FETAL CORD DNA METHYLOME IN ASSOCIATION WITH PARITY AND GDM

Ph.DDOCTOR OF PHILOSOPH

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 x 10(-4)), maternal height (P = 1.1 x 10(-4)), pregnancy weight gain (P = 2.2 x 10(-3)), prepregnancy alcohol consumption (P = 4.6 x 10(-4)), and tobacco exposure (P = 1.9 x 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.Peer reviewe

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 x 10(-4)), maternal height (P = 1.1 x 10(-4)), pregnancy weight gain (P = 2.2 x 10(-3)), prepregnancy alcohol consumption (P = 4.6 x 10(-4)), and tobacco exposure (P = 1.9 x 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.Peer reviewe

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

Comparison of Methyl-capture Sequencing vs. Infinium 450K methylation array for methylome analysis in clinical samples

<p>Interindividual variability in the epigenome has gained tremendous attention for its potential in pathophysiological investigation, disease diagnosis, and evaluation of clinical intervention. DNA methylation is the most studied epigenetic mark in epigenome-wide association studies (EWAS) as it can be detected from limited starting material. Infinium 450K methylation array is the most popular platform for high-throughput profiling of this mark in clinical samples, as it is cost-effective and requires small amounts of DNA. However, this method suffers from low genome coverage and errors introduced by probe cross-hybridization. Whole-genome bisulfite sequencing can overcome these limitations but elevates the costs tremendously. Methyl-Capture Sequencing (MC Seq) is an attractive intermediate solution to increase the methylome coverage in large sample sets. Here we first demonstrate that MC Seq can be employed using DNA amounts comparable to the amounts used for Infinium 450K. Second, to provide guidance when choosing between the 2 platforms for EWAS, we evaluate and compare MC Seq and Infinium 450K in terms of coverage, technical variation, and concordance of methylation calls in clinical samples. Last, since the focus in EWAS is to study interindividual variation, we demonstrate the utility of MC Seq in studying interindividual variation in subjects from different ethnicities.</p

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 .Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofReviewedFacult

Choice of surrogate tissue influences neonatal EWAS findings

Abstract 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