DNA Methylation Variation Is Identified in Monozygotic Twins Discordant for Non-syndromic Cleft Lip and Palate.

Juan I Young,Jacqueline T Hecht,Susan Slifer,Susan H Blanton

doi:10.3389/fcell.2021.656865

Abstract

Non-syndromic cleft lip with or without cleft palate (NSCLP) is the most common craniofacial birth defect. The etiology of NSCLP is complex with multiple genes and environmental factors playing causal roles. Although studies have identified numerous genetic markers associated with NSCLP, the role of epigenetic variation remains relatively unexplored. Because of their identical DNA sequences, monozygotic (MZ) twins discordant for NSCLP are an ideal model for examining the potential contribution of DNA methylation to non-syndromic orofacial clefting. In this study, we compared the patterns of whole genome DNA methylation in six MZ twin pairs discordant for NSCLP. Differentially methylated positions (DMPs) and regions (DMRs) were identified in NSCLP candidate genes, including differential methylation in MAFB and ZEB2 in two independent MZ twin pairs. In addition to DNA methylation differences in NSCLP candidate genes, we found common differential methylation in genes belonging to the Hippo signaling pathway, implicating this mechanosensory pathway in the etiology of NSCLP. The results of this novel approach using MZ twins discordant for NSCLP suggests that differential methylation is one mechanism contributing to NSCLP, meriting future studies on the role of DNA methylation in familial and sporadic NSCLP.

Highlights

Non-syndromic cleft lip with and without cleft palate (NSCLP) is a common birth defect occurring in 1/700-1000 livebirths, affecting 135,000 newborns worldwide
Methylome analysis was performed on saliva samples from six sets of male MZ twins discordant for NSCLP using whole-genome bisulfite sequencing (WGBS)
The mean methylation level correlation between Whole genome bisulfite sequencing (WGBS) and the microarray determinations for the six samples was r = 0.90 and, on average, 97.63% of loci have delta methylation levels < | 0.2|, indicating that the methylation values derived from the sequencing and the array methods are consistent across commonly interrogated loci

Summary

Introduction

Non-syndromic cleft lip with and without cleft palate (NSCLP) is a common birth defect occurring in 1/700-1000 livebirths, affecting 135,000 newborns worldwide. While candidate gene and DNA Methylation in Cleft/Lip Palate genome-wide studies of case-control and family-based datasets have identified causal/susceptibility loci and variants, the majority of the genetic causes remain elusive. Based on the analysis of multiplex families, in which there are multiple, but not necessarily first degree, affected relatives, there are likely extra-genic factors affecting penetrance and/or expression of the genetic liability. It is likely that variation in multiple genes is etiologically necessary, with extra-genic factors contributing with a threshold effect

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Results

Conclusion