Functional convergence following disruption of diverse genes associated with neurodevelopmental disorders

项目总结 遗传变异的复杂相互作用是自闭症谱系障碍(ASD)易感性的基础。的确有 现在，来自大型财团的强有力的证据表明，参与染色质修饰的基因突变， 转录调控和突触蛋白增加了ASD的实质性风险；然而， 这些基因是相互关联的，并最终汇聚在少数功能缺陷上 未知。因此，迫切需要对新的基因发现进行建模，以直接评估它们的功能 影响，并确定它们的汇合点。我们团队和其他人在高吞吐量方面的创新 CRISPR工程现在已经使并行的机械研究变得容易处理，并使人类诱导的多能性 干细胞(HiPSCs)来源的神经元非常适合测试ASD风险变量的影响，预计ASD风险变量将发挥其 对胎儿皮质发育的影响。在这里，我们的多元PI提案将进行一项雄心勃勃的、系统的 在48个最健壮的ASD风险基因概要中进行等基因功能丧失(LoF)机制筛选 是从迄今为止最大的基因研究中发现的。此外，我们令人兴奋的初步结果表明 ASD基因的神经元模型共享的转录签名在关键调控节点聚合 这会导致突触缺陷。目的1将鉴定同基因的谷氨酸和氨基丁酸能神经元 与ASD风险相关的48个基因中穿透性LOF突变达到全基因组显著阈值 在神经元中表达。这些分析将识别单个ASD的转录和功能签名 基因通过RNAseq和一系列高通量的表型分析在两个神经元亚型。目标2 将扩展我们的初步结果，以发现ASD风险基因下游的趋同基因，表征 来自单个基因和/或趋同的十个最引人注目的发现的突触后果 签名，并集成这些数据来探索融合网络的可吸入性。我们最重要的是 目标是定义ASD病因基础上的不同基因、途径和网络之间的任何共同点， 并大幅扩大ASD的可能治疗靶点列表。这些研究将产生一个 史无前例的CRISPR编辑的ASD基因的同源资源，并匹配RNAseq和细胞表型 在谷氨酸和GABA能神经元中，这将被提供给更广泛的社区开放分布 通过NIMH RUDCR资源对神经精神障碍产生新的见解。

PROJECT SUMMARY A complex interplay of genetic variation underlies predisposition for autism spectrum disorder (ASD). There is now strong evidence from large consortia studies that mutations in genes involved in chromatin modification, transcriptional regulation, and synaptic proteins confer substantial risk for ASD; however, the extent to which these genes are interconnected and ultimately converge on a small number of functional deficits is largely unknown. A critical need therefore exists to model new gene discoveries, to directly evaluate their functional impact, and to determine their points of convergence. Innovations from our team and others in high-throughput CRISPR-engineering have now made parallelized mechanistic studies tractable, and human induced pluripotent stem cell (hiPSCs) derived neurons are well-suited to test the impact of ASD risk variants predicted to exert their influence during fetal cortical development. Here, our multi-PI proposal will undertake an ambitious, systematic isogenic loss-of-function (LoF) mechanistic screen in a compendium of 48 of the most robust ASD risk genes discovered from the largest genetic studies to date. Moreover, our exciting preliminary results suggest that transcriptional signatures shared across neuronal models of ASD genes converge on critical regulatory nodes that result in synaptic deficits. Aim 1 will characterize isogenic glutamatergic and GABAergic neurons with highly penetrant LoF mutations in 48 genes associated with ASD risk at genome-wide significant thresholds and that are expressed in neurons. These analyses will identify transcriptional and functional signatures of individual ASD genes through RNAseq and a series of high-throughput phenotyping assays in both neuronal sub-types. Aim 2 will expand our Preliminary Results to discover convergent genes downstream of ASD risk loci, characterize the synaptic consequences of the ten most compelling discoveries from individual genes and/or convergent signatures, and integrate these data to explore the druggability of the convergent networks. Our overarching goal is to define any commonalities among diverse genes, pathways and networks that underlie ASD etiology, and to dramatically expand the list of possible therapeutic targets for ASD. These studies will generate an unprecedented isogenic resource of CRISPR-edited ASD genes, and matched RNAseq and cellular phenotyping in glutamatergic and GABAergic neurons, that will be provided for open distribution to the broader community through the NIMH RUDCR resource to yield new insights into neuropsychiatric disorders.