Paediatrics (RCH) - Theses
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ItemAssociations between executive function, adaptive behaviour, participation and self-reported quality of life in adolescents with autism spectrum disorder without intellectual disability( 2018)Background ASD is a neurodevelopmental condition that affects an individual’s ability to communicate and interact with others. There has been an increase in the prevalence of ASD diagnosis, with the most considerable increase in adolescents who are intellectually able. These adolescents have difficulties with adaptive behaviour and struggle to participate in society, even if their intelligence is in the average or superior range. A better understanding of the relationship between impairments associated with ASD such as social impairments, executive function, adaptive behaviour, and participation and quality of life (QoL) is needed in intellectually able adolescents with autism. Aims Using the International Classification of Functioning, Disability and Health (ICF), with subjective functioning added, as a framework, this thesis aimed to study the association between 1) executive function (EF) and adaptive behaviour, over and above the contribution to adaptive behaviour of social impairment and intelligence; 2) EF and participation and the mediating role of adaptive behaviour in their association; 3) participation and self-reported QoL, over and above the contribution to self-reported QoL of social impairment and symptoms of anxiety and depression, in adolescents with ASD and without ID. Methods To address these aims, a cross-sectional study of 39 adolescents with ASD, aged from 10 to 16 years and 11 months (M = 13 years 4 months; SD = 3 months) and with Full Scale Intelligence Quotient (FSIQ) ranging between 78 and 144 (M = 107.97; SD = 18.25) was conducted. Adolescents completed a battery of tests to assess the ICF domains of Health Condition (social impairment, anxiety and depressive symptoms), Body Functions/impairments (EF, such as cognitive flexibility, inhibition, generativity, planning), Activity (adaptive behaviour), Participation (participation in extra-curricular activities) as well as the domain of subjective functioning (self-reported QoL). The domains of Environmental Factors (family social advantage) and Personal Factors (sex, age and intelligence) were also considered for each participant. Measures were chosen based on their use in clinical practice and research with young people with ASD, with the exception of participation, as there was no clear understanding of how to measure this domain for young people with ASD. To identify how participation has been measured in young people with ASD a systematic review of the literature was performed, with consideration of the measurement properties of the tools identified. As published, nine studies out of the 2539 screened investigated the measurement properties of the tool to assess participation in young people with ASD. These nine studies included seven tests, namely Adolescent and Young Adult Activity Card Sort, the Children’s Assessment of Participation and Enjoyment/Preference for Activities of Children (CAPE/PAC); the Experience Sampling Method; the Pediatric Evaluation of Disability Inventory Computer Adaptive Test (PEDI-CAT) and the School Function Assessment. The tests’ measurement properties were investigated in studies with high risk of bias and therefore, the quality of the evidence for these tools is limited. The PEDI-CAT demonstrated adequate measurement properties but lacked the assessment of the subjective dimension of participation. For this reason, the CAPE was chosen in this study. Preliminary analysis of the data from the cross-sectional study comprised a description of the sample, univariate analysis of the association between independent and dependent variables by hypothesis, and explorative univariate analysis of the association between covariates and dependent variables. To address the three aims of this thesis, three hierarchical regressions were performed with covariates entered in the first steps, and the independent variables by hypothesis entered last. Results The descriptive statistics fit well with the conceptualisation of autism as a spectrum condition with scores on measures of the constructs mentioned earlier varying between participants. Hierarchical regression relative to aim 1 showed that inhibition was positively associated with adaptive behaviour, above and beyond social abilities (reversed social impairments) and intelligence. Inhibition and social abilities were positively associated with adaptive behaviour and explained 50% of its variance. There was no association between cognitive flexibility, generativity, planning and adaptive behaviour over and above the contribution of social abilities and intelligence. Hierarchical regression relative to aim 2 showed that there was no association between EF abilities and participation. Furthermore, there was no association between adaptive behaviour and participation and, as such, adaptive behaviour was not found to be a mediator in the association between EF and participation. Hierarchical regression relative to aim 3 showed that there was no association between participation and self-reported QoL, above and beyond reversed anxiety and depressive symptoms. Reversed depressive symptoms were positively associated with self-reported QoL and explained 49% of the variance in self-reported QoL. Discussion Assessment of EF abilities, together with the assessment of social abilities, should become routine in the care of adolescents with ASD who have difficulties with adaptive behaviour. However, there is a need to investigate other factors in addition to EF abilities and adaptive behaviour which may influence participation in extra-curricular activities as impairments in the former are not associated with those in participation. Treating comorbid mental health symptoms of young people with ASD, in particular depressive symptoms, may result in an improvement of these young people’s self-reported QoL.
