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: 10357952
• 负责人: Alexander Nord
• 金额: $ 23.55万
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357952
• 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; Proteins; Protocols documentation; Regulation; Regulatory Element; Reporter; Reporter Genes; Research; Resolution; Role; Sampling; Scientist; Siblings; Specificity; Structure; 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.

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.