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A putative disease-associated haplotype within the SCN1A gene in Dravet syndrome
Biochemical and Biophysical Research Communications 408 (2011) 654–657
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A putative disease-associated haplotype within the SCN1A gene in Dravet syndrome Nourhène Fendri-Kriaa a,⇑, Salma Boujilbene b, Fatma Kammoun b,c, Emna Mkaouar-Rebai a, Afif Ben Mahmoud a, Ines Hsairi b,c, Ahmed Rebai d, Chahnez Triki b,c, Faiza Fakhfakh a,⇑ a
Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax, Tunisia Service de Neurologie Infantile, C.H.U. Hédi Chaker de Sfax, Tunisia c Unité de recherche «Neuropédiatrie», Faculté de Médecine de Sfax, Tunisia d Centre de Biotechnologie de Sfax, Tunisia b
a r t i c l e
i n f o
Article history: Received 13 April 2011 Available online 21 April 2011 Keywords: Febrile seizures SCN1A SCN1B Dravet syndrome SMEI Haplotype
a b s t r a c t Dravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy, is one of the most severe forms of childhood epilepsy. DS is caused by a mutation in the neuronal voltage-gated sodiumchannel alpha-subunit gene (SCN1A). However, 25–30% of patients with DS are negative for the SCN1A mutation screening, suggesting that other molecular mechanisms may account for these disorders. Recently, the first case of DS caused by a mutation in the neuronal voltage-gated sodium-channel beta-subunit gene (SCN1B) was also reported. In this report we aim to make the molecular analysis of the SCN1A and SCN1B genes in two Tunisian patients affected with DS. The SCN1A and SCN1B genes were tested for mutations by direct sequencing. No mutation was revealed in the SCN1A and SCN1B genes by sequencing analyses. On the other hand, 11 known single nucleotide polymorphisms were identified in the SCN1A gene and composed a putative disease-associated haplotype in patients with DS phenotype. One of the two patients with putative disease-associated haplotype in SCN1A had also one known single nucleotide polymorphism in the SCN1B gene. The sequencing analyses of the SCN1A gene revealed the presence of a putative disease-associated haplotype in two patients affected with Dravet syndrome. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Dravet syndrome (DS), previously known as severe myoclonic epilepsy in infancy (SMEI), is a rare genetic disorder characterized by frequent generalized, unilateral clonic or tonic–clonic seizures that begin during the first year of life [1]. Later, patients also manifest other seizure types, including absence, myoclonic, and simple and complex partial seizures. Psychomotor development stagnates around the second year of life. DS is considered to be the most severe phenotype within the spectrum of generalized epilepsy with febrile seizures plus (GEFS+). DS is a malignant epileptic syndrome, while GEFS+ is usually benign [2]. The diagnosis can be confirmed by genetic testing that is now available, and can show mutations within the neuronal voltagegated sodium-channel alpha-subunit gene (SCN1A) [3]. In fact, in 25–70% of SMEI/DS patients, all types of mutations in the SCN1A gene have been identified [1,4–12]. Recently, Sun et al. identified pathogenic mutations in the SCN1A gene in 49 (77.8%) of 63 Chinese probands with Dravet syndrome. Most mutations were trun-
⇑ Corresponding authors. Address: Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Avenue Magida Boulila, 3029 Sfax, Tunisia. Fax: +216 74 46 14 03. E-mail addresses: [email protected] (N. Fendri-Kriaa), [email protected] (F. Fakhfakh). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.079
cating (61.2%). The mutations included 19 missense, 14 frameshift, six nonsense, and eight splice site alterations. They also identified deletions or duplications of SCN1A in two (12.5%) of 16 patients [13]. Though about a third of patients with SMEI/DS remained negative for mutations in SCN1A [4,14,9,15], some SNPs in the SCN1A gene were described to be associated with DS and epilepsy in several populations [9,16,17]. In addition, Patino et al. reported the first patient with Dravet syndrome due to a homozygous mutation in SCN1B (p.R125C), and showed that the consequence of this mutation was the inability of b1 polypeptides to be trafficked to the surface of transfected mammalian cells [18]. We reported here the SCN1A and SCN1B genes sequencing analyses of two Tunisian patients affected with Dravet syndrome and we revealed a putative disease-associated haplotype in the SCN1A gene.
