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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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ItemDNA methylation marks in peripheral blood and the risk of developing mature B cell neoplasms( 2018)Dysregulation of DNA methylation is a feature of mature B cell neoplasms (MBCN) but it is not known whether methylation changes can be detected in blood-derived DNA prior to MBCN diagnosis. In this prospective cohort study, peripheral blood was collected from healthy participants at recruitment (1990-1994). Participants who were subsequently diagnosed with MBCN (chronic lymphocytic lymphoma, B cell non-Hodgkin lymphoma and myeloma) up to 2012 were matched to the same number of controls based on age, sex, ethnicity, and type of blood sample (Guthrie cards, mononuclear cells, buffy coats). DNA methylation was measured using the Infinium®HumanMethylation450 BeadChip. Peripheral blood DNA was collected from 438 matched case-control pairs, a median of 10.6 years prior to diagnosis with MBCN. A series of analytical approaches was used in order to evaluate whether there was a distinct methylation profile associated with MBCN. First, global methylation analysis was performed, identifying increased methylation in CpG island and promoter-associated CpGs and widespread hypomethylation. Second, conditional logistic regression was used to identify differentially methylated CpG sites (DMPs) and kernel smoothing was used to identify differentially methylated regions (DMRs). Third, differential methylation variability, considered to be a distinctive feature in cancer, was assessed. In total, 1,338 DMPs were identified, of which 90 had gain of methylation in CpG sites associated with homeobox genes and 1,248 had loss of methylation in CpG sites associated with MAPK signaling pathway genes and genes involved in chemokine signaling pathways. There were 9,857 DMRs, with a cluster of 151 DMRs located in a 3.8kb region on 6p21.3, corresponding to the major histocompatibility locus. Differential methylation variability analysis identified 144 novel CpG sites distinctively located outside CpG islands. Conclusion: Distinctive changes in peripheral blood DNA methylation can be detected many years prior to diagnosis with MBCN, suggesting that changes in DNA methylation are an early epigenetic event. This contributes to our understanding of the timing of methylation changes in the development of MBCN.
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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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ItemMechanism of action of the potential tumour suppressor Csk homologous kinase (Chk) in colorectal cancer cells( 2018)Aberrant activation of Src family tyrosine kinases (SFKs) directs initiation of metastasis and development of drug resistance in multiple solid tumours and haematological cancers. Intriguingly, 80% of colorectal cancer (CRC) cells exhibit a 5 to 10-fold increase in SFK activity directly correlating with their oncogenic potential. Since oncogenic mutations of SFKs are rare events, aberrant activation of SFKs in cancer is likely due to dysregulation of the two major upstream inhibitors: C-terminal Src kinase (Csk) and its homolog Csk homologous kinase (Chk). SFKs are maintained in the inactive conformation by two major intermolecular interactions (i) binding of the SH3 domain to the SH2-kinase linker and (ii) binding of the C-terminal tail phosphotyrosine to the SH2 domain. Phosphorylation of the C-terminal tail tyrosine is a pre-requisite for SFKs to adopt this closed inactive conformation. Both Csk and Chk can phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Moreover, they can also employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types, suggesting that they perform overlapping as well as distinct functions to suppress SFK activity in cells. My PhD project aimed to delineate how Csk and Chk cooperate to inhibit SFK activity in cells. Specifically, I define (i) the contributions of the catalytic and non-catalytic inhibitory mechanisms towards the inhibitory activity of Csk and Chk and (ii) the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism. Using biochemical and biophysical approaches, I determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced CRC cells. Specifically, I determined the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and the SFK member, Src. We employed Surface plasmon resonance (SPR) spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of SFK. Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of phosphorylation of the SFK C-terminal tail tyrosine but it binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism. Next, I sought to determine the functional domain in Chk responsible for binding and inhibition of SFKs. Using an engineered Csk-Chk chimera and SPR, I mapped the major determinants in Chk governing its tight binding with Src to its kinase domain. Previous studies of the crystal structure of the Csk kinase domain in complex with Src revealed five basic arginine residues responsible for Csk binding to Src; mutations of any one of these basic residues significantly reduced the affinity of binding of Csk to Src and abolished the inhibitory activity of Csk1. Since these residues are conserved in the Chk kinase domain, we hypothesized that they are also determinants governing Chk binding and inhibition of SFKs. Studies of Chk mutants generated by site-directed mutagenesis revealed that mutations of two of the five arginine residues in Chk had a slight impact on its binding affinity to the SFK. However, the mutations did not impact the ability of Chk to inhibit SFKs by the non-catalytic mechanism. Based upon these findings, we propose a model in which the optimal alignment of the five basic residues in Csk and Chk is critical for their inhibition of SFKs. The model also suggests that the Chk kinase domain exhibits a much higher propensity than the Csk kinase domain to adopt this configuration. Chk can efficiently employ the non-catalytic inhibitory mechanism to inhibit multiple active forms of SFKs as compared to Csk, suggesting that Chk is a versatile tumour suppressor capable of constraining the activity of multiple active forms of SFKs. The lack of this non-catalytic inhibitory mechanism may account for SFK overactivation in the Chk-deficient CRC cells. Additionally, several pieces of evidence suggest Chk as a potential tumour suppressor down-regulated by epigenetic silencing and/or inactivated by missense mutations in several cancer cases such as colorectal and lung carcinoma. In spite of the potential significance of Chk in cancer development, convincing evidence supporting Chk as a tumour suppressor has not been presented. Our collaborators and us previously reported down-regulation of Chk expression in several CRC cell lines and in CRC biopsies obtained from several patients2, suggesting that Chk is a potential colorectal tumour suppressor and suppression of its expression contributes to cancer initiation and/or progression. In my PhD study, I conducted experiments to further confirm that Chk is a potential CRC tumour suppressor and aimed to delineate its tumour suppressive mechanism in CRC cells. Specifically, I used transduced human CRC cells expressing recombinant Chk under the control doxycycline as the model system and examined how recombinant Chk expression affects the oncogenic phenotypes and SFK activity in these cells. First, q-PCR further confirmed downregulation of Chk in a panel of CRC cell lines and that Chk expression is likely suppressed at the transcriptional level. To ascertain if Chk gene transcription is suppressed by epigenetic silencing, I performed genome-wide methylation and bisulfite sequencing studies and revealed hypermethylation of the Chk gene promoter in a panel of CRC cell lines. Our collaborators in the Walter and Eliza Hall Institute in Melbourne also discovered that treatment with a panel of CRC cell lines with inhibitors of epigenetic silencing led to an increase in the mRNA level of Chk. Collectively, these results strongly suggest that expression of Chk is down-regulated in CRC cells by an epigenetic silencing mechanism involving DNA methylation of the Chk gene promoter. Additionally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient CRC cells. Reintroduction of Chk in CRC cell lines resulted in robust inhibition of Src activity and suppression of anchorage-independent growth. As Chk is a protein kinase directly phosphorylating multiple substrates in cells, it may exert its tumour suppressive action by phosphorylating SFKs as well as other cellular proteins. Similar to protein kinases containing protein-protein interaction domains such as SH2 and SH3 domains, Chk may use its SH2 and/or SH3 domains to bind its non-SFK substrates prior to their phosphorylation. As an attempt to search for and identify these non-SFK substrates of Chk in transduced CRC cells, I used the DLD1-Chk-GFP expressing CRC cell line and an unbiased proteomics approach to identify the cellular proteins bound to recombinant Chk-GFP in the transduced DLD-1 cells. My analysis revealed co-immunoprecipitation of Chk-GFP with Src and many other non-SFK proteins. Besides acting as the direct substrates, some of the non-SFK cellular proteins bound to Chk may also be upstream regulators that bind and control Chk functions in CRC cells. Thus, further investigation focusing on these Chk-binding proteins will identify the potential upstream regulators and direct protein substrates of Chk. In summary, my findings strongly suggest that Chk is potential tumour suppressor inhibiting SFK activity by the non-catalytic inhibitory mechanism. Specifically, my research results provide biochemical and structural insights into the mechanism of the tumour suppressive action and regulation of Chk in CRC cells. Future studies to clearly define the molecular basis of the tumour suppressive action of Chk may benefit the development of new therapeutic strategies for the treatment of CRC.
