ABSTRACT
Accounting for cellular heterogeneity is essential in neonatal epigenome-wide association studies (EWAS) performed on heterogeneous tissues, such as umbilical cord tissue (CT) or cord blood (CB). Using a reference-panel-based statistical approach, the cell type composition of heterogeneous tissues can be estimated by comparison of whole tissue DNA methylation profiles with cell type-specific DNA methylation signatures. Currently, there is no adequate DNA methylation reference panel for CT, and existing CB panels have been generated on lower coverage Infinium HumanMethylation450 arrays. In this study, we generate a reference panel for CT and improve available CB panels by using the higher coverage Infinium MethylationEPIC arrays. We performed DNA methylation profiling of 9 cell types isolated from CT and CB samples from 14 neonates. In addition to these cell types, we profiled DNA methylation of unfractionated CT and CB. Cell type composition of these unfractionated tissue samples, as estimated by our reference panels, was in agreement with that obtained by flow cytometry. Expectedly, DNA methylation profiles from CT and CB were distinct, reflecting their mesenchymal and hematopoietic stem cell origins. Variable CpGs from both unfractionated CT and its isolated cell types were more likely to be located in open seas and intronic regions than those in CB. Cell type specific CpGs in CT were enriched in intercellular matrix pathways, while those from CB were enriched in immune-related pathways. This study provides an open source reference panel for estimation and adjustment of cellular heterogeneity in CT and CB, and broadens the scope of tissue utilization assessed in future neonatal EWAS studies.
Subject(s)
Blood Cells/metabolism , DNA Methylation , Epigenomics/standards , Fetal Blood/metabolism , Sequence Analysis, DNA/standards , Umbilical Cord/metabolism , Adult , CpG Islands , Female , Fetal Blood/cytology , Humans , Infant, Newborn , Organ Specificity , Pregnancy , Reference Standards , Sequence Analysis, DNA/methods , Umbilical Cord/cytologyABSTRACT
Experimental studies show a substantial contribution of early life environment to obesity risk through epigenetic processes. We examined inter-individual DNA methylation differences in human birth tissues associated with child's adiposity. We identified a novel association between the level of CpG methylation at birth within the promoter of the long non-coding RNA ANRIL (encoded at CDKN2A) and childhood adiposity at age 6-years. An association between ANRIL methylation and adiposity was also observed in three additional populations; in birth tissues from ethnically diverse neonates, in peripheral blood from adolescents, and in adipose tissue from adults. Additionally, CpG methylation was associated with ANRIL expression in vivo, and CpG mutagenesis in vitro inhibited ANRIL promoter activity. Furthermore, CpG methylation enhanced binding to an Estrogen Response Element within the ANRIL promoter. Our findings demonstrate that perinatal methylation at loci relevant to gene function may be a robust marker of later adiposity, providing substantial support for epigenetic processes in mediating long-term consequences of early life environment on human health.
