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HORT COMMUNICATION
e-novo mutations and genetic variation in theCN1A gene in Malaysian patients with generalizedpilepsy with febrile seizures plus (GEFS+)
mmilia Husni Tana, Salmi Abdul Razakb, Jafri Malin Abdullaha,bdul Aziz Mohamed Yusoffa,∗
Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, MalaysiaDepartment of Paediatric, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
eceived 21 March 2012; received in revised form 5 August 2012; accepted 10 August 2012vailable online 1 September 2012
Summary Generalized epilepsy with febrile seizures plus (GEFS+) comprises a group of clin-ically and genetically heterogeneous epilepsy syndrome. Here, we provide the first report ofclinical presentation and mutational analysis of SCN1A gene in 36 Malaysian GEFS+ patients.Mutational analysis of SCN1A gene revealed twenty seven sequence variants (missense muta-
he term ‘‘generalized epilepsy with febrile seizureslus’’(GEFS+) [MIM# 604236] was first described by Scheffernd Berkovic (1997) as comprising a group of clinicallynd genetically heterogeneous epilepsy syndrome. There is
variety of clinical phenotypes, including typical febrile
eizures (FS), febrile seizures plus (FS+), and other seizureypes such as absences, myoclonic, atonic or partial seizures
nd even refractory syndromes with developmental delayScheffer and Berkovic, 1997).
Many genetic defects have been reported that are asso-iated with GEFS+, since the first description of GEFS+ in5 years ago (Scheffer and Berkovic, 1997). Recent molec-lar genetic studies have revealed that mutations of theoltage gated sodium channel genes of �1-subunit (SCN1A)Escayg et al., 2001), �2-subunit (SCN2A) (Sugawara et al.,001a,b), �1-subunit (SCN1B) (Wallace et al., 1998) and the-aminobutyric acid (GABA) receptor genes of �2-subunitGABRG2) (Baulac et al., 2001) and � subunit (GABRD)
Dibbens et al., 2004) were associated with GEFS+ spectrum.he most clinically relevant gene which is widely reportedor GEFS+ spectrum is SCN1A gene which has 26 exons ands highly conserved across the species (Escayg et al., 2001).
In the present study we reported a comprehensive clinicaltudy and molecular analyses of Malaysian GEFS+ patientshere we determined the contribution of SCN1A mutations
n Malaysian population. There is no report published soar on the association between SCN1A gene mutation andalaysian GEFS+ patients. Interestingly, we also identified 2ovel de-novo missense mutations of the SCN1A gene amonghe studied patients.
aterials and methods
atients
his study recruited 36 unrelated Malaysian GEFS+ patients.hey were examined at Hospital Universiti Sains Malaysia.he patients in this study were fulfilled the inclusion criteriaor GEFS+ spectrum according to ILAE (Commission on Classi-cation and Terminology of the International League Againstpilepsy, 1989). This study was approved by the Ethics Com-ittee of the Universiti Sains Malaysia. Written informed
onsent was obtained from all participants.
NA isolation and PCR amplification
enomic DNA was isolated from EDTA-anticoagulated bloodsing QIAamp DNA Blood Kit (Qiagen Inc., Hilden, Germany).or PCR amplification, 32 sets of oligonucleotide primersere used to amplify 26 exons of SCN1A gene as previouslyescribed by Wallace et al. (2001).
HPLC analysis
eteroduplex formation was induced by heat denaturationf PCR products at 94 ◦C for 5 min, followed by gradual rean-ealing from 94 ◦C to 25 ◦C over 45 min. DHPLC analysis wasarried out on the WAVE automated instrument (Varian Inc.,SA). PCR products (4 �L per sample) were eluted at a flowate of 0.9 mL/min with a linear acetonitrile gradient. Analy-is per sample took ∼7 min. Data analysis was based on visualnspection of chromatograms, and comparison with normalontrols was included in each run. Heterozygous profilesere detected as distinct elution peaks from homozygousild type peaks.
alidation of mutations on DNA sequencing
ragments showing an aberrant DHPLC pattern were fur-her analyzed for identification of sequence variants by ABI700 automated sequencer (PE Applied Biosystems). Thisas performed according to manufacturer’s protocol using
reamplified and purified PCR product of the aberrant frag-ent (forward and reverse). The sequence variants were
lassified according to the nomenclature established by Den
unnen and Antonarakis (2001), and numbering was startedrom the initiating ATG codon on the basis of the Homo sapi-ns sodium channel, voltage gated, type 1, alpha subunitSCN1A), mRNA (GenBank accession number NM 006920).
