DNA Methylation Effects on Van der Woude Syndrome Phenotypic Variability

Objective Van der Woude Syndrome (VWS) presents with combinations of lip pits (LP) and cleft lip and/or cleft palate (CL/P, CPO). VWS phenotypic heterogeneity even amongst relatives, suggests that epigenetic factors may act as modifiers. IRF6, causal for 70% of VWS cases, and TP63 interact in a regulatory loop coordinating epithelial proliferation and differentiation in palatogenesis. We hypothesize that differential DNA methylation within IRF6 and TP63 regulatory regions underlie VWS phenotypic discordance.Methods DNA methylation of CpG sites in IRF6 and TP63 promoters and in an IRF6 enhancer element was compared amongst blood or saliva DNA samples of 78 unrelated cases. Analyses were done separately for blood and saliva, within each sex and in combination, and to address cleft type (CL/P +/- LP vs. CPO +/- LP) and phenotypic severity (any cleft + LP vs. any cleft only).Results For cleft type, blood samples showed higher IRF6 and TP63 promoter methylation on males with CPO +/- LP compared to CL/P +/- LP and on individuals with CPO +/- LP compared to those with CL/P +/- LP, respectively. Saliva samples showed higher IRF6 enhancer methylation on individuals with CPO +/- LP compared to CL/P +/- LP and contrary to above, lower TP63 promoter methylation on CPO +/- LP compared to CL/P +/- LP. For phenotypic severity, blood samples showed no differences; however, saliva samples showed higher IRF6 promoter methylation in individuals with any cleft + LP compared to those without lip pits.Conclusion We observed differential methylation in IRF6 and TP63 regulatory regions associated with cleft type and phenotypic severity, indicating that epigenetic changes in IRF6 and TP63 can contribute to phenotypic heterogeneity in VWS.