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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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ItemInvestigating epithelial-mesenchymal plasticity in breast cancer using circulating tumour cells and circulating tumour DNA( 2017)Breast cancer is the most frequent invasive cancer among women worldwide with mortality primarily caused by metastasis. One of the key proposed processes underlying metastasis, including the escape to the bloodstream, is epithelial- mesenchymal plasticity (EMP). This refers to the dynamic transition between epithelial and mesenchymal phenotypes of cells within the primary tumour mass. Within the bloodstream, tumour cells and tumour DNA, which are referred to as circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA), respectively, have showcased their potential use as liquid biopsy in cancer management. The presence of CTCs has been shown to associate with poor prognosis in metastatic cancers, which becomes worse with higher CTC numbers. By virtue of carrying genetic and epigenetic features of primary tumours, ctDNA has demonstrated its utility in detecting and monitoring cancer progression. Various studies conducted on the molecular characterisation of CTCs have generated data supporting the role of EMP in generating CTCs. However, the dynamic changes in expression, especially of genes associated with EMP, between primary tumours, CTCs and metastases remain far from conclusive. In keeping with this paradigm, as cancer cells in the primary tumours shift from the epithelial to the mesenchymal phenotype, any released ctDNA may have epithelial and/or mesenchymal features depending on its cellular origin. To date, research on the utility of EMP-associated methylation markers to detect ctDNA is lacking. In light of the suggested role of EMP in different key steps of cancer progression and metastasis, this thesis has aimed to study EMP reflected in CTCs and ctDNA to provide further insights into the CTC molecular characteristics and assist in the detection of ctDNA. This thesis is comprised of two principal areas of study: (1) the gene expression profiling of CTCs in two human breast cancer xenograft models, and (2) the locus- specific methylation profiling of breast cancer cell lines and breast tumours. Firstly, the expression profiles of EMP-associated genes were characterised in CTCs at the population level and the single cell level, and were compared with the expression profiles of primary tumours and (where possible) metastases, for two xenograft models, the MDA-MB-468 and ED-03. In pooled CTCs relative to primary tumours from both models, a significant increase in expression of mesenchymal markers (SNAI1, VIM, SERPINE1 and NOTCH1), and surprisingly, of a typical epithelial marker CDH1, were observed. A decrease/loss of EPCAM was reproducibly observed in CTCs of both models, while decreased CD24 and EGFR in CTCs were only seen in the MDA-MB-468 model. Genes associating with hypoxia (HIF1A, BNIP3 and APLN) and cellular metabolism (PPARGC1A) were also significantly elevated in CTCs of both models. In additional studies, a direct lysis method was successfully optimised to assist the gene expression study in single cells. The subsequent analysis of single CTCs revealed heterogeneity of CTCs, with co-expression of epithelial and mesenchymal markers, and high expression levels of epithelial markers in individual CTCs. The results reinforced the complex gene expression profiles and alterations seen in pooled CTCs. Secondly, a panel of DNA methylation markers, including those associated with EMP, was developed and tested in breast cancer cell lines, primary tumours and whole blood of normal controls to identify suitable markers for ctDNA detection. In a panel of breast cancer cell lines spanning the epithelial-mesenchymal spectrum, the majority of epithelial cell lines were methylated for cancer-associated markers (i.e., RASSF1A, RARß) and epithelial methylation-based markers (i.e. VIM, DKK3 and CRABP1). Mesenchymal cell lines were exclusively methylated for mesenchymal methylation-based markers (GRHL2, MIR200C and CDH1); however, the level of methylation was quite low. The methylation profiles of the studied genes classified primary tumours into intermediate phenotypes while few tumours were mesenchymal. In