2. Materials and methods 2.1. Patient ascertainment We selected two subjects with a clinical diagnosis of DS at the child neurology consulting room in Sfax, Tunisia. Epileptic seizures and epilepsy syndrome diagnoses were performed according to the
N. Fendri-Kriaa et al. / Biochemical and Biophysical Research Communications 408 (2011) 654–657
criteria established by the ‘‘International League Against Epilepsy’’ [19,20]. Peripheral blood samples were collected and genomic DNA was extracted by standard procedures [21]. Informed consent was obtained from individuals before being enrolled in the genetic study. In addition, DNAs from 10 healthy subjects were used. These controls were selected to have neither personal nor family history in their first degree relatives of neurodevelopmental disorder. 2.2. Mutation analyses All exons of SCN1A and SCN1B genes were amplified with touchdown program using the primers previously reported [22,23] to cover the coding exons and exon–intron boundaries. The PCR was performed in a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems) in a final volume of 50 lL containing 50 ng genomic DNA, 0.1 lM of each primer, 1 PCR buffer Go Taq (Promega), 1.2 mM MgCl2, 0.13 mM dNTP and 1 U Taq DNA polymerase. The amplified products were sequenced on an Applied Biosystems ABI 3100 sequencer (Alameda County, CA, USA). A Blast homology search was performed using the program blast2seq [24] at the National Center for Biotechnology Information Website to compare individual sequences to wild-type ones. 2.3. Genotyping Two fluorescent dye-labeled microsatellite markers D2S2330 and D2S335 distant of 7 cM intervals according to genome browser were used. After amplification, PCR products were pooled, with the Genescan 400HD size standard, and size fractionated on an ABI PRISM 3100-Avant automated DNA sequencer (Applied Biosystems, USA). The data were analyzed using ABI Prism GeneScan and Genotyper software version 3.7 (Applied Biosystems) to determine alleles sizes. 2.4. Biostatistic and bioinformatic tools Inference of haplotype frequencies was performed using Phase [25]. The prediction of a hypothetical polyadenylation site was carried out using the PolyA_SVM program (version 2.2) (http://polya.umdnj.edu/polya_svm/server/). The data of the hapmap project and haploview program were used to know if SNPs belonged to a linkage disequilibrium block (http://www.hapmap.org). Genome browser was used to choose microsatellite markers and to know the distance between them (http://genome.ucsc.edu/). 3. Results Sequencing analyses of the SCN1B gene in two patients with DS phenotype did not reveal any mutation but revealed in patient (1)
655
one known single nucleotide polymorphism (SNP) in a heterozygous state rs2278995 (T > C) in the exon 6 of the SCN1B gene containing the 30 untranslated region (30 UTR). According to the data of in silico prediction using PolyA_SVM, SNP rs2278995 is distant of 184 bp from a hypothetical polyadenylation site with a score of 1.204 in position 9213 (Fig. 1). In addition, both patients were tested for mutations in the SCN1A gene; however, no mutation was found in this gene in the tested patients. On the other hand, 11 known single nucleotide polymorphisms were identified in a homozygous state. One variation rs566839 A > T was revealed in the 50 untranslated region (50 UTR) of exon 1. Three exonic variations were detected: one SNP rs7580482 (A > G) in exon 9 (Val404Val), one SNP rs6432860 (T > C) in exon 13 (Val753Val) and one SNP rs2298771 (G > A) in exon 16 (Ala1056Thr). Seven intronic polymorphisms were also revealed: one SNP rs3812718 (G > A) in intron 4, one SNP rs994399 (C > T) in intron 6, two SNPs rs1542484 (T > C) and rs1461193 (C > T) in intron 7, one SNP rs11690959 (C > T) in intron 8, one SNP rs6432861 (G > A) in intron 9 and one SNP rs7601520 (C > T) in intron 15 (Fig. 2). The haplotypes construction and an analysis of recombination events were performed for all variants in the SCN1A gene. A haplotype was revealed and delimited by SNP rs566839 in 50 UTR of exon 1 and SNP rs2298771 in exon 16. To try to delimit the region of homozygosity, the microsatellite markers D2S2330 and D2S335 bordering the SCN1A gene were tested and revealed in a heterozygous state in both patients. In addition, according to the hapmap project data (http:// www.hapmap.org), these SNPs belong to a linkage disequilibrium block extending 37.426 kb and covering 16 exons of SCN1A gene (Fig. 3). We chose three SNPs from 11 SNPs in this block: rs3812718 (intron 4), rs6432860 (exon 13) and rs2298771 (exon 16) to test them in 10 controls from the Tunisian population and to estimate the haplotype frequencies (Fig. 4). Using Phase, the ACA haplotype frequency was 100% in patients, which was significantly higher than that in the control population estimated at 17 ± 11.4% (6–28%). This haplotype of SNPs was in a homozygous state in these two patients with DS and was considered as a putative disease-associated haplotype (Figs. 2 and 4).
