Cell-type specific and activity-dependent characterization of non-coding autism de novo variants in human stem cell-derived neurons

项目摘要 自闭症谱系障碍（ASD）是一种异质性神经发育障碍，具有复杂的遗传学特征。 架构ASD的有效治疗和诊断工具的开发受到我们的阻碍。 对潜在遗传变异的不完全理解。新生变异体（DNV），估计有助于 30-40%的病例主要在基因组的蛋白质编码区进行研究。数十万 的非编码变异已被确定，但破译其功能的贡献ASD病因仍然存在 挑战性顺式调节元件如启动子和增强子代表了一种测定表达的途径。 非编码DNV的潜在影响，但其调节活性取决于细胞环境，如细胞 类型和激活状态。ASD生物学中主要涉及的两种细胞类型是兴奋性（兴奋性）和 抑制性（GABA能）神经元，这两者都可以在体外从人多能干细胞产生， 去极化以模拟由神经元活化引起的转录组和表观遗传变化。我们实验室 注释了在基线和激活状态下存在于两种细胞类型中的增强子，并发现了2495个增强子 含有非编码ASD DNV，包括数百种细胞类型特异性或活性依赖性的。 使用大规模平行报告基因分析（MPRA），该提案将确定是否发现非编码DNV 在孤独症个体中， 基线或激活状态。此外，基因-增强子作图已经揭示了含有DNV的一个子集， 预计增强子可调节先前与ASD有关的基因。验证顺式调节活性， 为了比较对下游基因网络的反式效应，将在两种细胞中进行CRISPR抑制筛选， 类型和激活状态，靶向25个ASD基因及其含DNV的增强子。如果成功，这项工作 将证明非编码从头变异对ASD生物学的潜在功能贡献， 到目前为止，这仍然是该领域的一个悬而未决的问题。此外，这将生成转录组数据集， 在两种临床相关的细胞类型中，在静息和活跃状态下， 越来越多的功能基因组学ASD研究，强调收敛调节基因网络。这 研究将在西奈山的伊坎医学院进行，该学院拥有NIH资助的第二大医学院。 神经科学系和西弗自闭症中心的所在地，以连接基础科学和 进行临床试验以获得更有效的ASD治疗。科学的严谨，创新的技术，复杂的分析， 多学科的合作，以及这里概述的充足的导师机会将推动我走向一个 成功的职业生涯作为一个独立的研究教授研究的分子机制， 精神疾病，同时指导未来的科学家和倡导心理健康教育。

PROJECT SUMMARY Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder with a complex genetic architecture. The development of effective therapeutics and diagnostic tools for ASD has been hindered by our incomplete understanding of underlying genetic variation. De novo variants (DNVs), estimated to contribute to 30-40% of cases, have been primarily studied in protein-coding regions of the genome. Hundreds of thousands of non-coding variants have been identified but deciphering their functional contribution to ASD etiology remains challenging. Cis-regulatory elements such as promoters and enhancers represent one avenue to assay the potential impact of non-coding DNVs, but their regulatory activity is dependent on cellular contexts such as cell type and activation state. The two cell types primarily involved in ASD biology are excitatory (glutamatergic) and inhibitory (GABAergic) neurons, both of which can be generated in vitro from human pluripotent stem cells and depolarized to model the transcriptomic and epigenetic changes caused by neuronal activation. Our lab annotated the enhancers present in both cell types at baseline and activated states and found 2495 enhancers containing non-coding ASD DNVs, including several hundred that are cell-type specific or activity-dependent. Using a massively parallel reporter assay (MPRA), this proposal will determine whether non-coding DNVs found in individuals with autism alter cis-regulatory activity in glutamatergic or GABAergic human neurons in either baseline or activated states. Further, gene-enhancer mapping has revealed that a subset of DNV-containing enhancers is predicted to regulate genes previously implicated in ASD. To validate cis-regulatory activity and to compare trans-effects on downstream gene networks, a CRISPR inhibition screen will be performed in both cell types and activation states, targeting 25 ASD genes and their DNV-containing enhancers. If successful, this work will demonstrate the potential functional contribution of non-coding de novo variants to ASD biology, which has thus far remained an outstanding question in the field. Moreover, this will generate transcriptomic datasets for top ASD risk genes in two clinically relevant cell types at both resting and active states to expand upon the growing number of functional genomics ASD studies, emphasizing convergent regulatory gene networks. This research will take place at the Icahn School of Medicine at Mount Sinai, containing the 2nd best NIH-funded neuroscience department and home to the Seaver Autism Center, renowned for bridging basic science and clinical trials for more effective ASD care. The scientific rigor, innovative techniques, sophisticated analyses, multi-disciplinary collaborations, and ample mentorship opportunities outlined here would propel me towards a successful career as an independent research professor studying the molecular mechanisms underlying psychiatric disorders while mentoring future generations of scientists and advocating for mental health education.