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 建筑学。我们的ASD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。从头变体（DNV），估计有助于 30-40％的病例主要在基因组的蛋白质编码区域进行了研究。成千上万 已经确定了非编码变体的，但解释了其对ASD病因的功能贡献 具有挑战性的。顺式调节元素（例如发起人和增强剂）代表一个途径来测定 非编码DNV的潜在影响，但它们的调节活性取决于细胞环境，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（谷氨酸能）和 抑制性（GABA能）神经元，这两种神经元都可以在人类多能干细胞和 去极化以建模由神经元激活引起的转录组和表观遗传变化。我们的实验室 注释在基线和激活状态下两种细胞类型中存在的增强剂，发现2495个增强剂 包含非编码ASD DNV，其中包括几百个细胞类型的特异性或活性依赖性。 使用大量并联的记者测定法（MPRA），该提案将确定是否找到了非编码DNV 自闭症患者改变了谷氨酸能或GABA能的人类神经元中的顺式调节活性 基线或激活状态。此外，基因增强剂映射显示了含DNV的子集 预计增强子将调节先前与ASD有关的基因。验证顺式调节活动和 比较在下游基因网络上的跨效应，将在两个细胞中进行CRISPR抑制屏幕 针对25个ASD基因及其含DNV的增强子的类型和激活状态。如果成功，这项工作 将证明非编码从头变体对ASD生物学的潜在功能贡献，该生物具有 到目前为止，该领域仍然是一个杰出的问题。此外，这将生成转录组数据集的 在静止状态和活性状态下，两种临床相关细胞类型中的最高ASD风险基因，以扩展 越来越多的功能基因组学ASD研究强调了收敛的调节基因网络。这 研究将在西奈山的伊坎医学院进行，其中包括第二届NIH资助 神经科学系和Seaver自闭症中心的住所，以桥接基础科学和 临床试验，以进行更有效的ASD护理。科学严谨，创新技术，复杂的分析， 多学科的合作以及这里概述的充足的指导机会将使我走向 作为研究分子机制的独立研究教授的成功职业 精神疾病会在指导后代的科学家并倡导心理健康教育的同时。