Functional validation of sequence variants affecting neurodevelopmental and craniofacial phenotypes

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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.