Functional evaluation of catastrophic childhood epilepsy genes in zebrafish

项目摘要/摘要 患有灾难性癫痫的儿童发展为顽固性癫痫，具有严重的智力和 行为障碍，包括自闭症。这些儿童中的许多人在生命早期就被诊断出患有遗传形式。 癫痫的症状。随着基因测序的最新进展，大约60个功能丧失(LOF)单基因 已经在这一患者群体中发现了突变。斑马鱼LOF突变体旨在代表这些 人类的遗传条件将为阐明基本的疾病机制和药物提供宝贵的工具 发现号。方法，如CRISPR/CAS9系统(成簇规则间隔短回文 Repeat/Cas9)现在允许在一系列动物模型中快速而有效地修改内源性基因， 包括斑马鱼(Danio Rerio)。使用这项技术，我们最近开发并表征了STXBP1 突变型斑马鱼品系，代表具有爆发抑制的早期婴儿癫痫脑病儿童。 在这项建议中，我们将使用以下方法为所有已知的人类LOF突变生成临床前突变斑马鱼品系 CRISPR/Cas9基因编辑技术(目标I)。以确定这些突变体是否表现出功能性 表型(包括癫痫)，我们将仔细监测行为和神经活动使用独特的分析和 我们实验室开发的方法学(AIM II)。我们的策略包括：(I)对行为进行详细分析 使用自动运动跟踪和高速成像，(Ii)使用活体记录大脑活动 电生理学技术和(Iii)神经网络的全脑钙成像。斑马鱼纹路 癫痫的表型也将与一小部分现有的抗癫痫药物进行筛选。所有斑马鱼 在这里产生并仔细描述的突变系将通过以下方式提供给科学界 NINDS支持的俄勒冈州斑马鱼国际资源中心。既作为资源又作为产品 洞察这些单基因突变的功能后果，我们的建议建立了一个 以斑马鱼为基础的针对单基因癫痫疾病的综合精确医学方法 主要是在儿童中。

Project Summary/Abstract Children with catastrophic epilepsies develop intractable forms of epilepsy with severe intellectual and behavioral disabilities, including autism. Many of these children are diagnosed, early in life, with genetic forms of epilepsy. With recent advances in gene sequencing approximately 60 loss-of-function (LOF) single-gene mutations have been identified in this patient population. Zebrafish LOF mutants designed to represent these human genetic conditions would provide valuable tools for elucidating basic disease mechanisms and drug discovery. Methods such as the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat/Cas9) now allow for rapid and efficient modification of endogenous genes in a range of animal models, including zebrafish (Danio rerio). Using this technique, we recently developed and characterized a stxbp1 mutant zebrafish line representing children with early infantile epileptic encephalopathy with burst suppression. In this proposal, we will generate pre-clinical mutant zebrafish lines for all known human LOF mutations using CRISPR/Cas9 gene editing techniques (Aim I). To determine whether these mutants exhibit functional phenotypes (including epilepsy), we will carefully monitor behavior and neural activity using unique assays and methodologies developed in our laboratory (Aim II). Our strategy includes (i) detailed analysis of behavior using automated locomotion tracking and high-speed imaging, (ii) recording brain activity using in vivo electrophysiology techniques and (iii) brain-wide calcium imaging of neural networks. Zebrafish lines with epileptic phenotypes will also be screened against a small panel of existing antiepileptic drugs. All zebrafish mutant lines generated and carefully characterized here will be made available to the scientific community via the NINDS-supported Zebrafish International Resource Center in Oregon. Both as a resource and offering insight into functional consequences of these single gene mutations, our proposal establishes a comprehensive zebrafish-based precision medicine approach targeted to monogenic epilepsy disorders seen primarily in children.