- Email: [email protected]
CACNA1I Is Not Associated With Childhood Absence Epilepsy in the Chinese Han Population
CACNA1I Is Not Associated With Childhood Absence Epilepsy in the Chinese Han Population Juli Wang, PhD,* Yuehua Zhang, PhD,* Jianmin Liang, PhD,* Hong Pan, MD,* Husheng Wu, MD,† Keming Xu, MD,‡ Xiaoyan Liu, MD,* Yuwu Jiang, PhD,* Yan Shen, MD,§ and Xiru Wu, MD* This study investigated whether the T-type calcium channel gene CACNA1I causes susceptibility in the Chinese Han population to childhood absence epilepsy, a form of idiopathic generalized seizure disorder. For this investigation, we searched for mutations in the 35 exons and exon-intron boundaries of the CACNA1I gene in 50 Han Chinese patients with childhood absence epilepsy. Seventeen single nucleotide polymorphisms were identified in the 35 exons. Using six single nucleotide polymorphisms as markers, the allele and genotype distributions of all of the identified single nucleotide polymorphisms were examined; there was no significant difference between the childhood absence epilepsy cases and the control groups. Thus, we do not consider the CACNA1I gene to be an important susceptibility gene for childhood absence epilepsy in the Chinese Han population. © 2006 by Elsevier Inc. All rights reserved.
Childhood absence epilepsy, an idiopathic form of generalized epilepsy, is typified by sudden brief impairment of consciousness with 3-Hz spike and slow-wave discharges over both brain hemispheres. This disease constitutes 8-15% of total childhood epilepsies [1]. A family history of epilepsy is present in 15-44% of patients
with generalized absence seizures. Studies have demonstrated that the spike wave is inherited as a complex trait. The basic underlying mechanism of generalized absence seizures appears to involve thalamocortical circuitry and the generation of abnormal oscillatory rhythms from that particular neuronal network [2,3]. The three types of low-threshold T-type calcium channel genes, CACNA1G, CACNA1H, and CACNA1I, respectively encode T-type calcium channel ␣1G (Cav3.1, chromosome 17q22), ␣1H (Cav3.2, chromosome 16p13.3), and ␣1I (Cav3.3, chromosome 22q13.1) subunits. It has been proposed that lowthreshold T-type calcium channels may be related to absence seizures [4,5]. This hypothesis is supported by evidence that drugs effective in the treatment of absence seizures, such as ethosuximide, exert their antiepileptic action by reducing the T-type Ca2⫹ current IT (inward transient calcium current) in thalamic neurons [4,6,7]. T-type Ca2⫹ currents may be involved in the genesis of slow-wave discharges, which signify absence seizures [8]. Thalamic reticular neurons, which express high and moderate levels of ␣1I and ␣1H messenger ribonucleic acids, respectively, possess slower kinetics of activation and inactivation as compared with the thalamic relay neurons of the ventral basal complex, a region that appears to express ␣1G exclusively [9]. The ␣1I channels open after small membrane depolarizations in a manner similar to the stimulated ␣1G and ␣1H channels, but at more depolarized potentials. The ␣1I channel kinetics of activation and inactivation is dramatically slower, which allows it to act as a Ca2⫹ injector [10]. The CACNA1I gene contains at least 37 exons and spans over 115 kb. To date, there have been no reports on the association of T-type CACNA1I gene integrity and childhood absence epilepsy. To identify
From the *Department of Pediatrics, Peking University First Hospital, Beijing, P. R. China; †Beijing Children’s Hospital, Beijing; ‡Capital Institute of Pediatrics, Beijing; §National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing.
Communications should be addressed to: Dr. Wu; Department of Pediatrics; Peking University First Hospital; No. 8 Xishiku Street; Beijing, 100034, P. R. China. E-mail: [email protected] Received December 12, 2005; accepted March 14, 2006.
Wang J, Zhang Y, Liang J, Pan H, Wu H, Xu K, Liu X, Jiang Y, Shen Y, Wu X. CACNA1I Is Not Associated With Childhood Absence Epilepsy in the Chinese Han Population. Pediatr Neurol 2006;35:187-190.
