Epigenetic Signatures in Monozygotic and Dizygotic Twins Discordant for Orofacial Clefts

A. L. Petrin; H. L. Keen; L. Dunlay; X. J. Xie; E. Zeng; A. Butali; A. Wilcox; M. L. Marazita; J. C. Murray; L. M. Moreno-Uribe

doi:10.64898/2026.04.07.26350251

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Epigenetic Signatures in Monozygotic and Dizygotic Twins Discordant for Orofacial Clefts

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Epigenetic Signatures in Monozygotic and Dizygotic Twins Discordant for Orofacial Clefts

A. L. Petrin, H. L. Keen, L. Dunlay, X. J. Xie, E. Zeng, A. Butali, A. Wilcox, M. L. Marazita, J. C. Murray and L. M. Moreno-Uribe

medRxiv

Cold Spring Harbor Laboratory Preprints

04/08/2026

DOI: 10.64898/2026.04.07.26350251

PMCID: PMC13086122

PMID: 42006789

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https://doi.org/10.64898/2026.04.07.26350251View

Preprint (Author's original) This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

Introduction Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital malformation with complex etiology involving both genetic and environmental factors. Epigenetic mechanisms may mediate environmental contributions, but separating genetic from environmental effects remains challenging. Methods We present an epigenome-wide association study with 32 monozygotic and 22 dizygotic twin pairs discordant for NSCL/P on blood and saliva samples. Differential methylation analysis was conducted using linear models to identify CpG sites showing significant methylation differences between affected and unaffected twins followed by functional annotation and pathway enrichment analysis. Results The top-ranked finding is a differentially methylated region comprising two CpG sites at the CYP26A1 locus, cg12110262 (P = 3.21x10-7) and cg15055355 (P = 1.39x10-3). CYP26A1 is essential for retinoic acid catabolism and craniofacial patterning. The chromatin regulator ANKRD11, which causes KBG syndrome featuring cleft palate was the second best hit. Differentially methylated CpG sites showed significant enrichment in craniofacial enhancers and overlap with multiple GWAS-validated cleft genes including VAX1, PVRL1, SMAD3, and PRDM16. Conclusions Our findings implicate retinoic acid signaling and chromatin regulation in NSCL/P etiology and demonstrate the value of discordant twin designs for distinguishing environmental from genetic epigenetic contributions to complex malformations.

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Title: Subtitle: Epigenetic Signatures in Monozygotic and Dizygotic Twins Discordant for Orofacial Clefts
Creators: A. L. Petrin - University of Iowa
H. L. Keen - University of Iowa, Iowa Institute of Human Genetics
L. Dunlay - University of Iowa
X. J. Xie - Florida State University
E. Zeng - University of Iowa
A. Butali - University of Iowa
A. Wilcox - National Institute of Environmental Health Sciences
M. L. Marazita - University of Pittsburgh
J. C. Murray - University of Iowa, Craniofacial Anomalies Research Center
L. M. Moreno-Uribe - University of Iowa, Craniofacial Anomalies Research Center
Resource Type: Preprint
Publication Details: medRxiv
DOI: 10.64898/2026.04.07.26350251
PMID: 42006789
PMCID: PMC13086122
Publisher: Cold Spring Harbor Laboratory Preprints
Language: English
Date posted: 04/08/2026
Academic Unit: Preventive and Community Dentistry; Orthodontics; Oral Pathology, Radiology and Medicine; Anatomy and Cell Biology; Stead Family Department of Pediatrics; Epidemiology; Iowa Neuroscience Institute; Biostatistics; Pediatric Dentistry; Craniofacial Anomalies Research Center; Dental Research; Iowa Institute of Human Genetics
Record Identifier: 9985153548302771

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