4. Discussion We reported sequencing analyses of the SCN1A and SCN1B genes in two Tunisian patients affected with Dravet syndrome. The mutational analyses of the SCN1A gene revealed 11 known single nucleotide polymorphisms but no mutation. These SNPs were in a homozygous state and were assumed to compose a single haplotype block. We showed that these 11 SNPs belonged to a linkage disequilibrium block extending 37.426 kb and covering 16 exons of the SCN1A gene (http://www.hapmap.org). We chose three SNPs from them: rs3812718 (intron 4), rs6432860 (exon 13) and rs2298771 (exon 16) to test them in 10 controls from
Fig. 1. Prediction of a hypothetical polyadenylation site using the PolyA_SVM program. The (T) variant of SNP rs2278995 is boxed and is distant of 184 bp from the site 9213 which is underlined.
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N. Fendri-Kriaa et al. / Biochemical and Biophysical Research Communications 408 (2011) 654–657
Fig. 2. Genotypes of single nucleotide polymorphisms (SNPs) in the SCN1A gene.
Fig. 3. Result of haploview presenting a block of 25 kb containing four SNPs of our putative associated haplotype.
Fig. 4. Result comparison of the SCN1A gene sequencing in patients with febrile seizures. One individual with FS (1), one affected by epilepsy with generalized tonico-clonic seizures (2) and two individuals with FS+(3, 4) were reported recently [26]. Two patients were analyzed with DS (5, 6) in the current study. The putative disease-associated haplotype for single nucleotide polymorphisms (SNPs) in the SCN1A is boxed and indicated by gray bar. The three SNPs tested in controls to estimate the haplotype frequencies are horizontally boxed.
the Tunisian population and to estimate the haplotype frequencies. Using Phase, the frequency of ACA haplotype is 100% in patients significantly higher than that in the control population estimated at 17 ± 11.4% (6–28%). This haplotype of SNPs was in a homozygous state in these two patients with DS and was considered as a putative disease-associated haplotype (Figs. 2 and 4). Recently, we also reported this haplotype in a homozygous state only in one patient with DS. The same haplotype was present in the other four patients with febrile seizures but in a heterozygous state proving its association with the DS phenotype (Fig. 4) [26].
Among the 11 SNPs detected in our patients, rs3812718, rs1542484, rs6432861 and rs2298771 were also described to be associated with DS and epilepsy in several populations. In fact, in the Belgian population, these three SNPs rs3812718, rs1542484 and rs6432861 were also described in haplotype of 15 SNPs in a homozygous state in 11 patients affected with DS [9]. In addition, in the Caucasian population, Schlachter et al. found an association between SNP rs3812718 of SCN1A and febrile seizures (FS) [16]. This polymorphism has been hypothesized to be a candidate SNP for FS [16]. Specifically, this SNP (rs3812718) is believed to be a
N. Fendri-Kriaa et al. / Biochemical and Biophysical Research Communications 408 (2011) 654–657
candidate functional variant because it alters the proportions of the neonatal and adult transcripts of the gene [27] and has been reported to be associated with the maximal doses of carbamazepine and phenytoin, both sodium channel-blocking antiepileptic drugs, in patients with epilepsy [28]. The SNP rs2298771 belonging to the ACA haplotype was also tested in Iranian patients with epilepsy. The results revealed that SNP rs2298771, considered as common SNP, was present in 20/ 34 (0.588) probands with allelic frequency as 0.7058/0.2942 in patients and 0.515/0.485 in control group, respectively, for A/G but the statistical analysis did not show any significant differences between the groups. In fact, it was present in a heterozygous state in four patients with DS and in a homozygous state in the three others [17]. In the Indian population, the frequency of AG genotype of SNP rs2298771 was significantly higher in epilepsy patients in comparison to that in healthy controls (P = 0.005) and associated with epileptic patients. Thus AG genotype was suggested to be involved in increasing risk for developing epilepsy, but it did not modulate drug response [29]. The SNP rs2298771 results in change of amino acid at highly consensus conserved site in the coding region of the SCN1A, and possibly affects functioning of