent technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research (A) 21249062, Grant-in-Aid for Challenging Exploratory Research (23659529), Japan Society for the Promotion of Science (JSPS); Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) Exploratory Research, Japan Science and Technology Agency (JSP); Research Grants for Nervous and Mental Disorder (21B-5) and Health and Labor Science Research Grant KB230019, KB230004 from the Ministry of Health, Labor and Welfare; 2013–2017 “Central Research Institute for the Molecular Pathomechanisms of Epilepsy of Fukuoka University” Recommended Projects of Fukuoka University (117016); and Research foundation for Clinical Medical Promotion. References [1] Caraballo R, Pavek S, Lemainque A, Gastaldi M, Echenne B, Motte J, et al. Linkage of benign familial infantile convulsions to chromosome 16p12-q12 suggests allelism to the infantile convulsions and choreoathetosis syndrome. Am J Hum Genet (in eng) 2001;68:788–94. [2] Szepetowski P, Rochette J, Berquin P, Piussan C, Lathrop GM, Monaco AP. Familial infantile convulsions and paroxysmal choreoathetosis: a new neurological syndrome linked to the pericentromeric region of human chromosome 16. Am J Hum Genet (in Eng) 1997;61:889–98. [3] Chen WJ, Lin Y, Xiong ZQ, Wei W, Ni W, Tan GH, et al. Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia. Nat Genet (in Eng) 2011;43:1252–5. [4] Heron SE, Grinton BE, Kivity S, Afawi Z, Zuberi SM, Hughes JN, et al. PRRT2 mutations cause benign familial infantile epilepsy and infantile convulsions with choreoathetosis syndrome. Am J Hum Genet (in Eng) 2012;90:152–60. [5] Contino MF, Lebby T, Arcinue EL. Rotaviral gastrointestinal infection causing afebrile seizures in infancy and childhood. Am J Emerg Med (in Eng) 1994;12:94–5. [6] Kawano G, Oshige K, Syutou S, Koteda Y, Yokoyama T, Kim BG, et al. Benign infantile convulsions associated with mild gastroenteritis: a retrospective study of 39 cases including virological tests and efficacy of anticonvulsants. Brain Dev (in Eng) 2007;29:617–22. [7] Lin SC, Hsu HY, Wang PJ, Lee CN, Chang MH, Shen YZ, et al. Rotavirus gastroenteritis associated with afebrile seizure in childhood. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi (in Eng) 1996;37:204–7. [8] Biervert C, Schroeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ, et al. A potassium channel mutation in neonatal human epilepsy. Science (in Eng) 1998;279:403–6. [9] Charlier C, Singh NA, Ryan SG, Lewis TB, Reus BE, Leach RJ, et al. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat Genet (in Eng) 1998;18:53–5. [10] Claes LR, Ceulemans B, Audenaert D, Deprez L, Jansen A, Hasaerts D, et al. De novo KCNQ2 mutations in patients with benign neonatal seizures. Neurology (in Eng) 2004;63:2155–8. [11] de Haan GJ, Pinto D, Carton D, Bader A, Witte J, Peters E, et al. A novel splicing mutation in KCNQ2 in a multigenerational
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006
A. Ishii et al. / Brain & Development xxx (2012) xxx–xxx
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
family with BFNC followed for 25 years. Epilepsia (in Eng) 2006;47:851–9. Hirose S, Okada M, Kaneko S, Mitsudome A. Are some idiopathic epilepsies disorders of ion channels?: a working hypothesis. Epilepsy Res (in Eng) 2000;41:191–204. Hirose S, Zenri F, Akiyoshi H, Fukuma G, Iwata H, Inoue T, et al. A novel mutation of KCNQ3 (c.925T –>C) in a Japanese family with benign familial neonatal convulsions. Ann Neurol (in Eng) 2000;47:822–6. Ishii A, Fukuma G, Uehara A, Miyajima T, Makita Y, Hamachi A, et al. A de novo KCNQ2 mutation detected in non-familial benign neonatal convulsions. Brain Dev (in Eng) 2009;31:27–33. Singh NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R, et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat Genet (in Eng) 1998;18:25–9. Fukuma G, Oguni H, Shirasaka Y, Watanabe K, Miyajima T, Yasumoto S, et al. Mutations of neuronal voltage-gated Na+ channel alpha 1 subunit gene SCN1A in core severe myoclonic epilepsy in infancy (SMEI) and in borderline SMEI (SMEB). Epilepsia (in Eng) 2004;45:140–8. Hirose S, Iwata H, Akiyoshi H, Kobayashi K, Ito M, Wada K, et al. A novel mutation of CHRNA4 responsible for autosomal dominant nocturnal frontal lobe epilepsy. Neurology (in Eng) 1999;53:1749–53. Ishii A, Zhang B, Kaneko S, Hirose S. Positive association between benign familial infantile convulsions and LGI4. Brain Dev (in Eng) 2010;32:538–43. Cao L, Huang XJ, Zheng L, Xiao Q, Wang XJ, Chen SD. Identification of a novel PRRT2 mutation in patients with paroxysmal kinesigenic dyskinesias and c.649dupC as a mutation hot-spot. Parkinsonism Relat Disord (in Eng) 2012.
7
[20] Li J, Zhu X, Wang X, Sun W, Feng B, Du T, et al. Targeted genomic sequencing identifies PRRT2 mutations as a cause of paroxysmal kinesigenic choreoathetosis. J Med Genet (in Eng) 2012;49:76–8. [21] Liu Q, Qi Z, Wan XH, Li JY, Shi L, Lu Q, et al. Mutations in PRRT2 result in paroxysmal dyskinesias with marked variability in clinical expression. J Med Genet (in Eng) 2012;49:79–82. [22] Wang JL, Cao L, Li XH, Hu ZM, Li JD, Zhang JG, et al. Identification of PRRT2 as the causative gene of paroxysmal kinesigenic dyskinesias. Brain (in Eng) 2011;134:3493–501. [23] Ono S, Yoshiura K, Kinoshita A, Kikuchi T, Nakane Y, Kato N, et al. Mutations in PRRT2 responsible for paroxysmal kinesigenic dyskinesias also cause benign familial infantile convulsions. J Hum Genet (in Eng) 2012;57:338–41. [24] Lee H-Y, Huang Y, Bruneau N, Roll P, Roberson Elisha DO, Hermann M, et al. Mutations in the gene PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions. Cell Rep 2012;1:2–12. [25] Zhao N, Hashida H, Takahashi N, Sakaki Y. Cloning and sequence analysis of the human SNAP25 cDNA. Gene (in Eng) 1994;145:313–4. [26] Seagar M, Takahashi M. Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis. J Bioenerg Biomembr (in Eng) 1998;30:347–56. [27] Sheng ZH, Rettig J, Cook T, Catterall WA. Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature (in Eng) 1996;379:451–4. [28] Verderio C, Pozzi D, Pravettoni E, Inverardi F, Schenk U, Coco S, et al. SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization. Neuron (in Eng) 2004;41:599–610.
Please cite this article in press as: Ishii A et al. Genetic analysis of PRRT2 for benign infantile epilepsy, infantile convulsions with choreoathetosis syndrome, and benign convulsions with mild gastroenteritis.. Brain Dev (2012), http://dx.doi.org/10.1016/j.braindev.2012.09.006