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: 10457436
• 负责人: ARNOLD KRIEGSTEIN
• 金额: $ 74.99万
• 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/10457436
• 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; Methods; 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; Small Nuclear RNA; Source; Spliced Genes; Statistical Data Interpretation; Syndrome; Systems Analysis; Techniques; Testing; Therapeutic; Tissue Sample; Tissues; Untranslated RNA; Validation; Variant; Work; autism spectrum disorder; base; biobank; brain cell; brain tissue; case control; cell type; comorbidity; 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; 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.

摘要