the inactivation gate in the cytosol regulating efflux and influx of sodium ions. Another possibility is that this polymorphism is in linkage disequilibrium with some other genetic variants of the same gene that imparts risk for epilepsy [29]. In addition, the sequencing results of the SCN1B gene did not reveal any mutation in this gene for any of the tested patients but revealed SNP rs2278995 (T > C) in the exon 6 of the SCN1B gene in a heterozygous state in one patient (1). This SNP was recently reported and was a part of putative disease-associated haplotype in Tunisian patients with febrile seizures [30]. This SNP is located in the 30 UTR of SCN1B and might have an effect on the stability and maturity of mRNA. In fact, in silico prediction using PolyA_SVM (version 2.2) shows that this SNP is located in a neighbouring hypothetical polyadenylation site (Score of 1.204 in position 9213) and thus might affect the expression of the SCN1B gene. We noticed that the patient (1), who had the SNP in SCN1B and the putative disease-associated haplotype in SCN1A at the same time, was the first case of DS which combined polymorphisms in two neuronal voltage-gated sodium-channel subunit genes. In conclusion, the SCN1A gene sequencing analyses revealed the presence of a putative disease-associated haplotype in two patients affected with Dravet syndrome. Acknowledgments We thank the patients and their families for their cooperation in the present study and for giving informed consent. This work was supported by the Ministry of Higher Education and Scientific Research in Tunisia. The proper institutional approval was obtained as well as the disclosure of sources of funding and other disclosure of conflict of interest related to the submitted manuscript. We extend our thanks to Mr. Jamil JAOUA, founder and former coordinator of the English Unit at the Sfax Faculty of Science for having proofread this paper. References [1] L. Claes, J. Del-Favero, B. Ceulemans, et al., De novo mutations in the sodiumchannel gene SCN1A cause severe myoclonic epilepsy of infancy, Am. J. Hum. Genet. 68 (2001) 1327–1332. [2] I. Ohmori, M. Ouchida, Y. Ohtsuka, et al., Significant correlation of the SCN1A mutations and severe myoclonic epilepsy in infancy, Biochem. Biophys. Res. Commun. 295 (2002) 17–23.
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[3] J.J. Millichap, S. Koh, L.C. Laux, et al., Child neurology: Dravet syndrome: when to suspect the diagnosis, Neurology 73 (13) (2009) 59–62. [4] C. Depienne, O. Trouillard, C. Saint-Martin, et al., Spectrum of SCN1A gene mutations associated with Dravet syndrome analysis of 333 patients, J. Med. Genet. 46 (3) (2009) 183–191. [5] L.A. Harkin, J.M. McMahon, X. Iona, et al., The spectrum of SCN1A-related infantile epileptic encephalopathies, Brain 130 (2007) 843–852. [6] F. Madia, P. Striano, E. Gennaro, et al., Cryptic chromosome deletions involving SCN1A in severe myoclonic epilepsy of infancy, Neurology 67 (2006) 1230– 1235. [7] J.C. Mulley, P. Nelson, S. Guerrero, et al., A new molecular mechanism for severe myoclonic epilepsy of infancy: exonic deletions in SCN1A, Neurology 67 (2006) 1094–1095. [8] J.C. Mulley, I.E. Scheffer, S. Petrou, et al., SCN1A mutations and epilepsy, Hum. Mutat. 25 (2005) 535–542. [9] A. Suls, K.G. Claeys, D. Goossens, et al., Microdeletions involving the SCN1A gene may be common in SCN1A-mutation-negative SMEI patients, Hum. Mutat. 2 (2006) 914–920. [10] J.W. Wang, H. Kurahashi, A. Ishii, et al., Microchromosomal deletions involving SCN1A and adjacent genes in severe myoclonic epilepsy in infancy, Epilepsia 49 (2008) 1528–1534. [11] S. Gökben, A. Berdeli, G. Serdarog˘lu, An inherited nonsense R1645X mutation in neuronal sodium channel alpha1-subunit gene in a Turkish patient with severe myoclonic epilepsy of infancy, Neuropediatrics 40 (2) (2009) 82–84. [12] Z. Arlier, Y. Bayri, L.E. Kolb, et al., Four Novel SCN1A mutations in Turkish patients with severe myoclonic epilepsy of infancy (SMEI), J. Child Neurol. 25 (2010) 1265–1268. [13] H. Sun, Y. Zhang, X. Liu, et al., Analysis of SCN1A mutation and parental origin in patients with Dravet syndrome, J. Hum. Genet. 55 (2010) 421–427. [14] R. Nabbout, O. Dulac, Epileptic encephalopathies: a brief overview, J. Clin. Neurophysiol. 