eOIb
AA, amino acid; NA, nucleotide; V, valine; A, alanine; E, glutamicacid; K, lysine; P, proline; N, asparagine; L, leucine; H, histidine;R, arginine; D, aspartic acid; G, glycine.
esults
linical features
he clinical features of 36 patients are summarized inable 1. The clinical features of affected individualsbtained by reviewing medical records and EEG, marked aslinical heterogeneity with a variety of seizure types andeurological deficits. All clinically affected individuals hadeizure disorders starting in childhood and mostly had gener-lized tonic clonic seizures (GTCS) which occurred with andithout fever. Regarding seizure presentation, four had mul-
iple seizures. Other neuropsychiatric disorders were alsorominent, with twelve individuals presenting intellectualisabilities (mental retardation, learning difficulty, ADHD,evelopmental delay, autism), four with debilitating neu-opsychiatric disease (behavioral problem) and one withtaxia.
olecular analysis
total of 36 unrelated patients with GEFS+ were screenedor mutations in SCN1A. We found 9 point mutations in SCN1Aonsisted of 7 missense mutations and 2 silent mutationsTable 2). Two novel missense mutations were also discov-red which were c.4748A>G transition in exon 25 effecting ahange from histidine to arginine at codon 1583 (Fig. 1) and.5197A>G of exon 26 bringing a substitution asparagine tospartate at codon 1733 (in the loop between transmem-rane segments S5 and S6 of the sodium channel protein),espectively. None of the above identified mutations wereetected in the control population of 60 healthy controls asetermined by molecular analyses.
In this study, we also detected eighteen single nucleotideolymorphisms (SNPs) (coding and non coding region). Oneoding variant, A1212G (V404) in exon 9 was polymorphic inatients and controls. These SNPs were identified in mostases of GEFS+ spectrum cases (Escayg et al., 2001; Claes
t al., 2003; Bonanni et al., 2004; Mahoney et al., 2009).ther non coding SNPs including IVS5 + 33 A>C, IVS7 + 21 T>C,
VS9 + 52 G>A and IVS23 + 33 G>A have previously describedy many researchers (Escayg et al., 2001; Claes et al., 2003;
Clinical and genetic results of SCN1A gene mutations in 36 GEFS+
Figure 1 DNA sequencing of the SCN1A gene at exon 25. Toppanel: A at the codon 1583 (arrow) in a normal control. Lowerpanel: sequencing result showed a heterozygous A-to-G tran-
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ioppofiSZvameaic
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sition (arrow) at codon 1583 resulting in amino acid exchange(CAT → CGT; His → Arg).
Depienne et al., 2009). Three SNPs from this study werenovel (IVS8 + 13 G>T, IVS14 + 22 A>C and IVS22 − 58 A>T).Some non coding SNPs were present at polymorphic frequen-cies (>1%) and some at lower frequencies. One non codingSNPs identified in the patient with autistic features in thisstudy, IVS7 + 21 T>C were also observed in six patients withautism (Weiss et al., 2003; Osaka et al., 2007). Further stud-ies in independent samples required to determine whetherthe SNPs are associated with predisposition to epilepsy.
Discussion
The current study was designed to determine whether SCN1A
mutations affect the development of GEFS+ in the Malaysianpopulation. We have identified for the first time mutationsof SCN1A in 9/36 (25%) of patients with GEFS+, of whichtwo were novel. All of these nine mutations detected were
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Malaysian patients 213
e novo mutation. The mutation rate (25%) observed inur study is much higher than the value of 3—10% previ-usly reported (Escayg et al., 2001; Wallace et al., 2001;eulemans et al., 2004; Scheffer et al., 2009). We believehat these SCN1A mutations might represent one of theenetic susceptibility factors for GEFS+ in the Malaysian pop-lation.
In this study we identified two novel missense muta-ions in SCN1A in two Malaysian patients with GEFS+.e have previously submitted these mutations, as ‘‘novel
CN1A mutations’’, using the National Center for Biotech-ology Information (NCBI) database (GenBank accessionumbers HQ726795 and HQ726796). Both of the novel muta-ions (His1583Arg and Asn1733Asp) were predicted to beathogenic because they are located at conserved amino-cid residues at the domain IV of SCN1A and were not seenn 60 healthy controls. The first mutation was a heterozy-ous c.4748A>G transition in exon 25 which is located at S2f domain IV of SCN1A. This patient was diagnosed as hav-ng GEFS+, and reported having frequent generalized toniclonic seizures with neurological deficits. According to aeport by Kanai et al. (2004) the compound heterozygousutation found in patients may have cumulative effects that
xplained the severity of the disease which caused GEFS+ith a heterogeneous phenotype that includes mental retar-ation, developmental delay, ataxia, anxiety disorder andDHD. The variable phenotype in the study by Kanai et al.2004) is similar to that in the present study.
