Mapping human brain cell type-specific isoform usage in ASD

绘制 ASD 中人脑细胞类型特异性亚型的使用情况

• 批准号: 10433311
• 负责人: Dalila Pinto
• 金额: $ 21.13万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-11 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433311
• 关键词: Address; Affect; Autopsy; Bar Codes; Bayesian Modeling; Biological; Biological Assay; Brain; Brain Diseases; Brain region; Catalogs; Cell Nucleus; Communities; Complement; Complementary DNA; Corpus striatum structure; Data; Data Set; Disease; Etiology; Exhibits; Family; Female; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic; Genetic Risk; Genetic Transcription; Genetic Variation; Genetic study; Goals; Hippocampus (Brain); Human; Individual; Length; Link; Maps; Mental disorders; Methodology; Methods; Molecular; Molecular Target; Neuroglia; Neurons; Neurosciences; Pathogenesis; Pathway interactions; Pattern; Play; Population; Prefrontal Cortex; Prevention strategy; Protein Isoforms; RNA; Regulation; Resolution; Resources; Risk; Role; Sampling; Sorting - Cell Movement; Specificity; Spliced Genes; Structure; Switch Genes; Technology; Therapeutic; Tissues; Transcript; Transcriptional Regulation; Translations; Work; autism spectrum disorder; base; brain cell; brain tissue; case control; cell type; disorder control; disorder risk; exome; genetic risk factor; genome annotation; genome sequencing; genome wide association study; genomic locus; improved; innovation; mRNA Expression; male; mind control; neurobiological mechanism; neuropsychiatric disorder; new therapeutic target; novel; novel strategies; prevent; reference genome; risk variant; transcriptome; transcriptome sequencing

Psychiatric disorders such as autism spectrum disorder (ASD) affect millions of individuals and their families worldwide. Genetic risk plays an important role in the etiology of ASD, and we have shown that changes in brain mRNA expression at the isoform-level, rather than overall changes in gene expression, show the largest effect sizes and genetic enrichments. However, the extent of isoform diversity in brain, and its dysregulation in disorders such as ASD, is vastly underexplored because many isoforms cannot be resolved at the cell type-level by commonly used short-read RNAseq technologies and are yet to be directly profiled. Thus, there is an urgent need to assess the role of isoform expression and its relation to ASD-associated genetic variation − with precise cell-type and spatial specificity to understand the neurobiological mechanisms through which it confers disease risk. Our goal is to address these and other deficits in our understanding of the landscape of isoform expression in neurotypical and diseased brain. As such, we will use long-read RNA isoform sequencing to generate isoform maps and characterize the structure, expression abundance and usage of full-length isoforms in 3 brain regions implicated in ASD. We will further complement by deeply profiling 50 high-confidence ASD risk genes with evidence of isoform dysregulation, to identify isoform expression patterns at the cellular level. In Aim 1 we will perform whole-transcriptome full-length (FL) RNA isoform sequencing (IsoSeq) of postmortem hippocampus (HC) and striatum (STR) brain tissues of ASD cases and neurotypical controls, and combine the data with our existing data for prefrontal cortex (PFC) to construct a comprehensive map of isoform expression across the 3 brain regions. Notably, we will also use our reference maps of normal and dysregulated full-length isoform expression as priors for analyses of a compendium of >2,000 RNAseq datasets from ASD cases and controls amassed by the PsychENCODE consortium and other efforts, and identify dysregulated isoforms and isoform co-expression network modules. In Aim 2 we will use a complementary approach to profile single-nuclei of the same PFC, HC and STR tissues in ASD and controls to disambiguate isoform expression differences of 50 ASD risk genes at the cell-type level using 10X snIsoSeqCap, which is a novel assay to sequence FL- transcripts of selected genes across their entire length at the single nucleus/cell level. We will perform integrative analyses to identify isoform expression differences between tissues and cell types, and selected isoform changes will be validated by RNA fluorescent in situ hybridization (FISH) and quantitative isoform-specific PCR in selected neuronal and non-neuronal cell types. The maps generated here will improve existing reference genome annotations, and allow to address major outstanding questions regarding isoform expression in human brain. The new data and methodologies will provide a tremendous resource for the neuroscience community, paving the way to better inform neurobiological mechanisms of genetic risk for ASD.

自闭症谱系障碍（ASD）等精神疾病会影响数百万个人及其家人 全世界。遗传风险在ASD的病因中起着重要作用，我们已经表明大脑的变化 同工型水平的mRNA表达，而不是基因表达的总体变化，显示出最大的作用 大小和遗传富集。但是，大脑中同工型多样性的程度及其失调 诸如ASD之类的疾病被大大流失了，因为在单元格级别无法解决许多同工型 通过常用的短阅读RNASEQ技术，尚未直接介绍。那是紧急的 需要评估同工型表达的作用及其与ASD相关遗传变异的关系 - 精确 细胞类型和空间特异性，以了解其供认疾病的神经生物学机制 风险。我们的目标是解决这些和其他定义在我们对同工型表达的景观的理解中 在神经型和解剖的大脑中。因此，我们将使用长阅读的RNA同工型测序来生成同工型 地图并表征3个大脑区域中全长同工型的结构，表达抽象和使用 在ASD中实施。我们将通过深入分析50个高信心ASD风险基因的进一步完成 同工型失调的证据，以鉴定细胞水平的同工型表达模式。 在AIM 1中，我们将执行全转录组全长（FL）RNA同工型测序（ISOSEQ） ASD病例和神经型对照的海马（HC）和纹状体（Str）脑组织，并结合 带有我们现有数据前额叶皮层（PFC）的数据，以构建同工型表达式的综合图 跨三个大脑区域。值得注意的是，我们还将使用正常和失调的全长参考图 同工型表达作为对ASD案例的2,000个RNASEQ数据集的纲要分析的先验。 由心理码联盟和其他努力积累的控制物，并确定失调的同工型和 同工型共表达网络模块。在AIM 2中，我们将使用完整的方法来概况单核 ASD中的同一PFC，HC和Str组织以及对照组，以消除歧义的同工型表达差异 50使用10倍Snisoseqap在细胞类型水平的ASD风险基因，这是对序列序列的新测定 选定基因在单核/细胞水平上的整个长度的转录本。我们将执行集成 分析以鉴定组织和细胞类型之间的同工型表达差异，并选择的同工型变化 RNA荧光原位杂交（FISH）和定量同工型特异性PCR将得到验证 神经元和非神经元细胞类型。 这里生成的地图将改善现有的参考基因组注释，并允许解决专业 关于人脑中同工型表达的杰出问题。新数据和方法将 为神经科学社区提供巨大的资源，为更好地告知神经生物学的道路 ASD遗传风险的机制。