Functional analysis ofESRP1/2gene variants andCTNND1isoforms in orofacial cleft pathogenesis

Abstract

Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulatorsESRP1andESRP2regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Usingesrp1/2mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of humanESRP1/2gene variants. We found that many variants predicted byin silicomethods to be pathogenic were functionally benign.Esrp1also regulates the alternative splicing ofCtnnd1and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression ofctnnd1is sufficient to rescue morphogenesis of epithelial-derived structures inesrp1/2zebrafish mutants. Additionally, we identified 13CTNND1variants from genome sequencing of OFC cohorts, confirmingCTNND1as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement ofEsrp-Ctnnd1acting in the embryonic epithelium to regulate palatogenesis.