Introduction
© 2006 by Elsevier Inc. All rights reserved. doi:10.1016/j.pediatrneurol.2006.03.006 ● 0887-8994/06/$—see front matter
Wang et al: Childhood Absence Epilepsy in Chinese Han 187
whether such an association exists, we sequenced 35 exons and the exon-intron boundaries of CACNA1I in 50 Han Chinese patients diagnosed with childhood absence epilepsy. Methods There were 50 consecutive patients with childhood absence epilepsy (21 males and 29 females) ranging in age from 4 to 14 years (mean 7.2, S.D. 2.1; mean age at onset 6.3, S.D. 1.7); all were of Han ethnicity of China. Informed consent was obtained from their parents. The patients were eligible for the study if they fulfilled the criteria for the diagnosis of childhood absence epilepsy [11]. The clinical inclusion criteria for childhood absence epilepsy patients are as follows: (1) typical absence seizures appear as the initial seizure type at 3 to 12 years of age; (2) frequent absence seizures occur multiple times per day; (3) absence seizures are associated with bilateral, symmetric, and synchronous discharge of regular 3-Hz slow-wave discharge in the electroencephalogram with normal background; (4) general physical and neurologic examinations are normal; (5) normal neuroradiologic examinations including computed tomography or magnetic resonance imaging (if needed). We created an identical special questionnaire for each center, and organized meetings of principal persons of the three centers (the authors of this paper from the Department of Pediatrics, Peking University First Hospital; Beijing Children’s Hospital; Capital Institute of Pediatrics) once in 2 months to discuss the clinical and electroencephalographic manifestations of all the patients to ensure that they met the inclusion criteria. The deoxyribonucleic acid from 50 unrelated healthy adult blood donors from the central blood bank of our hospital, with no history (family or personal) of epilepsy, was used as negative control. Genomic deoxyribonucleic acid was extracted from peripheral blood leukocytes from all subjects using the method recommended by Miller et al. [12]. The same primers were used for intron amplification and sequencing the 35 target exons (primers are available on request). The polymerase chain reaction amplifications were performed in a 15-L reaction volume containing 50 ng of genomic deoxyribonucleic acid, 10 mM Tris (pH 8.4), 50 mM KCl, 3.0 mM MgCl2, 200 M of each deoxyribonucleoside triphosphate, 0.6 units HotStartaq DNA Polymerase (Qiagen, Cologne, Germany), and 0.3 M of each primer. The polymerase chain reaction thermocycles were conducted with a GeneAmp PCR System 2400 (Perkin Elmer). An initial denaturation was performed for 15 minutes at 95°C, followed by a second denaturation for 30 seconds at 94°C, annealing for 1 minute at 62°C, decreasing the annealing temperature by 1°C for 2 of the initial 14 cycles, and extension for 1 minute 30 seconds at 72°C. The annealing temperature then remained at 56°C for 40 seconds of the subsequent 26 cycles, and the extension parameters were reduced to 60 seconds at 72°C. A final extension was performed at 72°C for 10 minutes. The amplification products were purified with a Multiscreen filter plate (Millipore, Inc.). Cycle sequencing was performed according to the instructions using the Big Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems, Chiba, Japan). All sequencing was conducted on an ABI PRISM 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). The ABI sequence software was used for lane tracking and for first pass base calling (Perkin Elmer, Boston, MA). The tests for Hardy-Weinberg equilibrium, allele and genotype frequencies, and chi-square tests were all performed using the software package SPSS 10.0.
