部分性癫痫伴热性惊厥附加症患者SCN1A基因突变筛查及内含子突变对剪切影响的体外分析
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摘要
目的
在部分性癫痫伴热性惊厥附加症(PEFS+)患者中筛查SCN1A基因突变,研究SCN1A突变与PEFS+的关系;研究不同内含子突变对SCN1A基因剪切的影响;总结PEFS+的SCN1A突变特点,从突变的角度探讨PEFS+作为Dravet综合征(DS)和全面性癫痫伴热性惊厥附加症(GEFS+)过渡表型的可能。
研究对象和方法
收集符合以下标准的病例的临床资料及基因组DNA:(1)起病前精神运动发育正常,起病后无明显精神发育迟滞或仅有轻度异常;(2)于3个月至6岁间开始出现热性惊厥,随后可出现非热性癫痫发作或6岁以后仍有热性痫性发作;(3)除GTCS外,非热性发作仅表现为部分性发作或部分继发全面性发作,无其它痫性发作形式;(4)排除肿瘤、外伤、感染、局灶性皮质发育异常等症状性癫痫;(5)排除Dravet综合征等特发性癫痫。采用高效液相色谱仪(DHPLC)与直接测序结合的方法,对患者SCN1A基因外显子和剪切相关内含子区进行突变筛查。对新发现的突变,通过检索多态性位点数据库及设置正常对照组(100例)进行验证;通过构建迷你基因模型转染真核细胞的方法研究内含子突变c.473+5 G>A、c.473+110 A>G对剪切的影响;通过检索SCN1A突变数据库及复习文献,结合本研究结果总结PEFS+的SCN1A突变特点,对比GEFS+和DS,分析PEFS+作为GEFS+和DS过渡表型的可能性。
结果
共收集PEFS+病例37例,发现4个SCN1A基因突变:c.473+5 G>A、c.473+110 A>G、c.1028+35 T>C、c.2378 C>T,其中c.473+5 G>A可引起前体mRNA异常剪切导致蛋白质截短;c.473+110 A>G对剪切无影响,可能为非致病性突变。截止目前,共报道PEFS+表型SCN1A突变20个,包括:错义突变17个、剪切位点突变2个、移码突变1个。PEFS+主要由错义突变引起,52%错义突变位于电压感受区和中央孔区(S4～S6),比例明显高于GEFS+(27.3%)接近于DS(60.2%))。剪切位点突变和移码突变虽然可以导致PEFS+,但不是其主要突变形式。
结论
(1)SCN1A很可能是PEFS+致病基因。(2)PEFS+主要由SCN1A错义突变引起,突变位点主要位于重要功能区域(S4-S6);剪切位点突变和移码突变亦可导致PEFS+,但不是其主要突变形式。(3)PEFS+在SCN1A突变形式和突变位点方面的特点提示它可能是DS和GEFS+间的一种过渡表型。(4)SCN1A剪切位点突变可能通过引起蛋白质截短使钠通道功能丧失,最终导致PEFS+患者发病。
Purpose
To screen SCN1A mutations in patients with partial epilepsy with febrile seizures plus(PEFS+)and investigate the mechanism of intronic mutations effecting on precursor mRNA(pre-mRNA) splicing. To characterize SCN1A mutations of PEFS+ and discuss the possibility that PEFS+ is a distinct phenotype of SCN1A mutation and different from both Dravet syndrome (DS) and generalized epilepsy with febrile seizures plus (GEFS+).
Patients and methods
37 patients with PEFS+ were collected. All the 26 exons and splicing regions of SCN1A were screened with denaturing high performance liquid chromatography (DHPLC) and direct sequencing. A splicing assay by mini-gene construction was conducted to determine the effect of the c.473+5 G>A and c.473+110A>G mutation on pre-mRNA splicing. All the update SCN1A mutations which were identified in PEFS+ patients had been reviewed to detect a common feature and to find the relationship between PEFS+、GEFS+ and DS.
