E. Li1,2,3, B. Garrity1,2,3, D. Truong1,2,3, K. Mukherjee1,2,3, E. C. Liao1,2,3 1Massachusetts General Hospital,Boston, MA, USA 2Harvard Medical School,Boston, MA, USA 3Harvard Stem Cell Institute,Cambridge, MA, USA
Introduction:
Cleft lip and/or palates (CL/P) are among the most common congenital malformations. Mutations in the transcription factor gene IRF6 represent the most significant genetic contributors to orofacial clefts. Hundreds of novel sequence variations in IRF6 have been reported. However, the functional significances of the myriad of IRF6 mutations remain difficult to ascertain as in silico prediction programs have poor predictive power and often provide conflicting results. To address this gap in knowledge, we hypothesized that the protein function of IRF6 gene variants could be rapidly assessed by their ability to rescue the irf6-/- mutant phenotype in animal models.
Methods:
CRISPR genome editing was used to generate irf6-/- zebrafish. Previously uncharacterized IRF6 variants were identified and categorized by computational predictions from Polyphen2 and SIFT. Variants were introduced to wildtype irf6 cDNA by site-directed mutagenesis; mRNA was produced by in vitro transcription and microinjected into irf6-/- embryos to assess for phenotypic and functional rescue.
Results:
All offspring of irf6-/- females displayed an embryonic rupture phenotype caused by defects in the embryonic periderm, a simple squamous epithelium surrounding the embryo that models the human oral epithelium. The periderm rupture model is validated for use in the analysis of human IRF6 function, as injection of human IRF6 mRNA into mutant zebrafish embryos can fully rescue the mutant phenotype. Human IRF6 gene variant mRNAs were then injected in a rapid and accurate rescue assay to assess the effects of human IRF6 mutations on protein function.
The IRF6 gene variant rescue results revealed that neither location of the mutations within the IRF6 protein nor computational programs were accurate predictors of whether observed human IRF6 mutations actually resulted in defective proteins. Several variants predicted to be benign failed to rescue, while others predicted to be deleterious by both computational programs were able to rescue not only periderm rupture, but also normal craniofacial development.
Conclusion:
The zebrafish irf6-/- rescue assay rapidly addressed the functional significance of novel IRF6 mutations and overcame computational limitations for predicting variant pathogenicity. These validated biological results can help improve future computational predictions, and moreover, unlock the possibility for rapidly characterizing yet undiscovered IRF6 variants from orofacial cleft patients, illustrating a generalizable functional genomics paradigm of personalized medicine.