Results Seventeen single nucleotide polymorphisms (SNPs are named according to the nomenclature recommended by Antonarakis [13]) were identified throughout the CACNA1I gene (Table 1), 4 and 13 of which are located in exons and introns, respectively. One of the four coding
188
PEDIATRIC NEUROLOGY
Vol. 35 No. 3
single nucleotide polymorphisms results in a nonsynonymous amino acid replacement. The frequencies of the minor alleles of six of the single nucleotide polymorphisms [99C ⬎ T (Ser⬎Ser), IVS1-51A ⬎ G, IVS2⫹37insGCCCT, IVS4⫹81C ⬎ T, IVS8-15A ⬎ G, and IVS28⫹158T ⬎ C] were higher than 6%. Consequently, these six markers were used to study the association of single nucleotide polymorphism occurrence in the CACNA1I gene in patients with or without childhood absence epilepsy. None of the observed single nucleotide polymorphism genotypes deviated significantly (P ⬍ 0.05) from those predicted by the Hardy-Weinberg equilibrium calculation. Furthermore, the allele and genotype frequencies of the six single nucleotide polymorphisms did not differ significantly between the childhood absence epilepsy patients and control subjects. Discussion Neurons in the reticular thalamic nucleus, thalamic relay neurons, and neocortical pyramidal cells comprise a circuit that sustains the thalamocortical oscillatory burstfiring of absence seizures [2,3]. Reticular thalamic neurons are endowed with large T-type currents that mediate bursting behavior associated with slow-wave discharges. The critical role of the T-type channels in slow-wave discharge epilepsies is also supported by treatment of absence seizures using ethosuximide, an inhibitor of Ttype Ca2⫹ currents [4]. Chen et al. [14,15] studied two T-type calcium channel CACNA1H and CACNA1G genes in childhood absence epilepsy patients. They identified 12 mutations in the CACNA1H gene in 14 of 188 children with childhood absence epilepsy, and found no mutations in 230 control subjects. Khosravani and coworkers [16] generated five of these mutations in the rat ␣1H T-type channel homologue and characterized them functionally in HEK293 cells. They found that three of the missense mutations mediate significant gain-of-function effects on T-type channel activity. This increase in activity may play a role in altering seizure threshold levels in patients with childhood absence epilepsy. Chen et al. [15] also found that T-type calcium channel gene ␣1G is not associated with childhood absence epilepsy in the Chinese Han population. The gene effects in the context of childhood absence epilepsy may occur through synergistic interactions with other factors such as additional ion channels and intracellular modulators, all of which are capable of numerous activity modes in the epileptic brain [16]. Low-voltage-activated T-type Ca2⫹ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy [8]. Vitko et al. [17] found that many of these single nucleotide polymorphisms alter gating in a manner that would increase the propensity of neurons to fire. The CACNA1I (␣1I) gene is the last member of T-type
Table 1.
PCR primers for the identified SNPs of the alpha (11) gene and summary of the SNPs*
Region Exon 1 Exon 2 Exon 3 Exon 4 Exon 8 Exon 9 Exon 11 Exon 12 Exon 13–14 Exon 16 Exon 17 Exon 19–20 Exon 28 Exon 31 Exon 32
Primers 5=-gcatccgtccacttattggt 5=-ctctgagctcgctcaccac 5=-aatggggtgtatgcctgtgt 5=-ggtagtgcagcgcaggat 5=-aaatgactgcattggactgg 5=-ggacgcacaacttaggaagg 5=-cgccatttacagaggaggag 5=-gcacatcactggcacacaat 5=-atctccatctccccatctcc 5=-gggctgtgaaggaaacactt 5=-tctcaaactcctgggctcac 5=-ggcccctgtcctcactttat 5=-tatggtgaggacaccgacct 5=-atttatgcccatcccttcct 5=-gagcccctgattagcttgtg 5=-cactgaggcttaggggttca 5=-cccctaagcctcagtgttca 5=-gggataggttgcctgctaga 5=-ggaagcctccttagaaaagctc 5=-caggaatgcaacagacatgg 5=-tctagggtcaagcccattcc 5=-cgcacccacttctgtcctat 5=-gaggagaggataggggttgc 5=-atggggctacgagtgaaatg 5=-cagccacacagcctaatgg 5=-tgcaaagttgcctgtgtgtt 5=-ctccccgagcctcagtttat 5=-ctgttctggctcagctctcc 5=-gggatgtctccgtcctcat 5=-ggcccagaaagacagaggtc
PCR Product Size (bp)
Polymorphisms (Amino Acid Change)
498
99C⬎T(Ser⬎Ser)
382 500
IVS1-51A⬎G IVS2⫹37insGCCCT IVS 3⫹34 T⬎C
474
IVS 4⫹81C⬎T
586
IVS 7–27 C⬎T
453
IVS8-15A⬎G
500
IVS 11⫹120 T⬎G
534 692
2157T⬎C(Ile⬎Ile) IVS12⫹129T⬎C IVS14⫹40T⬎C
472
2859T⬎C(Ser⬎Ser)
675
3118A⬎G(I⬎V)
689
IVS 18-18 C⬎T
495
IVS 28⫹158 T⬎C
461
IVS 31⫹125 C⬎T
493
IVS32⫹23 T⬎C
Abbreviations: IVS ⫽ Intervening sequence PCR ⫽ Polymerase chain reaction SNP ⫽ Single nucleotide polymorphism * SNPs are named according to the nomenclature recommended by Antonarakis [13]. Exon SNPs are numbered according to their positions in the coding sequence (NM_021096). Intron SNPs are designated by IVS (intervening sequence), positive numbers start from the G of the donor site GT.
