Paediatrics (RCH) - Theses
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ItemInvestigating the DNA methylation profiles of children with oligoarticular juvenile idiopathic arthritis (JIA)( 2019)Juvenile idiopathic arthritis (JIA) is a complex autoimmune disease affecting children aged between 6 months and 16 years. JIA represents a group of 7 subtypes of disease, with the most common being oligoarticular JIA (oJIA). Despite a prevalence of up to 1 in 400, rates similar to those in T1D, JIA research is relatively sparse. Research into disease pathogenesis has largely focussed on genetic risk factors, and has also identified CD4+ T-cells as likely to mediate the autoimmune process. However, research is particularly needed regarding diagnosis and prognosis of disease and its outcomes. Currently, diagnosis is almost entirely dependent on clinical observation and history, with little in the way of biomarkers to classify patients or to guide clinical management. Epigenetics represent biological modifications to DNA and chromatin that control gene expression and chromatin structure. DNA methylation is perhaps the most accessible modification available for study, and is known to modulate immune cell function particularly amongst CD4+ T-cell subsets. A number of autoimmune diseases have reported significant DNAm associations, and have also provided intriguing data on the potential of DNAm to predict clinical outcomes. This study hypothesised that DNAm is important in oJIA pathogenesis, and potentially provides a biological basis for the diagnosis and prognosis of disease. This study utilised CD4+ T-cells and a case-control study design to analyse the associations between DNAm and oJIA, with data generated from the Illumina Infinium HumanMethylation450 BeadChip array. Cases were matched with controls according to age and sex. Further, cases were subtyped according to current diagnostic criteria and had active disease, both of which attempted to ensure all cases were clinically homogeneous. The first aim was to profile DNAm in oJIA cases compared to controls. Processing of data through analysis pipelines resulted in high quality data. Differential methylation analysis suggested that oJIA cases and controls could be segregated in cluster analysis using DNAm data, despite no genome wide significant hits being produced. Immune system pathways analysis suggested the top hits were relevant to disease, being enriched for receptor binding of cytokines such as IL6, IL17 as well as MHC class II. In addition, a number of top ranking probes were enriched within cell death and survival functions. Indeed, gene expression data suggested genes within those pathways were also correlated with DNAm. Technical validation of a selection of probes was highly successful, with all probes validating. A small replication study, however, was not able to reproduce these findings. Of particular note, a wide distribution of DNAm values was observed for many of the validated probes. Since technical validation was so successful, this DNAm heterogeneity potentially derived from sample group heterogeneity, which may well have played a part in difficulties replicating data. Therefore, biological sources of heterogeneity were explored in chapter 5, focussing primarily on the genetic associations with DNAm. Probes utilised for technical validation were analysed for genetic associations associating with either mean or variable DNAm. Both analyses suggested that the most robust associations were for known mQTLs and enhancer SNPs. Indeed, DNAm differences according to genotype were up to 13% and 27% for 2 probes analysed, representing a many-fold difference over case-control differences (typically approximately 5%). Combined with an intermediate level of minor allele frequency for many of these robustly associated SNPs, these mQTLs represent a likely source of biological variation contributing to oJIA DNAm variation. These minor allele frequencies increase the likelihood of inadvertent sampling bias, potentially resulting in difficulties in replicating DNAm data. Deeper analysis provided some initial indication that these mQTLs may also be potential oJIA risk loci, with the most significant associations again coming from known mQTL or enhancer SNPs. This also suggested DNAm data may well identify regions of interest for genetic risk loci discovery. The final chapter hypothesised that sources of potential clinical heterogeneity not captured within current classification criteria may well lead to DNAm heterogeneity, as could recognised subgroups within oJIA. Of primary focus, age of disease diagnosis was assessed for associations with DNAm. This study found that case-control analyses of older diagnosed samples (greater than or equal to 6 years) resulted in case-control clustering using far fewer probes. Indeed, the reduction of probes required for clustering was more pronounced in the analysis of younger diagnosed samples (less than 6 years of age), and also resulted in a genome wide significant hit. These subgroups represented 