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)等精神疾病影响着数以百万计的个人和他们的家庭。 全世界。遗传风险在ASD的病因中起着重要的作用，我们已经证明了大脑的变化 在异构体水平上的mRNA表达，而不是基因表达的整体变化，显示出最大的影响 大小和基因丰富。然而，脑内异构体多样性的程度及其在脑内的失调 像asd这样的疾病，由于许多异构体不能在细胞类型水平上解决，所以被极大地未被研究。 通过常用的短读RNAseq技术，尚未直接进行分析。因此，有一个紧急的 需要准确评估异构体表达的作用及其与自闭症相关遗传变异−的关系 细胞类型和空间特异性，以了解其致病的神经生物学机制 风险。我们的目标是解决我们对异形表达图景的理解中的这些和其他缺陷 在神经典型的和患病的大脑中。因此，我们将使用长阅读的RNA亚型测序来产生亚型 3个脑区全长异构体的结构、表达丰度和用途的图谱和特征 与自闭症有关。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 异构体失调的证据，以确定细胞水平上的异构体表达模式。 在目标1中，我们将对身体进行全转录组全长(FL)RNA异构体测序(IsoSeq ASD患者和神经典型对照的海马区(HC)和纹状体(STR)脑组织，并结合 与我们现有的前额叶皮质(PFC)数据一起构建一张全面的亚型表达图 横跨三个大脑区域。值得注意的是，我们还将使用正常和失调全长的参考地图 以异构体表达为先验分析ASD病例和ASD病例的2,000个RNAseq数据集 心理编码联盟和其他努力积累的控制，并确定失调的异构体和 异构体共表达网络模块。在目标2中，我们将使用一种补充方法来分析单核 ASD和对照组中相同的PFC、HC和STR组织的差异以消除其异构体表达差异 在细胞水平上使用10X SNIsoSeqCap对50个ASD危险基因进行测序，这是一种新的测序FL-SeqCap的方法 选定基因在单核/细胞水平上的全长转录本。我们将综合表演 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聚合酶链式反应(PCR)在选定的 神经细胞和非神经细胞类型。 这里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 关于人脑亚型表达的悬而未决的问题。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。