calcium channels that we studied in childhood absence epilepsy. This gene (exons 1-37) encodes the ␣1I isoform, which activates and inactivates much more slowly than the other T-type Ca2⫹ channels. These distinctive kinetics features, along with its brain region–specific expression, suggest that ␣1I channels endow neurons with the ability to generate long-lasting bursts of firing [10]. The ␣1I isoform is similar to high-voltage-activated calcium channels in that the depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity [18]. No mutations were identified in the CACNA1I gene among the 50 childhood absence epilepsy patients. Using the six single nucleotide polymorphism CACNA1I gene markers, we found that the allele and genotype distributions of all the single nucleotide polymorphisms studied were not significantly different among the childhood absence epilepsy patients and the control subjects. The small sample size of the childhood absence epilepsy patients provided only limited statistical power to detect gene variants of small effects or
those that are rare major determinants of risk due to allelic and locus heterogeneity. In addition, we did not screen all of the introns and the 5=untranslated region, which might affect ribonucleic acid splicing or regulatory elements for gene transcription, respectively. We also did not screen exons 21 and 37, which are rich in guanine-cytosine content. We therefore suggest that the CACNA1I is not an important susceptibility gene for childhood absence epilepsy, at least at the coding level in the Chinese population. Further studies are recommended in larger cohorts to verify these results.
We are very thankful to all the patients and their families who participated in this work. We thank all the clinicians who contributed cases to the study, and Professor Yu Qi for valuable discussions. This work was supported by grants from the National Natural Science Foundation of China (No. 30371494), China National “211 Project” in Peking University (No. 205), and National High-Tech Research & Development Program of China (No. 2002AA223011 and 2002BA711A07).
Wang et al: Childhood Absence Epilepsy in Chinese Han 189
References [1] Robinson R, Gardiner M. Genetics of childhood epilepsy. Arch Dis Child 2000;82:121-5. [2] Snead OC 3rd. Basic mechanisms of generalized absence seizures. Ann Neurol 1995;37:146-57. [3] Crunelli V, Leresche N. Childhood absence epilepsy: Genes, channels, neurons and networks. Nat Rev Neurosci 2002;3:371-82. [4] Coulter DA, Huguenard JR, Prince DA. Characterization of ethosuximide reduction of low-threshold calcium current in thalamic neurons. Ann Neurol 1989;25:582-93. [5] Crunelli V, Leresche N. A role for GABAB receptors in excitation and inhibition of thalamocortical cells. Trends Neurosci 1991;14:16-21. [6] Gomora JC, Daud AN, Weiergraber M, Perez-Reyes E. Block of cloned human T-type calcium channels by succinimide antiepileptic drugs. Mol Pharmacol 2001;60:1121-32. [7] Kostyuk PG, Molokanova EA, Pronchuk NF, Savchenko AN, Verkhratsky AN. Different action of ethosuximide on low- and highthreshold calcium current in rat sensory neurons. Neuroscience 1992;51:755-8. [8] Avanzini G, Panzica F, de Curtis M. The role of the thalamus in vigilance and epileptogenic mechanisms. Clin Neurophysiol 2000;111: S19-26. [9] Talley EM, Cribbs LL, Lee JH, Daud A, Perez-Reyes E, Bayliss DA. Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 1999;19: 1895-911. [10] Lee JH, Daud AN, Cribbs LL, et al. Cloning and expression of
190
PEDIATRIC NEUROLOGY
Vol. 35 No. 3
a novel member of the low voltage-activated T-type calcium channel family. J Neurosci 1999;19:1912-21. [11] Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia 1989;30:389-99. [12] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988;16:1215. [13] Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 1998;11:1-3. [14] Chen Y, Lu J, Pan H, et al. Association between genetic variation of CACNA1H and childhood absence epilepsy. Ann Neurol 2003;54:239-43. [15] Chen Y, Lu J, Zhang Y, et al. T-type calcium channel gene alpha (1G) is not associated with childhood absence epilepsy in the Chinese Han population. Neurosci Lett 2003;341:29-32. [16] Khosravani H, Altier C, Simms B, et al. Gating effects of mutations in the Cav3.2 T-type calcium channel associated with childhood absence epilepsy. J Biol Chem 2004; 279:9681-4. [17] Vitko I, Chen Y, Arias JM, Shen Y, Wu XR, Perez-Reyes E. Functional characterization and neuronal modeling of the effects of childhood absence epilepsy variants of CACNA1H, a T-type calcium channel. J Neurosci 2005;25:4844-55. [18] Gomora JC, Murbartian J, Arias JM, Lee JH, Perez-Reyes E. Cloning and expression of human T-type channel Cav3.3: Insights into prepulse facilitation. Biophys J 2002;83:229-41.