2 highly divergent populations, since top ranking probes from each subgroup had virtually no overlapping probes. This data suggested that age subgroups in oJIA represent sources of sample heterogeneity, leading to DNAm heterogeneity. Technical validation for a large majority of the select probes from the younger-diagnosed analysis was also successful. However, a small replication study could not reproduce these initial findings. In light of the potential for mQTLs to have pronounced effects on DNAm, as explored in chapter 5, larger replication groups (or, indeed, discovery groups) will likely be needed to mitigate the risk of sampling error to enable reproduction of findings. OJIA heterogeneity was also explored by looking at known subgroups, Persistent vs Extended disease. A number of oJIA cases would go on to develop extended disease, and the possibility existed for DNAm signatures to identify these cases prior to disease extension. This was indeed the case, with an exploratory analysis suggesting a number of probes can cluster persistent cases from extended-to-be cases. Further, these probes were able to produce a highly sensitive and specific test to predict disease extension, thereby providing a proof of principle for a prediction test using DNAm data. This study is the largest case-control analysis of JIA DNAm to date, and provided insights into the potential for DNAm to identify pathogenic pathways, identify sources of oJIA heterogeneity, and opened the possibility for biological markers of disease to be used in clinical management. The findings regarding the pronounced effect of mQTLs on DNAm also suggest that genetics is a large source of DNAm variability, far larger than group differences typically found in a complex diseases (such as oJIA). The identification of subgroup specific differences, even with a clinically homogeneous subtype, warrants further investigation to explore potential differences in pathogenesis between age groups and the use of DNAm as biomarkers for classification or disease management.
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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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ItemIntragenic DNA methylation and neurodevelopmental outcomes in children with fragile X-related disorders( 2019)The type and severity of clinical involvement in children with fragile X syndrome (FXS) and disorders related to premutations (PM) of the fragile X mental retardation 1 gene (FMR1), herein collectively denoted as fragile X-related disorders (FXDs), is highly variable. Multiple molecular factors contribute to the heterogeneity of neurodevelopmental outcomes. Increased intragenic DNA methylation (DNAm) in blood of the fragile X-related epigenetic element 2 (FREE2) region, located at the FMR1 exon 1/intron 1 boundary, was associated with lower intellectual functioning in a cohort of female children and adults with FXS and with neuro-cognitive and psychiatric phenotypes in women with PM. Nevertheless, FREE2 DNAm has not yet been investigated in exclusively paediatric male and female FXDs cohorts. The overarching aim of this thesis was to explore FREE2 DNAm and neurodevelopmental outcomes of Australian male and female children with FXDs. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry and methylation specific-quantitative melt analysis were used to analyse FREE2 DNAm in venous blood, buccal epithelial cells (BEC) and retrieved newborn blood spots (NBS). In addition, FMR1 mRNA levels in blood were assessed using real-time polymerase chain reaction (PCR) relative standard curve method. The evaluation of the neurodevelopmental outcomes concentrated on direct clinical assessment of intellectual functioning and autism spectrum disorder (ASD) symptom severity. Intelligence Quotient (IQ) scores were corrected for floor effect using the Whitaker and Gordon (WG) extrapolation method. The findings highlighted the variability of the clinical presentation of children with PM. Results also showed that compared to sex-matched paediatric controls, children with FXS had significantly higher levels of FREE2 DNAm levels in blood and BEC and, within the FXS group, higher FREE2 DNAm levels in blood correlated with lower FMR1 mRNA levels. In children with FXS, the application of the WG method effectively addressed the floor effect inherent in standardised intelligence scales, unmasked inter-individual variability in IQ scores and uncovered significant associations between intragenic DNAm and neurodevelopmental outcomes. Strength and statistical significance of these epigenotype-phenotype relationships varied based on sex, position of the differentially methylated sites, tissue analysed, assay used and neurodevelopmental domain investigated. The most significant finding was in males with FXS, for whom higher levels of BEC FREE2 DNAm were associated with lower WG-corrected Full Scale IQ (cFSIQ) and Performance IQ (cPIQ) scores. Finally, findings showed that the best-performing FREE2 biomarker had sensitivity, specificity, negative