Parallel assessment of neurodevelopment genes implicated in autism using zebrafish

项目总结/摘要 在患有自闭症谱系障碍（ASD）的个体中，一些最严重的疾病来自合并症。 大脑生长加速，称为不成比例的巨脑畸形（DM）。ASD-DM与 退化性自闭症，智商增长较慢，语言表达困难更大，认知能力更严重， 缺陷最近对ASD先证者的基因组测序研究发现， 在早期胎儿发育中表达的影响细胞周期和增殖的基因的杂合突变。 尽管在少数已知的ASD-DM基因（包括CHD 8）中已发现了复发性变异， 和PTEN，许多基因在ASD-DM患者中受到新生变异的影响， 报道，因此需要筛选数百到数千个具有未知意义的候选基因。 为了最终确认疾病基因，实验验证是必要的。这项研究假设， ASD-DM候选基因直系同源物的敲除将导致特定细胞的丰度改变， 在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 脑类器官模型。由于其体积小，繁殖能力强，胚胎透明， 斑马鱼的发育基因非常适合于发育基因的功能研究。虽然淘汰赛的 斑马鱼中的单个基因已经成功地确定了缺陷，没有系统的研究表征多个基因的缺陷。 基因的平行研究已经用于ASD。一个限制是缺乏更高通量的定量 用于表征神经发育的测定。此外，很少有研究评估导致疾病的误解， 用鱼代替。拟议项目的主要目标是在功能上表征ASD-DM 候选基因，并开发体内策略，以快速测定已鉴定的患者突变， 对神经发育的影响。为实现这一目标，该项目将重点实现以下目标：（1） 功能性测定患者功能丧失和错义变异的未知意义， 单个ASD-DM基因CHD 8的保守的人/鱼直系同源物;和（2）靶向多个ASD-DM 使用高通量基因编辑从疾病测序研究中识别的候选基因 方法来表征它们对斑马鱼大脑发育的影响。随着突变体的识别，未来 工作包括开发小分子筛选，以拯救斑马鱼携带 我们的研究产生的候选基因的突变。这些研究途径区分了我们对 正在进行的小鼠研究中的斑马鱼。如果成功，开发的方法将大大改善我们的 查明疾病基因的能力，对改善诊断措施至关重要，有助于早期干预， 治疗以及有助于更好地了解ASD中巨脑畸形的病因。

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.