The second mutation, a c.5197A>G transition in exon 26s located between S5 and S6 in the pore forming regionf domain IV of SCN1A. The majority of mutations in theore forming loop regions have been identified in SMEIatients (Kanai et al., 2004). To date, there have been fourther reports of missense mutations in SCN1A gene identi-ed in patients with GEFS+ located in the loop of S5 and6 (Sugawara et al., 2001a,b; Pineda-Trujillo et al., 2005;ucca et al., 2008; Mahoney et al., 2009). According to pre-ious report, mutation in the loop of S5 and S6 has beenssociated with severity of clinical manifestations includedultiple seizures and mental retardation (Pineda-Trujillo
t al., 2005). This observation is consistent with our findings the patient, who presented with the amino acid substitut-ons (N1733D) in the pore region of SCN1A, can lead to alinically more severe epileptic syndrome.
The 36 patients were screened for all 26 exons of theCN1A, and variants were found in 9 introns. The 60 healthyontrols were then screened for these 9 introns. Interest-ngly one of the variants from this study (IVS7 + 21 T>C) waslso found in autism cases as previously reported by Weisst al. (2003) and Osaka et al. (2007), this finding can be fur-her evaluated to correlate the role of SCN1A gene, whetherhe efficiency maybe altered by the changes of nucleotiden autism susceptibility. Four other variants (SNPs) identi-ed in this study are potentially associated with disease andollow up studies to detect possible disease association withhis variant are permitted. The SNPs of the sodium channelene cluster will be useful for testing the contributions thathese genes make to other inherited disorder.
A silent mutation is a mutation in human gene that doesot change the resulting amino acid sequences (Chamarynd Hurst, 2009). It is well known that silent mutationas not much effect to diseases. But, recent studies have
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ndicated that silent mutation might lead to differentlyolded proteins and cause disease (Richards et al., 2007;ansen et al., 2009). In the previous report by Stanford et al.
1999), a novel S305S silent mutation was identified in pro-ressive supranuclear palsy, which resulted in an increase ofhe splicing of exon 10 in the tau gene. Since then, numeroustudies have showed that silent mutations have been linkedith more than 50 genetic disorders (Chamary and Hurst,009). In 2010, a silent mutation of SCN1A was detectedn an Indonesian patient with GEFS+ by Herini et al. How-ver, this mutation was not linked to the disruption of anyxonic splicing enhancer motifs of SNCIA gene (Herini et al.,010). In the present study, we detected two silent muta-ions (c.2439T>A → P814P and c.5118A>G → L1707L) whichre both mutations translated into the same amino acid.t is considered essential that further studies be conductedhether theses silent mutations may affect the translationrocess of the mRNA into the protein. Even though this studyoes not address the issue specifically but the idea of silentutation might play a role in the expression of the epilepsyhenotypes is important to discover soon.
In conclusion, our findings suggest that genetic defectsf the SCN1A gene are likely to play a role in the etiologyf GEFS+ in our Malaysian patients and alterations in therder of pore forming region of SCN1A protein that is mis-ense mutations can lead to GEFS+ with a variable neurologichenotype that includes mood and anxiety disorder. Fur-her genotype/phenotype association studies and additionalolecular investigations in more extended GEFS+ popula-
ions will be needed to elucidate the cause of the variabilityf the disease. The present study adds another novel muta-ion to the collection of mutations causing GEFS+.
cknowledgements
e sincerely thank to our patient and their family mem-ers for their helpful cooperation in our research. Here, welso would like to thank Universiti Sains Malaysia for provid-ng the Research University Grant (PSKBP/1001/8120195) asart of Brain Cluster Research and for awarding me the Post-raduate Scholarship Scheme (USM Fellowship). This studyas approved by the Human Ethics Committee Universitiains Malaysia.
eferences
aulac, S., Huberfeld, G., Gourfinkel-An, q.I., Mitropoulou, G.,Beranger, A., Prud’-homme, J.F., Baulac, M., Brice, A., Bruz-zone, R., LeGuern, E., 2001. First genetic evidence of GABAAreceptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene. Nat. Genet. 28, 46—48.
onanni, P., Malcarne, M., Moro, F., Veggiotti, P., Buti, D., Ferrari,A.R., Parrini, E., Mei, D., Volzone, A., Zara, F., Heron, S.E.,Bordo, L., Marini, C., Guerrini, R., 2004. Generalized epilepsywith febrile seizures plus (GEFS+): clinical spectrum in sevenItalian families unrelated to SCN1A, SCN1B and GABRG2 genemutations. Epilepsia 45 (2), 149—158.
eulemans, B.P., Claes, L.R., Lagae, L.G., 2004. Clinical correla-tions of mutations in the SCN1A gene: from febrile seizuresto severe myoclonic epilepsy in infancy. Pediatr. Neurol. 30,236—243.
