Paediatrics (RCH) - Theses
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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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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.