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ItemCombined genetic and epigenetic analysis to identify early life determinants of complex phenotype( 2019)There is now considerable evidence indicating that risk of many complex diseases in adulthood may be influenced by exposure to environmental exposures in utero. A growing number of studies suggest epigenetic markers, including DNA methylation, are involved in this process. Understanding how DNA methylation is impacted by pregnancy exposures, and related to later health, may both contribute to unravelling the aetiology of complex disease risk in later life and provide a potential early-life biomarker for risk prediction. However, current evidence is limited. There has been a predominance of small, poorly powered studies, failure to consider the effects of genetic variation, and limited replication of previous findings. In addition, previous studies investigating the relationship between DNA methylation and offspring health have been primarily cross-sectional. For these reasons, I investigated the associations between pregnancy exposures (in particular, maternal smoking, nutrition and metabolic health, psychosocial stress, and adverse pregnancy conditions), birth outcomes, and offspring blood DNA methylation of the insulin-like growth factor 2 (IGF2) and H19, hypoxia-inducible factor 3A (HIF3A), leptin (LEP) genes. I also considered how genetic variation impacted on these associations. I then investigated the longitudinal relationship between early life methylation and anthropometry, as well as the association between early life methylation and later childhood measures of weight, adiposity, and cardiovascular health. To do this, the large, population-based longitudinal Barwon Infant Study pre-birth cohort (n=1,074) was used, with clinical and questionnaire measures from 28 weeks pregnancy, birth, 12 months post-birth and 4 years post-birth time points. DNA methylation of candidate regions was measured using the Sequenom EpiTyper mass-spectrometry platform in cord (birth) and peripheral (12-month) blood. Infant genetic variation in and near the candidate genes was considered. Infant adiposity was assessed as sum of triceps and subscapular skinfold thicknesses in infancy, and with DEXA scanning at 4 years of age. We found evidence that exposure to maternal psychosocial stress, gestational diabetes, and pre-eclampsia was associated with differences in offspring methylation at the candidate regions, as was infant sex. Genetic variation showed strong effects on DNA methylation levels, with some evidence for the associations of pre-eclampsia and infant adiposity with LEP methylation differing by infant genotype. Early life methylation of HIF3A and LEP showed modest associations with four-year blood pressure and BMI, respectively. While these associations persisted with adjustment for potential confounding factors, they explained relatively little variance in the four-year phenotypes compared to traditional predictors, such as weight. These findings suggest that offspring DNA methylation of these candidate genes involved in regulation of growth and metabolism are sensitive to several environmental exposures and genetic factors. While there is modest evidence for methylation in infant blood associating with later phenotypes, methylation of these genes appears unlikely to have useful predictive utility in isolation. This study is the first to perform early life longitudinal analysis to investigate the association between anthropometry and methylation in infancy. It is also the first to report evidence of earlier methylation associating with later cardiovascular phenotypes. However, as gene expression data was not available, the functional consequences of the altered methylation observed in blood is unclear. Further work is required to replicate these findings in independent cohorts, to determine the nature of expression of these genes in blood, and to investigate if the relationship between early life methylation and later health persists into adulthood.
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ItemDNA methylation and breast cancer risk factors: insights from twin and family studies( 2018)DNA methylation, the most studied epigenetic mechanism regulating gene expression, has been thought to play a critical role in the aetiology of complex diseases and traits, including breast cancer. Twin and family studies have investigated the unmeasured causes of DNA methylation variation; there are, however, several research gaps, such as interpreting the average heritability across sites as the heritability of genome-wide DNA methylation level and assuming DNA methylation variation across sites to have the same causes. Two DNA methylation features, genome-wide average DNA methylation (GWAM) and epigenetic age acceleration, are putatively associated with breast cancer risk. The causes of variation remain unknown for GWAM and have not been well investigated for epigenetic age acceleration. Understanding the potential causality between DNA methylation and conventional breast cancer risk factors, which has been rarely investigated for body mass index (BMI), mammographic density and smoking, might bring a better understanding of breast cancer aetiology. To address current research gaps, my thesis used DNA methylation data from multiple twin and family studies to investigate the causes of variation in GWAM, in site-specific DNA methylation and in epigenetic age acceleration, and to investigate the potential causality between DNA methylation and BMI, mammographic density and smoking with a novel analytic method using data for related individuals - Inference about Causation through Examination of FAmiliaL CONfounding (ICE FALCON). My thesis found that: a) genome-wide methylation level, measured as GWAM, is determined by prenatal environmental factors acting in utero, the effects of which last into old age, and by environmental factors shared by cohabitating family members, including spouse pairs (Chapter 4); b) site-specific variation DNA methylation has specific causes, and substantial variation is explained by measurement error and environmental factors (Chapter 5); c) evidence consistent with twin birth changing the intrauterine environment such that sibling pairs both born after a twin birth are correlated in DNA methylation while sibling pairs both born before a twin birth are not (Chapter 6); d) variation in epigenetic age acceleration is caused by shared environmental factors as well as genetic factors (Chapter 7); and e) BMI, mammographic dense area and smoking are associated with DNA methylation at several genetic loci, and these associations are likely due to the causal effects of the three factors on DNA methylation, the same conclusion to those made by Mendelian randomisation analysis (Chapters 8 to 10). The findings of my thesis suggest that DNA methylation appears to be fundamentally about the way the environment influences the way genes work. Although there might be methylation sites at which the variation has a genetic basis, these are rare. The effects of the environment can start from the time of conception, or at least while in the womb, and continue into adulthood. Some of those environmental effects are shared by family members, even spouse pairs, and these effects can potentially influence breast cancer risk in adulthood. Conventional breast cancer risk factors can cause changes to DNA methylation, indicating that DNA methylation might explain in part why these factors modifying risk. Most of the novel results of this thesis could not have come about without the use of data from twin pairs, or of data from other pairs of relatives including spouse pairs. The thesis has also shown the value of ICE FALCON in making inference about observational epigenetic association based on considering familial confounding - ICE FALCON gives the same conclusion as those being found by Mendelian randomisation. More importantly, ICE FALCON does not require extensive genome knowledge and data that are required by Mendelian randomisation.