Results
4 heterozygous mutations were detected: c.473+5 G>A、c.473+110 A>G、c.1028+35 T>C、c.2378 C>T. The splice site mutation c.473+5 G>A could disrupt normal splcing and finally result in a truncation of SCN1A. The intron mutation c.473+110 A>G did not effect on splicing. 20 SCN1A mutations in PEFS+ were reviewed and PEFS+ was mainly induced by missense mutations. 52% of these mutations located in the S4-S6 segments. The proportion of mutations in the S4-S6 segments in PEFS+ is much higher than GEFS+(27.3%) and close to DS(60.2%). Splicing-site mutation and frame-shift mutation could also lead to PEFS+,but they were not the main mutation types for PEFS+.
Conclusion
SCN1A is a candidate gene for PEFS+. Missense mutation of SCN1A is the most common mutation type for PEFS+. Most of missense mutations in PEFS+ were located in the S4-S6 segments. Splicing-site mutations and frame-shift mutations were also related to PEFS+. The pathogenesis of intron mutations for PESF+ was to result in SCN1A truncation by altering splicing sites of SCN1A pre-mRNA. According to the mutation characteristics, PEFS+ seems to be a transition phenotype between DS and GEFS+.
在部分性癫痫伴热性惊厥附加症(PEFS+)患者中筛查SCN1A基因突变,研究SCN1A突变与PEFS+的关系;研究不同内含子突变对SCN1A基因剪切的影响;总结PEFS+的SCN1A突变特点,从突变的角度探讨PEFS+作为Dravet综合征(DS)和全面性癫痫伴热性惊厥附加症(GEFS+)过渡表型的可能。
研究对象和方法
收集符合以下标准的病例的临床资料及基因组DNA:(1)起病前精神运动发育正常,起病后无明显精神发育迟滞或仅有轻度异常;(2)于3个月至6岁间开始出现热性惊厥,随后可出现非热性癫痫发作或6岁以后仍有热性痫性发作;(3)除GTCS外,非热性发作仅表现为部分性发作或部分继发全面性发作,无其它痫性发作形式;(4)排除肿瘤、外伤、感染、局灶性皮质发育异常等症状性癫痫;(5)排除Dravet综合征等特发性癫痫。采用高效液相色谱仪(DHPLC)与直接测序结合的方法,对患者SCN1A基因外显子和剪切相关内含子区进行突变筛查。对新发现的突变,通过检索多态性位点数据库及设置正常对照组(100例)进行验证;通过构建迷你基因模型转染真核细胞的方法研究内含子突变c.473+5 G>A、c.473+110 A>G对剪切的影响;通过检索SCN1A突变数据库及复习文献,结合本研究结果总结PEFS+的SCN1A突变特点,对比GEFS+和DS,分析PEFS+作为GEFS+和DS过渡表型的可能性。
结果
共收集PEFS+病例37例,发现4个SCN1A基因突变:c.473+5 G>A、c.473+110 A>G、c.1028+35 T>C、c.2378 C>T,其中c.473+5 G>A可引起前体mRNA异常剪切导致蛋白质截短;c.473+110 A>G对剪切无影响,可能为非致病性突变。截止目前,共报道PEFS+表型SCN1A突变20个,包括:错义突变17个、剪切位点突变2个、移码突变1个。PEFS+主要由错义突变引起,52%错义突变位于电压感受区和中央孔区(S4～S6),比例明显高于GEFS+(27.3%)接近于DS(60.2%))。剪切位点突变和移码突变虽然可以导致PEFS+,但不是其主要突变形式。
结论
(1)SCN1A很可能是PEFS+致病基因。(2)PEFS+主要由SCN1A错义突变引起,突变位点主要位于重要功能区域(S4-S6);剪切位点突变和移码突变亦可导致PEFS+,但不是其主要突变形式。(3)PEFS+在SCN1A突变形式和突变位点方面的特点提示它可能是DS和GEFS+间的一种过渡表型。(4)SCN1A剪切位点突变可能通过引起蛋白质截短使钠通道功能丧失,最终导致PEFS+患者发病。
Purpose
To screen SCN1A mutations in patients with partial epilepsy with febrile seizures plus(PEFS+)and investigate the mechanism of intronic mutations effecting on precursor mRNA(pre-mRNA) splicing. To characterize SCN1A mutations of PEFS+ and discuss the possibility that PEFS+ is a distinct phenotype of SCN1A mutation and different from both Dravet syndrome (DS) and generalized epilepsy with febrile seizures plus (GEFS+).