and positive predictive values of 100% for detection of full mutation alleles in NBS of males and females with FXS. Additionally, this study revealed that for males with FXS, FREE2 DNAm in NBS was significantly associated with lower cFSIQ and cPIQ scores obtained in childhood and adolescence. This is the first study in any monogenic neurodevelopmental disorder associated with intellectual disability, showing that a perinatal epigenetic biomarker is significantly associated with paediatric neuropsychological outcomes. In conclusion, the results of this thesis contribute to the characterisation of the neurodevelopmental outcomes in children with FXDs, provide evidence that FREE2 DNAm is a sensitive epigenetic biomarker significantly associated with the variability of intellectual functioning in male children with FXS, and may have implications for the development of new methylation specific tests for earlier diagnosis with potential prognostic applications.
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ItemInvestigating genomic and environmental risk factors and their interactions in juvenile idiopathic arthritis( 2017)Juvenile idiopathic arthritis (JIA) is a paediatric autoimmune disease arising from an abnormal immune response to self. It is the most common childhood rheumatic disease, with a prevalence of around 1 in 1000 Caucasian children. Disease prevalence is biased towards females, with around 2–3 females affected for every male. Due to the young age of onset, JIA can have a severe effect on a child’s growing skeleton and cause serious functional disability. And though onset is in childhood, the morbidity associated with JIA can be life-long as currently there is no cure for the disease, treatments are imperfect and preventative measures aren’t available – largely due to the limited understanding of disease pathogenesis. We hypothesised that genetic and environmental risk factors contribute individually and through interaction to cause JIA, and contribute to the sex bias in disease prevalence. The first aim of this study was to replicate the association of genetic variants that had previously been associated with JIA, in our independent sample. We confirmed the association of seven risk loci in our sample, six replicated for the first time. Our findings significantly strengthen the evidence that these loci harbour true JIA risk variants. The second aim of this study was to investigate whether autosomal genetic variants confer sex-specific risk for JIA. We established that of the 68 JIA risk loci tested, eight conferred sex-specific risk for JIA. Of these, three had statistically significant evidence of sex modifying the effect of that SNP on JIA. Of note, we replicated the femalespecific association of PTPN22 rs2476601 across two independent samples. Our findings illustrate that the genetic architecture of JIA differs between the sexes. Our third aim was to investigate whether the Y chromosome contributes to JIA risk in males. We determined that genetic variation captured by Y chromosome haplogroup I was associated with JIA risk, in males over the age of 6. We also demonstrated that there was an increased risk of JIA for males that had a father with autoimmune disease. Our findings are the first to suggest that the Y chromosome may play a role in JIA risk and provide further evidence that JIA has sex-specific genetic architecture. Next we considered the role of the environment in JIA risk. The fourth aim of this study was to assess the association between factors that impact vitamin D status and JIA. We identified a protective association between increasing UVR exposure over the life course and at 12 weeks of pregnancy, and JIA. Our findings are the first to implicate insufficient UVR exposure in the development of JIA. We then considered mechanisms through which genetic and environmental risk may be mediated, such as DNA methylation and gene expression. Our fifth aim was to identify sex-specific DNA methylation differences in CD4+ T cells between oligoarticular JIA cases and healthy controls. Oligoarticular JIA cases did not have substantial sex-specific DNA methylation differences when compared to controls, but there was evidence of modest case–control differences and these were more prominent in males than females. Our findings suggest that DNA methylation is not a significant driver of the sex bias in JIA. The final aim of this study was to investigate whether CD4+ T cell gene expression profiles differed between oligoarticular JIA cases and healthy controls. Oligoarticular JIA cases had aberrant gene expression relative to controls, suggesting that disease processes are in part driven by gene regulatory differences in CD4+ T cells. In conclusion, the cumulative findings of this study improve our understanding of the aetiology of JIA by revealing sex-specific genetic architecture for the disease, establishing UVR exposure as an environmental risk factor for JIA, and characterising the DNA methylation and gene expression signatures of the active disease state.