S
E.H. Tan et al.
hamary, J.V., Hurst, L.D., 2009. The price of silent mutations. Sci.Am. 300 (6), 46—53.
laes, L., Ceulemans, B., Audenaert, D., Smets, K., Löfgren, A., Del-Favero, J., Ala-Mello, S., Basel-Vanagaite, L., Plecko, B., Raskin,S., Thiry, P., Wolf, N.I., Van Broeckhoven, C., De Jonghe1, P.,2003. De novo SCN1A mutations are a major cause of severemyoclonic epilepsy of infancy. Hum. Mutat. 21, 615—621.
en Dunnen, J.T., Antonarakis, E., 2001. Nomenclature for adescription of human sequence variations. Hum. Genet. 109,121—124.
epienne, C., Trouillard, O., Saint-Martin, C., Gourfinkel-An, I.,Bouteiller, D., Carpentier, W., Keren, B., Abert, B., Gautier,A., Baulac, S., Arzimanoglou, A., Cazeneuve, C., Nabbout, R.,LeGuern, E., 2009. Spectrum of SCN1A gene mutations associ-ated with Dravet syndrome: analysis of 333 patients. J. Med.Genet. 46 (3), 183—191.
ibbens, L.M., Feng, H.J., Richards, M.C., Harkin, L.A., Hodgson,B.L., Scott, D., Jenkins, M., Petrou, S., Sutherland, G.R., Schef-fer, I.E., Berkovic, S.F., Macdonald, R.L., Mulley, J.C., 2004.GABRD encoding a protein for extra- or perisynaptic GABAAreceptors is a susceptibility locus for generalized epilepsies.Hum. Mol. Genet. 13, 1315—1319.
scayg, A., Heils, A., MacDonald, B.T., Haug, K., Sander, T., Meisler,M.H., 2001. A novel SCN1A mutation associated with generalizedepilepsy with febrile seizures plus — and prevalence of variantsin patients with epilepsy. Am. J. Hum. Genet. 68, 866—873.
ansen, T.V.O., Steffensen, A.Y., Jonson, L., Andersen, M.K., Ejlert-sen, B., Nielsen, F.C., 2009. The silent mutation nucleotide744G → A, Lys172Lys, in exon 6 of BRCA2 result in exon skipping.Breast Cancer Res. Treat., March (Epub ahead of print).
erini, E.S., Gunadi, Harahap, I.S., Yusoff, S., Morikawa, S.,Patria, S.Y., Nishimura, N., Sunartini, Sutaryo, Takada, S., Mat-suo, M., Nishio, H., 2010. Generalized epilepsy with febrileseizures plus (GEFS+) spectrum: clinical manifestations andSCN1A mutations in Indonesian patients. Epilepsy Res. 90 (1—2),132—139.
anai, K., Hirose, S., Oguni, H., Fukuma, G., Shirasaka, Y., Miya-jima, T., Wada, K., Iwasa, H., Yasumoto, S., Matsuo, M., Ito,M., Mitsudome, A., Kaneko, S., 2004. Effect of localization ofmissense mutations in SCN1A on epilepsy phenotype severity.Neurology 63, 329—334.
ahoney, K., Moore, S.J., Buckley, D., Alam, M., Parfrey, P., Pen-ney, S., Merner, N., Hodgkinson, K., Young, T.L., 2009. Variableneurologic phenotype in a GEFS+ family with a novel mutationin SCN1A. Seizure 18 (7), 492—497.
saka, H., Ogiwara, I., Mazaki, E., Okamura, N., Yamashita, S.,Iai, M., Yamada, M., Kurosawa, K., Iwamoto, H., Yasui-Furukori,N., Kaneko, S., Fujiwara, T., Inoue, Y., Yamakawa, K., 2007.Patients with a sodium channel alpha 1 gene mutation show widephenotypic variation. Epilepsy Res. 75, 46—51.
ineda-Trujillo, N., Carrizosa, J., Cornejo, W., Arias, W., Franco, C.,Cabrera, D., Bedoya, G., Ruíz-Linares, A., 2005. A novel SCN1Amutation associated with severe GEFS+ in a large South Americanpedigree. Seizure 14 (2), 123—128.