Childhood absence epilepsy, an idiopathic form of generalized epilepsy, is typified by sudden brief impairment of consciousness with 3-Hz spike and slow-wave discharges over both brain hemispheres. This disease constitutes 8-15% of total childhood epilepsies [1]. A family history of epilepsy is present in 15-44% of patients
with generalized absence seizures. Studies have demonstrated that the spike wave is inherited as a complex trait. The basic underlying mechanism of generalized absence seizures appears to involve thalamocortical circuitry and the generation of abnormal oscillatory rhythms from that particular neuronal network [2,3]. The three types of low-threshold T-type calcium channel genes, CACNA1G, CACNA1H, and CACNA1I, respectively encode T-type calcium channel ␣1G (Cav3.1, chromosome 17q22), ␣1H (Cav3.2, chromosome 16p13.3), and ␣1I (Cav3.3, chromosome 22q13.1) subunits. It has been proposed that lowthreshold T-type calcium channels may be related to absence seizures [4,5]. This hypothesis is supported by evidence that drugs effective in the treatment of absence seizures, such as ethosuximide, exert their antiepileptic action by reducing the T-type Ca2⫹ current IT (inward transient calcium current) in thalamic neurons [4,6,7]. T-type Ca2⫹ currents may be involved in the genesis of slow-wave discharges, which signify absence seizures [8]. Thalamic reticular neurons, which express high and moderate levels of ␣1I and ␣1H messenger ribonucleic acids, respectively, possess slower kinetics of activation and inactivation as compared with the thalamic relay neurons of the ventral basal complex, a region that appears to express ␣1G exclusively [9]. The ␣1I channels open after small membrane depolarizations in a manner similar to the stimulated ␣1G and ␣1H channels, but at more depolarized potentials. The ␣1I channel kinetics of activation and inactivation is dramatically slower, which allows it to act as a Ca2⫹ injector [10]. The CACNA1I gene contains at least 37 exons and spans over 115 kb. To date, there have been no reports on the association of T-type CACNA1I gene integrity and childhood absence epilepsy. To identify
From the *Department of Pediatrics, Peking University First Hospital, Beijing, P. R. China; †Beijing Children’s Hospital, Beijing; ‡Capital Institute of Pediatrics, Beijing; §National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing.
Communications should be addressed to: Dr. Wu; Department of Pediatrics; Peking University First Hospital; No. 8 Xishiku Street; Beijing, 100034, P. R. China. E-mail: [email protected] Received December 12, 2005; accepted March 14, 2006.
Wang J, Zhang Y, Liang J, Pan H, Wu H, Xu K, Liu X, Jiang Y, Shen Y, Wu X. CACNA1I Is Not Associated With Childhood Absence Epilepsy in the Chinese Han Population. Pediatr Neurol 2006;35:187-190.
