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

人类干细胞源性神经元中非编码自闭症从头变异的细胞类型特异性和活性依赖性表征

• 批准号: 10677459
• 负责人: Sarah Elizabeth Williams
• 金额: $ 4.77万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-24 至 2026-04-23
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677459
• 关键词: Advocate; Affect; Basic Science; Binding; Biological Assay; Biology; Caring; Child; Chromatin; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Complex; Copy Number Polymorphism; Data; Data Set; Dependence; Development; Diagnosis; Engineering; Enhancers; Epigenetic Process; Etiology; Funding; Future Generations; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Genetic Variation; Genome; Genomics; Glutamates; Goals; Guide RNA; Home; Human; Impairment; In Vitro; Individual; Interdisciplinary Study; Laboratory Research; Maps; Membrane; Mental disorders; Mentors; Mentorship; Modeling; Molecular; Neurodevelopmental Disorder; Neurons; Neurosciences; Open Reading Frames; Pathway interactions; Point Mutation; Psychiatry; Regulatory Element; Reporter; Research; Rest; Role; Scientist; Siblings; Symptoms; Techniques; Training; United States National Institutes of Health; Untranslated RNA; Validation; Variant; Work; autism spectrum disorder; career; cell type; clinically relevant; de novo mutation; design; diagnostic tool; early childhood; excitatory neuron; exome sequencing; experimental study; functional genomics; gene network; gene regulatory network; genetic architecture; human pluripotent stem cell; human stem cells; individuals with autism spectrum disorder; inhibitory neuron; innovation; medical schools; mental health education; neurodevelopment; professor; promoter; psychogenetics; repetitive behavior; risk variant; single-cell RNA sequencing; skills; social; stem cells; therapeutically effective; transcription factor; transcriptomics

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.

项目概要 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 建筑学。自闭症谱系障碍 (ASD) 有效治疗和诊断工具的开发一直受到我们的阻碍 对潜在遗传变异的不完全了解。新生变异（DNV），估计有助于 30-40% 的病例主要在基因组的蛋白质编码区域进行研究。数十万 的非编码变异已被识别，但仍需破译它们对 ASD 病因的功能贡献 具有挑战性的。顺式调控元件（例如启动子和增强子）代表了检测顺式调控元件的一种途径 非编码 DNV 的潜在影响，但其调节活动取决于细胞环境，例如细胞 类型和激活状态。主要参与 ASD 生物学的两种细胞类型是兴奋性（谷氨酸）和 抑制性（GABAergic）神经元，这两种神经元都可以在体外由人类多能干细胞产生 去极化以模拟神经元激活引起的转录组和表观遗传变化。我们的实验室 注释了两种细胞类型在基线和激活状态下存在的增强子，并发现了 2495 个增强子 包含非编码 ASD DNV，其中包括数百个细胞类型特异性或活性依赖性的 DNV。 该提案将使用大规模并行报告分析 (MPRA) 来确定是否发现非编码 DNV 自闭症患者的谷氨酸能或 GABA 能人类神经元的顺式调节活性发生改变 基线或激活状态。此外，基因增强子图谱显示，含有 DNV 的子集 增强子预计可以调节先前与自闭症谱系障碍有关的基因。验证顺式监管活动并 比较对下游基因网络的反式效应，将在两个细胞中进行 CRISPR 抑制筛选 类型和激活状态，针对 25 个 ASD 基因及其包含 DNV 的增强子。如果成功的话，这项工作 将展示非编码从头变异对自闭症谱系障碍生物学的潜在功能贡献 迄今为止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 两种临床相关细胞类型在静息和活动状态下的顶级 ASD 风险基因，以扩展 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 研究将在西奈山伊坎医学院进行，该学院包含 NIH 资助的第二好的 神经科学系和西弗自闭症中心的所在地，该中心以桥接基础科学和 更有效的 ASD 护理的临床试验。科学严谨、创新技术、精密分析、 这里概述的多学科合作和充足的指导机会将推动我走向 作为一名独立研究教授，研究潜在的分子机制，取得了成功的职业生涯 精神疾病，同时指导未来几代科学家并倡导心理健康教育。