Paediatrics (RCH) - Theses
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ItemEpigenetics of human placental development and pregnancy-associated disease( 2013)INTRODUCTION: Epigenetics literally means ‘above DNA’ and refers to the study of molecular modifications that control gene expression and chromatin structure. DNA methylation, the most extensively studied epigenetic modification, is involved in both the maintenance of chromosome stability and gene expression. Due to its role in gene expression, tissue specific DNA methylation patterns are assumed to reflect the function of a specific gene in a particular tissue. The human placenta facilitates the interaction between the mother and the fetus, including nutrient and oxygen exchange, waste removal and the protection of the fetus from the maternal immune response. Due to its role at the feto-maternal interface, the placenta is exposed to several environmental factors with the capacity to alter placental function and fetal development. Many of these effects are likely to be mediated by epigenetic change. Linking specific environmental exposures, genetic, and epigenetic variation to maternal and neonatal outcomes may provide valuable mechanistic insights into the role of placental dysfunction in pregnancy-associated disease and later health. Therefore, DNA methylation studies in healthy and disease placentas have the potential to identify new genes associated with placental function. The aim of this PhD was to take a genome-scale approach to characterise gene promoter methylation in the normal human placenta. MATERIALS AND METHODS: Several different tissues, primary cells and cell lines were used in this study. These included placental villi from first, second and third trimester, purified first trimester villous and extravillous cytotrophoblasts, choriocarcinoma and trophoblast-derived cell lines. Placental tissue, neonatal cord blood and maternal peripheral blood serum from twin births, collected as part of the Peri/post-natal Epigenetic Twins Study (PETS) cohort, were used for two aims of this project. Environmental data on maternal nutrition and supplementation during pregnancy were collected through questionnaires or measured in maternal blood serum. DNA methylation levels were analysed on the genome-scale level using the Illumina Infinium HumanMethylation27 BeadChip, and at the gene-specific level using the SEQUENOM MassARRAY EpiTYPER platform. RESULTS: Genome-scale DNA methylation analysis of normal human placenta from first to third trimester identified dynamic changes in DNA methylation patterns in response to increasing gestation and environmental/stochastic factors. Most of the changes were observed at genes involved in immune cell communication and signalling, which likely reflects the change in cell composition as well as the differing immunological interactions between the mother and the fetus as the pregnancy progresses. Furthermore, increasing inter-individual variation in methylation level at certain CpG sites over gestation suggests an accumulation of environmental and/or stochastic influences during intrauterine development. Next, the twin model was employed to quantify the relative influence of the underlying genetic and environmental/stochastic factors on placental methylation at term. Genome-scale methylation analysis of placentas from 8 monozygotic (MZ) and 6 dizygotic (DZ) twin births identified widespread differences in methylation within MZ twin pairs, supporting a role of the intrauterine environment in shaping the placental methylation patterns at term. In general MZ twins were more epigenetically similar than DZ pairs, underlining the influence of DNA sequence on methylation patterns. In the subsequent attempt to tease out the association between a specific environment (maternal and neonatal vitamin D) and placental CYP24A1 methylation in 32 MZ and 54 DZ pairs, no link was identified. Finally, a comparison between first trimester cytotrophoblasts and several widely used trophoblast-derived and choriocarcinoma cell lines identified widespread differences in DNA methylation patterns. Almost all gene families tested showed significant differences in methylation between primary cells and transformed cell lines, with choriocarcinoma lines showing the largest differences. This information may be useful when deciding which cell line to use for functional analysis. CONCLUSIONS: This study revealed that placental DNA methylation patterns are dynamic during pregnancy, likely reflecting placental function at specific points in gestation. Furthermore, the intrauterine environment was shown to shape the placental DNA methylation profile through a combination of environmental and stochastic influences. The identification of environmentally sensitive CpG sites across gestation and within MZ twin pairs warrants further investigation.