Introduction
© 2006 by Elsevier Inc. All rights reserved. doi:10.1016/j.pediatrneurol.2006.03.006 ● 0887-8994/06/$—see front matter
Wang et al: Childhood Absence Epilepsy in Chinese Han 187
whether such an association exists, we sequenced 35 exons and the exon-intron boundaries of CACNA1I in 50 Han Chinese patients diagnosed with childhood absence epilepsy. Methods There were 50 consecutive patients with childhood absence epilepsy (21 males and 29 females) ranging in age from 4 to 14 years (mean 7.2, S.D. 2.1; mean age at onset 6.3, S.D. 1.7); all were of Han ethnicity of China. Informed consent was obtained from their parents. The patients were eligible for the study if they fulfilled the criteria for the diagnosis of childhood absence epilepsy [11]. The clinical inclusion criteria for childhood absence epilepsy patients are as follows: (1) typical absence seizures appear as the initial seizure type at 3 to 12 years of age; (2) frequent absence seizures occur multiple times per day; (3) absence seizures are associated with bilateral, symmetric, and synchronous discharge of regular 3-Hz slow-wave discharge in the electroencephalogram with normal background; (4) general physical and neurologic examinations are normal; (5) normal neuroradiologic examinations including computed tomography or magnetic resonance imaging (if needed). We created an identical special questionnaire for each center, and organized meetings of principal persons of the three centers (the authors of this paper from the Department of Pediatrics, Peking University First Hospital; Beijing Children’s Hospital; Capital Institute of Pediatrics) once in 2 months to discuss the clinical and electroencephalographic manifestations of all the patients to ensure that they met the inclusion criteria. The deoxyribonucleic acid from 50 unrelated healthy adult blood donors from the central blood bank of our hospital, with no history (family or personal) of epilepsy, was used as negative control. Genomic deoxyribonucleic acid was extracted from peripheral blood leukocytes from all subjects using the method recommended by Miller et al. [12]. The same primers were used for intron amplification and sequencing the 35 target exons (primers are available on request). The polymerase chain reaction amplifications were performed in a 15-L reaction volume containing 50 ng of genomic deoxyribonucleic acid, 10 mM Tris (pH 8.4), 50 mM KCl, 3.0 mM MgCl2, 200 M of each deoxyribonucleoside triphosphate, 0.6 units HotStartaq DNA Polymerase (Qiagen, Cologne, Germany), and 0.3 M of each primer. The polymerase chain reaction thermocycles were conducted with a GeneAmp PCR System 2400 (Perkin Elmer). An initial denaturation was performed for 15 minutes at 95°C, followed by a second denaturation for 30 seconds at 94°C, annealing for 1 minute at 62°C, decreasing the annealing temperature by 1°C for 2 of the initial 14 cycles, and extension for 1 minute 30 seconds at 72°C. The annealing temperature then remained at 56°C for 40 seconds of the subsequent 26 cycles, and the extension parameters were reduced to 60 seconds at 72°C. A final extension was performed at 72°C for 10 minutes. The amplification products were purified with a Multiscreen filter plate (Millipore, Inc.). Cycle sequencing was performed according to the instructions using the Big Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems, Chiba, Japan). All sequencing was conducted on an ABI PRISM 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). The ABI sequence software was used for lane tracking and for first pass base calling (Perkin Elmer, Boston, MA). The tests for Hardy-Weinberg equilibrium, allele and genotype frequencies, and chi-square tests were all performed using the software package SPSS 10.0.
Results Seventeen single nucleotide polymorphisms (SNPs are named according to the nomenclature recommended by Antonarakis [13]) were identified throughout the CACNA1I gene (Table 1), 4 and 13 of which are located in exons and introns, respectively. One of the four coding
188
PEDIATRIC NEUROLOGY
Vol. 35 No. 3
single nucleotide polymorphisms results in a nonsynonymous amino acid replacement. The frequencies of the minor alleles of six of the single nucleotide polymorphisms [99C ⬎ T (Ser⬎Ser), IVS1-51A ⬎ G, IVS2⫹37insGCCCT, IVS4⫹81C ⬎ T, IVS8-15A ⬎ G, and IVS28⫹158T ⬎ C] were higher than 6%. Consequently, these six markers were used to study the association of single nucleotide polymorphism occurrence in the CACNA1I gene in patients with or without childhood absence epilepsy. None of the observed single nucleotide polymorphism genotypes deviated significantly (P ⬍ 0.05) from those predicted by the Hardy-Weinberg equilibrium calculation. Furthermore, the allele and genotype frequencies of the six single nucleotide polymorphisms did not differ significantly between the childhood absence epilepsy patients and control subjects. Discussion Neurons in the reticular thalamic nucleus, thalamic relay neurons, and neocortical pyramidal cells comprise a circuit that sustains the thalamocortical oscillatory burstfiring of absence seizures [2,3]. Reticular thalamic neurons are endowed with large T-type currents that mediate bursting behavior associated with slow-wave discharges. The critical role of the T-type channels in slow-wave discharge epilepsies is also supported by treatment of absence seizures using ethosuximide, an inhibitor of Ttype Ca2⫹ currents [4]. Chen et al. [14,15] studied two T-type calcium channel CACNA1H and CACNA1G genes in childhood absence epilepsy patients. They identified 12 mutations in the CACNA1H gene in 14 of 188 children with childhood absence epilepsy, and found no mutations in 230 control subjects. Khosravani and coworkers [16] generated five of these mutations in the rat ␣1H T-type channel homologue and characterized them functionally in HEK293 cells. They found that three of the missense mutations mediate significant gain-of-function effects on T-type channel activity. This increase in activity may play a role in altering seizure threshold levels in patients with childhood absence epilepsy. Chen et al. [15] also found that T-type calcium channel gene ␣1G is not associated with childhood absence epilepsy in the Chinese Han population. The gene effects in the context of childhood absence epilepsy may occur through synergistic interactions with other factors such as additional ion channels and intracellular modulators, all of which are capable of numerous activity modes in the epileptic brain [16]. Low-voltage-activated T-type Ca2⫹ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy [8]. Vitko et al. [17] found that many of these single nucleotide polymorphisms alter gating in a manner that would increase the propensity of neurons to fire. The CACNA1I (␣1I) gene is the last member of T-type
Table 1.