Patients and methods
37 patients with PEFS+ were collected. All the 26 exons and splicing regions of SCN1A were screened with denaturing high performance liquid chromatography (DHPLC) and direct sequencing. A splicing assay by mini-gene construction was conducted to determine the effect of the c.473+5 G>A and c.473+110A>G mutation on pre-mRNA splicing. All the update SCN1A mutations which were identified in PEFS+ patients had been reviewed to detect a common feature and to find the relationship between PEFS+、GEFS+ and DS.
Results
4 heterozygous mutations were detected: c.473+5 G>A、c.473+110 A>G、c.1028+35 T>C、c.2378 C>T. The splice site mutation c.473+5 G>A could disrupt normal splcing and finally result in a truncation of SCN1A. The intron mutation c.473+110 A>G did not effect on splicing. 20 SCN1A mutations in PEFS+ were reviewed and PEFS+ was mainly induced by missense mutations. 52% of these mutations located in the S4-S6 segments. The proportion of mutations in the S4-S6 segments in PEFS+ is much higher than GEFS+(27.3%) and close to DS(60.2%). Splicing-site mutation and frame-shift mutation could also lead to PEFS+,but they were not the main mutation types for PEFS+.
Conclusion
SCN1A is a candidate gene for PEFS+. Missense mutation of SCN1A is the most common mutation type for PEFS+. Most of missense mutations in PEFS+ were located in the S4-S6 segments. Splicing-site mutations and frame-shift mutations were also related to PEFS+. The pathogenesis of intron mutations for PESF+ was to result in SCN1A truncation by altering splicing sites of SCN1A pre-mRNA. According to the mutation characteristics, PEFS+ seems to be a transition phenotype between DS and GEFS+.
引文
[1] Meisler MH, O'Brien JE, Sharkey LM. (2010) Sodium channel gene family: epilepsy mutations, gene interactions and modifier effects. J Physiol 588: 1841-1848.
[2] Catterall WA. (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26:13-25.
[3] Andavan GS, Lemmens-Gruber R. (2011) Voltage-gated sodium channels: mutations, channelopathies and targets. Curr Med Chem 18:377-397.
[4] Dravet C, Bureau M, Oguni H, Fukuyama Y, Cokar O. (2005) Severe myoclonic epilepsy in infancy: Dravet syndrome. Adv Neurol 95:71-102.
[5] De Jonghe P. (2011) Molecular genetics of Dravet syndrome. Dev Med Child Neurol 53 Suppl 2:7-10.
[6] Gambardella A, Marini C. (2009) Clinical spectrum of SCN1A mutations. Epilepsia 50 Suppl 5:20-23.
[7] Lin H, Wang YP, Wang MY, Wu LW. (2008) [Linkage location and mutation analysis of generalized epilepsy with febrile seizures plus]. Zhonghua Yi Xue Za Zhi 88:3177-3181.
[8] Baulac S, Gourfinkel-An I, Nabbout R, Huberfeld G, Serratosa J, Leguern E, Baulac M. (2004) Fever, genes, and epilepsy. Lancet Neurol 3:421-430.
[9] Zupanc ML. (2009) Clinical evaluation and diagnosis of severe epilepsy syndromes of early childhood. J Child Neurol 24:6S-14S.
[10] Yu FH, Mantegazza M, Westenbroek RE, Robbins CA, Kalume F, Burton KA, Spain WJ, McKnight GS, Scheuer T, Catterall WA. (2006) Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci 9:1142-1149.
[11] Claes L, Del-Favero J, Ceulemans B, Lagae L, Van Broeckhoven C, De Jonghe P. (2001) De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am J Hum Genet 68:1327-1332.
