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。虽然癫痫可以在家族中遗传，但更常见的是“散发性”或自发性，没有明确的家族史，散发性癫痫病例的病因大多是未知的。由于癫痫的病因常常指导治疗的选择，对患者进行分类在治疗上是非常重要的。许多癫痫综合征似乎与“体细胞”突变有关，在这种突变中，脑细胞的一个亚群显示出一种不为身体所有细胞共有的突变，因为这种突变发生在受精后胚胎体细胞有丝分裂期间。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，但是直到现在，技术限制已经阻止了对癫痫的体细胞突变的系统搜索。全基因组或全外显子组测序将无法识别体细胞突变，除非该研究是为了检测它们而设计的：负责任的突变可能不存在于大多数血细胞中，而是仅限于大脑细胞。即使是局限于神经元的相关突变也可能在大量脑测序中被遗漏，因为1]神经元被具有不同胚胎起源的胶质细胞包围，它们不会与这些细胞共享突变，2]神经元本身来源于两个胚胎起源，锥体神经元来源于皮层增殖区域，抑制性非锥体神经元从遥远的皮层下区域迁移到皮层。因此，对死后脑组织的大量测序也可能无法检测到局限于特定神经元群的突变。我们最近开发了一种技术，可以对单个或少量的大脑皮层神经元核进行分类，并对其基因组进行扩增，其数量足以进行任何下一代测序。我们建议使用这项技术来检查死后大脑或癫痫患者皮质切除的皮层神经元，以便1)对大脑神经元的拷贝数变异（CNV）进行系统评估；2)进行全外显子组测序，以确定已知癫痫基因的突变。将单个神经元序列与来自身体其他细胞的序列进行比较，可以确定皮层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类大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.