20 (2003) 393–397. [15] R.H. Wallace, B.L. Hodgson, B.E. Grinton, et al., Sodium channel alpha1-subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms, Neurology 61 (2003) 765–769. [16] K. Schlachter, U. Gruber-Sedlmayr, E. Stogmann, et al., A splice site variant in the sodium channel gene SCN1A confers risk of febrile seizures, Neurology 72 (2009) 974–978. [17] A. Ebrahimi, M. Houshmand, S.H. Tonekaboni, et al., Two novel mutations in SCN1A gene in Iranian patients with epilepsy, Arch. Med. Res. 41 (2010) 207– 214. [18] G.A. Patino, L.R. Claes, L.F. Lopez-Santiago, et al., A functional null mutation of SCN1B in a patient with Dravet syndrome, J. Neurosci. 29 (2009) 10764– 10778. [19] Commission on Classification and Terminology of the International League against Epilepsy, Proposal for revised classification of epilepsies and epileptic syndromes, Epilepsia 30 (1989) 389–399. [20] J.R. Engel, A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE task force on classification and terminology, Epilepsia 42 (2001) 796–803. [21] E. Kawasaki, H. Erlich, Polymerase chain reaction and analysis of cancer cell markers, J. Natl. Cancer Inst. 82 (1990) 806–807. [22] T. Sugawara, Y. Tsurubuchi, K.L. Agarwala, et al., A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction, Proc. Natl. Acad. Sci. USA 98 (2001) 6384–6389. [23] M. Raudenská, A. Bittnerová, T. Novotny´, et al., Mutation analysis of candidate genes SCN1B, KCND3 and ANK2 in patients with clinical diagnosis of long QT syndrome, Physiol. Res. 57 (2008) 857–862. [24] T.A. Tatusova, T.L. Madden, Blast 2 sequences – a new tool for comparing protein and nucleotide sequences, FEMS Microbiol. Lett. 174 (1999) 247–250. [25] M. Stephens, N. Smith, P. Donnelly, A new statistical method for haplotype reconstruction from population data, Am. J. Hum. Genet. 68 (2001) 978–989. [26] N. Fendri-Kriaa, F. Kammoun, A. Rebai, et al., Genetic screening of two Tunisian families with generalized epilepsy with febrile seizures plus, Eur. J. Neurol. 16 (2009) 697–704. [27] E.L. Heinzen, W. Yoon, S.K. Tate, et al., Nova2 interacts with a cis-acting polymorphism to influence the proportions of drug responsive splice variants of SCN1A, Am. J. Hum. Genet. 80 (2007) 876–883. [28] S.K. Tate, C. Depondt, S.M. Sisodiya, et al., Genetic predictors of the maximum doses patients receive during clinical use of the anti-epileptic drugs carbamazepine and phenytoin, Proc. Natl. Acad. Sci. USA 102 (2005) 5507– 5512. [29] R. Lakhan, R. Kumari, U.K. Misra, et al., Differential role of sodium channels SCN1A and SCN2A gene polymorphisms with epilepsy and multiple drug resistance in the north Indian population, Br. J. Clin. Pharmacol. 68 (2009) 214–220. [30] N. Fendri-Kriaa, F. Kammoun, I.H. Salem, et al., Eur. J. Neurol. (2010), doi:10.1111/j.1468-1331.2010.03216.x.
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A putative disease-associated haplotype within the SCN1A gene in Dravet syndrome Nourhène Fendri-Kriaa a,⇑, Salma Boujilbene b, Fatma Kammoun b,c, Emna Mkaouar-Rebai a, Afif Ben Mahmoud a, Ines Hsairi b,c, Ahmed Rebai d, Chahnez Triki b,c, Faiza Fakhfakh a,⇑ a
Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax, Tunisia Service de Neurologie Infantile, C.H.U. Hédi Chaker de Sfax, Tunisia c Unité de recherche «Neuropédiatrie», Faculté de Médecine de Sfax, Tunisia d Centre de Biotechnologie de Sfax, Tunisia b
a r t i c l e
i n f o
Article history: Received 13 April 2011 Available online 21 April 2011 Keywords: Febrile seizures SCN1A SCN1B Dravet syndrome SMEI Haplotype
a b s t r a c t Dravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy, is one of the most severe forms of childhood epilepsy. DS is caused by a mutation in the neuronal voltage-gated sodiumchannel alpha-subunit gene (SCN1A). However, 25–30% of patients with DS are negative for the SCN1A mutation screening, suggesting that other molecular mechanisms may account for these disorders. Recently, the first case of DS caused by a mutation in the neuronal voltage-gated sodium-channel beta-subunit gene (SCN1B) was also reported. In this report we aim to make the molecular analysis of the SCN1A and SCN1B genes in two Tunisian patients affected with DS. The SCN1A and SCN1B genes were tested for mutations by direct sequencing. No mutation was revealed in the SCN1A and SCN1B genes by sequencing analyses. On the other hand, 11 known single nucleotide polymorphisms were identified in the SCN1A gene and composed a putative disease-associated haplotype in patients with DS phenotype. One of the two patients with putative disease-associated haplotype in SCN1A had also one known single nucleotide polymorphism in the SCN1B gene. The sequencing analyses of the SCN1A gene revealed the presence of a putative disease-associated haplotype in two patients affected with Dravet syndrome. