Distinct DNA methylation profiles in subtypes of orofacial cleft

Abstract

Background: Epigenetic data could help identify risk factors for orofacial clefts, either by revealing a causal role for epigenetic mechanisms in causing clefts or by capturing information about causal genetic or environmental factors. Given the evidence that different subtypes of orofacial cleft have distinct aetiologies, we explored whether children with different cleft subtypes showed distinct epigenetic profiles.

Methods: In whole-blood samples from 150 children from the Cleft Collective cohort study, we measured DNA methylation at over 450,000 sites on the genome. We then carried out epigenome-wide association studies (EWAS) to test the association between methylation at each site and cleft subtype (cleft lip only (CLO) n = 50; cleft palate only (CPO) n = 50; cleft lip and palate (CLP) n = 50). We also compared methylation in the blood to methylation in the lip or palate tissue using genome-wide data from the same 150 children and conducted an EWAS of CLO compared to CLP in lip tissue.

Results: We found four genomic regions in blood differentially methylated in CLO compared to CLP, 17 in CPO compared to CLP and 294 in CPO compared to CLO. Several regions mapped to genes that have previously been implicated in the development of orofacial clefts (for example, TBX1, COL11A2, HOXA2, PDGFRA), and over 250 associations were novel. Methylation in blood correlated with that in lip/palate at some regions. There were 14 regions differentially methylated in the lip tissue from children with CLO and CLP, with one region (near KIAA0415) showing up in both the blood and lip EWAS.

Conclusions: Our finding of distinct methylation profiles in different orofacial cleft (OFC) subtypes represents a promising first step in exploring the potential role of epigenetic modifications in the aetiology of OFCs and/or as clinically useful biomarkers of OFC subtypes.

Keywords: Cleft Collective; Cleft lip; Cleft palate; DNA methylation; EWAS; Epigenome-wide association study; Orofacial clefts.

Fig. 1

Orofacial cleft subtypes. Orofacial clefts are traditionally categorised as either cleft lip only (CLO; a, b), cleft palate only (CPO; c–f) or cleft lip with cleft palate (CLP; g–j). Further subtyping can be made according to laterality and whether the soft and/or hard palate is affected. The dark bars represent the cleft

Fig. 2

Age at sampling and OFC subtype. Children with CPO were older on average than children with CLO or CLP because surgery for palate repair usually occurs later than surgery for lip repair

Fig. 3

Manhattan plots of the three pairwise epigenome-wide studies of DNA methylation in whole-blood samples from children with CLO, CLP and CPO. P values for age-related CpGs have been set to 1 (i.e. −log10 P value of 0) in the comparisons involving CPO. The red line indicates the threshold where P = 1 × 10⁻⁷ (i.e. a Bonferroni-corrected P value of 0.05)

Fig. 4

Blood DNA methylation levels at the top differentially methylated regions. DMRs were selected based on largest effect size and a Sidak-corrected P value <0.05 for each pairwise epigenome-wide study in blood

Fig. 5

A Venn diagram to show the crossover in CpGs within DMRs associated with each subtype comparison. Arrows show the direction of association, i.e. hyper- (up) or hypo- (down) methylation