PCR primers for the identified SNPs of the alpha (11) gene and summary of the SNPs*
Region Exon 1 Exon 2 Exon 3 Exon 4 Exon 8 Exon 9 Exon 11 Exon 12 Exon 13–14 Exon 16 Exon 17 Exon 19–20 Exon 28 Exon 31 Exon 32
Primers 5=-gcatccgtccacttattggt 5=-ctctgagctcgctcaccac 5=-aatggggtgtatgcctgtgt 5=-ggtagtgcagcgcaggat 5=-aaatgactgcattggactgg 5=-ggacgcacaacttaggaagg 5=-cgccatttacagaggaggag 5=-gcacatcactggcacacaat 5=-atctccatctccccatctcc 5=-gggctgtgaaggaaacactt 5=-tctcaaactcctgggctcac 5=-ggcccctgtcctcactttat 5=-tatggtgaggacaccgacct 5=-atttatgcccatcccttcct 5=-gagcccctgattagcttgtg 5=-cactgaggcttaggggttca 5=-cccctaagcctcagtgttca 5=-gggataggttgcctgctaga 5=-ggaagcctccttagaaaagctc 5=-caggaatgcaacagacatgg 5=-tctagggtcaagcccattcc 5=-cgcacccacttctgtcctat 5=-gaggagaggataggggttgc 5=-atggggctacgagtgaaatg 5=-cagccacacagcctaatgg 5=-tgcaaagttgcctgtgtgtt 5=-ctccccgagcctcagtttat 5=-ctgttctggctcagctctcc 5=-gggatgtctccgtcctcat 5=-ggcccagaaagacagaggtc
PCR Product Size (bp)
Polymorphisms (Amino Acid Change)
498
99C⬎T(Ser⬎Ser)
382 500
IVS1-51A⬎G IVS2⫹37insGCCCT IVS 3⫹34 T⬎C
474
IVS 4⫹81C⬎T
586
IVS 7–27 C⬎T
453
IVS8-15A⬎G
500
IVS 11⫹120 T⬎G
534 692
2157T⬎C(Ile⬎Ile) IVS12⫹129T⬎C IVS14⫹40T⬎C
472
2859T⬎C(Ser⬎Ser)
675
3118A⬎G(I⬎V)
689
IVS 18-18 C⬎T
495
IVS 28⫹158 T⬎C
461
IVS 31⫹125 C⬎T
493
IVS32⫹23 T⬎C
Abbreviations: IVS ⫽ Intervening sequence PCR ⫽ Polymerase chain reaction SNP ⫽ Single nucleotide polymorphism * SNPs are named according to the nomenclature recommended by Antonarakis [13]. Exon SNPs are numbered according to their positions in the coding sequence (NM_021096). Intron SNPs are designated by IVS (intervening sequence), positive numbers start from the G of the donor site GT.