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Dravet syndrome (DS), previously known as severe myoclonic epilepsy in infancy (SMEI), is a rare genetic disorder characterized by frequent generalized, unilateral clonic or tonic–clonic seizures that begin during the first year of life [1]. Later, patients also manifest other seizure types, including absence, myoclonic, and simple and complex partial seizures. Psychomotor development stagnates around the second year of life. DS is considered to be the most severe phenotype within the spectrum of generalized epilepsy with febrile seizures plus (GEFS+). DS is a malignant epileptic syndrome, while GEFS+ is usually benign [2]. The diagnosis can be confirmed by genetic testing that is now available, and can show mutations within the neuronal voltagegated sodium-channel alpha-subunit gene (SCN1A) [3]. In fact, in 25–70% of SMEI/DS patients, all types of mutations in the SCN1A gene have been identified [1,4–12]. Recently, Sun et al. identified pathogenic mutations in the SCN1A gene in 49 (77.8%) of 63 Chinese probands with Dravet syndrome. Most mutations were trun-
⇑ Corresponding authors. Address: Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Avenue Magida Boulila, 3029 Sfax, Tunisia. Fax: +216 74 46 14 03. E-mail addresses: [email protected] (N. Fendri-Kriaa), [email protected] (F. Fakhfakh). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.079
cating (61.2%). The mutations included 19 missense, 14 frameshift, six nonsense, and eight splice site alterations. They also identified deletions or duplications of SCN1A in two (12.5%) of 16 patients [13]. Though about a third of patients with SMEI/DS remained negative for mutations in SCN1A [4,14,9,15], some SNPs in the SCN1A gene were described to be associated with DS and epilepsy in several populations [9,16,17]. In addition, Patino et al. reported the first patient with Dravet syndrome due to a homozygous mutation in SCN1B (p.R125C), and showed that the consequence of this mutation was the inability of b1 polypeptides to be trafficked to the surface of transfected mammalian cells [18]. We reported here the SCN1A and SCN1B genes sequencing analyses of two Tunisian patients affected with Dravet syndrome and we revealed a putative disease-associated haplotype in the SCN1A gene.
2. Materials and methods 2.1. Patient ascertainment We selected two subjects with a clinical diagnosis of DS at the child neurology consulting room in Sfax, Tunisia. Epileptic seizures and epilepsy syndrome diagnoses were performed according to the
N. Fendri-Kriaa et al. / Biochemical and Biophysical Research Communications 408 (2011) 654–657
criteria established by the ‘‘International League Against Epilepsy’’ [19,20]. Peripheral blood samples were collected and genomic DNA was extracted by standard procedures [21]. Informed consent was obtained from individuals before being enrolled in the genetic study. In addition, DNAs from 10 healthy subjects were used. These controls were selected to have neither personal nor family history in their first degree relatives of neurodevelopmental disorder. 2.2. Mutation analyses All exons of SCN1A and SCN1B genes were amplified with touchdown program using the primers previously reported [22,23] to cover the coding exons and exon–intron boundaries. The PCR was performed in a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems) in a final volume of 50 lL containing 50 ng genomic DNA, 0.1 lM of each primer, 1 PCR buffer Go Taq (Promega), 1.2 mM MgCl2, 0.13 mM dNTP and 1 U Taq DNA polymerase. The amplified products were sequenced on an Applied Biosystems ABI 3100 sequencer (Alameda County, CA, USA). A Blast homology search was performed using the program blast2seq [24] at the National Center for Biotechnology Information Website to compare individual sequences to wild-type ones. 2.3. Genotyping Two fluorescent dye-labeled microsatellite markers D2S2330 and D2S335 distant of 7 cM intervals according to genome browser were used. After amplification, PCR products were pooled, with the Genescan 400HD size standard, and size fractionated on an ABI PRISM 3100-Avant automated DNA sequencer (Applied Biosystems, USA). The data were analyzed using ABI Prism GeneScan and Genotyper software version 3.7 (Applied Biosystems) to determine alleles sizes. 