Somatic mutations in epilepsy: whole genome sequence analysis of single neurons

描述（由申请人提供）： 虽然癫痫有很大的遗传贡献，但大多数癫痫患者的致病基因尚不清楚。虽然癫痫可以在家庭中遗传，但更常见的是“散发性”或自发性，没有明确的家族史，散发性癫痫病例的原因大多未知。由于癫痫的病因通常指导治疗方法的选择，因此对患者进行分类在治疗上非常重要。 许多癫痫综合征似乎涉及“体细胞”突变，其中脑细胞亚群显示出不为身体所有细胞所共有的突变，因为突变发生在受精后胚胎体细胞的有丝分裂期间。我们的实验室和其他实验室已经报道了几种已知引起癫痫的基因的体细胞突变，然而到目前为止，技术限制阻止了对癫痫体细胞突变的系统研究。全基因组或全外显子组测序将无法识别体细胞突变，除非研究旨在检测它们：负责的突变可能不存在于大多数血细胞中，而是仅限于大脑中的细胞。甚至限于神经元的相关突变也可能被大量脑测序遗漏，因为1]神经元被具有不同胚胎学起源的神经胶质细胞包围，并且它们不会被预期与神经胶质细胞共享突变，并且2]神经元本身衍生自两个胚胎学起源，其中锥体神经元衍生自皮质增殖区域，而抑制性非锥体神经元从遥远的皮质下区域迁移到皮质中，因此死后脑组织的批量测序也可能检测不到限于特定神经元群体的突变。 我们最近开发了一种技术，可以分选单个或少量的大脑皮层神经元细胞核，并扩增其基因组，其数量足以进行任何下一代测序。我们建议使用这种技术来检查癫痫患者死后大脑或皮层切除的皮层神经元，以便1）对大脑神经元中的拷贝数变异（CNV）进行系统评估; 2）进行全外显子组测序以识别已知癫痫基因中的突变。然后，将单个神经元序列与身体其他细胞的序列进行比较，可以确定皮质神经元中自发突变发生的频率，并识别和分类这些突变。因此，我们提出的研究能够系统地分析癫痫人脑中体细胞突变的所有机制。

DESCRIPTION (provided by applicant): Although epilepsy has a large genetic contribution, causative genes are not known for most epilepsy patients. And while epilepsy can be inherited in families, it is more commonly 'sporadic' or spontaneous, without a clear family history, and causes of sporadic epilepsy cases are mostly unknown. Since the cause of epilepsy often directs the choice of treatments, categorizing patients is of great importance therapeutically. Many epilepsy syndromes appear to involve 'somatic' mutations, in which a subpopulation of brain cells shows a mutation not shared by all cells of the body, because the mutation occurred during mitosis of somatic cells of the embryo after fertilization. Our lab and other have reported on somatic mutations is several genes known to cause epilepsy, however until now, technological limitations have prevented a systematic search for somatic mutations in epilepsy. Whole genome or whole exome sequencing will not identify somatic mutations unless the study is designed to detect them: responsible mutations may not be present in most blood cells, but would instead be limited to cells in the brain. Even relevant mutations limited to neurons may be missed by bulk brain sequencing because 1] neurons are surrounded by glial cells with distinct embryological origins, and with whom they would not be expected to share mutations, and 2] neurons themselves are derived from two embryological origins, with pyramidal neurons derived from cortical proliferative regions, and inhibitory nonpyramidal neurons migrating into cortex from distant subcortical regions, so that bulk sequencing of postmortem brain tissue may also not detect mutations limited to specific neuronal populations. We have recently developed technology that allows sorting of single or small numbers of cerebral cortical neuronal nuclei, and amplification of their genomes in quantities sufficient for any next-generation sequencing. We are proposing to use this technology to examine cortical neurons from postmortem brains or cortical resections from epilepsy patients in order to 1) perform a systematic assessment of copy number variation (CNV) in cerebral neurons; and 2) perform whole exome sequencing to identify mutations in known epilepsy genes. Comparison of single neuron sequence to that from other cells of the body could then determine how frequently spontaneous mutations occur in cortical neurons, and identify and catalogue these mutations. Our proposed study thus enables a systematic analysis of all mechanisms of somatic mutation in the epileptic human brain.