Functional validation of sequence variants affecting neurodevelopmental and craniofacial phenotypes

影响神经发育和颅面表型的序列变异的功能验证

基本信息

批准号：
10701310
负责人：
Victor G. Corces
金额：
$ 72.54万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-26 至 2024-09-25
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10701310
关键词：
3-Dimensional ATAC-seq Affect Affinity Alleles Amino Acids Atrial Heart Septal Defects Behavioral Binding Binding Sites Brain CCCTC-binding factor Cell Differentiation process Cell Line Cell Lineage Cell Nucleus Cell physiology Cells Cellular Assay Cerebrum Chromatin Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Craniofacial Abnormalities DNA DNA Binding DNA Sequence DNA-Binding Proteins Defect Development Developmental Delay Disorders Developmental Process Disease Distant Embryo Enhancers Face Fingers Gene Expression Gene Expression Alteration Gene Expression Profile Genes Genetic Transcription Genome Genomics Goals Heart Abnormalities Impaired cognition Individual Intellectual functioning disability Lead Location Mediating Methods Microcephaly Modeling Molecular Mouse Strains Mutation Nature Neural Crest Neural Crest Cell Neurons Neurophysiology - biologic function Organoids Outcome Pathogenicity Patients Pattern Phenotype Property Protein Region Proteins RNA Role Severities Site Skeleton Structure Suggestion Symptoms Time Tissues Tooth structure Validation Variant Vertebral column Work base brain cell cell motility cell type cohesin craniofacial experimental study gene interaction human disease in vivo individual patient induced pluripotent stem cell inorganic phosphate insight model development mouse model mutant neurodevelopment promoter protein function repaired residence single cell technology transcription factor transcriptome sequencing

3-Dimensional ATAC-seq Affect Affinity Alleles Amino Acids Atrial Heart Septal Defects Behavioral Binding Binding Sites Brain CCCTC-binding factor Cell Differentiation process Cell Line Cell Lineage Cell Nucleus Cell physiology Cells Cellular Assay Cerebrum Chromatin Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Craniofacial Abnormalities DNA DNA Binding DNA Sequence DNA-Binding Proteins Defect Development Developmental Delay Disorders Developmental Process Disease Distant Embryo Enhancers Face Fingers Gene Expression Gene Expression Alteration Gene Expression Profile Genes Genetic Transcription Genome Genomics Goals Heart Abnormalities Impaired cognition Individual Intellectual functioning disability Lead Location Mediating Methods Microcephaly Modeling Molecular Mouse Strains Mutation Nature Neural Crest Neural Crest Cell Neurons Neurophysiology - biologic function Organoids Outcome Pathogenicity Patients Pattern Phenotype Property Protein Region Proteins RNA Role Severities Site Skeleton Structure Suggestion Symptoms Time Tissues Tooth structure Validation Variant Vertebral column Work base brain cell cell motility cell type cohesin craniofacial experimental study gene interaction human disease in vivo individual patient induced pluripotent stem cell inorganic phosphate insight model development mouse model mutant neurodevelopment promoter protein function repaired residence single cell technology transcription factor transcriptome sequencing

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项目摘要

ABSTRACT Mutations in the CTCF gene result in CTCF-Related Disorders (CRD), a group of conditions characterized by neurodevelopmental delays, intellectual disability, and digestive, cardiac, and craniofacial abnormalities. These phenotypes can be attributed to alterations in the differentiation or function of cells of the brain or cells of the neural crest. The CTCF protein is involved in the establishment of the 3D organization of the chromatin in the nucleus by interfering with cohesin extrusion, which results in the formation of stable loops between distant sites in the genome. Through this organization, CTCF modulates interactions between regulatory sequences and their cognate promoters. Consequently, disruption of CTCF function may lead to alterations of gene expression during development and defects in cell lineage specification. This application is based on the hypothesis that functional analyses of different CTCF mutations present in patients with CRD will give important insights into the mechanisms by which these pathogenic variants affect gene expression during cell differentiation, and how these alterations lead to the variety of phenotypes observed in CRD patients. CTCF variants found in these patients are located in regions of the protein important for different aspects of CTCF function. This includes DNA sequence recognition, affinity for the DNA binding motif, interactions with RNA to stabilize binding at a subset of genomic sites, non-specific interactions with the DNA phosphate backbone that may affect CTCF residence time on DNA, and interactions with cohesin that may affect loop formation or stability. We therefore hypothesize that variants affecting these different functions are involved in targeting CTCF to specific genomic locations or its interaction with cohesin to elicit distinct altered patterns of gene expression. This in turn may affect cell function or differentiation into specific lineages to cause variant-specific phenotypes in CRD patients. Using these mutations, which affect single amino acids, we thus propose to examine correlations between patient phenotypes and different aspects of CTCF function. We will use patient-derived iPSCs, cerebral organoids, induced neural crest cells (iNCCs), and single-cell technologies to gain insights into the molecular and cellular processes altered by specific CTCF variants in the brain and neural crest. We will also use mouse models carrying the same mutations to determine the effects of these CTCF variants on brain and neural crest development in vivo and compare these results with those obtained using iNCCs and cerebral organoids. Results from this work will fill an important gap in our understanding of the fundamental principles by which alterations of different aspects of 3D chromatin organization result in human disease with different phenotypic outcomes.

抽象的 CTCF基因的突变导致CTCF相关疾病（CRD），这是一组表征的疾病通过神经发育延迟，智力残疾以及消化，心脏和颅面异常。这些表型可以归因于大脑细胞分化或功能的改变或神经rest的细胞。 CTCF蛋白参与建立3D组织通过干扰粘着蛋白的挤出，细胞核中的染色质，从而导致稳定环的形成在基因组中的遥远位点之间。通过该组织，CTCF调节调节序列及其同源启动子。因此，CTCF功能的破坏可能导致在发育过程中基因表达的改变和细胞谱系规范中的缺陷。此应用程序基于以下假设：在患者患者中存在不同CTCF突变的功能分析 CRD将对这些致病性变异的机制有重要见解细胞分化过程中的表达以及这些改变如何导致观察到的多种表型在CRD患者中。在这些患者中发现的CTCF变体位于蛋白质的区域中 CTCF功能的不同方面。这包括DNA序列识别，对DNA结合的亲和力基序，与RNA的相互作用以稳定基因组位点的子集的结合，非特异性相互作用与可能影响DNA上CTCF停留时间的DNA磷酸主链，并与粘蛋白相互作用这可能会影响循环形成或稳定性。因此，我们假设影响这些不同的变体功能涉及将CTCF靶向特定的基因组位置或其与粘着素的相互作用以引起基因表达的明显改变的模式。反过来，这可能会影响细胞功能或分化为特定谱系在CRD患者中引起变异特异性表型。使用这些突变，影响单个突变因此，我们建议检查患者表型与不同方面之间的相关性 CTCF功能。我们将使用患者来源的IPSC，脑器官，诱导的神经rest细胞（INCC），和单细胞技术，以洞悉特定的分子和细胞过程大脑和神经rest中的CTCF变体。我们还将使用带有相同突变的鼠标模型确定这些CTCF变体对体内大脑和神经rest发育的影响，并比较这些结果是使用INCC和大脑器官获得的结果。这项工作的结果将填补我们了解基本原则的重要差距 3D染色质组织导致人类疾病具有不同的表型结果。