ichards, A.J., Laidlaw, M., Meredith, S.P., Shankar, P., Poulson,A.V., Scott, J.D., Snead, M.P., 2007. Missense and silent mutationin COL2A1 result in stickler syndrome but via different molecularmechanism. Hum. Mutat. 28 (6), 639—647.
cheffer, I.E., Berkovic, S.F., 1997. Generalized epilepsy with febrileseizures plus. A genetic disorder with heterogeneous clinicalphenotypes. Brain 120, 479—490.
tanford, P.M., Halliday, G.M., Brooks, W.S., Kwok, J.B., Storey,C.E., Creasey, H., Morris, J.G., Fulham, M.J., Schofield, P.R.,1999. Progressive supranuclear palsy pathology caused by anovel silent mutation in exon 10 of the tau gene. Expansion of
EFS+
W
Z
E
Gsm
etB
e
Clinical and genetic results of SCN1A gene mutations in 36 G
the disease phenotype caused by tau gene mutations. Brain 123(5), 880—893.
Sugawara, T., Mazaki-Miyazaki, E., Ito, M., Nagafuji, H.,Fukuma, G., Mitsudome, A., Wada, K., Kaneko, S., Hirose,S., Yamakawa, K., 2001a. Nav1.1 mutations cause febrileseizures associated with afebrile partial seizures. Neurology 57,703—705.
Sugawara, T., Tsurubuchi, Y., Agarwala, K.L., Ito, M., Fukuma, G.,Mazaki-Miyazaki, E., Nagafuji, H., Noda, M., Imoto, K., Wada,K., Mitsudome, A., Kaneko, S., Montal, M., Nagata, K., Hirose,S., Yamakawa, K., 2001b. A missense mutation of the Na+ chan-nel alpha II subunit gene Na(v)1.2 in a patient with febrile andafebrile seizures causes channel dysfunction. Proc. Natl. Acad.Sci. U. S. A. 98, 6384—6389.
Wallace, R.H., Scheffer, I.E., Barnett, S., Richards, M., Dibbens,L., Desai, R.R., Lerman-Sagie, T., Lev, D., Mazarib, A., Brand,N., Ben-Zeev, B., Goikhman, I., Singh, R., Kremmidiotis, G.,Gardner, A., Sutherland, G.R., George Jr., A.L., Mulley, J.C.,Berkovic, S.F., 2001. Neuronal sodium-channel �1-subunit muta-tions in generalized epilepsy with febrile seizure plus. Am. J.Hum. Genet. 68, 859—865.
Wallace, R.H., Wang, D.W., Singh, R., Scheffer, I.E., GeorgeJr., A.L., Phillips, H.A., Saar, K., Reis, A., Johnson, E.W.,
Sutherland, G.R., Berkovic, S.F., Mulley, J.C., 1998. Febrileseizures and generalized epilepsy associated with a mutation inthe Na+-channel beta1 subunit gene SCN1B. Nat. Genet. 19 (4),366—370.
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eiss, L.A., Escayg, A., Kearney, J.A., Trudeau, M., MacDonald,B.T., Mori, M., Reichert, J., Buxbaum, J.D., Meisler, M.H., 2003.Sodium channels SCN1A, SCN2A and SCN3A in familial autism.Mol. Psychiatry 8, 186—194.
ucca, C., Redaelli, F., Epifanio, R., Zanotta, N., Romeo, A., Lodi,M., Veggiotti, P., Airoldi, G., Panzeri, C., Romaniello, R., DePolo, G., Bonanni, P., Cardinali, S., Baschirotto, C., Martorell,L., Borgatti, R., Bresolin, N., Bassi, M.T., 2008. Cryptogenicepileptic syndromes related to SCN1A: twelve novel mutationsidentified. Arch. Neurol. 65, 489—494.
lectronic database information
enBank, http://www.ncbi.nlm.nih.gov/GenBank/ Homo sapiensodium channel, voltage gated, type 1, alpha subunit (SCN1A),RNA (GenBank accession number NM 006920).
GenBank, http://www.ncbi.nlm.nih.gov/GenBank/ Homo sapi-ns sodium channel, voltage-gated, type 1, alpha subunit (SCN1A),ranscript variant 2, mRNA. Base substitution usm gefs0109 (Gen-ank, accession number HQ726795).
GenBank, http://www.ncbi.nlm.nih.gov/GenBank/ Homo sapi-ns sodium channel, voltage-gated, type 1, alpha subunit (SCN1A),
ranscript variant 2, mRNA. Base substitution usm gefs0209 (Gen-ank, accession number HQ726796).
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.lm.nih.gov/Omim/ GEFS+ [MIM 604236].