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ItemThe statistical analysis of high-throughput assays for studying DNA methylation( 2015)DNA methylation is an epigenetic modification that plays an important role in X-chromosome inactivation, genomic imprinting and the repression of repetitive elements in the genome. It must be tightly regulated for normal mammalian development and aberrant DNA methylation is strongly associated with many forms of cancer. This thesis examines the statistical and computational challenges raised by high-throughput assays of DNA methylation, particularly the current gold standard assay of whole-genome bisulfite-sequencing. Using whole-genome bisulfite-sequencing, we can now measure DNA methylation at individual nucleotides across entire genomes. These experiments produce vast amounts of data that require new methods and software to analyse. The first half of the thesis outlines the biological questions of interest in studying DNA methylation, the bioinformatics analysis of these data, and the statistical questions we seek to address. In discussing these bioinformatics challenges, we develop software to facilitate novel analyses of these data. We pay particular attention to analyses of methylation patterns along individual DNA fragments, a novel feature of sequencing-based assays. The second half of the thesis focuses on co-methylation, the spatial dependence of DNA methylation along the genome. We demonstrate that previous analyses of co-methylation have been limited by inadequate data and deficiencies in the applied statistical methods. This motivates a study of co-methylation from 40 whole-genome bisulfite-sequencing samples. These 40 samples represent a diverse range of tissues, from embryonic and induced pluripotent stem cells, through to somatic cells and tumours. Making use of software developed in the first half of the thesis, we explore different measures of co-methylation and relate these to one another. We identify genomic features that influence co-methylation and how it varies between different tissues. In the final chapter, we develop a framework for simulating whole-genome bisulfite-sequencing data. Simulation software is valuable when developing new analysis methods since it can generate data on which to assess the performance of the method and benchmark it against competing methods. Our simulation model is informed by our analyses of the 40 whole-genome bisulfite-sequencing samples and our study of co-methylation.
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ItemNo Preview AvailableAssociation between hypermethylation of DNA repetitive elements in white blood cell DNA and early-onset colorectal cancer(TAYLOR & FRANCIS INC, 2013-07-01)Changes in the methylation levels of DNA from white blood cells (WBCs) are putatively associated with an elevated risk for several cancers. The aim of this study was to investigate the association between colorectal cancer (CRC) and the methylation status of three DNA repetitive elements in DNA from peripheral blood. WBC DNA from 539 CRC cases diagnosed before 60 years of age and 242 sex and age frequency-matched healthy controls from the Australasian Colorectal Cancer Family Registry were assessed for methylation across DNA repetitive elements Alu, LINE-1 and Sat2 using MethyLight. The percentage of methylated reference (PMR) of cases and controls was calculated for each marker. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multivariable logistic regression adjusted for potential confounders. CRC cases demonstrated a significantly higher median PMR for LINE-1 (p < 0.001), Sat2 (p < 0.001) and Alu repeats (p = 0.02) when compared with controls. For each of the DNA repetitive elements, individuals with PMR values in the highest quartile were significantly more likely to have CRC compared with those in the lowest quartile (LINE-1 OR = 2.34, 95%CI = 1.48-3.70; p < 0.001, Alu OR = 1.83, 95%CI = 1.17-2.86; p = 0.01, Sat2 OR = 1.72, 95%CI = 1.10-2.71; p = 0.02). When comparing the OR for the PMR of each marker across subgroups of CRC, only the Alu marker showed a significant difference in the 5-fluoruracil treated and nodal involvement subgroups (both p = 0.002). This association between increasing methylation levels of three DNA repetitive elements in WBC DNA and early-onset CRC is novel and may represent a potential epigenetic biomarker for early CRC detection.
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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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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.