Paediatrics (RCH) - Theses
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ItemDNA methylation patterns in paediatric acute lymphoblastic leukaemia( 2014)Introduction: Disruption of DNA methylation is the most common molecular alteration in human cancers. Paediatric Acute Lymphoblastic Leukaemia (ALL) is the most prevalent childhood cancer and strong evidence indicates that DNA methylation alterations exist within this disease. Several genetic mutations have been described that contribute to the malignant transformation within the B-cell subtypes of ALL (B-ALL), however many of the malignant phenotypes are unexplained by genetic mutations alone. DNA methylation has the ability to alter gene expression and thus DNA methylation alterations may contribute to observed malignant phenotypes, potentially activating oncogenes or inactivating tumour suppressor genes analogous to genetic mutations. Furthermore, DNA methylation alterations represent viable clinical biomarkers for disease diagnosis, prognosis and disease tracking. At the start of this project, preliminary genome-scale DNA methylation profiling had been performed on paediatric B-ALL with appropriate B-cell controls to identify contributing DNA methylation alterations and only limited studies had investigated techniques, thresholds and assays for the clinical implementation of DNA methylation biomarkers. Materials and Methods: Two approaches were used to characterise genome-scale DNA methylation alterations in 69 paediatric B-ALL patients; the Illumina Infinium HumanMethylation BeadChip arrays HM27 and HM450. Validation of B-ALL DNA methylation alterations was conducted using the SEQUENOM MassARRAY EpiTYPER. Genome-scale analysis of gene expression (Affymetrix microarray) was also performed in 17 B-ALL cases and integrated with B-ALL methylome data. The study also developed novel techniques for the analysis of DNA methylation using MALDI-TOF Mass Spectrometry (SEQUENOM). Results: Genome-scale disruptions in DNA methylation were characterised in paediatric B-ALL, validating a number of previous small scale experiments and identifying hundreds of genes with associated DNA methylation disruption. DNA methylation alterations were found to be prevalent in all paediatric B-ALL subtypes and stable biomarkers of disease. Two highly differentially methylated sites in the gene promoters of FOXE3 and TLX3 were used as targets to establish new MALDI-TOF Mass Spectrometry techniques that could 1) analyse multiple DNA methylation regions in single reaction and 2) sensitively detect rare DNA methylation events. The techniques were applied to patient samples and enabled high sensitivity and specificity measurements for disease diagnosis. Furthermore, these techniques enabled sensitive disease tracking and insights into the detection of minimal residual disease by DNA methylation analysis. Integration of genome-scale DNA methylation and gene expression data identified common and subtype-specific epigenetic disruption in paediatric B-ALL effecting known tumour suppressors and genes implicated in apoptosis, cellular proliferation and cell signalling. Furthermore, this study uncovered prognostic DNA methylation signatures associated with B-ALL relapse, present across several B-ALL subtypes. Conclusions: The findings of this study have revealed common alterations to DNA methylation across the genomes of paediatric B-ALL that establish a mechanism for clonal inheritance of gene deregulation integral to malignant phenotype. Additionally, the study establishes targets, techniques and thresholds for clinical implementation of DNA methylation loci as biomarkers for disease diagnosis, prognosis and tracking.
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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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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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ItemHuman centromere organisation and function( 2012)Centromeres are essential for correct chromosome segregation during cell division. Whilst centromeric function is conserved throughout eukaryotes, the profile of centromeric DNA, although generally repetitive, is not, and neocentromeres have been reported at various euchromatic sites devoid of satellite DNA. The lack of DNA sequence conservation has led to the notion that centromere identity is maintained by epigenetic mechanisms. The repetitive nature of canonical centromeres has made studying the organization of the centromere difficult, although the discovery of neocentromeres has provided a useful model for studying the function and organization of the centromere domain, and several studies have used neocentromeres to investigate several proteins required for successful centromere formation and function. One such neocentromere model is the 10q25 neocentromere of Mardel(10), which has previously been used to map the binding domains of several centromere-associated proteins. This study utilized the 10q25 neocentromere model to map the binding domains of three global proteins reported to be enriched at canonical centromeres. The linker histone, histone H1 and the chromatin assembly protein, HMGA1 are enriched at the 10q25 domain following neocentromere formation, suggesting a possible role for each protein at active centromeres. The transcriptional regulator, CTCF, was present at the 10q25 domain both prior to and after neocentromere formation. However, neocentromere formation altered the binding profile of CTCF at the 10q25 domain, with an additional binding cluster observed overlapping the core CENP-A containing domain on the Mardel(10) neocentromere, suggesting a role for CTCF at active centromeres unrelated to the DNA sequence. This study also looked at the role CTCF may be playing at the centromere. Depletion of CTCF resulted in a decrease in transcription at canonical mouse centromeres and at the 10q25 neocentromere, and also resulted in an increase in mitotic segregation defects. Whilst it is evident that the presence of CTCF is important at the centromere for correct centromeric function, the exact mechanism by which CTCF acts at the centromere is still uncertain. Neocentromere-derived mini-chromosomes (NC-MiCs) were developed by telomere associated chromosome truncation (TACT) of the arms of the Mardel(10) marker chromosome. These NC-MiCs provide a useful model for studying the effects large-scale chromosomal rearrangements have on the size and organization of chromatin domains as they can be compared to the progenitor Mardel(10). This study used NC-MiC6, a 1.4Mb mini-chromosome derived from Mardel(10) and still containing the entire Mardel(10) CENP-A binding domain, to investigate the effect of severe chromosomal alterations on the size and organization of the centromere by analysis of the binding domain of CENP-A on NC-MiC6. The CENP-A binding domain on NC-MiC6 was reduced to one-third the size of the Mardel(10) CENP-A binding domain, despite the chromosome size being reduced by 98%. This reduction in size did not dramatically alter the stability of the chromosome, and no correlation was found between the chromosome size and the CENP-A domain size for neocentromeres. Whilst the ratio between chromosome size and centromere size is unlikely to be the trigger for the shrinkage and reorganization of the chromatin domain observed in the 10q25 CENP-A binding domain, the disruption of the chromatin scaffold matrix (S/MAR) or the flanking pericentric heterochromatin in the formation of NC-MiC6 may be responsible for this alteration. Whilst the evidence presented in this study provide further insights into the organization and function of the centromere, our knowledge is still far from complete and further studies are required before we will fully understand this complex and essential domain. Neocentromeres devoid of α-satellite DNA provide a useful model to obtain this information as they can be used to generate a linear ‘road map’ of protein binding at the neocentromere. This information can be used to further understand the organization of the centromere, and can be used in conjunction with three dimensional studies to understand the higher-order chromatin organization of the centromere, thus shedding some light on this complex domain.
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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.