calcium channels that we studied in childhood absence epilepsy. This gene (exons 1-37) encodes the ␣1I isoform, which activates and inactivates much more slowly than the other T-type Ca2⫹ channels. These distinctive kinetics features, along with its brain region–specific expression, suggest that ␣1I channels endow neurons with the ability to generate long-lasting bursts of firing [10]. The ␣1I isoform is similar to high-voltage-activated calcium channels in that the depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity [18]. No mutations were identified in the CACNA1I gene among the 50 childhood absence epilepsy patients. Using the six single nucleotide polymorphism CACNA1I gene markers, we found that the allele and genotype distributions of all the single nucleotide polymorphisms studied were not significantly different among the childhood absence epilepsy patients and the control subjects. The small sample size of the childhood absence epilepsy patients provided only limited statistical power to detect gene variants of small effects or
those that are rare major determinants of risk due to allelic and locus heterogeneity. In addition, we did not screen all of the introns and the 5=untranslated region, which might affect ribonucleic acid splicing or regulatory elements for gene transcription, respectively. We also did not screen exons 21 and 37, which are rich in guanine-cytosine content. We therefore suggest that the CACNA1I is not an important susceptibility gene for childhood absence epilepsy, at least at the coding level in the Chinese population. Further studies are recommended in larger cohorts to verify these results.
We are very thankful to all the patients and their families who participated in this work. We thank all the clinicians who contributed cases to the study, and Professor Yu Qi for valuable discussions. This work was supported by grants from the National Natural Science Foundation of China (No. 30371494), China National “211 Project” in Peking University (No. 205), and National High-Tech Research & Development Program of China (No. 2002AA223011 and 2002BA711A07).
Wang et al: Childhood Absence Epilepsy in Chinese Han 189
References [1] Robinson R, Gardiner M. Genetics of childhood epilepsy. Arch Dis Child 2000;82:121-5. [2] Snead OC 3rd. Basic mechanisms of generalized absence seizures. Ann Neurol 1995;37:146-57. [3] Crunelli V, Leresche N. Childhood absence epilepsy: Genes, channels, neurons and networks. Nat Rev Neurosci 2002;3:371-82. [4] Coulter DA, Huguenard JR, Prince DA. Characterization of ethosuximide reduction of low-threshold calcium current in thalamic neurons. Ann Neurol 1989;25:582-93. [5] Crunelli V, Leresche N. A role for GABAB receptors in excitation and inhibition of thalamocortical cells. Trends Neurosci 1991;14:16-21. [6] Gomora JC, Daud AN, Weiergraber M, Perez-Reyes E. Block of cloned human T-type calcium channels by succinimide antiepileptic drugs. Mol Pharmacol 2001;60:1121-32. [7] Kostyuk PG, Molokanova EA, Pronchuk NF, Savchenko AN, Verkhratsky AN. Different action of ethosuximide on low- and highthreshold calcium current in rat sensory neurons. Neuroscience 1992;51:755-8. [8] Avanzini G, Panzica F, de Curtis M. The role of the thalamus in vigilance and epileptogenic mechanisms. Clin Neurophysiol 2000;111: S19-26. [9] Talley EM, Cribbs LL, Lee JH, Daud A, Perez-Reyes E, Bayliss DA. Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 1999;19: 1895-911. [10] Lee JH, Daud AN, Cribbs LL, et al. Cloning and expression of
190
PEDIATRIC NEUROLOGY
Vol. 35 No. 3
a novel member of the low voltage-activated T-type calcium channel family. J Neurosci 1999;19:1912-21. [11] Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia 1989;30:389-99. [12] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988;16:1215. [13] Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 1998;11:1-3. [14] Chen Y, Lu J, Pan H, et al. Association between genetic variation of CACNA1H and childhood absence epilepsy. Ann Neurol 2003;54:239-43. [15] Chen Y, Lu J, Zhang Y, et al. T-type calcium channel gene alpha (1G) is not associated with childhood absence epilepsy in the Chinese Han population. Neurosci Lett 2003;341:29-32. [16] Khosravani H, Altier C, Simms B, et al. Gating effects of mutations in the Cav3.2 T-type calcium channel associated with childhood absence epilepsy. J Biol Chem 2004; 279:9681-4. [17] Vitko I, Chen Y, Arias JM, Shen Y, Wu XR, Perez-Reyes E. Functional characterization and neuronal modeling of the effects of childhood absence epilepsy variants of CACNA1H, a T-type calcium channel. J Neurosci 2005;25:4844-55. [18] Gomora JC, Murbartian J, Arias JM, Lee JH, Perez-Reyes E. Cloning and expression of human T-type channel Cav3.3: Insights into prepulse facilitation. Biophys J 2002;83:229-41.