Assessing Genomic, Regulatory and Transcriptional Variation at Single Nuclei Resolution in the Brains of Individuals with Autism Spectrum Disorder

评估自闭症谱系障碍患者大脑中单核分辨率的基因组、调控和转录变异

• 批准号: 10657693
• 负责人: ARNOLD KRIEGSTEIN
• 金额: $ 70.87万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-27 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657693
• 关键词: 3-Dimensional; ATAC-seq; Affect; Age; Anterior; Autopsy; BRAIN initiative; Bioinformatics; Biological Assay; Biological Markers; Biological Models; Biology; Brain; Brain Diseases; Brain region; Cell Culture Techniques; Cell Nucleus; Cell model; Cell physiology; Cells; Censuses; Child; Chromatin; Collaborations; Communication; Corpus striatum structure; Cytoskeleton; DNA; DNA Sequence Alteration; Data; Data Set; Development; Diagnostic; Disease; Etiology; Exhibits; Experimental Models; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Frequency; Genes; Genetic; Genetic Diseases; Genetic Models; Genetic Transcription; Genomics; Genotype; Genotype-Tissue Expression Project; Goals; Heritability; Heterogeneity; Human; Human Genetics; In Situ Hybridization; Individual; Lentivirus; Loss of Heterozygosity; Methods; Methyl-CpG-Binding Protein 2; Modality; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Mutation; Neurobiology; Neurodevelopmental Disorder; Neurons; Neurosciences; Nucleic Acid Regulatory Sequences; Organoids; Pathology; Phenotype; Pilot Projects; Pluripotent Stem Cells; Prefrontal Cortex; Process; Protein Isoforms; Protocols documentation; RNA Splicing; Reporter; Research; Resolution; Risk Factors; Rodent; Sample Size; Sampling; Second Pregnancy Trimester; Seizures; Single Nucleotide Polymorphism; Source; Spliced Genes; Statistical Data Interpretation; Systems Analysis; Techniques; Testing; Therapeutic; Tissue Sample; Tissues; Untranslated RNA; Validation; Variant; Work; autism spectrum disorder; biobank; brain cell; brain tissue; cell type; comorbidity; data integration; de novo mutation; diagnostic signature; differential expression; experience; functional genomics; functional group; genetic analysis; genetic disorder diagnosis; genetic risk factor; genetic variant; genome sequencing; genomic data; genomic locus; human model; individuals with autism spectrum disorder; insertion/deletion mutation; insight; loss of function mutation; neuropsychiatric disorder; polygenic risk score; repository; sex; single nucleus RNA-sequencing; stem cell model; stem cells; therapeutically effective; transcriptome sequencing; transcriptomics; whole genome

ABSTRACT Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder of unknown etiology and with limited effective therapeutic options that affects millions of individuals. Our research team has a longstanding commitment to understanding the cause of ASD and the molecular processes underlying brain development, function, and pathology. We will use this experience to apply the latest molecular techniques to samples from a new repository of brain tissue from individuals with ASD to create the largest and most detailed analysis of the molecular consequences of ASD. Genetic analyses of gene disrupting de novo mutations have identified over one hundred genes associated with ASD with three main functional groups: regulation of gene expression, neuronal communication, and cytoskeleton. Prior analyses of brain tissue from individuals with ASD have identified a group of downregulated neuronal communication genes, that overlap with ASD-associated genes, and a group of upregulated glial genes that do not overlap with ASD-associated genes or variants. It is unclear if these changes reflect altered cell composition or cell function and how they relate to genetic factors. We propose to analyze post-mortem brain samples from 40 individuals with ASD and 40 unaffected controls, sourced from the Autism BrainNet BioBank, to assess the molecular changes that occur. We will use whole-genome sequencing to identify gene disruptive variants in genes previously associated with ASD and to identify rare and common variants that may alter gene expression or splicing. In tissue samples the prefrontal cortex and striatum in from 40 cases and 40 controls, we will use recently developed single-nuclei methods to perform RNA-seq and ATAC-seq at single-cell resolution to identify ASD-related changes in gene regulation and expression in specific cell types and brain regions. For tissue samples from the prefrontal cortex of 20 cases and 20 controls we will also use cutting-edge single nuclei long-read RNA-seq (Iso-seq), along with bulk tissue RNA-seq, for an in-depth analysis of how gene isoforms differ between ASD cases and controls. Finally, we will assess how single-nuclei gene expression varies in brain organoids grown from pluripotent stem cells edited to contain mutations in three ASD-associated genes. Integrating these data, we will profile the molecular changes associated with ASD and assess how these changes vary by cell type, brain region, age, sex, seizure status, and genotype. We will use RNAscope in situ hybridization to validate the molecular and cell composition changes we observe and a lentivirus-based massively parallel reporter assay to test the function of regulatory regions or variants in proximity to genes with ASD-related differences in expression to validate these effects and assess causality. We hope that these insights will provide a basis for understanding the heterogeneity of ASD and the neurobiological features of this disorder and provide molecular signatures that could be developed into future biomarkers for ASD model systems.

