Dominant mutations in GRHL3 cause Van der Woude syndrome and disrupt oral periderm development. M. Peyrard1, E. J. Leslie2, Y. A. Kousa3, T. L. Smith4, M. Dunnwald2, M. Magnusson5, B. A. Lentz2, P. Unneberg6, I. Fransson1, H. K. Koillinen7, J. Rautio8, M. Pegelow9, A. Karsten9, L. Basel-Vanagaite10,11, W. Gordon12, B. Andersen12, T. Svensson5, J. C. Murray2, R. A. Cornell4, J. Kere1,5,13, BC. Schutte14 1) Department of Biosciences and Nutrition, Karolinska Institutet, and Center for Biotechnology, 14183 Huddinge, Sweden; 2) Department of Pediatrics and Interdisciplinary Program in Genetics, University of Iowa, Iowa City 52242, Iowa, USA; 3) Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing 48824, Michigan, USA; 4) Department of Anatomy and Cell Biology, University of Iowa, Iowa City 52242, Iowa, USA; 5) Department of Biosciences and Nutrition, Science for Life Laboratory, Karolinska Institutet, 17121 Solna, Sweden; 6) Department of Biochemistry and Biophysics Science for Life Laboratory, Stockholm University, 17121 Solna, Sweden; 7) Department of Clinical Genetics, Helsinki University Hospital, 00029 Helsinki, Finland; 8) Cleft Palate and Craniofacial Center, Department of Plastic Surgery, Helsinki University Hospital, 00029 Helsinki, Finland; 9) Department of Orthodontics, Stockholm Craniofacial Team, Institute of Odontology, Karolinska Institutet, 17177 Stockholm, Sweden; 10) Pediatric Genetics, Schneider Children's Medical Center of Israel and Raphael Recanati Genetic Institute, Rabin Medical Center, Petah Tikva 49100, Israel; 11) Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; 12) Department of Biological Chemistry, University of California Irvine, Irvine 92697, California, USA; 13) Research Programs Unit, University of Helsinki, and Folkhälsan Institute of Genetics, 00014 Helsinki, Finland; 14) Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing 48824, Michigan, USA.

   Van der Woude syndrome (VWS) is an autosomal dominant form of cleft lip and palate (CL/P). About 70% of VWS cases are caused by mutations in the gene encoding for the transcription factor Interferon Regulatory Factor 6 (IRF6). Because IRF6 is essential for development of the oral periderm in mouse and zebrafish, we hypothesized that a subset of the remaining 30% of VWS cases are caused by mutations in other genes regulating periderm development. Supporting locus heterogeneity in VWS, one large pedigree where the proband has lip pits, the hallmark of VWS, showed linkage to a locus on 1p33-p36 (VWS2) rather than to IRF6 at 1q32-q41. From this pedigree, we selected 8 affected individuals, including the proband, and 3 healthy individuals, for whole-exome sequencing of the ~700 genes in this linkage interval. We identified a likely causative mutation, F324Lfs, in the Grainy head-like 3 (GRHL3) gene. GRHL3 is a transcription factor with a deeply conserved role in formation of epithelial barriers. We also found likely disease-causing mutations in GRHL3 in seven additional families with VWS, of variable ethnicity and where no causative IRF6 mutations had been detected. While we observed no consistently unique phenotypes in these families, individuals with a GRHL3 mutation are more likely to have cleft palate and less likely to have cleft lip or lip pits than individuals with an IRF6 mutation. In a zebrafish-based assay, we found that all five human GRHL3 variants tested, F324Lfs included, disrupted periderm development during gastrulation. These results suggest that VWS is caused by dominant-negative alleles of GRHL3, in contrast to haploinsufficiency of IRF6 seen in most cases. Using murine models, we observed that Grhl3-/- embryos, similar to Irf6-/- embryos, have oral epithelial adhesions, a phenotype seen occasionally in human VWS, and exhibit abnormal development of the oral periderm. Finally, the oral phenotype of double heterozygous (Irf6+/-; Grhl3+/-) murine embryos is consistent with an additive rather than a synergistic interaction between the two genes, suggesting that Irf6 and Grhl3 function in separate but converging pathways in periderm development. Together our data demonstrate that mutations in two genes, IRF6 and GRHL3, lead to nearly identical phenotypes of CL/P, and support the hypothesis that failure of oral periderm development contributes to VWS. This work will increase the specificity and sensitivity of clinical diagnostics for VWS.

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