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ItemNovel therapeutic approaches to paediatric cancer( 2019)The overall cure rates for paediatric cancer have improved dramatically over the last 60 years. There is, however, a subset of paediatric embryonal tumours that carry a poor prognosis. In particular, metastatic neuroblastoma, in association with MYCN amplification, remains difficult to cure. The phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (AKT)/mammalian target of rapamycin (mTOR) cell signalling pathway, as well as epigenetic factors, play a key role in numerous cellular functions including cell growth, survival and angiogenesis. Activation of the PI3K/AKT/mTOR pathway is common in a variety of tumours including those associated with the MYC family of proto-oncogenes. As a result of its multiple cellular functions it is not surprising that deregulation of this pathway is frequently observed in cancer. The importance of MYCN as a therapeutic target, and the impact of PI3K inhibitors in MYCN amplified neuroblastoma has been well documented. Rapamycin and its analogues everolimus (RAD001) and temsirolimus (CCI-779) exert selective cytostatic/cytotoxic effects on by targeting mTORC1. PF-502 is a combined PI3K/mTOR inhibitor that serially inhibits multiple targets within this pathway. Accordingly, interest in both mTOR inhibitors and combined PI3K/mTOR inhibitors as anticancer drugs, with particular applications to neuroblastoma, has recently surged. When used in the aggressive TH-MYCN murine model of neuroblastoma, PF-502 produced a significant survival benefit by both apoptotic and anti-angiogenesis effects whereas temsirolimus worked primarily by an anti-angiogenic effect. Epigenetic modulation of tumours has recently received increasing attention recently. Histone deacetylase inhibitors have shown pre-clinical promise in a variety of paediatric tumours. Specifically, they have been shown to promote both tumour apoptosis and terminal differentiation in other paediatric embryonal tumour pre-clinical models. Accordingly, the role of agents targeting these pathways was studied in the aggressive TH-MYCN mouse model of neuroblastoma. Panobinostat, when given as a continuous, low dose, resulted in a significant survival benefit as a result of both apoptosis and differentiation, with terminal differentiation achieved by prolonged exposure to the drug. Following on from these pre-clinical studies, an open label, Phase I (3+3 design), multi-centre study evaluating panobinostat in pediatric patients with refractory solid tumours, including neuroblastoma, was conducted. Primary endpoints were to establish MTD, define and describe associated toxicities, including dose limiting toxicities (DLT) and to characterise its pharmacokinetics (PK). Secondary endpoints included assessing the anti-tumour activity of panobinostat, and its biologic activity, by measuring acetylation of histones in peripheral blood mononuclear cells (PBMNC). Panobinostat significantly induced acetylation of histone H3 and H4 at all time points measured when compared to pre-treatment samples. A significant biological effect of panobinostat, measured by acetylation status of histone H3 and H4, was achieved at a dose of 15mg/m2 and was well tolerated. PK data and drug tolerability at 15mg/m2 was similar to that previously published. In summary, pre-clinical experiments support serial inhibition of the PI3K/AKT/mTOR pathway and the use of histone deacetylase inhibitors as treatment approaches in neuroblastoma.