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ItemMicroRNA expression and genome-wide epigenetic analysis of paediatric acute myeloid leukaemia( 2014)Introduction: Paediatric Acute Myeloid Leukaemia (AML) is the third most prevalent cancer in Australian children under the age of 14. Chromosomal and genetic lesions are commonly found within specific subtypes of paediatric AML and these help to direct treatments and prognostic predictions. However, there are no common genetic lesions across subtypes of paediatric AML and subtype-associated genetic changes may fail to induce leukaemogenesis. DNA methylation is the most commonly studied molecular alteration in cancer. Strong evidence indicates that methylation variations exist within paediatric cancer known to modify gene expression. Similarly, microRNA are small non-coding RNA that also regulate gene expression and whose dysregulation within paediatric cancer is now beginning to be appreciated. Therefore altered methylation and miRNA expression may contribute to malignancy through activating oncogenes or inactivating tumour suppressor genes, similar to genetic mutations. Furthermore, both DNA methylation and microRNA expression represent viable epigenetic mechanisms for application to clinical biomarkers of disease diagnosis, prognosis and disease tracking. At the beginning of this study, limited interrogations of genome-scale DNA methylation and microRNA expression had been conducted within paediatric AML with appropriate non-leukaemic controls. Furthermore, there has been few extensive studies evaluating the utility of current epigenetic techniques on primary clinical specimens. Therefore I chose to study genome-wide DNA methylation and large-scale miRNA expression changes in paediatric AML, as well as interrogating techniques for using archived primary patient samples. Materials and Methods: Two approaches were used to interrogate large-scale DNA methylation and microRNA expression; TaqMan OpenArray Human MicroRNA Panel (Life Technologies) was used to interrogate 5 primary paediatric AML patients with matched controls; and Illumina Infinium HumanMethylation450 BeadChip array (HM450) was used to interrogate 18 primary AML alongside matched non-leukaemic samples. Validation of AML DNA methylation alterations was conducted using the SEQUENOM MassARRAY EpiTYPER with an expanded cohort of 28 patients, in conjunction with validation of microRNA expression utilising quantitative real-time PCR (qRT-PCR) and TaqMan singleplex assays on 32 primary paediatric AML, together with matched control groups. This study also developed extensive extraction techniques for the analysis of microRNA expression within a range of samples, including archived bone marrow aspirate smear slides. Results: Genome-scale DNA methylation disruptions were characterised within paediatric AML, and identified hundreds of genes associated with disease compared to matched control samples. Interrogation of a refined subset of target genes also identified gene expression alterations within these regions, which were further associated with patient disease onset and predicted outcome. Conversely, large-scale microRNA expression disruptions were characterised within paediatric AML, whereby a small number of reliable targets were identified. Such microRNA disruption was found to be associated with DNA methylation regulation on the microRNA gene, and could also be used as reliable biomarkers to predict disease onset and patient outcome in connection with identified patient cytogenetic abnormalities. Integration of data from genome-scale DNA methylation and combined gene and microRNA expression analysis, identified common epigenetic disruptions within paediatric AML affecting known tumour suppressor genes, cytoskeletal organisation, cellular proliferation and immune function. Conclusions: The findings of this study reveal that DNA methylation alterations within paediatric AML can associate with gene disruptions with the potential to initiate and perpetuate malignant phenotypes. Likewise, microRNA deregulation can establish widespread gene disruptions as a catalyst for leukaemogenesis. Interestingly we establish that combined deregulation of epigenetic mechanisms, and the occurrence of one epigenetic mechanism working to deregulate another, may be a common feature of paediatric AML. Lastly this study identifies targets and techniques to extend such studies into the clinic and provides attractive targets for therapeutic intervention.
