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

• 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
• 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

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.

摘要 CTCF基因的突变导致CTCF相关疾病（CRD），这是一组特征为 神经发育迟缓、智力残疾、消化系统、心脏和颅面畸形。 这些表型可归因于脑细胞分化或功能的改变， 神经嵴的细胞CTCF蛋白参与了细胞3D结构的建立， 通过干扰粘着蛋白的挤出，从而导致稳定环的形成， 在基因组中遥远的位点之间。通过这个组织，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 发育过程中基因表达的改变和细胞谱系特化的缺陷。本申请 基于以下假设，即对存在于患有以下疾病的患者中的不同CTCF突变进行功能分析： CRD将对这些致病性变异影响基因表达的机制提供重要的见解。 细胞分化过程中的表达，以及这些变化如何导致观察到的各种表型 在CRD患者中。在这些患者中发现的CTCF变体位于蛋白质的重要区域， 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的相互作用以稳定在基因组位点的子集处的结合，与 可能影响CTCF在DNA上停留时间的DNA磷酸骨架，以及与粘附素的相互作用 这可能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附素的相互作用， 基因表达模式的明显改变。这反过来又可能影响细胞功能或分化成特定的细胞。 在CRD患者中引起变体特异性表型的谱系。利用这些突变， 因此，我们建议检查患者表型和不同方面的相关性， CTCF功能。我们将使用患者来源的iPSC、脑类器官、诱导的神经嵴细胞（iNCC）， 和单细胞技术，以深入了解由特定的细胞因子改变的分子和细胞过程。 脑和神经嵴中的CTCF变体。我们还将使用携带相同突变的小鼠模型 为了确定这些CTCF变体对体内脑和神经嵴发育的影响， 这些结果与使用iNCC和脑类器官获得的结果一致。这项工作的结果将填补一个 我们对基本原则的理解存在重大差距， 3D染色质组织导致具有不同表型结果的人类疾病。