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

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.

Keywords: NSCLP; methylation; non-syndromic cleft lip and cleft palate; twins; whole genome bisulfite sequencing.

FIGURE 1

MZ co-twins are highly correlated for DNA methylation but have site-specific differences. (A) Pearson’s correlation coefficients derived from comparing intra-twin, parent-child and parent to parent (unrelated) methylation levels at 100X (blue) or 30X (brown) genome coverage. (B) Correlation coefficients using only variable CpGs as identified by Feinberg et al. (2010) at 30X genome coverage. (C) Comparison between DNA methylation values for 16,920 individual CpG loci generated by MethylationEPIC microarray and whole-genome bisulfite sequencing. Methylation values generated by Rnbeads for the MethylationEPIC microarray were subtracted from methylation levels calculated from the sequencing data for the same sample (MZ1A to MZ3U). Averaged comparisons contrasting EPIC and WGBS data from unpaired (Unp.) samples is also included (black line). (D) Distribution of average absolute differences in DNA methylation within the MZ twins as well as comparisons of unrelated individuals. A significantly (P<1.2e-10 for WGBS and P<1.9E-12 for EPIC, Kolmogorov–Smirnov test) smaller number of CpG sites with a large average within-twin differences in methylation level was observed in within twin comparisons as contrasted with comparisons of unrelated individuals (unaffected-unaffected, NSCLP-unaffected (inter-twins) or NSCLP-NSCLP).