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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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ItemThe role of filaggrin in food allergy and eczema in the first year of life( 2012)Eczema and IgE-mediated food allergy often develop early in life and have a substantial impact on the quality of life of a child for many years. Family history is a strong risk factor for the development of allergic disease and the strongest genetic association with eczema to date is the filaggrin gene (FLG), which produces a key structural protein in the epidermis. Eczema, food sensitisation and food allergy commonly co-associate in early childhood. Although many studies have now identified the link between FLG and eczema, to date only one study has looked at the association between FLG and peanut allergy. No other study has investigated whether the presence of FLG mutations increases the risk of developing food allergy in early life. The recent rise in the prevalence of eczema and food allergy has occurred too rapidly to be explained by genetic factors alone, suggesting that environmental factors play a role. However, the rise might only be occurring amongst those genetically at risk. This warrants the investigation of the role of gene-environment interactions, in particular epigenetic regulation, which remains largely unexplored. Within this thesis, I investigated the association of FLG mutations with infantile eczema, food sensitisation and food allergy in one-year-old infants, using samples from the HealthNuts study. HealthNuts is a large population-based cohort study of 5276 infants recruited at 12 months of age in Melbourne, Australia. During recruitment, all infants were examined for eczema and underwent a skin prick test to four common food allergens. Those with a wheal size ≥ 1mm underwent a hospital-based oral food challenge (the gold standard for food allergy diagnosis) and bloods were taken. Non-sensitised infants were randomly invited to clinic to act as negative biological controls. I also investigated the role of DNA methylation in regulating FLG expression and the outcome of allergic disease using a small pilot study and the HealthNuts study. The association of FLG mutations with infantile eczema was replicated in this study, with an odds ratio [OR] of 4.2 (95% CI, 2.0-8.8; P <0.001) for broad definition of eczema; an OR of 2.4 (95% CI, 1.5-4.1; P=0.001) for diagnosed eczema; and OR of 2.0 (95% CI, 1.3-3.3; P=0.003) for current eczema. FLG mutations were associated with atopic eczema (eczema with IgE-mediated food sensitisation) with an OR of 4.4 (95% CI, 2.1-9.6; P< 0.001), but not non-atopic eczema (p=0.984). FLG mutations increased the risk of IgE-mediated food sensitisation in the first year of life, after adjusting for the presence of eczema (adjusted OR [aOR], 3.0; 95% CI, 1.0-8.7; p= 0.043). However, there was no significant difference in FLG mutations when comparing food-tolerant infants with those with food allergy (OR, 0.7; 95% CI, 0.3-1.7; P= 0.478). This indicates that FLG mutations do not further increase the risk of IgE-mediated food allergy over and above that of IgE-mediated food sensitisation. Together these results support the biologically plausible hypothesis that decreased skin barrier function increases the risk of food sensitisation in early life, but other as yet undetermined factors must be important in the conversion from food sensitisation to allergy. FLG was found to be expressed in adult buccal epithelia, ~19% relative to FLG expression in adult skin tissue. FLG expression in infant buccal epithelia was found to be twice as high as those of adult buccal epithelia. Methylation was measured at three CpG sites upstream of FLG transcription start site. However, there was no clear correlation between methylation and FLG expression. Therefore, although associations between FLG methylation and various allergic outcomes were identified, the clinical relevance of these findings is unclear. Nevertheless, this epigenetic study has contributed novel data to add to our understanding of the role of gene-environment interaction involving FLG in allergic disease. In summary, this study has generated evidence for an important role for FLG in the establishment of food sensitisation and food allergy in infants.
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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.