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

项目总结 自闭症谱系障碍(ASD)是一种具有复杂遗传因素的异质性神经发育障碍 建筑。ASD的有效治疗和诊断工具的开发一直受到我们的 对潜在的遗传变异的不完全理解。新变种(DNV)，估计有助于 30%-40%的病例主要是在基因组的蛋白质编码区进行研究。数十万人 的非编码变体已经被识别，但破译它们对ASD病因学的功能贡献仍然存在 很有挑战性。顺式调控元件，如启动子和增强子，是分析 非编码DNV的潜在影响，但它们的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(谷氨酸能)和 抑制性(GABA能)神经元，两者都可以在体外从人类多能干细胞和 去极化以模拟神经元激活引起的转录和表观遗传变化。我们的实验室 注释了基线和激活状态下两种细胞类型中存在的增强子，发现了2495个增强子 包含非编码的ASD DNV，包括数百个特定于细胞类型或依赖于活动的DNV。 使用大规模平行报告分析(MPRA)，这一建议将确定是否发现非编码DNV 在自闭症患者中，改变谷氨酸或GABA能人类神经元的顺式调节活动 基线或激活状态。此外，基因增强子图谱显示，含有DNV的子集 据预测，增强剂将调节先前与ASD有关的基因。验证顺式监管活动并 比较对下游基因网络的反式影响，将在两个细胞中进行CRISPR抑制筛查 类型和激活状态，靶向25个ASD基因及其含有DNV的增强子。如果成功，这项工作 将展示非编码从头变体对ASD生物学的潜在功能贡献，ASD生物学已经 到目前为止，这仍然是该领域一个悬而未决的问题。此外，这将为以下内容生成转录数据集 在两种临床相关的细胞类型中，处于静息和活动状态的顶级ASD风险基因在 越来越多的功能基因组学ASD研究，强调趋同的调控基因网络。这 研究将在西奈山的伊坎医学院进行，该学院拥有美国国立卫生研究院资助的第二名 神经科学系和Seaver自闭症中心的所在地，该中心以连接基础科学和 更有效的ASD护理的临床试验。科学的严谨性，创新的技术，复杂的分析， 多学科协作和这里列出的充足的指导机会将推动我走向 作为一名独立研究教授，成功地研究了潜在的分子机制 在指导下一代科学家和倡导心理健康教育的同时，还在精神疾病方面做出了贡献。

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.