Nonlinear gene expression‐phenotype relationships contribute to variation and clefting in the A/WySn mouse

Background Cleft lip and palate is one of the most common human birth defects, but the underlying etiology is poorly understood. The A/WySn mouse is a spontaneously occurring model of multigenic clefting in which 20% to 30% of individuals develop an orofacial cleft. Recent work has shown altered methylation at a specific retrotransposon insertion downstream of the Wnt9b locus in clefting animals, which results in decreased Wnt9b expression. Results Using a newly developed protocol that allows us to measure morphology, gene expression, and DNA methylation in the same embryo, we relate gene expression in an individual embryo directly to its three‐dimensional morphology for the first time. We find that methylation at the retrotransposon relates to Wnt9b expression and morphology. IAP methylation relates to shape of the nasal process in a manner consistent with clefting. Embryos with low IAP methylation exhibit increased among‐individual variance in facial shape. Conclusions Methylation and gene expression relate nonlinearly to nasal process morphology. Individuals at one end of a continuum of phenotypic states display a clinical phenotype and increased phenotypic variation. Variable penetrance and expressivity in this model is likely determined both by among‐individual variation in methylation and changes in phenotypic robustness along the underlying liability distribution for orofacial clefting. Key Findings The A/WySn model allows us to study how variable penetrance cleft lip and palate may arise in a stochastic manner. Loss of methylation at the Wnt9bIAP locus relates to changes in Wnt9b gene expression and non‐linearly to changes in mean phenotype. The changes in Wnt9b alone are not sufficient to explain how the loss of methylation leads to clefting Phenotypic variance increases as methylation levels decrease, implying a possible explanation for the variable expressivity in clefting.