IADR Abstract Archives
DNA Methylation and Van der Woude Syndrome Phenotypic Variability
Objectives: Van der Woude Syndrome (VWS) is an autosomal dominant disorder and the most common syndromic form of orofacial clefting. VWS is caused by mutations in IRF6 (70%) and GRHL3 (5%) and classically presents with combinations of lip pits (LP), cleft lip (CL), cleft lip and palate (CLP), or cleft palate (CPO), with markedly phenotypic discordance even amongst individuals that carry the same mutation. Such discordance suggests a possible role for epigenetic factors as phenotypic modifiers. Both IRF6 and TP63 interact in a regulatory loop to coordinate epithelial proliferation and differentiation which are critical for palatogenesis. We hypothesize that differential DNA methylation (DNAm) in CpG sites within regulatory regions of IRF6 and TP63 are associated with VWS phenotypic discordance.
Methods: We measured DNAm levels of CpG sites located in the promoter regions of IRF6 and TP63, and in a known IRF6 enhancer element (MCS9.7) in 83 individuals with VWS grouped within 5 phenotypes: 1=CL/P+LP, 2=CL/P, 3=CPO+LP, 4=CPO, 5=LP. Blood DNA samples were bisulfite converted and pyrosequenced with primers specific to the target regions. We compared the methylation levels of each CpG amongst phenotypes using one-way ANOVA on ranks with the post-hoc Tukey-Kramer test (alpha=0.05).
Results: CpG sites in the promoter region of IRF6 showed statistically significant differences in methylation between phenotypic groups (P<0.05). Individuals with any form of cleft (groups 1-4) had significantly higher mean/median methylation levels than individuals with lip pits only (group 5), while no difference was found among phenotype groups with a cleft (groups 1-4). Significant differences in methylation in the TP63 promoter and IRF6 enhancer were not observed.
Conclusions: Results indicated that hypermethylation of the IRF6 promoter is associated with more severe phenotypes (any cleft +/- lip pits); thus, possibly impacting an already weakened IRF6 protein due to a genetic mutation and leading to a more severe phenotype.
Methods: We measured DNAm levels of CpG sites located in the promoter regions of IRF6 and TP63, and in a known IRF6 enhancer element (MCS9.7) in 83 individuals with VWS grouped within 5 phenotypes: 1=CL/P+LP, 2=CL/P, 3=CPO+LP, 4=CPO, 5=LP. Blood DNA samples were bisulfite converted and pyrosequenced with primers specific to the target regions. We compared the methylation levels of each CpG amongst phenotypes using one-way ANOVA on ranks with the post-hoc Tukey-Kramer test (alpha=0.05).
Results: CpG sites in the promoter region of IRF6 showed statistically significant differences in methylation between phenotypic groups (P<0.05). Individuals with any form of cleft (groups 1-4) had significantly higher mean/median methylation levels than individuals with lip pits only (group 5), while no difference was found among phenotype groups with a cleft (groups 1-4). Significant differences in methylation in the TP63 promoter and IRF6 enhancer were not observed.
Conclusions: Results indicated that hypermethylation of the IRF6 promoter is associated with more severe phenotypes (any cleft +/- lip pits); thus, possibly impacting an already weakened IRF6 protein due to a genetic mutation and leading to a more severe phenotype.