2.4. Biostatistic and bioinformatic tools Inference of haplotype frequencies was performed using Phase [25]. The prediction of a hypothetical polyadenylation site was carried out using the PolyA_SVM program (version 2.2) (http://polya.umdnj.edu/polya_svm/server/). The data of the hapmap project and haploview program were used to know if SNPs belonged to a linkage disequilibrium block (http://www.hapmap.org). Genome browser was used to choose microsatellite markers and to know the distance between them (http://genome.ucsc.edu/). 3. Results Sequencing analyses of the SCN1B gene in two patients with DS phenotype did not reveal any mutation but revealed in patient (1)
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one known single nucleotide polymorphism (SNP) in a heterozygous state rs2278995 (T > C) in the exon 6 of the SCN1B gene containing the 30 untranslated region (30 UTR). According to the data of in silico prediction using PolyA_SVM, SNP rs2278995 is distant of 184 bp from a hypothetical polyadenylation site with a score of 1.204 in position 9213 (Fig. 1). In addition, both patients were tested for mutations in the SCN1A gene; however, no mutation was found in this gene in the tested patients. On the other hand, 11 known single nucleotide polymorphisms were identified in a homozygous state. One variation rs566839 A > T was revealed in the 50 untranslated region (50 UTR) of exon 1. Three exonic variations were detected: one SNP rs7580482 (A > G) in exon 9 (Val404Val), one SNP rs6432860 (T > C) in exon 13 (Val753Val) and one SNP rs2298771 (G > A) in exon 16 (Ala1056Thr). Seven intronic polymorphisms were also revealed: one SNP rs3812718 (G > A) in intron 4, one SNP rs994399 (C > T) in intron 6, two SNPs rs1542484 (T > C) and rs1461193 (C > T) in intron 7, one SNP rs11690959 (C > T) in intron 8, one SNP rs6432861 (G > A) in intron 9 and one SNP rs7601520 (C > T) in intron 15 (Fig. 2). The haplotypes construction and an analysis of recombination events were performed for all variants in the SCN1A gene. A haplotype was revealed and delimited by SNP rs566839 in 50 UTR of exon 1 and SNP rs2298771 in exon 16. To try to delimit the region of homozygosity, the microsatellite markers D2S2330 and D2S335 bordering the SCN1A gene were tested and revealed in a heterozygous state in both patients. In addition, according to the hapmap project data (http:// www.hapmap.org), these SNPs belong to a linkage disequilibrium block extending 37.426 kb and covering 16 exons of SCN1A gene (Fig. 3). We chose three SNPs from 11 SNPs in this block: rs3812718 (intron 4), rs6432860 (exon 13) and rs2298771 (exon 16) to test them in 10 controls from the Tunisian population and to estimate the haplotype frequencies (Fig. 4). Using Phase, the ACA haplotype frequency was 100% in patients, which was significantly higher than that in the control population estimated at 17 ± 11.4% (6–28%). This haplotype of SNPs was in a homozygous state in these two patients with DS and was considered as a putative disease-associated haplotype (Figs. 2 and 4).
4. Discussion We reported sequencing analyses of the SCN1A and SCN1B genes in two Tunisian patients affected with Dravet syndrome. The mutational analyses of the SCN1A gene revealed 11 known single nucleotide polymorphisms but no mutation. These SNPs were in a homozygous state and were assumed to compose a single haplotype block. We showed that these 11 SNPs belonged to a linkage disequilibrium block extending 37.426 kb and covering 16 exons of the SCN1A gene (http://www.hapmap.org). We chose three SNPs from them: rs3812718 (intron 4), rs6432860 (exon 13) and rs2298771 (exon 16) to test them in 10 controls from
Fig. 1. Prediction of a hypothetical polyadenylation site using the PolyA_SVM program. The (T) variant of SNP rs2278995 is boxed and is distant of 184 bp from the site 9213 which is underlined.
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Fig. 2. Genotypes of single nucleotide polymorphisms (SNPs) in the SCN1A gene.
Fig. 3. Result of haploview presenting a block of 25 kb containing four SNPs of our putative associated haplotype.
