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

描述(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的，没有明确的家族史，散发性癫痫的原因大多未知。由于癫痫的病因通常指导着治疗的选择，因此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都与体细胞突变有关，其中一部分脑细胞显示出一种并非所有身体细胞都共有的突变，因为这种突变发生在受精后胚胎体细胞的有丝分裂过程中。我们的实验室和其他实验室已经报道了几个已知导致癫痫的基因的体细胞突变，然而，到目前为止，技术限制阻碍了对癫痫体细胞突变的系统研究。全基因组或整个外显子组测序不会识别体细胞突变，除非这项研究旨在检测它们：负责任的突变可能不存在于大多数血细胞中，而将仅限于大脑中的细胞。即使是仅限于神经元的相关突变也可能被批量脑测序遗漏，因为1)神经元被具有不同胚胎起源的神经胶质细胞包围，并且它们不会被期望与其共享突变，2)神经元本身来自两个胚胎起源，锥体神经元来自皮质增殖区，抑制性非锥体神经元从遥远的皮质下区域迁移到皮质，因此对死后脑组织的批量测序也可能不会检测到仅限于特定神经元群体的突变。我们最近开发了一种技术，可以对单个或少量的大脑皮层神经元核进行分选，并扩增其基因组的数量，足以进行任何下一代测序。我们建议使用这项技术来检测死后大脑的皮质神经元或癫痫患者的皮质切除，以便1)对大脑神经元的拷贝数变异(CNV)进行系统评估；2)执行整个外显子组测序，以识别已知癫痫基因的突变。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发生自发突变的频率，并对这些突变进行识别和分类。因此，我们提议的研究使得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.