Distinct DNA methylation profiles in subtypes of orofacial cleft

Sharp et al. Clinical Epigenetics (2017) 9:63 DOI 10.1186/s13148-017-0362-2 RESEARCH Open Access Distinct DNA methylation profiles in subtypes of orofacial cleft Gemma C. Sharp1*, Karen Ho2, Amy Davies3, Evie Stergiakouli1, Kerry Humphries3, Wendy McArdle4, Jonathan Sandy3, George Davey Smith2, Sarah J. Lewis2 and Caroline L. Relton2 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, DNA methylation, Epigenome-wide association study, EWAS, Cleft lip, Cleft palate, Orofacial clefts Background Orofacial clefts (OFCs) are a set of common birth defects that affect roughly 15 in every 10,000 births in Europe [1]. There are three main subtypes of OFC: cleft palate only (CPO), cleft lip only (CLO) and cleft lip with cleft palate (CLP) (Fig. 1). Non-syndromic cases, which comprise around 70% of cases of cleft lip with or without cleft palate, have a complex aetiology involving both genetic and environmental factors [2]. * Correspondence: 1 MRC Integrative Epidemiology Unit, School of Oral and Dental Sciences, University of Bristol, Bristol, England Full list of author information is available at the end of the article A child born with an OFC may face difficulties with feeding, speech, dental development, hearing and social adjustment. At considerable health, emotional and financial costs, they undergo surgery in the first year of life and many need additional surgical procedures later in life. They may experience low self-esteem, psychosocial problems and poor educational attainment, and the condition can harm the emotional wellbeing of the whole family [2–4]. In 2012, the James Lind Alliance identified the top 10 priorities in OFC research, which includes (1) identifying the genetic and environmental causes of OFCs and (2) © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sharp et al. Clinical Epigenetics (2017) 9:63 Page 2 of 17 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 identifying strategies to improve diagnosis of CPO. Epigenetic data might help to address both of these. Firstly, given the key role of epigenetic processes such as DNA methylation in embryonic development, we and others have hypothesised that aberrant epigenetic mechanisms might play a role in causing OFCs [2, 5, 6]. This hypothesis has been supported by data suggesting an important role for DNA methylation and other epigenetic processes in regulating normal orofacial development and OFCs in mice [7–12], but published epigenetic data for OFCs in humans is lacking. The three major subtypes of OFC (CPO, CLO, CLP) appear to be aetiologically distinct, for example, lip and palate formation occur at different times during embryogenesis, and there is a higher risk of familial recurrence of the same subtype compared with risk of recurrence of a different subtype [13, 14]. Therefore, if epigenetic mechanisms play a causal role in OFC aetiology, the precise role may differ by subtype. Secondly, regardless of whether epigenetics plays any causal role in OFC development, epigenetic data ‘captures’ information about the underlying genetic architecture and historical prenatal environmental exposures and could therefore be a useful measure of genetic and prenatal environmental influences that do cause OFCs. Again, we might expect these epigenetic indicators to differ by subtype, reflecting differential influence by different risk factors. Thirdly, if epigenetic profiles differ by OFC subtype, epigenetic measures could be developed into a biomarker to improve diagnosis of OFCs, either pre- or postnatally. This would be particularly useful for diagnosing CPO, which is often undetected on ultrasound and can go undiagnosed after birth, resulting in impaired feeding and growth, poorer outcomes and distress for families [15]. Additionally, epigenetic data could be useful in studying later-life outcomes associated with OFCs. For example, DNA methylation in cord blood has been associated with childhood IQ [16], so future studies could explore whether methylation mediates reported associations between OFCs and poor educational attainment [4]. Alternatively, even in the absence of a mechanistic role for epigenetic processes, epigenetic data might predict later-life outcomes caused by genetic and/or environmental factors. As a first step towards exploring the (...truncated)