Fig. 4. Result comparison of the SCN1A gene sequencing in patients with febrile seizures. One individual with FS (1), one affected by epilepsy with generalized tonico-clonic seizures (2) and two individuals with FS+(3, 4) were reported recently [26]. Two patients were analyzed with DS (5, 6) in the current study. The putative disease-associated haplotype for single nucleotide polymorphisms (SNPs) in the SCN1A is boxed and indicated by gray bar. The three SNPs tested in controls to estimate the haplotype frequencies are horizontally boxed.
the Tunisian population and to estimate the haplotype frequencies. Using Phase, the frequency of ACA haplotype is 100% in patients significantly higher than that in the control population estimated at 17 ± 11.4% (6–28%). This haplotype of SNPs was in a homozygous state in these two patients with DS and was considered as a putative disease-associated haplotype (Figs. 2 and 4). Recently, we also reported this haplotype in a homozygous state only in one patient with DS. The same haplotype was present in the other four patients with febrile seizures but in a heterozygous state proving its association with the DS phenotype (Fig. 4) [26].
Among the 11 SNPs detected in our patients, rs3812718, rs1542484, rs6432861 and rs2298771 were also described to be associated with DS and epilepsy in several populations. In fact, in the Belgian population, these three SNPs rs3812718, rs1542484 and rs6432861 were also described in haplotype of 15 SNPs in a homozygous state in 11 patients affected with DS [9]. In addition, in the Caucasian population, Schlachter et al. found an association between SNP rs3812718 of SCN1A and febrile seizures (FS) [16]. This polymorphism has been hypothesized to be a candidate SNP for FS [16]. Specifically, this SNP (rs3812718) is believed to be a
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candidate functional variant because it alters the proportions of the neonatal and adult transcripts of the gene [27] and has been reported to be associated with the maximal doses of carbamazepine and phenytoin, both sodium channel-blocking antiepileptic drugs, in patients with epilepsy [28]. The SNP rs2298771 belonging to the ACA haplotype was also tested in Iranian patients with epilepsy. The results revealed that SNP rs2298771, considered as common SNP, was present in 20/ 34 (0.588) probands with allelic frequency as 0.7058/0.2942 in patients and 0.515/0.485 in control group, respectively, for A/G but the statistical analysis did not show any significant differences between the groups. In fact, it was present in a heterozygous state in four patients with DS and in a homozygous state in the three others [17]. In the Indian population, the frequency of AG genotype of SNP rs2298771 was significantly higher in epilepsy patients in comparison to that in healthy controls (P = 0.005) and associated with epileptic patients. Thus AG genotype was suggested to be involved in increasing risk for developing epilepsy, but it did not modulate drug response [29]. The SNP rs2298771 results in change of amino acid at highly consensus conserved site in the coding region of the SCN1A, and possibly affects functioning of the inactivation gate in the cytosol regulating efflux and influx of sodium ions. Another possibility is that this polymorphism is in linkage disequilibrium with some other genetic variants of the same gene that imparts risk for epilepsy [29]. In addition, the sequencing results of the SCN1B gene did not reveal any mutation in this gene for any of the tested patients but revealed SNP rs2278995 (T > C) in the exon 6 of the SCN1B gene in a heterozygous state in one patient (1). This SNP was recently reported and was a part of putative disease-associated haplotype in Tunisian patients with febrile seizures [30]. This SNP is located in the 30 UTR of SCN1B and might have an effect on the stability and maturity of mRNA. In fact, in silico prediction using PolyA_SVM (version 2.2) shows that this SNP is located in a neighbouring hypothetical polyadenylation site (Score of 1.204 in position 9213) and thus might affect the expression of the SCN1B gene. We noticed that the patient (1), who had the SNP in SCN1B and the putative disease-associated haplotype in SCN1A at the same time, was the first case of DS which combined polymorphisms in two neuronal voltage-gated sodium-channel subunit genes. In conclusion, the SCN1A gene sequencing analyses revealed the presence of a putative disease-associated haplotype in two patients affected with Dravet syndrome. Acknowledgments We thank the patients and their families for their cooperation in the present study and for giving informed consent. This work was supported by the Ministry of Higher Education and Scientific Research in Tunisia. The proper institutional approval was obtained as well as the disclosure of sources of funding and other disclosure of conflict of interest related to the submitted manuscript. We extend our thanks to Mr. Jamil JAOUA, founder and former coordinator of the English Unit at the Sfax Faculty of Science for having proofread this paper. References [1] L. Claes, J. Del-Favero, B. Ceulemans, et al., De novo mutations in the sodiumchannel gene SCN1A cause severe myoclonic epilepsy of infancy, Am. J. Hum. Genet. 68 (2001) 1327–1332. [2] I. Ohmori, M. Ouchida, Y. Ohtsuka, et al., Significant correlation of the SCN1A mutations and severe myoclonic epilepsy in infancy, Biochem. Biophys. Res. Commun. 295 (2002) 17–23.
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