Thesis
DNA methylation effects on Van der Woude Syndrome phenotypic variability
University of Iowa
Master of Science (MS), University of Iowa
Spring 2026
DOI: 10.25820/etd.008436
Abstract
BACKGROUND/OBJECTIVE: 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%), GRHL3 (5%), and PRKCI (2-3%) and classically presents with combinations of lip pits (LP), cleft lip (CL), cleft lip and palate (CLP), or cleft palate (CP), with markedly phenotypic discordance even amongst individuals carrying the same mutation, suggesting a role for epigenetic factors as phenotypic modifiers. IRF6, GRHL3, and TP63 interact during craniofacial development to coordinate epithelial proliferation and differentiation. We hypothesize that changes in DNA methylation (DNAm) within regulatory regions of IRF6, GRHL3 and TP63 are associated with VWS phenotypic variability.
METHODS: We used pyrosequencing to measure DNAm levels of cpg sites located in the promoters of IRF6, GRHL3, and TP63, and in a known IRF6 enhancer element (MCS9.7) in 141 individuals with VWS (Blood DNA=104; Saliva DNA=37) grouped within 5 phenotypes: 1=CL/P+LP, 2=CL/P, 3=CP+LP, 4=CP, 5=LP. Secondary analyses were conducted based on cleft type: Group I=CL/P +/- LP and Group II=CP +/- LP; and by the presence of lip pits: Group III=Any cleft + LP and Group IV=Any cleft only. DNAm levels at each site were compared using one-way ANOVA followed by the post-hoc Tukey-Kramer test (alpha=0.05). RESULTS: Significant DNAm differences were observed in saliva samples in two cpg sites located in IRF6 promoter (p=0.015 and p=0.024). Post-hoc analyses further revealed that individuals with CP+LP phenotype (Group 3) had higher DNAm compared to individuals with LP alone(Group 5) (p=0.0096) and compared to individuals with CP alone (Group 4)(p=0.038). Significant DNAm differences were observed in saliva samples in one cpg site (p=0.009) and blood samples in 2 cpg sites (p=0.033 and p=0.018) in the IRF6 enhancer, MCS9.7. Post-hoc analyses further revealed that in saliva samples, individuals with CP (Group 4) had higher DNAm compared to individuals with CL/P + LP (Group 1)(p=0.012), individuals with Any cleft (Group IV) had higher DNAm compared to individuals with Any cleft + LP (Group III)(p=0.022), and individuals with CP +/- LP (Group II) had higher DNAm compared to individuals with CL/P +/- LP (Group I)(p=0.028). In blood samples, individuals with CL/P + LP (Group 1) and CP + LP (Group 3) had higher DNAm compared to individuals with CL/P (Group 2)(p=0.012, p=0.006) and (p=0.027); and individuals with Any cleft + LP (Group III) had higher DNAm compared to individuals with Any cleft only (Group IV)(p=0.008 and p=0.004). For GRHL3, significant DNAm differences were observed in blood samples in three cpg sites. Post-hoc analyses further revealed that individuals with CP +/- LP (Group II) had higher DNAm compared to individuals with CL/P +/- LP (Group I)(p=0.044 and p=0.048). No significant DNAm differences were observed in TP63.
CONCLUSION: Our findings indicate that hypermethylation of the IRF6 promoter is associated with a more severe phenotype (CP+LP) compared to LP only and CP only in saliva samples. Differential methylation at the MCS9.7 enhancer of IRF6 distinguishes orofacial cleft subtypes, with markedly higher CpG1 methylation in CPO versus CL/P in saliva and opposite directional patterns compared to promoter methylation, indicating complex, site-specific regulation. In blood, consistent hypermethylation at adjacent CpG2/3 sites in individuals with lip pits suggests a reproducible, systemic epigenetic signature independent of cleft type. Results indicated that hypermethylation of GRHL3 is more associated with cleft palate (CP +/- LP) compared with cleft lip with or without cleft palate (CL/P +/- LP) in blood samples We demonstrate an epigenetic contribution to VWS clinical variability and provide key findings to understand disease expressivity that can ultimately inform genetic counseling for affected families.
Keywords: Van der Woude Syndrome, Orofacial Clefts, Cleft lip, Cleft palate, DNA methylation, epigenetics, phenotypic variability
Details
- Title: Subtitle
- DNA methylation effects on Van der Woude Syndrome phenotypic variability
- Creators
- Amanda M. Seaberg
- Contributors
- Aline L Petrin (Advisor)Lina M Moreno Uribe (Committee Member)Kyungsup Shin (Committee Member)Azeez Butali (Committee Member)Fang Qian (Committee Member)
- Resource Type
- Thesis
- Degree Awarded
- Master of Science (MS), University of Iowa
- Degree in
- Orthodontics
- Date degree season
- Spring 2026
- DOI
- 10.25820/etd.008436
- Publisher
- University of Iowa
- Number of pages
- xi, 43 pages
- Copyright
- Copyright 2026 Amanda M Seaberg
- Language
- English
- Date submitted
- 04/21/2026
- Description illustrations
- Illustrations, graphs, charts, tables
- Description bibliographic
- Includes bibliographical references (pages 37-43).
- Public Abstract (ETD)
BACKGROUND/OBJECTIVE: Van der Woude Syndrome (VWS) is an autosomal dominant disorder and the most common syndromic form of orofacial clefting, with identified causal mutations in IRF6, GRHL3, and PRKCI. Despite shared mutations, individuals show marked phenotypic variability, suggesting epigenetic modifiers. We hypothesize that differential DNA methylation (DNAm) in regulatory regions of IRF6, GRHL3, and TP63 contributes to this variability.
METHODS: DNAm was quantified by pyrosequencing in 141 individuals with VWS (Blood DNA=104; Saliva DNA=37) across phenotypic groups based on cleft type and presence of lip pits. One-way ANOVA with post-hoc Tukey-Kramer tests (α=0.05) was used.
RESULTS: Significant DNAm differences were identified in IRF6 promoter CpG sites in saliva, with higher methylation in individuals with cleft palate and lip pits (CP+LP) compared to lip pits alone and cleft palate alone. In the IRF6 enhancer (MCS9.7), DNAm differences distinguished cleft subtypes across both saliva and blood, with consistent patterns linking lip pits to increased methylation. In blood, individuals with lip pits showed higher enhancer methylation than those without. Additionally, hypermethylation at GRHL3 CpG sites in blood was associated with cleft palate compared to cleft lip with/without palate. No significant differences were observed for TP63.
CONCLUSION: Hypermethylation of IRF6 regulatory regions is associated with more severe VWS phenotypes and distinguishes cleft subtypes, while GRHL3 methylation further differentiates cleft palate presentations. These findings support a role for site-specific epigenetic regulation in VWS phenotypic variability and may inform genetic counseling.
- Academic Unit
- Orthodontics
- Record Identifier
- 9985176975402771
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