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

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 基因突变会导致 CTCF 相关疾病 (CRD)，这是一组以神经发育迟缓、智力障碍以及消化、心脏和颅面异常。这些表型可归因于大脑或细胞的分化或功能的改变。神经嵴细胞。 CTCF蛋白参与3D组织的建立通过干扰粘连蛋白挤出来破坏细胞核中的染色质，从而形成稳定的环基因组中相距较远的位点之间。通过这个组织，CTCF 调节了之间的互动调控序列及其同源启动子。因此，CTCF 功能的破坏可能会导致发育过程中基因表达的改变和细胞谱系规范的缺陷。这个应用程序基于以下假设：对患有以下疾病的患者中存在的不同 CTCF 突变进行功能分析： CRD 将为了解这些致病变异影响基因的机制提供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表型变化 CRD 患者。在这些患者中发现的 CTCF 变异位于对生命至关重要的蛋白质区域。 CTCF 功能的不同方面。这包括 DNA 序列识别、DNA 结合亲和力基序、与 RNA 相互作用以稳定在基因组位点子集上的结合、与 DNA 磷酸主链可能会影响 CTCF 在 DNA 上的停留时间以及与粘连蛋白的相互作用这可能会影响环的形成或稳定性。因此，我们假设影响这些不同的变异功能涉及将 CTCF 靶向特定的基因组位置或其与粘连蛋白的相互作用以引发基因表达的明显改变模式。这反过来可能会影响细胞功能或分化为特定的谱系在 CRD 患者中引起变异特异性表型。使用这些影响单个氨基酸，因此我们建议检查患者表型和不同方面之间的相关性 CTCF 功能。我们将使用患者来源的 iPSC、脑类器官、诱导神经嵴细胞 (iNCC)、和单细胞技术，以深入了解特定因素改变的分子和细胞过程大脑和神经嵴中的 CTCF 变异。我们还将使用携带相同突变的小鼠模型确定这些 CTCF 变体对体内大脑和神经嵴发育的影响并进行比较这些结果与使用 iNCC 和大脑类器官获得的结果相比较。这项工作的结果将填补我们对改变事物不同方面的基本原则的理解存在重大差距 3D 染色质组织导致人类疾病具有不同的表型结果。