Choice of surrogate tissue influences neonatal EWAS findings

Abstract

BackgroundEpigenomes are tissue specific and thus the choice of surrogate tissue can play a critical role in interpreting neonatal epigenome-wide association studies (EWAS) and in their extrapolation to target tissue. To develop a better understanding of the link between tissue specificity and neonatal EWAS, and the contributions of genotype and prenatal factors, we compared genome-wide DNA methylation of cord tissue and cord blood, two of the most accessible surrogate tissues at birth.MethodsIn 295 neonates, DNA methylation was profiled using Infinium HumanMethylation450 beadchip arrays. Sites of inter-individual variability in DNA methylation were mapped and compared across the two surrogate tissues at birth, i.e., cord tissue and cord blood. To ascertain the similarity to target tissues, DNA methylation profiles of surrogate tissues were compared to 25 primary tissues/cell types mapped under the Epigenome Roadmap project. Tissue-specific influences of genotype on the variable CpGs were also analyzed. Finally, to interrogate the impact of the in utero environment, EWAS on 45 prenatal factors were performed and compared across the surrogate tissues.ResultsNeonatal EWAS results were tissue specific. In comparison to cord blood, cord tissue showed higher inter-individual variability in the epigenome, with a lower proportion of CpGs influenced by genotype. Both neonatal tissues were good surrogates for target tissues of mesodermal origin. They also showed distinct phenotypic associations, with effect sizes of the overlapping CpGs being in the same order of magnitude.ConclusionsThe inter-relationship between genetics, prenatal factors and epigenetics is tissue specific, and requires careful consideration in designing and interpreting future neonatal EWAS.Trial registrationThis birth cohort is a prospective observational study, designed to study the developmental origins of health and disease, and was retrospectively registered on 1 July 2010 under the identifier NCT01174875.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-017-0970-x) contains supplementary material, which is available to authorized users.

Cell type-specific DNA methylation in neonatal cord tissue and cord blood: a 850K-reference panel and comparison of cell types

“…Interindividual variation in DNA methylation was generally modest across all 9 isolated cell types from CB and CT, with more variation among CT cell types (median IQR: Endothelial 1.7%, Stromal 1.7%, Epithelial 2.1%) than CB cell types (median IQR ranged from 1.1% to 1.2%). Consistent with our earlier finding [17], we found greater interindividual variation across whole CT samples (median IQR: 2.0%, 2.2% for USCS and frozen CT) than CB (median IQR: 1.4% and 1.6% for unsorted leukocytes and buffy coat).

10.1080/15592294.2018.1522929-F0005Figure 5.Comparison of interindividual variation in unfractionated cord tissue (CT), cord blood (CB), and their constituent cell types: CT showed more variable CpGs than CB. (a) Distribution of DNA methylation inter-quartile ranges (IQR) in each cell type/tissue.

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“…As we quantified interindividual variation in a small sample size (n = 10), we expect the overall proportion of useful CpGs to increase with increasing sample size, with a retained trend for CT to still have a higher proportion of variable CpGs than CB. We have also previously reported this observation from a comparison of whole CT and CB DNA methylation profiles from a larger cohort drawn from the same population [17]. …”

“…Our previous study on the choice of surrogate tissue for neonatal EWAS compared frozen CT with CB buffy coat and found higher interindividual variability in DNA methylation in CT than CB [17]. However, in that study we were unable to conclusively exclude the possibility that this was due to cell type heterogeneity.…”

“…Thus, it is helpful to have an updated catalog of cell type-specific CpGs on the expanded array. Third, while we have previously reported a comparison between the DNA methylation profiles of whole CT and CB [17], it was unclear how the constituent cell types of these tissues compare and contributed to these differences. Hence, here we generate a reference panel to deconvolute cell type constituents of both CB and CT. We also benchmark our reference panel by comparing cell type proportions as estimated from the panel with cell type proportions measured using flow cytometry on the same samples.…”

Epigenetic programming—The important first 1000 days

“…This project showed that tissues could be clustered according to their germinal origin: ectodermal, such as brain and skin, endodermal, such as lung and gastrointestinal, and mesodermal, such as blood and muscle . Cord blood was clustered with hematopoietic stem cell tissues and cord tissue was clustered with mesenchymal stem cell tissues . Saliva is a mixture of cells, mainly buccal cells and leucocytes, and has been shown to be a fairly representative surrogate tissue for the brain .…”