[12] Escayg A, MacDonald BT, Meisler MH, Baulac S, Huberfeld G, An-Gourfinkel I, Brice A, LeGuern E, Moulard B, Chaigne D, Buresi C, Malafosse A. (2000) Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2. Nat Genet 24:343-345.
[13] Ceulemans BP, Claes LR, Lagae LG. (2004) Clinical correlations of mutations in the SCN1A gene: from febrile seizures to severe myoclonic epilepsy in infancy. Pediatr Neurol 30:236-243.
[14] Dravet C, Bureau M, Dalla Bernardina B, Guerrini R. (2011) Severe myoclonic epilepsy in infancy (Dravet syndrome) 30 years later. Epilepsia 52 Suppl 2:1-2.
[15] Liao WP, Shi YW, Long YS, Zeng Y, Li T, Yu MJ, Su T, Deng P, Lei ZG, Xu SJ, Deng WY, Liu XR, Sun WW, Yi YH, Xu ZC, Duan S. (2010) Partial epilepsy with antecedent febrile seizures and seizure aggravation by antiepileptic drugs: associated with loss of function of Na(v) 1.1. Epilepsia 51:1669-1678.
[16] Shapiro MB, Senapathy P. (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155-7174.
[17] Ward AJ, Cooper TA. (2010) The pathobiology of splicing. J Pathol 220:152-163.
[18] Baralle D, Baralle M. (2005) Splicing in action: assessing disease causing sequence changes. J Med Genet 42:737-748.
[19] Desmet FO, Hamroun D, Lalande M, Collod-Beroud G, Claustres M, Beroud C. (2009) Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37:e67.
[20] Krawczak M, Thomas NS, Hundrieser B, Mort M, Wittig M, Hampe J, Cooper DN. (2007) Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat 28:150-158.
[21] Das S, Levinson B, Vulpe C, Whitney S, Gitschier J, Packman S. (1995)Similar splicing mutations of the Menkes/mottled copper-transporting ATPase gene in occipital horn syndrome and the blotchy mouse. Am J Hum Genet 56:570-576.
[22] Kaler SG, Gallo LK, Proud VK, Percy AK, Mark Y, Segal NA, Goldstein DS, Holmes CS, Gahl WA. (1994) Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at the MNK locus. Nat Genet 8:195-202.
[23] Depienne 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 associated with Dravet syndrome: analysis of 333 patients. J Med Genet 46:183-191.
[24] Kumakura A, Ito M, Hata D, Oh N, Kurahashi H, Wang JW, Hirose S. (2009) Novel de novo splice-site mutation of SCN1A in a patient with partial epilepsy with febrile seizures plus. Brain Dev 31:179-182.
[25] Korff C, Laux L, Kelley K, Goldstein J, Koh S, Nordli D, Jr. (2007) Dravet syndrome (severe myoclonic epilepsy in infancy): a retrospective study of 16 patients. J Child Neurol 22:185-194.
[26] Martin, M. S.Tang, B.Papale, L. A.Yu, F. H.Catterall, W. A.Escayg.(2007) The voltage-gated sodium channel Scn8a is a genetic modifier of severe myoclonic epilepsy of infancy. Hum Mol Genet 23: 2892-2899
[2] Catterall WA. (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26:13-25.
[3] Andavan GS, Lemmens-Gruber R. (2011) Voltage-gated sodium channels: mutations, channelopathies and targets. Curr Med Chem 18:377-397.
[4] Dravet C, Bureau M, Oguni H, Fukuyama Y, Cokar O. (2005) Severe myoclonic epilepsy in infancy: Dravet syndrome. Adv Neurol 95:71-102.
[5] De Jonghe P. (2011) Molecular genetics of Dravet syndrome. Dev Med Child Neurol 53 Suppl 2:7-10.
[6] Gambardella A, Marini C. (2009) Clinical spectrum of SCN1A mutations. Epilepsia 50 Suppl 5:20-23.
[7] Lin H, Wang YP, Wang MY, Wu LW. (2008) [Linkage location and mutation analysis of generalized epilepsy with febrile seizures plus]. Zhonghua Yi Xue Za Zhi 88:3177-3181.
