Parallel assessment of neurodevelopment genes implicated in autism using zebrafish

使用斑马鱼并行评估与自闭症有关的神经发育基因

• 批准号: 10842174
• 负责人: Megan Y Dennis
• 金额: $ 9.45万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10842174
• 关键词: Acceleration; Biological; Biological Assay; Brain; Candidate Disease Gene; Cell Cycle; Cell Proliferation; Cerebrum; Cognitive deficits; Defect; Development; Developmental Gene; Diagnostic; Diagnostic Procedure; Disease; Early Intervention; Early treatment; Embryo; Etiology; Fetal Development; Fishes; Future; Gene Mutation; Genes; Genetic; Goals; Growth; Heterozygote; Human; Instruction; Knock-out; Language; Life; Measures; Megalencephaly; Methods; Modeling; Mus; Mutation; Neurodevelopmental Disorder; Organoids; Orthologous Gene; PTEN gene; Patients; Phenotype; Population; Prognosis; Recurrence; Reporting; Reproduction; Research; Societies; Validation; Variant; Work; Zebrafish; autism spectrum disorder; career; cell type; comorbidity; de novo mutation; genome sequencing; improved; in vivo; individuals with autism spectrum disorder; loss of function; mutant; neurodevelopment; proband; rapid test; small molecule; variant of unknown significance

PROJECT SUMMARY/ABSTRACT Among individuals with autism spectrum disorder (ASD), some of the worst prognoses come from comorbidity with accelerated brain growth, known as disproportionate megalencephaly (DM). ASD-DM is associated with regressive autism, slower gains in IQ, greater difficulties with expressive language, and more severe cognitive defects. Recent genome sequencing studies of probands with ASD have identified an excess of rare de novo heterozygous mutations of genes expressed in early fetal development that impact cell cycle and proliferation. Although recurrent variants have been identified in a handful of well-known ASD-DM genes, including CHD8 and PTEN, many genes impacted by de novo variants in patients with ASD-DM have never before been reported, thus requiring sifting through hundreds to thousands of candidate genes with unknown significance. To ultimately confirm disease genes, experimental validation is necessary. The proposed study hypothesizes that knockout of ASD-DM candidate gene orthologs will result in alterations in the abundance of specific cell types in the developing zebrafish brain, reminiscent of those observed in human patients as well as mouse and cerebral organoid models. Due to their small size, robust reproduction, embryonic transparency, and rapid development, zebrafish are well suited for functional studies of developmental genes. Although knockouts of single genes in zebrafish have successfully pinpointed defects, no systematic study characterizing multiple genes in parallel has been performed for ASD. One limitation is the lack of higher-throughput quantitative assays to characterize neurodevelopment. Further, very few studies have assessed disease-causing missense substitutions using fish. The primary goal of the proposed project is to functionally characterize ASD-DM candidate genes and develop an in vivo strategy to rapidly assay identified patient mutations to measure their impact on neurodevelopment. To achieve this goal, the project will focus on the following aims: (1) functionally assay patient loss-of-function and missense variants of unknown significance in the conserved human/fish ortholog of a single ASD-DM gene, CHD8; and (2) target multiple ASD-DM candidate genes identified from disease sequencing studies using a higher-throughput gene editing method to characterize their impacts on brain development in zebrafish. As mutants are identified, future work includes developing small-molecule screens to rescue quantitative phenotypes of zebrafish carrying mutations of candidate genes generated from our study. These avenues of research differentiate our use of zebrafish from ongoing mouse studies. If successful, the developed approaches will significantly improve our ability to pinpoint disease genes critical in improving diagnostic measures facilitating earlier interventions and treatments as well as contributing to a better understanding of the etiology underlying megalencephaly in ASD.

项目概要/摘要 在自闭症谱系障碍 (ASD) 患者中，一些最糟糕的预后来自于合并症 大脑生长加速，称为不成比例的巨脑畸形（DM）。 ASD-DM 与 退行性自闭症、智商增长缓慢、语言表达困难更大、认知能力更严重 缺陷。最近对患有自闭症谱系障碍（ASD）先证者的基因组测序研究发现了过量的罕见从头基因 胎儿早期发育中表达的基因杂合突变，影响细胞周期和增殖。 尽管在一些众所周知的 ASD-DM 基因中发现了复发性变异，包括 CHD8 和 PTEN，自闭症谱系障碍 (ASD-DM) 患者中受新生变异影响的许多基因以前从未被研究过。 报道，因此需要筛选数百到数千个意义未知的候选基因。 为了最终确认疾病基因，需要进行实验验证。拟议的研究假设 敲除 ASD-DM 候选基因直系同源物将导致特定细胞丰度的改变 发育中的斑马鱼大脑中的类型，让人想起在人类患者以及小鼠和小鼠中观察到的类型 脑类器官模型。由于其体型小、繁殖能力强、胚胎透明、生长速度快 发育过程中，斑马鱼非常适合发育基因的功能研究。虽然淘汰赛 斑马鱼的单个基因已成功查明缺陷，但尚无系统研究表征多个缺陷 已针对自闭症谱系障碍 (ASD) 进行了并行基因研究。一个限制是缺乏更高通量的定量方法 表征神经发育的测定。此外，很少有研究评估致病错义 用鱼代替。拟议项目的主要目标是从功能上表征 ASD-DM 候选基因并开发体内策略来快速分析已识别的患者突变以测量其 对神经发育的影响。为实现这一目标，项目将重点实现以下目标：（1） 对患者的功能丧失和意义不明的错义变异进行功能分析 单个 ASD-DM 基因 CHD8 的保守人类/鱼类直系同源物； (2) 针对多个 ASD-DM 使用高通量基因编辑从疾病测序研究中鉴定出候选基因 表征它们对斑马鱼大脑发育影响的方法。随着突变体的识别，未来 工作包括开发小分子筛选来拯救斑马鱼携带的定量表型 我们的研究产生的候选基因的突变。这些研究途径使我们的使用与众不同 来自正在进行的小鼠研究的斑马鱼。如果成功的话，所开发的方法将显着改善我们的 查明疾病基因的能力对于改进诊断措施至关重要，促进早期干预和 治疗以及有助于更好地了解 ASD 巨脑畸形的病因学。