DNA methylation effects on Van der Woude Syndrome phenotypic variability

Abstract

AbstractOBJECTIVEVan der Woude Syndrome (VWS) classically presents with combinations of lip pits (LP) and orofacial clefts, with marked phenotypic discordance even amongst individuals carrying the same mutation. Such discordance suggests a possible role for epigenetic factors as phenotypic modifiers. BothIRF6, causal for 70% of VWS cases, andTP63interact in a regulatory loop to coordinate epithelial proliferation and differentiation for palatogenesis. We hypothesize that differential DNA methylation (DNAm) in CpG sites within regulatory regions ofIRF6andTP63are associated with VWS phenotypic discordance.METHODSWe measured DNAm levels of CpG sites located in the promoter regions ofIRF6andTP63and in anIRF6enhancer element (MCS9.7) in 83 individuals with VWS grouped within 5 phenotypes for primary analysis: 1=CL+/-P+LP, 2=CL+/-P, 3=CP+LP, 4=CP, 5=LP and 2 phenotypes for secondary analysis: 1=any cleft and LP, 2= any cleft without LP. DNA samples were bisulfite converted and pyrosequenced with target-specific primers. Methylation levels were compared amongst phenotypes.RESULTSCpG sites in theIRF6promoter showed statistically significant differences in methylation among phenotypic groups in both analyses (P<0.05). Individuals with any form of cleft (Groups 1-4) had significantly higher methylation levels than individuals with lip pits only (Group 5). In the secondary analysis, individuals in Group 1 (cleft+LP) had significantly higher methylation than Group 2 (cleft only).CONCLUSIONResults indicated that hypermethylation of theIRF6promoter is associated with more severe phenotypes (any cleft +/- lip pits); thus, possibly impacting an already genetically weakenedIRF6protein and leading to a more severe phenotype.