[8] Baulac S, Gourfinkel-An I, Nabbout R, Huberfeld G, Serratosa J, Leguern E, Baulac M. (2004) Fever, genes, and epilepsy. Lancet Neurol 3:421-430.
[9] Zupanc ML. (2009) Clinical evaluation and diagnosis of severe epilepsy syndromes of early childhood. J Child Neurol 24:6S-14S.
[10] Yu FH, Mantegazza M, Westenbroek RE, Robbins CA, Kalume F, Burton KA, Spain WJ, McKnight GS, Scheuer T, Catterall WA. (2006) Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci 9:1142-1149.
[11] Claes L, Del-Favero J, Ceulemans B, Lagae L, Van Broeckhoven C, De Jonghe P. (2001) De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am J Hum Genet 68:1327-1332.
[12] Escayg A, MacDonald BT, Meisler MH, Baulac S, Huberfeld G, An-Gourfinkel I, Brice A, LeGuern E, Moulard B, Chaigne D, Buresi C, Malafosse A. (2000) Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2. Nat Genet 24:343-345.
[13] Ceulemans BP, Claes LR, Lagae LG. (2004) Clinical correlations of mutations in the SCN1A gene: from febrile seizures to severe myoclonic epilepsy in infancy. Pediatr Neurol 30:236-243.
[14] Dravet C, Bureau M, Dalla Bernardina B, Guerrini R. (2011) Severe myoclonic epilepsy in infancy (Dravet syndrome) 30 years later. Epilepsia 52 Suppl 2:1-2.
[15] Liao WP, Shi YW, Long YS, Zeng Y, Li T, Yu MJ, Su T, Deng P, Lei ZG, Xu SJ, Deng WY, Liu XR, Sun WW, Yi YH, Xu ZC, Duan S. (2010) Partial epilepsy with antecedent febrile seizures and seizure aggravation by antiepileptic drugs: associated with loss of function of Na(v) 1.1. Epilepsia 51:1669-1678.
[16] Shapiro MB, Senapathy P. (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155-7174.
[17] Ward AJ, Cooper TA. (2010) The pathobiology of splicing. J Pathol 220:152-163.
[18] Baralle D, Baralle M. (2005) Splicing in action: assessing disease causing sequence changes. J Med Genet 42:737-748.
[19] Desmet FO, Hamroun D, Lalande M, Collod-Beroud G, Claustres M, Beroud C. (2009) Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37:e67.
[20] Krawczak M, Thomas NS, Hundrieser B, Mort M, Wittig M, Hampe J, Cooper DN. (2007) Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat 28:150-158.
[21] Das S, Levinson B, Vulpe C, Whitney S, Gitschier J, Packman S. (1995)Similar splicing mutations of the Menkes/mottled copper-transporting ATPase gene in occipital horn syndrome and the blotchy mouse. Am J Hum Genet 56:570-576.
[22] Kaler SG, Gallo LK, Proud VK, Percy AK, Mark Y, Segal NA, Goldstein DS, Holmes CS, Gahl WA. (1994) Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at the MNK locus. Nat Genet 8:195-202.
[23] Depienne 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 associated with Dravet syndrome: analysis of 333 patients. J Med Genet 46:183-191.
[24] Kumakura A, Ito M, Hata D, Oh N, Kurahashi H, Wang JW, Hirose S. (2009) Novel de novo splice-site mutation of SCN1A in a patient with partial epilepsy with febrile seizures plus. Brain Dev 31:179-182.
[25] Korff C, Laux L, Kelley K, Goldstein J, Koh S, Nordli D, Jr. (2007) Dravet syndrome (severe myoclonic epilepsy in infancy): a retrospective study of 16 patients. J Child Neurol 22:185-194.
[26] Martin, M. S.Tang, B.Papale, L. A.Yu, F. H.Catterall, W. A.Escayg.(2007) The voltage-gated sodium channel Scn8a is a genetic modifier of severe myoclonic epilepsy of infancy. Hum Mol Genet 23: 2892-2899