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ItemWhole-genome expression profiling of cord blood mononuclear cells from monozygotic twin pairs( 2010)Substantial variation in gene expression levels exists between individuals, within specific tissues or cell types. With gene expression being the primary mechanism through which genetic information is translated into phenotype, the extent, nature and sources of this variation constitutes an important aspect of human biology. Variation in gene expression levels reflects a complex interplay of genetic and environmental factors. Certain environmental factors and exposures can modify gene expression through epigenetic modifications of DNA and chromatin, thus regulating transcription in a manner largely independent of genetic variation. The sensitivity of epigenetic mechanisms to these factors offers a means through which the environment can modulate expression of the genotype, with effects upon gene expression and ultimately, the phenotype. Studies of variation in gene expression in monozygotic (genetically identical) twins support a substantial environmental contribution to variation in gene expression levels. It is known that the epigenetic and gene expression profiles of monozygotic twins diverge throughout life. Mounting evidence suggests that the period of pre-natal development represents a particularly sensitive one for the occurrence of environmentally induced changes to epigenetic status and gene activity. The current study forms part of a larger research program investigating epigenetic variation in twins and its association with birth weight, maternal nutrition and foetal genotype. The Peri-/Post-natal Epigenetic Twins Study (PETS) builds upon the ‘developmental origins of adult disease’ hypothesis, based on the association between low birth weight and increased risk of cardiovascular and metabolic disease in later life, and focuses on epigenetic changes occurring in utero as the basis of the ‘foetal programming’ phenomenon. The current study is based on the hypothesis that divergence of epigenetic and gene expression profiles occurs from conception in monozygotic twins, in response to differing environments as experienced in utero. This study investigates the gene expression profiles of the cord blood mononuclear cells (CBMCs) of twelve newborn monozygotic twin pairs, including six pairs with birth weight discordance at greater than 15%. Genome-wide expression profiling was performed using the Illumina® Human-6 v2 BeadChip system. Gene expression discordance within twin pairs was assessed using three measures. Of these, the measure of Euclidean distance was considered to be the most systematic and useful. Expression discordance was found to vary substantially across pairs in our sample, with expression discordance being generally lower within twin pairs than between unrelated individuals. A significant correlation was identified between expression discordance and chorionicity, with greater expression discordance in dichorionic pairs compared to monochorionic pairs. The measure of Euclidean distance was also applied to publicly available datasets from genome-wide expression profiling of comparable tissues from adult twin pairs, revealing higher levels of expression discordance within the adult pairs relative to the newborn pairs. All genes surveyed by the microarray analysis were ranked according to the degree of within-pair variation shown across twin pairs. This ranked gene list was subjected to gene ontology analysis to identify gene ontology (GO) terms for which the corresponding ranks were higher than expected. Of the 27 GO terms ranked significantly higher than expected, one third related to immune response or response to other external signals. This data supports our hypothesis that divergence of gene expression profiles occurs from conception in monozygotic twins, and is reflected in differential expression phenotypes detectable at birth. Taken together, these results highlight the role of environment in determining gene expression profiles, and the contribution of environmentally induced changes in gene expression to expression discordance within monozygotic twin pairs. The increased variation observed within dichorionic twin pairs, coupled with the prominence of genes involved in immune/external signal response amongst those showing increased variation across pairs, implies that this variation may arise in response to subtly differing environments experienced by co-twins in utero. Birth weight is a phenotype of particular interest in the Peri-/Post-natal Epigenetic Twins Study (PETS), due to the association of low birth weight with an elevated risk of cardiovascular and metabolic disease in later life. In this study, linear modelling identified 342 genes whose expression levels showed a significant association with birth weight in dichorionic twin pairs. Gene ontology analysis of these genes revealed significant over-representation of GO terms relating to protein dephosphorylation, a process intrinsic to many forms of signal transduction. These data imply a link between the environmental modulation of gene activity via signal response/transduction and the phenotype of birth weight. Due to the myriad number of signal transduction pathways and physiological processes regulated by protein phosphorylation and dephosphorylation, it is not possible to pinpoint with certainty those that may be linked to birth weight or implicated in the association between low birth weight and elevated disease risk from our data. However, these results do provide a basis for further investigation of the specific environmental factors involved in the determination of gene expression variants associated with birth weight. It is to be hoped that future analyses will assist in the identification of the mechanisms underlying the correlation between low birth weight and an elevated risk of cardiovascular and metabolic disease in later life.