addition, MIR200C, RASSF1A, AKR1B1 and TWIST1 were methylated at high frequency in our cohort. Among these, RASSF1A and AKR1B1 showed no methylation in whole blood of normal controls, suggesting their potential use as markers for ctDNA detection from plasma of the breast cancer patients in our cohort. Preliminary experiments established ddPCR assays for these two markers, allowing further testing on patient cell-free DNA samples for the detection of ctDNA. The results of this thesis challenge the conventional model of EMT, where cells in epithelial tumours become mesenchymal, with associated migratory properties, and later re-epithelialise (MET) at a distant metastasis. Firstly, the complex and consistent alterations in the epithelial and mesenchymal markers in CTCs across the two models is suggestive of a ‘hybrid’ phenotype. The overall findings from the CTC work that CTCs were not as mesenchymal as expected also suggest that other processes than EMP directly influence the generation and survival of CTCs. Secondly, nearly all the examined breast tumours exhibited an intermediate rather than a strong epithelial phenotype based on the methylation profiles. This suggested that a plasticity is already present at the solid tumour state. These findings provide an alternative view of EMP in both primary breast cancer and the disseminated forms, and provide an important platform for further research in this field.
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ItemBiomarkers in ductal carcinoma in situ( 2016)Ductal carcinoma in situ (DCIS), a non-invasive form of breast cancer and a non-obligate precursor of invasive carcinoma of the breast, displays heterogeneous behaviour. Most DCIS are adequately managed by local surgical excision alone, but in 20-30% of cases, disease recurrence occurs after local surgical excision either as DCIS or invasive carcinoma. Accurate identification of these two clinical outcome groups at the time of diagnosis is desirable to allow appropriate treatment allocation. In this thesis, genomic and epigenetic alterations in DCIS epithelium, including copy number aberrations, somatic mutations, and DNA methylation were investigated as markers of DCIS biology and outcome. In addition, the expression and significance of LRH-1, a nuclear receptor which acts as a transcription factor, was investigated in both invasive carcinoma and DCIS. Copy number analysis of DCIS of known clinical outcome identified amplification of 20q13 to be associated with disease recurrence, but this was unable to be validated on an independent cohort. Targeted next generation sequencing of a panel of breast cancer-relevant genes revealed that the mutational profile of DCIS was similar to that reported for invasive carcinomas, with the most frequently mutated genes being GATA3, PIK3CA, and TP53. A high prevalence of GATA3 mutations in DCIS was observed and TP53-mutant DCIS was associated with high stromal tumour infiltrating lymphocytes. Mutations of RUNX1 were a novel finding, not previously reported in DCIS. Promoter methylation of a candidate gene panel, consisting predominantly of known tumour suppressor genes, was associated with adverse tumour features in DCIS. Methylation load varied among DCIS cases, suggesting that methylation differs in importance in the tumorigenesis of DCIS, and that assessment of methylation may be useful as a biological classifier of DCIS. Finally, LRH-1 mRNA expression patterns in breast cancers was similar to that reported for breast cancer cell lines and distinct LRH-1 immunohistochemical staining patterns were associated with tumour phenotype in both invasive breast carcinoma and DCIS. The results of this thesis demonstrate that copy number alterations, somatic mutations, DNA methylation, and LRH-1 expression are indicative of DCIS phenotype and hence biology. These markers showed promise as prognostic biomarkers, although validation of their utility was hampered by the small number of pure DCIS cases with both adequate genomic material and long-term clinical outcome data. Nonetheless, the findings of this thesis indicate that assessment of these biomarkers can be performed in routine diagnostic tissue material and provide several avenues for future research.
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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.