Functional validation of sequence variants affecting neurodevelopmental and craniofacial phenotypes

摘要 CTCF基因突变导致CTCF相关疾病(CRD)，这是一组特征为 由神经发育迟缓、智力残疾以及消化、心脏和颅面畸形所致。 这些表型可归因于脑细胞分化或功能的改变或 神经脊的细胞。CTCF蛋白参与了3D组织的建立 核内的染色质通过干扰粘附素的挤出而导致稳定环的形成 在基因组中遥远的位置之间。通过这个组织，CTCF调节了 调控序列及其同源启动子。因此，CTCF功能的中断可能会导致 发育过程中基因表达的变化和细胞谱系特征的缺陷。此应用程序 是基于一种假设，即不同CTCF突变的功能分析存在于慢性粒细胞白血病患者中 CRD将为这些致病变异影响基因的机制提供重要的见解 在细胞分化过程中的表达，以及这些变化是如何导致观察到的表型变化的 在CRD患者中。在这些患者中发现的CTCF变异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、对DNA结合的亲和力 基序，与RNA的相互作用以稳定在基因组部位的子集的结合，非特异性相互作用 DNA磷酸骨架可能影响CTCF在DNA上的滞留时间，以及与粘附素的相互作用 这可能会影响环路的形成或稳定性。因此我们假设，影响这些不同的变种 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以诱导 基因表达的不同变化模式。这反过来可能会影响细胞功能或分化为特定的 导致CRD患者变异特异性表型的谱系。使用这些突变，它会影响单个 氨基酸，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IPSCs、脑器官、诱导神经脊细胞(INCC)， 和单细胞技术，以深入了解由特定基因改变的分子和细胞过程 大脑和神经脊中的CTCF变体。我们还将使用携带相同突变的小鼠模型 确定这些CTCF变异体在体内对脑和神经脊发育的影响并进行比较 这些结果与使用iNCCs和脑器官获得的结果一致。这项工作的结果将填补一个 在我们对基本原则的理解上的重大差距，根据这些原则，不同方面的变化 3D染色质组织导致人类疾病，具有不同的表型结果。

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.