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Abstract：
OBJECTIVE: The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses.
METHODS: We performed WES (mean coverage approximately 40×) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing.
RESULTS: In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl(-) /H(+) -exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures.
CONCLUSIONS: The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.
METHODS: We performed WES (mean coverage approximately 40×) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing.
RESULTS: In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl(-) /H(+) -exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures.
CONCLUSIONS: The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.
摘要：
目的：当癫痫发作对抗癫痫药物耐药时，儿童癫痫的治疗尤其具有挑战性。或者随着时间的推移癫痫发作类型发生了许多变化，或者有共同的认知，行为，或运动障碍。尽管努力根据临床和脑电图标准对此类癫痫进行分类，许多儿童从未接受过明确的病因诊断.全外显子组测序（WES）被证明是鉴定引起神经系统疾病的从头变异的一种非常有效的方法。尤其是那些与大脑发育异常有关的.在本文中，我们探讨了WES在无病因诊断的异质性散发性癫痫患儿队列中识别候选因果从头变异的实用性。
方法：我们对10个三人组进行了WES（平均覆盖率约为40倍），这些三人组包括未受影响的父母和一个以难以控制的癫痫发作和一些发育迟缓为特征的散发性癫痫患儿。癫痫性脑病,自闭症特征，认知障碍，或运动障碍。使用标准生物信息学工具进行序列加工和变异识别。使用定制过滤系统来优先考虑可能的功能意义的从头变体,以通过Sanger测序进行验证。
结果：在10个先证者中，有9个，我们确定了一个或多个预测改变蛋白质功能的从头变体，总共15个。四个先证者在先前显示患有严重，早发性癫痫（SCN1A中的两个，CDKL5和EEF1A2各一个)。三个孩子,从头变异体位于具有与癫痫相关的功能作用的基因中(KCNH5,CLCN4和ARHGEF15).KCNH5中的变体改变了编码的电压门控钾通道的电压传感器的高度保守的精氨酸残基之一。使用基于细胞的测定法进行的体外分析显示，CLCN4突变极大地损害了ClC-42Cl（-）/H（）交换剂的离子转运，并且ARHGEF15中的突变降低了基因产物的GEF交换活性，Ephexin5，约50％。感兴趣的，这七个先证者都在生命的头六个月内出现癫痫发作，其中六例有顽固性癫痫发作。
结论：发现10名儿童中有7名携带对神经元兴奋性具有已知或似是而非的临床意义的基因的从头突变，这表明WES将用于儿童散发性癫痫的分子遗传学诊断。特别是当癫痫发作早期发作且难以控制时。
方法：我们对10个三人组进行了WES（平均覆盖率约为40倍），这些三人组包括未受影响的父母和一个以难以控制的癫痫发作和一些发育迟缓为特征的散发性癫痫患儿。癫痫性脑病,自闭症特征，认知障碍，或运动障碍。使用标准生物信息学工具进行序列加工和变异识别。使用定制过滤系统来优先考虑可能的功能意义的从头变体,以通过Sanger测序进行验证。
结果：在10个先证者中，有9个，我们确定了一个或多个预测改变蛋白质功能的从头变体，总共15个。四个先证者在先前显示患有严重，早发性癫痫（SCN1A中的两个，CDKL5和EEF1A2各一个)。三个孩子,从头变异体位于具有与癫痫相关的功能作用的基因中(KCNH5,CLCN4和ARHGEF15).KCNH5中的变体改变了编码的电压门控钾通道的电压传感器的高度保守的精氨酸残基之一。使用基于细胞的测定法进行的体外分析显示，CLCN4突变极大地损害了ClC-42Cl（-）/H（）交换剂的离子转运，并且ARHGEF15中的突变降低了基因产物的GEF交换活性，Ephexin5，约50％。感兴趣的，这七个先证者都在生命的头六个月内出现癫痫发作，其中六例有顽固性癫痫发作。
结论：发现10名儿童中有7名携带对神经元兴奋性具有已知或似是而非的临床意义的基因的从头突变，这表明WES将用于儿童散发性癫痫的分子遗传学诊断。特别是当癫痫发作早期发作且难以控制时。
