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: 10620755
• 负责人: Dalila Pinto
• 金额: $ 25.35万
• 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/10620755
• 关键词: Address; Affect; Autopsy; Bar Codes; Bayesian Modeling; Biological; Biological Assay; Brain; Brain Diseases; Brain region; Catalogs; Cell Nucleus; Cells; Communities; Complement; Complementary DNA; Corpus striatum structure; Data; Data Set; Disease; Etiology; Exhibits; Family; Female; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic; Genetic Complementation Test; Genetic Risk; Genetic Transcription; Genetic Variation; Genetic study; Goals; Hippocampus; 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; Specificity; Spliced Genes; Structure; Switch Genes; Technology; Therapeutic; Tissues; Transcript; Transcriptional Regulation; Translations; Work; autism spectrum disorder; brain cell; brain tissue; cell type; comparison 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) 等精神疾病影响着数百万人及其家庭 全世界。遗传风险在 ASD 的病因学中起着重要作用，我们已经证明大脑的变化 同工型水平的 mRNA 表达，而不是基因表达的整体变化，显示出最大的影响 大小和遗传富集。然而，大脑中亚型多样性的程度及其在大脑中的失调 由于许多异构体无法在细胞类型水平上解析，因此诸如自闭症谱系障碍（ASD）之类的疾病尚未得到充分研究 通过常用的短读长 RNAseq 技术进行分析，但尚未直接进行分析。因此，有一个紧急的 需要评估亚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，以了解其导致疾病的神经生物学机制 风险。我们的目标是解决我们对异构体表达景观的理解中的这些和其他缺陷 在神经正常和患病的大脑中。因此，我们将使用长读长 RNA 同工型测序来生成同工型 绘制并表征 3 个大脑区域全长亚型的结构、表达丰度和用途 与自闭症谱系障碍有关。我们将通过深入分析 50 个高置信度 ASD 风险基因来进一步补充 同工型失调的证据，以确定细胞水平上的同工型表达模式。 在目标 1 中，我们将进行死后全转录组全长 (FL) RNA 同工型测序 (IsoSeq) ASD 病例和神经典型对照的海马 (HC) 和纹状体 (STR) 脑组织，并结合 数据与我们现有的前额皮质 (PFC) 数据一起构建异构体表达的综合图谱 跨越3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 亚型表达作为先验，用于分析 ASD 病例中超过 2,000 个 RNAseq 数据集的概要 PsychENCODE 联盟和其他努力积累的控制，并识别失调的亚型和 同工型共表达网络模块。在目标 2 中，我们将使用补充方法来分析单核 ASD 和对照中相同的 PFC、HC 和 STR 组织，以消除亚型表达差异的歧义 使用 10X snIsoSeqCap 在细胞类型水平上检测 50 个 ASD 风险基因，这是一种对 FL- 进行测序的新型检测方法 在单核/细胞水平上选定基因的全长转录本。我们将进行综合性的 分析以确定组织和细胞类型之间的异构体表达差异以及选定的异构体变化 将通过 RNA 荧光原位杂交 (FISH) 和定量异构体特异性 PCR 在选定的 神经元和非神经元细胞类型。 此处生成的图谱将改进现有的参考基因组注释，并允许解决主要问题 关于人脑中异构体表达的突出问题。新的数据和方法将 为神经科学界提供了巨大的资源，为更好地了解神经生物学铺平了道路 ASD 的遗传风险机制。