Testing naturally-occurring mutations for impact on brain enhancer function

测试自然发生的突变对大脑增强功能的影响

• 批准号: 10207123
• 负责人: Alexander Nord
• 金额: $ 19.63万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207123
• 关键词: Address; Adopted; Alleles; Biological Assay; Brain; Brain Diseases; Cells; Clinical; Cloning; Code; Codon Nucleotides; Collection; Consensus; DNA; DNA Sequence; Data; Development; Diagnosis; Disease; Distal; Elements; Enhancers; Evaluation; Evolution; Fluorescence; Gene Expression; Genes; Genetic Code; Genetic Enhancer Element; Genetic Risk; Genetic Transcription; Genome; Genomics; Human; Human Cloning; Image; Individual; Investigation; Label; Length; Link; Measurement; Mediating; Methods; Modeling; Mus; Mutagenesis; Mutation; Mutation Analysis; Neurodevelopmental Disorder; Neuroglia; Neurons; Nucleotides; Oligonucleotides; Patients; Performance; Population; Process; Protocols documentation; Regulation; Regulatory Element; Reporter; Reporter Genes; Research; Resolution; Role; Sampling; Scientist; Siblings; Specificity; Structural Protein; System; Testing; Triplet Multiple Birth; Untranslated RNA; Validation; Variant; Work; autism spectrum disorder; base; cell type; clinical care; cohort; de novo mutation; disorder risk; excitatory neuron; experimental study; genetic disorder diagnosis; genetic variant; genome sequencing; genome-wide; human DNA; in vivo; inhibitory neuron; innovation; insight; large datasets; neonatal mice; novel; novel strategies; postnatal; proband; screening; whole genome

SUMMARY Enhancers are distal non-coding regulatory DNA elements that contribute to the activation of target genes in a developmental, cell-type, and context-dependent manner. In other words, enhancers act as the genomic switches that enable the precise control of gene expression required for the development and function of the brain. There is a consensus that enhancers are a critical for gene expression and that sequence variation within enhancers contributes to genetic risk. However, there are major barriers towards being able to predict which non-coding mutations matter in the context of regulatory function and disease risk. To date, the role of non-coding regions in brain disorders, including autism spectrum disorder, has been largely impenetrable due to the quantity of non-coding DNA and the difficulty characterizing functional impact of variants outside of coding sequence in the brain. Whole genome sequencing (WGS) efforts promise comprehensive genome-wide mutation analysis, and this method has been adopted at scale in ASD. However, computational prediction of regulatory variant function alone is currently insufficient for functional variant identification, including in ASD. Massively parallel reporter assays (MPRAs) provide a solution via enabling functional screening of enhancers and their variants, and quantitative measurement of the regulatory capacity of hundreds to thousands of individual candidate sequences in a single experiment. For such functional assays to be relevant to the brain and ASD, it is critical to use models that capture the complexity and organization of the brain. We implemented a large-scale screen of de novo regulatory variants using in vivo deployment of an MPRA in postnatal mouse brain. From a pool of ~1000 de novo variants assayed in early postnatal mouse cortex using our innovative function-based test, we identified strong and weak enhancers, and putative allele-specific activity associated with these naturally occurring de novo regulatory mutations. Although a significant demonstration of assay potential, our preliminary results fail to fully take advantage of the ability to interrogating context-dependent function in the complexity of the brain. Here we proposed work to verify in vivo MPRA performance and extend this method to generate cell-type specific enhancer readout. In doing this, we will define the regulatory capacity of candidate enhancers harboring de novo variants from ASD proband and control genomes. In Aim 1, we propose a screen-centered approach, using our MPRA to define cell-type and allele-specific enhancer activity. In Aim 2, we propose an enhancer-centered approach, defining in vivo activity of individual enhancers in mouse postnatal brain via image-based analysis. Our results will generate function-based evaluations of naturally occurring de novo regulatory mutations, enabling statistical testing of functionally-defined enhancer activity in tandem with deep single enhancer functional investigations. If successful, our work will set the stage for usage of function-based screening of disease-relevant variants across AAV-accessible CNS systems and generate novel insights regarding the function of de novo enhancer variants from ASD WGS data.

摘要 增强子是远端的非编码调控DNA元件，有助于激活靶基因 发育的、细胞类型的和上下文相关的方式。换句话说，增强剂就像基因组一样 能够精确控制细胞发育和功能所需的基因表达的开关 大脑。人们一致认为增强剂对基因表达和序列变异至关重要 在增强剂中会增加遗传风险。然而，在能够进行预测方面存在重大障碍。 哪些非编码突变在调节功能和疾病风险方面具有重要意义。到目前为止， 大脑障碍中的非编码区，包括自闭症谱系障碍，在很大程度上是无法渗透的 对非编码DNA的数量和对外部突变体的功能影响进行表征的难度 大脑中的编码序列。全基因组测序(WGS)的努力有望在全基因组范围内实现全面 突变分析，该方法已在ASD中大规模采用。然而，计算预测 目前仅有调节变异功能是不足以识别功能变异的，包括在ASD中。 大规模平行报告分析(MPRA)通过实现增强子的功能性筛选提供了解决方案 及其变种，以及对成百上千个 单个实验中的单个候选序列。为了让这种功能分析与大脑相关 对于ASD来说，使用能够捕捉大脑复杂性和组织的模型是至关重要的。我们实施了 在生后小鼠体内部署MPRA大规模筛选从头调控变异体 大脑。从出生后早期小鼠皮质中分析的约1000个新变异体中使用我们的创新技术 基于功能的测试，我们确定了强和弱的增强子，并推测与等位基因特异的活性相关 这些自然发生的从头开始的调节突变。虽然这是化验的重要证明 潜在的，我们的初步结果未能充分利用询问上下文相关的能力 在大脑的复杂性中发挥作用。在此，我们提出了在体内验证MPRA性能并扩展的工作 这种方法可以生成细胞类型的特定增强子读数。在此过程中，我们将定义监管能力 含有ASD先证者和对照基因组从头开始变异的候选增强子。在目标1中，我们 提出一种以屏幕为中心的方法，使用我们的MPRA来定义细胞类型和等位基因特异的增强子活性。 在目标2中，我们提出了一种以增强子为中心的方法，定义了单个增强子在体内的活性 通过基于图像的分析小鼠出生后的大脑。我们的结果将生成基于函数的评估 自然发生从头开始的调控突变，使得能够对功能定义的增强子进行统计测试 活性与深入的单增强子功能研究同步进行。如果成功，我们的工作将为 在AAV可访问的CNS系统中使用基于功能的疾病相关变体筛查 从ASD WGS数据中产生关于从头开始增强子变体的功能的新见解。