Functional analysis of gene variants and isoforms in orofacial cleft pathogenesis.

Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators and regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human gene variants. We found that many variants predicted by methods to be pathogenic were functionally benign. also regulates the alternative splicing of and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of is sufficient to rescue morphogenesis of epithelial-derived structures in zebrafish mutants. Additionally, we identified 13 variants from genome sequencing of OFC cohorts, confirming as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of acting in the embryonic epithelium to regulate palatogenesis.