Paediatrics (RCH) - Theses
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ItemInfluence of maternal diet and lifestyle on twins DNA methylation at birth and the changes that occur within the first 18 months of life( 2013)The Developmental Origins of Heath and Disease (DOHaD) concept describes the importance and impact of prenatal environment on later health and disease risk. One of the mechanisms of how prenatal environment can influence the development of the offspring is epigenetics, defined as the study of mitotically and/or meiotically heritable changes in the gene function independent of DNA sequence. The term “epigenetics” was coined by Conrad Waddington as an important mechanism that shapes the development of an organism. In this PhD, DNA methylation was used as a representative of epigenetic status, as it is the most robust epigenetic mark. There is mounting evidence for associations between maternal factors and DNA methylation at gene-specific, global, and genome-wide levels. However, most studies to date have been limited in scope, examining DNA methylation in specific regions in one or two cell types. Newborn twins’ samples from the Peri/postnatal Epigenetic Twins Study (PETS) cohort were used for all analyses in this thesis. The benefit of using twins is the ability to investigate shared and non-shared maternal factors (e.g., the fetal ‘supply line’) on DNA methylation, which until now, have not been studied. The major aim of my PhD was to investigate associations of various shared and non-shared maternal factors with DNA methylation at a gene-specific (Chapter 3 and 4), global (Chapter 4) and genome-wide level (Chapter 5) in various cell types. At a gene-specific level, IGF2 and H19 were the chosen as candidate genes because they are known to be important in embryonic growth. Repeat elements Alu and LINE-1 were used as surrogates of global methylation. Epigenetic profile was hypothesised to be dynamic in the first few months of life, due to its critical developmental stages in early childhood. Previous studies have used a cross-sectional model to investigate DNA methylation changes over time at various ages, but very few studies have used a longitudinal model for this purpose from birth to infancy. My PhD also aimed to fill this gap by using a longitudinal study of methylation in twins from birth to 18 months of age at a genome-wide level (Chapter 6). Using the Sequenom MassARRAY EpiTYPER platform to measure DNA methylation of genomic regions controlling expression at IGF2/H19, and in the Alu and LINE-1 interspersed repeats in five different cell types of newborn twins, it was found that both shared and non-shared maternal factors associate with DNA methylation at birth, and often in a cell type- and region-specific manner, even with the surrogates of global methylation. These are valuable findings, as they further inform that associations of specific maternal factors with certain regions and cell types cannot always be extrapolated to other regions or cell types. This study further reports that false positives can occur, most likely due to small sample size. Methylation analysis at a genome-wide level found that maternal factors were more likely to influence genes involved in metabolic pathways, especially amino acid metabolism. It was also found that maternal factors were less likely to affect regions of functional importance. These findings are important for future studies of prenatal environment. Finally, it was reported that one third of the genome in buccal epithelium rapidly changes over time in the first 18 months of life. This study has revealed the complexity of the epigenome in the newborn in response to the influence of maternal factors, and the dynamic changes over time in the epigenome from birth to infancy. Validation of associations in larger sample sizes in various cell types, and identification of the legacy of such influences in the long-term health of the offspring warrant further investigation. Furthermore, expression analysis on these findings would further solidify the clinical relevance of the associations seen.
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ItemEpigenetic discordance in newborn monozygotic and dizygotic twin pairs( 2012)Introduction: There is strong evidence that the intrauterine environment can program the long-term health outcomes of the developing fetus. Adverse fetal programming is often also accompanied by low birth weight and this can act as a predictor for later health complications (e.g. hypertension). Although substantiated by numerous animal studies and a small number of human studies, the mechanisms underlying this phenomenon (known as “fetal programming”), remain to be elucidated. Interestingly, epigenetic marks are reprogrammed during early development and subject to change more frequently than genetic mutations. Additionally, epigenetic marks are sensitive to a myriad of environmental influences, suggesting that environmentally mediated epigenetic change during early development may underpin the phenomenon of fetal programming. In order to increase our understanding of this potential mechanistic link, the current study measured aspects of intrauterine environment and epigenetic profile in Human Umbilical Vascular Endothelial Cells (HUVECs) collected from healthy twins at birth as a part of the recently established Pre/Post-natal Epigenetic Twins Study (PETS). HUVECs provide an insight into the fetal programming hypothesis because this cell type is an important mediator in both controlling fetal growth and maintaining cardiovascular health. Furthermore, this study utilised a twin design, controlling for genetic influences (monozygotic twins) or major shared environmental factors (dizygotic twins) on epigenetic profile. Epigenetic profile was measured on a genome-scale using a recently developed DNA methylation microarray and gene expression arrays (as a proxy sum of all epigenetic marks). In addition, the H19/IGF2 imprinted region was examined at a high resolution as an example of a genomic region subject to epigenetic control, also implicated in fetal growth. Materials and Methods: Three approaches were employed to measure within-twin-pair epigenetic discordance in this study: 1. Genome-scale gene expression analysis of 10 MZ pairs; 2. Genome-scale DNA methylation analysis of 13 MZ and 11 DZ pairs; and 3. DNA methylation analysis of 33 MZ and 26 DZ pairs on H19/IGF2 imprinted locus. Genome-scale analyses of gene expression and DNA methylation were performed using Illumina BeadChip expression and Infinium methylation microarrays, whilst our H19/IGF2 locus methylation analysis was performed using the Sequenom MassARRAY EpiTYPER platform. Results: Both genome-scale and locus specific analyses identified a range of within-pair epigenetic discordance within MZ twin pairs at birth, indicating epigenetic drift in utero most likely due to subtle differences in the in utero environment together with stochastic factors. However, evidence of a genetic influence on epigenetic profile was also found, as within twin pair discordances were generally lower for MZ twins relative to DZ twins and unrelated individuals. By regressing within-pair discordance for gene expression and DNA methylation with birth weight discordance, we were able to identify a number of genes which may play an important role in fetal growth and which provide a potential mechanism for the fetal programming hypothesis. In addition, we show common involvement of genes which are discordantly expressed (i.e. hypervariable genes) in immune reponse and response to external signals and differently methylated genes in cell death and proliferation. This study also shows a greater variation in DNA methylation in regions distant from CpG islands than the islands themselves, providing compelling evidence in support of the important role of DNA methylation at CpG dinucleotides proximal to CpG islands (CpG island shores and shelves). We also utilised publically available gene expression microarray data of twins of different ages and compared their gene expression discordance with those detected at birth in our twins and found an increasing epigenetic discordance associated with the age. Finally, the data from our concurrent studies of additional tissues (cord blood mononuclear cells, buccal, placental cells) revealed a highly tissue specific DNA methylation pattern in the H19/IGF2 region. Conclusions: The findings of this study have revealed multiple levels of regulation of epigenetic profile occurring in humans prior to birth. It supports a role for the in utero period specifying the epigenetic profile in response to maternal nutrition and other environmental exposures (in addition to other stochastic influences), with implications for the fetus’ immediate, as well as long-term health outcomes.
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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.