抽象的 自闭症谱系障碍（ASD）是一种高度可遗传的神经发育障碍 有限的有效治疗选择会影响数百万个人。我们的研究团队长期存在 承诺了解ASD的原因和大脑发育的分子过程， 功能和病理。我们将利用这种经验将最新分子技术应用于来自 来自ASD患者的脑组织的新存储库，以创建最大，最详细的分析 ASD的分子后果。基因破坏从头突变的遗传分析已经确定 与ASD相关的一百个与三个主要官能团相关的基因：基因表达的调节， 神经元通信和细胞骨架。先前对ASD个体的脑组织进行的先前分析 确定了一组下调的神经元通信基因，该基因与ASD相关基因重叠， 一组上调的神经基因与与ASD相关的基因或变体不重叠。目前尚不清楚 如果这些变化反映了细胞组成或细胞功能的改变以及它们与遗传因素的关系。我们 提议分析来自40个ASD和40个未受影响对照的40个个体的验尸后脑样本， 从自闭症Brainnet生物库，以评估发生的分子变化。我们将使用全基因组 测序以鉴定先前与ASD相关的基因中的基因破坏性变异，并确定稀有和 可能改变基因表达或剪接的常见变体。在组织样品中，前额叶皮层和纹状体 在40例和40个对照中，我们将使用最近开发的单核方法执行RNA-Seq和 在单细胞分辨率下的ATAC-SEQ，以鉴定基因调控和表达的ASD相关变化。 细胞类型和大脑区域。对于来自20例和20个对照的前额叶皮层的组织样品，我们将 还使用尖端的单核长读RNA-Seq（ISO-Seq）以及散装组织RNA-Seq，以深入 分析基因同工型在ASD病例和对照之间如何差异。最后，我们将评估单核如何 基因表达在从多能干细胞中生长的脑器官中有所不同，以包含三个突变 ASD相关基因。整合这些数据，我们将介绍与ASD和ASD相关的分子变化 评估这些变化如何随细胞类型，大脑区域，年龄，性别，癫痫发作状态和基因型而变化。我们将使用 rnascope原位杂交以验证我们观察到的分子和细胞组成变化和A 基于慢病毒的大规模平行记者测定法，以测试调节区域或近端变体的功能 与ASD相关的表达差异的基因验证这些作用并评估因果关系。我们希望那样 这些见解将为理解ASD的异质性和神经生物学特征提供基础 这种疾病并提供了可以发展为ASD模型未来生物标志物的分子特征 系统。

项目成果

Neuropsychiatric biomarker discovery: go big or go home.

神经精神生物标志物的发现：要么做大，要么回家。

• DOI: 10.1016/j.molmed.2023.09.002
• 发表时间: 2023
• 期刊: Trends in molecular medicine
• 影响因子: 13.6
• 作者: Ljungdahl,Alicia;Sanders,StephanJ
• 通讯作者: Sanders,StephanJ

AlphaMissense is better correlated with functional assays of missense impact than earlier prediction algorithms.

与早期的预测算法相比，AlphaMissense 与错义影响的功能分析具有更好的相关性。

• DOI: 10.1101/2023.10.24.562294
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Ljungdahl,Alicia;Kohani,Sayeh;Page,NicholasF;Wells,EloiseS;Wigdor,EmilieM;Dong,Shan;Sanders,StephanJ
• 通讯作者: Sanders,StephanJ