Functional Characterization of a Novel IRF6 Frameshift Mutation From a Van Der Woude Syndrome Family.

Background Loss-of-function mutations in interferon regulatory factor-6 (IRF6) are responsible for about 70% of cases of Van Der Woude Syndrome (VWS), an autosomal dominant developmental disorder characterized by pits and/or sinuses of the lower lip and cleft lip, cleft palate, or both. Methods We collected a Chinese Han VWS pedigree, performed sequencing and screening for the causal gene mutant. Initially, species conservation analysis and homology protein modeling were used to predict the potential pathogenicity of mutations. To test whether a VWS family-derived mutant variant ofIRF6retained function, we carried out rescue assays inirf6maternal-null mutant zebrafish embryos. To assess protein stability, we overexpressed reference and family-variants of IRF6in vitro. Results We focused on a VWS family that includes a son with bilateral lip pits, uvula fissa and his father with bilateral cleft lip and palate. After sequencing and screening, a frameshift mutation ofIRF6was identified as the potential causal variant (NM.006147.3, c.1088-1091delTCTA; p.Ile363ArgfsTer33). The residues in this position are strongly conserved among species and homology modeling suggests the variant alters the protein structure. Inirf6maternal-null mutant zebrafish embryos the periderm differentiates abnormally and the embryos rupture and die during gastrulation. Injection of mRNA encoding the reference variant of human IRF6, but not of the frame-shift variant, rescued such embryos through gastrulation. Upon overexpression in HEK293FT cells, the IRF6 frame-shift mutant was relatively unstable and was preferentially targeted to the proteasome in comparison to the reference variant. Conclusion In this VWS pedigree, a novel frameshift ofIRF6was identified as the likely causative gene variant. It is a lost function mutation which could not rescue abnormal periderm phenotype inirf6maternal-null zebrafish and which causes the protein be unstable through proteasome-dependent degradation.