Functional evaluation of catastrophic childhood epilepsy genes in zebrafish

斑马鱼灾难性儿童癫痫基因的功能评估

• 批准号: 9905567
• 负责人: Scott C Baraban
• 金额: $ 34.67万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905567
• 关键词: 3-Dimensional; Animal Model; Antiepileptic Agents; Basic Science; Behavior; Behavior monitoring; Behavioral; Benchmarking; Biological Assay; Brain; Brain imaging; CRISPR/Cas technology; Calcium; Cardiovascular Diseases; Cell Culture Techniques; Child; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Consumption; DNA Sequence Alteration; Databases; Diabetes Mellitus; Diagnosis; Disease; Early Infantile Epileptic Encephalopathy; Electrophysiology (science); Epilepsy; Etiology; Evaluation; Exhibits; Functional disorder; Funding; Gene Mutation; Generations; Genes; Genetic; Genetic Diseases; Goals; Home environment; Human; Human Genetics; Image; International; Laboratories; Lead; Life; Locomotion; Malignant Neoplasms; Methodology; Methods; Mission; Modeling; Modification; Mutant Strains Mice; National Institute of Neurological Disorders and Stroke; Neurons; Oregon; Patients; Phenotype; Pre-Clinical Model; Research; Resistance; Resistance profile; Resources; Seizures; Speed; System; Techniques; Time; United States National Institutes of Health; Universities; Vertebrates; Vision; Zebrafish; autism spectrum disorder; childhood epilepsy; design; disability; drug discovery; effective therapy; gene function; genome wide association study; improved; in vivo; induced pluripotent stem cell; insight; loss of function; loss of function mutation; multidisciplinary; mutant; neural network; next generation sequencing; open source; patient population; pre-clinical; precision medicine; preclinical development; relating to nervous system; side effect; stem cell technology; tool

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.

项目总结/摘要 患有灾难性癫痫的儿童会发展成难治性癫痫， 行为障碍包括自闭症这些孩子中的许多人在生命的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 已经在该患者群体中鉴定了突变。斑马鱼LOF突变体设计代表这些 人类遗传条件将为阐明基本疾病机制和药物治疗提供有价值的工具。 的发现方法如CRISPR/Cas9系统（成簇的规则间隔的短回文序列）， 重复/Cas9）现在允许在一系列动物模型中快速有效地修饰内源基因， 包括斑马鱼（Danio rerio）。利用这种技术，我们最近开发和表征了一种stxbp 1 突变斑马鱼线代表儿童早期婴儿癫痫性脑病爆发抑制。 在这个提议中，我们将使用以下方法产生所有已知人类LOF突变的临床前突变斑马鱼品系： CRISPR/Cas9基因编辑技术（Aim I）。为了确定这些突变体是否表现出 表型（包括癫痫），我们将使用独特的检测方法仔细监测行为和神经活动， 在我们的实验室开发的方法（目标II）。我们的策略包括（i）详细分析行为 使用自动运动跟踪和高速成像，（ii）使用体内记录大脑活动 电生理学技术和（iii）神经网络的全脑钙成像。斑马鱼线与 还将针对一小组现有的抗癫痫药物筛选癫痫表型。所有斑马鱼 这里产生并仔细表征的突变株系将通过以下方式提供给科学界： NINDS支持的位于俄勒冈州的斑马鱼国际资源中心。作为一种资源和提供 深入了解这些单基因突变的功能后果，我们的建议建立了一个 以斑马鱼为基础的综合精准医学方法针对单基因癫痫疾病 主要是儿童。

项目成果

Phenotypic analysis of catastrophic childhood epilepsy genes.

• DOI: 10.1038/s42003-021-02221-y
• 发表时间: 2021-06-03
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Griffin A;Carpenter C;Liu J;Paterno R;Grone B;Hamling K;Moog M;Dinday MT;Figueroa F;Anvar M;Ononuju C;Qu T;Baraban SC
• 通讯作者: Baraban SC