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.

项目摘要/摘要 在自闭症谱系障碍（ASD）的个体中，一些最糟糕的预后来自合并症 随着脑生长的加速，被称为不成比例的大脑（DM）。 ASD-DM与 回归自闭症，智商的收益较慢，表现力的语言更大的困难以及更严重的认知能力 缺陷。 ASD概率的最新基因组测序研究已经确定了过量的稀有de从 基因的杂合突变在早期影响细胞周期和增殖的早期发育中表达。 尽管在包括CHD8在内的少数知名的ASD-DM基因中已经鉴定了复发变体 和PTEN，ASD-DM患者受到从头变异的许多基因从未有过 报道，因此需要筛分数百至数千个具有未知意义的候选基因。 为了最终确认疾病基因，需要实验验证。拟议的研究假设 ASD-DM候选基因直系同源物的敲除将导致丰富的特定细胞的改变 发育中的斑马鱼大脑中的类型，让人联想到人类患者以及小鼠和小鼠和 大脑器官模型。由于它们的尺寸小，稳健的繁殖，胚胎透明度和快速 发育，斑马鱼非常适合发育基因的功能研究。虽然淘汰赛 斑马鱼中的单个基因已成功查明缺陷，没有系统的研究表征多个 并行对ASD进行了基因。一个限制是缺乏高通量定量 特征神经发育的测定。此外，很少有研究评估了引起疾病的错义 用鱼取代。拟议项目的主要目标是在功能上表征ASD-DM 候选基因并制定一种体内策略，以快速测定确定患者突变以测量其突变 对神经发育的影响。为了实现这一目标，该项目将集中在以下目标上：（1） 在功能上测定患者的功能丧失和错义变体，其意义不明 单个ASD-DM基因CHD8的保守人/鱼类直系同源物； （2）目标多个ASD-DM 使用高通量基因编辑从疾病测序研究中鉴定出的候选基因 表征它们对斑马鱼中大脑发育的影响的方法。如发现突变体的未来 工作包括开发小分子屏幕以挽救斑马鱼的定量表型 我们研究产生的候选基因的突变。这些研究的途径使我们对 正在进行的小鼠研究中的斑马鱼。如果成功，开发的方法将显着改善我们的 能够确定疾病基因在改善诊断措施中至关重要的疾病基因，从而促进早期干预措施和 疗法以及对ASD中大脑潜在的病因的更好理解。