Dichotomy in the Impact of Elevated Maternal Glucose Levels on Neonatal Epigenome.

CONTEXT: Antenatal hyperglycemia is associated with increased risk of future adverse health outcomes in both mother and child. Variations in offspring's epigenome can reflect the impact and response to in utero glycemic exposure, and may have different consequences for the child. OBJECTIVE: We examined possible differences in associations of basal glucose status and glucose handling during pregnancy with both clinical covariates and offspring cord tissue DNA methylation. RESEARCH DESIGN AND METHODS: This study included 830 mother-offspring dyads from the Growing Up in Singapore Towards Healthy Outcomes cohort. The fetal epigenome of umbilical cord tissue was profiled using Illumina HumanMethylation450 arrays. Associations of maternal mid-pregnancy fasting (fasting plasma glucose [FPG]) and 2-hour plasma glucose (2hPG) after a 75-g oral glucose challenge with both maternal clinical phenotypes and offspring epigenome at delivery were investigated separately. RESULTS: Maternal age, prepregnancy body mass index, and blood pressure measures were associated with both FPG and 2hPG, whereas Chinese ethnicity (P = 1.9 × 10-4), maternal height (P = 1.1 × 10-4), pregnancy weight gain (P = 2.2 × 10-3), prepregnancy alcohol consumption (P = 4.6 × 10-4), and tobacco exposure (P = 1.9 × 10-3) showed significantly opposite associations between the 2 glucose measures. Most importantly, we observed a dichotomy in the effects of these glycemic indices on the offspring epigenome. Offspring born to mothers with elevated 2hPG showed global hypomethylation. CpGs most associated with the 2 measures also reflected differences in gene ontologies and had different associations with offspring birthweight. CONCLUSIONS: Our findings suggest that 2 traditionally used glycemic indices for diagnosing gestational diabetes may reflect distinctive pathophysiologies in pregnancy, and have differential impacts on the offspring's DNA methylome.

Analysis of two birth tissues provides new insights into the epigenetic landscape of neonates born preterm.

BACKGROUND: Preterm birth (PTB), defined as child birth before completion of 37 weeks of gestation, is a major challenge in perinatal health care and can bear long-term medical and financial burden. Over a million children die each year due to PTB complications, and those who survive can face developmental delays. Unfortunately, our understanding of the molecular pathways associated with PTB remains limited. There is a growing body of evidence suggesting the role of DNA methylation (DNAm) in mediating the effects of PTB on future health outcomes. Thus, epigenome-wide association studies (EWAS), where DNAm sites are examined for associations with PTB, can help shed light on the biological mechanisms linking the two. RESULTS: In an Asian cohort of 1019 infants (68 preterm, 951 full term), we examined and compared the associations between PTB and genome-wide DNAm profiles using both cord tissue (n = 1019) and cord blood (n = 332) samples on Infinium HumanMethylation450 arrays. PTB was significantly associated (P < 5.8e-7) with DNAm at 296 CpGs (209 genes) in the cord blood. Over 95% of these CpGs were replicated in other PTB/gestational age EWAS conducted in (cord) blood. This replication was apparent even across populations of different ethnic origin (Asians, Caucasians, and African Americans). More than a third of these 296 CpGs were replicated in at least 4 independent studies, thereby identifying a robust set of PTB-linked epigenetic signatures in cord blood. Interrogation of cord tissue in addition to cord blood provided novel insights into the epigenetic status of the neonates born preterm. Overall, 994 CpGs (608 genes, P < 3.7e-7) associated with PTB in cord tissue, of which only 10 of these CpGs were identified in the analysis using cord blood. Genes from cord tissue showed enrichment of molecular pathways related to fetal growth and development, while those from cord blood showed enrichment of immune response pathways. A substantial number of PTB-associated CpGs from both the birth tissues were also associated with gestational age. CONCLUSIONS: Our findings provide insights into the epigenetic landscape of neonates born preterm, and that its status is captured more comprehensively by interrogation of more than one neonatal tissue in tandem. Both these neonatal tissues are clinically relevant in their unique ways and require careful consideration in identification of biomarkers related to PTB and gestational age. TRIAL REGISTRATION: This 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 .

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

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.

Choice of surrogate tissue influences neonatal EWAS findings.

BACKGROUND: Epigenomes 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. METHODS: In 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. RESULTS: Neonatal 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. CONCLUSIONS: The inter-relationship between genetics, prenatal factors and epigenetics is tissue specific, and requires careful consideration in designing and interpreting future neonatal EWAS. TRIAL REGISTRATION: This 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 .

Developmental pathways to adiposity begin before birth and are influenced by genotype, prenatal environment and epigenome.

BACKGROUND: Obesity is an escalating health problem worldwide, and hence the causes underlying its development are of primary importance to public health. There is growing evidence that suboptimal intrauterine environment can perturb the metabolic programing of the growing fetus, thereby increasing the risk of developing obesity in later life. However, the link between early exposures in the womb, genetic susceptibility, and perturbed epigenome on metabolic health is not well understood. In this study, we shed more light on this aspect by performing a comprehensive analysis on the effects of variation in prenatal environment, neonatal methylome, and genotype on birth weight and adiposity in early childhood. METHODS: In a prospective mother-offspring cohort (N = 987), we interrogated the effects of 30 variables that influence the prenatal environment, umbilical cord DNA methylation, and genotype on offspring weight and adiposity, over the period from birth to 48 months. This is an interim analysis on an ongoing cohort study. RESULTS: Eleven of 30 prenatal environments, including maternal adiposity, smoking, blood glucose and plasma unsaturated fatty acid levels, were associated with birth weight. Polygenic risk scores derived from genetic association studies on adult adiposity were also associated with birth weight and child adiposity, indicating an overlap between the genetic pathways influencing metabolic health in early and later life. Neonatal methylation markers from seven gene loci (ANK3, CDKN2B, CACNA1G, IGDCC4, P4HA3, ZNF423 and MIRLET7BHG) were significantly associated with birth weight, with a subset of these in genes previously implicated in metabolic pathways in humans and in animal models. Methylation levels at three of seven birth weight-linked loci showed significant association with prenatal environment, but none were affected by polygenic risk score. Six of these birth weight-linked loci continued to show a longitudinal association with offspring size and/or adiposity in early childhood. CONCLUSIONS: This study provides further evidence that developmental pathways to adiposity begin before birth and are influenced by environmental, genetic and epigenetic factors. These pathways can have a lasting effect on offspring size, adiposity and future metabolic outcomes, and offer new opportunities for risk stratification and prevention of obesity. CLINICAL TRIAL REGISTRATION: This 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 .