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ItemVariation of the Microbiome in Paediatric Crohn’s Disease( 2019)Introduction: Crohn’s Disease (CD) is a chronic, relapsing condition of the human gastrointestinal system with several significant extraintestinal manifestations. It can affect any age group; 10% of patients being diagnosed prior to their 18th birthday. Pediatric CD is known to have a more severe and difficult to manage phenotype. A rising incidence has been observed in recent years although the disease phenotype has remained largely unchanged. There is a growing body of literature describing dysbiosis in patients with CD. Despite extensive research, the role and behaviour of the gut microbiome in pediatric CD is not well understood. Aims: We aimed to characterize the microbiome in patients with CD longitudinally and compare it with non-IBD patients. We also sought to explore if there were any microbiome differences between ASCA positive and ASCA negative patients. Methods: We analysed the microbiome of 345 biopsies from 204 patients, including 88 CD first diagnosis (CDFD) patients, 38 patients in relapse (CDRL), 12 patients in remission (CDRM), and 66 controls. Species identification was conducted using oligotyping in combination with ARB/SILVA taxonomic annotation. Comparison was also made between ASCA status, microbial diversity and clinical characteristics. Results: We observed 45 bacteria to be statistically different between CDFD samples and controls, with Fusobacterium being the most implicated species in CDFD patients. We also identified gender specific differences in CD. Five species showed a strong association with patients with CD in relapse and 10 species in patients with CD in remission. Three taxa showed a positive co-occurrence across the two groups. Hespellia porcina (closest taxonomic neighbor to Clostridium oroticum) had the strongest association with samples from patients with CD in relapse. Interestingly, Fusobacterium was not part of the CD relapse associated taxa group. Faecalibacterium prausnitzii was equally present in CDFD and in control samples. ASCA was highly specific but poorly sensitive for the diagnosis of CD. In patients with CD, ASCA positivity was more likely to be present in patients older than 10 years, and associated with increased likelihood of ileocolonic disease distribution and long-term risk of surgery. Microbial alpha and beta diversity were similar in patients with CD with or without ASCA, but significantly less when compared to non-IBD controls. 14 bacterial species were statistically associated with ASCA positive patients with CD and 14 species with ASCA negative patients (p< 0.05). By using a false discovery rate corrected P value, two species remained statistically associated with both the groups. Ruminococcus torques and bacterium Yersinia enterocolitica were statistically associated with CD ASCA positive patients (p = 0.0178). Enterobacter cloacae and Faecalibacterium prausnitzii were statistically associated with CD ASCA negative patients (p = 0.0178 and 0.0342, respectively). Conclusions: This is the first study to investigate gut mucosal microbiome in a pediatric CD cohort with longitudinal sampling. Significant differences in microbiome were observed between treatment naïve patients with CD, patients with CD in relapse, patients with CD in remission and non-IBD patients. We also identified differences in the gut microbiome between patients with CD depending on presence of ASCA.
