High-throughput modeling of autism risk genes using zebrafish

自闭症谱系障碍(ASD)是由环境因素和遗传因素共同引起的，具有遗传性 贡献率估计为60%-80%。数十个增加ASD风险的基因已经被发现，其中大多数是基于 新生突变，但这些突变预计只占ASD病例的15%-20%。因此， 据预测，ASD的大部分遗传因素是由常见和罕见的遗传变异造成的，但 几乎没有这样的基因被发现。最近，利用全基因组测序，我们报道了全基因组 60个ASD风险基因的证据，其中26个是ASD的新基因，信号来自遗传和新生 蛋白质截断或错义突变。大多数这些基因的功能是未知的，所以一个关键的和 必要的下一步是使用模型来探索它们对神经发育和神经元功能的影响 有机体。目前将遗传风险因素转化为表型、机制和治疗的速度是 部分受限于体内哺乳动物模型系统的低效，这使得它们不适用于创建 并对大量突变品系进行行为测试。在这里，我们利用斑马鱼，它占据了一个独特的 生态位作为脊椎动物模型，具有适合活体筛选和机械理解的特征， 包括体外发育、透明、体积小、发育快、保守而又相对简单 脊椎动物的大脑，与自闭症相关的行为，以及相对于哺乳动物模型的成本效益。而当 斑马鱼不能概括ASD，而且在模拟人类疾病方面存在局限性，这是一种新兴的文献 支持这一观点，即这是一个有用的模型来研究导致自闭症风险的基因的功能。宁可 而不是一次一个地评估ASD风险基因，我们将通过以下方式加速机械性理解的进展 高通量的检测和分析。在特定的目标1中，我们将在斑马鱼同源基因中产生零突变 24个高度可信的、新颖的、全基因组显著的ASD风险基因，并系统地测试每个突变体 神经发育、行为、神经网络和转录的表型。在具体目标2中，我们将 使用转录分析，在整个大脑和单细胞水平上，将ASD风险基因整合到功能性 网络，并测试跨基因和物种的融合，包括自闭症死后大脑。我们还将 测试通常在ASD中并存的行为表型之间的功能关联，如中断 睡眠和社交行为缺陷。在具体目标3中，我们将进行机械研究，以了解如何 特定的ASD风险基因突变会导致表型。该项目将高效且具有成本效益地创建 以及描述脊椎动物模型中大量新的ASD风险基因的特征。这些动物模型 将是社区的宝贵资源，特别是用于大规模体内药物筛查以识别新的 自闭症的治疗方法。

Autism spectrum disorder (ASD) is caused by both environmental and genetic factors, with the genetic contribution estimated at 60-80%. Dozens of genes that increase risk for ASD have been identified, most based on de novo mutations, but these mutations are predicted to account for only 15-20% of ASD cases. Thus, the majority of the genetic contribution to ASD is predicted to result from common and rare inherited variation, but few such genes have been identified. Recently, using whole genome sequencing, we reported genome wide evidence for >60 ASD risk genes, 26 of them novel for ASD, with signals derived from inherited and de novo protein truncating or missense mutations. The functions of most of these genes are unknown, so a crucial and necessary next step is to explore their impact on neurodevelopment and neuronal function using a model organism. The current pace of translating genetic risk factors into phenotypes, mechanisms and therapies is limited in part by inefficiencies with in vivo mammalian model systems, which makes them impractical for creating and behaviorally testing large numbers of mutant lines. Here, we leverage the zebrafish, which occupies a unique niche as a vertebrate model with features amenable to both in vivo screening and mechanistic understanding, including ex utero development, transparency, small size, rapid development, a conserved yet relatively simple vertebrate brain, behaviors relevant to ASD, and cost-effectiveness relative to mammalian models. While the zebrafish cannot recapitulate ASD and has limitations for modeling a human disorder, an emerging literature supports the notion that it is a useful model to study the functions of genes that contribute to ASD risk. Rather than assess ASD-risk genes one at a time, we will accelerate progress towards mechanistic understanding via high-throughput assays and analyses. In Specific Aim 1 we will generate null mutations in the zebrafish orthologs of 24 high confidence, novel, genome-wide significant ASD risk genes, and systematically test each mutant for neurodevelopmental, behavioral, neuronal network, and transcriptomic phenotypes. In Specific Aim 2, we will use transcriptomic analyses, at the whole brain and single cell levels, to integrate ASD risk genes into functional networks, and test for convergence across genes and species, including ASD post mortem brain. We will also test for functional associations among behavioral phenotypes that are often co-morbid in ASD, such as disrupted sleep and social behavioral deficits. In Specific Aim 3 we will perform mechanistic studies to understand how mutation of specific ASD-risk genes leads to phenotypes. This project will efficiently and cost-effectively create and characterize vertebrate animal models for a large number of novel ASD risk genes. These animal models will be a valuable resource for the community, particularly for large-scale in vivo drug screens to identify new therapies for ASD.