Functional analysis of ESRP1/2 and CTNND1 gene variants in orofacial cleft

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Technical abstract Genetic study of orofacial clefts (OFC) is foundational to genetics of congenital structural birth defects. Most OFC cases are non-syndromic and involve complex genetic mechanisms that are yet to be fully elucidated. Currently, genetic diagnosis for cleft anomalies is hampered by two critical knowledge gaps. First, genes essential for palate formation are incompletely identified. Second, even when the cleft risk genes are associated, algorithms used to impute deleterious from benign gene variants via computational and statistical methods remain unreliable. There is a critical need to translate genome sequencing to clinically actionable data, where functional studies provide the highest-level evidence to impute pathogenicity. We showed that Esrp1 and its paralog Esrp2 (hereafter Esrp1/2) operate in the periderm of mouse and zebrafish to regulate craniofacial development. Esrp1/2 mediates alternative splicing of RNA transcripts, creating epithelial isoforms that function in oral epithelium and periderm. This proposal tests the central hypothesis that Esrp1/2 is required to generate epithelial isoform of Ctnnd1, which maintains periderm integrity necessary for craniofacial morphogenesis. We will impute pathogenicity of ESRP1/2 and CTNND1 human gene variants associated with OFC. Using completed genome sequencing projects and projects in progress, we curate large numbers of ESRP1, ESRP2 and CTNND1 gene variants to ascertain their function. We employ complementary in vivo zebrafish esrp1/2 mutant assay and in vitro Esrp1/2 murine Py2T cell lines to optimize rigor of approach. We will also discover and functionally validate Esrp-regulated genes, using zebrafish epithelial transgenic reporter lines. We discovered that Esrp1/2 regulates alternative splicing of CTNND1 and will functionally interrogate human CTNND1 gene variants in the zebrafish ctnnd1 mutant in a rescue assay. The expected outcome of this project is to gain mechanistic insights by leveraging genome sequencing data associated with orofacial cleft cohorts, to functionally analyze ESRP1/2 and CTNND1 gene variants in craniofacial development. We will also identify and functionally validate Esrp-regulated genes acting in the periderm. This work will have broader impact by elucidating how regulation of RNA alternative splicing and cell signaling mechanisms are important in periderm and craniofacial morphogenesis.