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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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ItemImproving the quality of Essential Newborn Care in Solomon Islands( 2019)Gaps in the quality of hospital care in low- and middle-income countries contribute to neonatal death and morbidity. Most neonatal deaths occur in the first few days of life, many from preventable or treatable causes. Essential newborn care consists of low-cost interventions, such as basic resuscitation, early breastfeeding and skin-to-skin contact, which have been shown to improve outcomes. Successful essential newborn care implementation requires understanding of the setting in which it is being delivered and the contextual factors that enable healthcare workers to provide quality care for newborns. Solomon Islands is a low-resource country in the Western Pacific and a Small Island Developing State with high neonatal mortality rates, increasing births per capita and limited healthcare resources and personnel. The Ministry of Health and Medical Services (MHMS) and stakeholders implemented an intervention to improve newborn quality of care in Solomon Islands. This thesis evaluates the quality of care in hospitals of Solomon Islands and describes contextual factors that enabled successful implementation of a multifaceted intervention to improve essential newborn care. This thesis used a mixed methods design comprising the following sequential studies: (1) Quality of hospital care for newborns was described through a cross-sectional study using a structured assessment tool to identify strengths and limitations in structure and process components of existing care. (2) Three years of perinatal outcomes were audited to determine stillbirth, perinatal and neonatal mortality rates and the main causes of neonatal morbidity and mortality. (3) The impact of the World Health Organization Early Essential Newborn Care training program on knowledge and skills of healthcare workers was assessed, using a before-and-after study that identified the factors associated with improved retention of knowledge and skills. (4) The implementation process was described through interviews of healthcare workers, and interview data were triangulated with quantitative results to describe features of the intervention that supported implementation. This thesis demonstrated gaps in structure and process elements in quality of newborn care, which limited provision of appropriate, timely clinical care in the hospitals. Very high perinatal mortality rates, mainly owing to stillbirths, were identified. The provinces had higher rates of perinatal mortality than the National Referral Hospital. The main causes of neonatal mortality were complications of prematurity, birth asphyxia and infection. Knowledge and skills of healthcare workers significantly increased following the coaching program. At the time of evaluation, some loss of skills over time had occurred, particularly in cadres of healthcare workers that did not routinely use relevant skills. The evaluation of the implementation process reflected strengths of the intervention, specifically the training methods (content, short duration and practical approach) and the impact of a small training team of MHMS midwives and nurses who delivered the program independently. The challenges and sense of anxiety faced by healthcare workers in remote, isolated settings with infrequent exposure to resuscitation were highlighted. The barriers to establishing independent, ongoing quality improvement activities reflect the challenge of sustaining action across a large geographical area that has a dispersed health workforce when there is little capacity for regular oversight and support. This thesis highlights the potential impact from a multifaceted intervention to improve essential newborn care. Together with efforts to address stillbirths and improve intrapartum quality of care and quality of care in the neonatal period, essential newborn care may form an important part of a strategy to improve outcomes for newborns.
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ItemINVESTIGATING THE ROLE OF INNATE IMMUNITY IN MEDIATING THE NON-SPECIFIC EFFECTS OF BACILLE CALMETTE-GUÉRIN VACCINE( 2019)Background: Epidemiological evidence suggests that Bacillus Calmette-Guerin (BCG) vaccine exerts non-specific (heterologous) effects in infants; decreasing neonatal mortality in high-mortality settings and preventing allergy and morbidity from infection in developed countries. New tuberculosis (TB) vaccines could potentially lack these beneficial effects. Immune mechanisms underlying the non-specific effects of BCG vaccine have been linked to ‘trained immunity’ or innate immune memory. Aims: Part 1: To investigate whether neonatal BCG vaccination alters the immune response to heterologous pathogens and Toll-like receptor (TLR) ligands in (i) the neonatal period and (ii) infancy. Part 2: To use a systems vaccinology approach to identify innate immune signatures underlying the non-specific effects of BCG vaccine. Methods: Part 1: In the Melbourne Infant Study: BCG for allergy and infection reduction (MIS BAIR), 1272 infants were randomised to receive BCG vaccine or no BCG vaccine within the first 10 days of life. A subset of participants was recruited to an immunological sub-study. A whole blood stimulation assay of ‘specific’ mycobacterial antigens, heterologous bacterial or fungal antigens and TLR ligands was used to interrogate cytokine responses at 7 days (n=212) and 7 months (n=167) post randomisation. Part 2: In the BabyBAIR study, 44 infants who were BCG vaccinated prior to travel to a TB-endemic area were recruited. Blood was collected from participants prior to BCG vaccination, and at 7 days and 3 months post vaccination. Cytokine responses and cell populations were analysed following in vitro stimulation of whole blood as above. RNASeq was also done on whole blood and the transcriptome was analysed for differentially expressed genes. Pathway analysis was done using functional gene set enrichment analysis (fGSEA) at each time point compared to baseline. Results: In the MIS BAIR study, infants who were BCG-vaccinated had significant differences in their heterologous cytokine responses at both 7 days and 7 months post randomisation compared to BCG-naive infants. At 7 days post randomisation, compared to BCG-naive neonates, BCG-vaccinated neonates had evidence of a pro-inflammatory bias in RPMI-stimulated (nil) samples. Following heterologous stimulation, BCG-vaccinated neonates had decreased chemokine (MCP-1, MIP-1alpha, MIP-1beta, IL-1RA, IL-6 and IL-10 responses following stimulation of TLR2 (PEPG) and TLR7/8 (R848). At 7 months post randomisation, compared to BCG-naive infants, BCG-vaccinated infants, had decreased IFN-gamma responses to stimulation with heterologous pathogens. Decreased IFN-γ responses in the BCG-vaccinated group were attributable to a reduction in the proportion of individuals mounting an IFN-gamma response. Heterologous cytokine responses were modified by sex and maternal BCG vaccination status in both neonates and older infants. In the BabyBAIR study, longitudinal cytokine analysis showed BCG vaccination to be associated with a pro-inflammatory bias at baseline (prior to BCG vaccination), a robust pro-inflammatory heterologous response at 7 days post BCG vaccination and downregulation of pro-inflammatory cytokines 3 months post BCG vaccination compared to baseline. Flow cytometry suggested that both myeloid and monocyte derived dendritic cells were associated with the observed heterologous cytokine responses. RNASeq analysis of the whole blood transcriptome following BCG vaccination indicated widespread changes in innate immune signalling pathways and identified several potential mechanisms by which BCG vaccine could mediate its beneficial heterologous effects. Conclusions: Neonatal BCG vaccination leads to significant changes in the immune phenotype of vaccinated individuals. Following in vitro stimulation with heterologous pathogens, BCG-vaccinated infants have altered immune responses which might improve regulation of the inflammatory response during acute infection and a subsequent reduction in all-cause mortality. These results support the paradigm of BCG-induced trained immunity and provide additional information regarding the nature of the response in neonates and between different classes of pathogens.
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ItemThe epigenetic landscape of paediatric acute myeloid leukaemia( 2019)Paediatric acute myeloid leukaemia (AML) is a cancer of the blood and bone marrow. It is currently one of the leading causes of cancer-related mortality in children. While induction therapy is largely successful in achieving patient remission, the relatively high mortality rate is driven by the large genetic heterogeneity of AML and recurrence of disease. Disease relapse rate is higher than other childhood leukaemias, is fast acting and often chemotherapy resistant. While much of the genetic contribution to disease has been described, there is still a component of AML pathogenesis that has yet to be discovered. Many of the genetic lesions found in adult AML directly affect epigenetic modifying genes, however this is not the case in children. Despite this, previous research has shown vast epigenetic alteration in paediatric AML. As such it is possible that some of the unexplained pathogenesis in childhood AML can be elucidated by modulation of gene activity via aberrant changes in the most widely studied epigenetic process, DNA methylation (DNAm). Few studies have comprehensively interrogated the DNA methylome of paediatric AML, nor has the prognostic utility or biomarker potential of DNAm been explored. In this study, we explored the global methylation profile of paediatric AML in comparison to non-leukaemic controls and subtype-dependant and independent biomarkers of disease that may have functional relevance. Furthermore, we described DNAm signatures with potential prognostic utility, to accurately identify predisposition to relapse at diagnosis. Genome-wide DNAm was interrogated via the HumanMethylation450 BeadChip Array (HM450K) on a cohort comprising of 128 archival and fresh bone marrow tissue sourced from multiple hospitals around Australia. This data was then combined with the TARGET AML cohort comprising of a further 231 bone marrow samples. Targeted replication and validation of findings was undertaken on a reduced cohort using SEQUENOM MassArray EpiTYPER. Bioinformatic and machine learning analyses were undertaken in R. The findings revealed subtype-independent genome-wide average methylation (GWAM) to be increased in diagnostic samples compared to non-leukaemic controls. This was further verified by differences in the global methylation proxy genes known as LINE1 and Alu. Deeper interrogation of these differences demonstrated wide-spread differential methylation in previously implicated genes in AML pathogenesis including WT1 and DGKG, both of which were validated in an independent cohort. Other genes identified to be differentially methylated included ZSCAN1, REC8 and IRX1. Subtype analysis validated previous studies showing inv(16)-specific differential methylation in MN1 and MEIS1. Finally, DNAm was used as the primary feature for a machine learning model designed to predict patient relapse at diagnosis. The final model achieved an area under the curve (AUC) of 94% with correct identification of 91% of all cases involved (F-measure=0.914). To date, this study represents the largest and most comprehensive insight into aberrant DNAm in paediatric AML. Results have increased our understanding of genes that are differentially methylated and highlight the potential utility of DNAm as a future prognostic biomarker. It is anticipated that these findings will serve as a foundation for future functional studies aimed at delivering truly personalised treatment regimens for children with AML.
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ItemModelling Inherited Kidney Diseases with Kidney Organoids Derived by Directed Differentiation of Patient Induced Pluripotent Stem Cells( 2019)Genetic kidney diseases are a heterogeneous group of disorders with varying phenotypes dependent on the affected nephron segment. Next generation sequencing has increased our appreciation of the breadth of gene variants associated with these diseases. It has also identified large numbers of variants of unknown significance (VUS), which require functional genomic validation. There is an unmet need for novel therapies for genetic kidney diseases as most invariably progress to dialysis or transplantation without any form of targeted treatment. Laboratory based research of genetic kidney disease requires the recapitulation of a disease phenotype in animal and/or in vitro cellular disease models. Interspecies variation in anatomy, physiology and gene function limits the translation of animal models to human disease and clinical care. Classical two dimensional cell cultures lack the complexity and intercellular cross-talk of in vivo three dimensional tissue. Kidney organoids are three dimensional, miniature, multicellular, human, in vitro micro-tissues, offering distinct disease modelling advantages over other models. Furthermore, kidney organoids can be regenerated from induced pluripotent stem cells (iPSC) reprogrammed from patients with genetic kidney disease, potentially providing outcomes with personalised clinical relevance. As a novel platform, the capabilities and limitations of kidney organoids as disease models are not well understood. By differentiating and characterising kidney organoids from the iPSC of patients with inherited kidney diseases, this thesis aims to explore the application of kidney organoids to disease modelling. As proof of concept, kidney organoids were first generated from iPSC reprogrammed from a patient with compound heterozygous variants in IFT140, an already validated nephronophthisis (NPHP) genotype. An isogenic control was generated by precision CRISPR-Cas9 gene editing. In this project, differential primary ciliary morphology within organoid tubules and transcriptional profiling of organoid epithelium validated the ability of the organoids to model genetic disease. Attempts were then made to validate novel, candidate variants for other pedigrees with unresolved trio whole exome sequencing. In a proband with clinically suspected NPHP, DNAH5 was selected as a candidate gene, despite previously association with a motile ciliary phenotype. In this project, kidney organoids were unable to validate the patient variant as pathogenic. In addition, a number of lessons were learned regarding the necessary variant curation process prior to making a commitment to modelling with kidney organoids. In the final chapter, kidney organoids validated a novel genotype for the glomerular disease steroid resistant nephrotic syndrome, via international collaboration with the laboratory of Prof Friedhelm Hildebrandt. Glomeruli within kidney organoids differentiated from iPSC expressing a patient-derived, homozygous variant in NOS1AP, displayed aberrant development, increased podocyte apoptosis and reduced expression of PAR polarity proteins. Together these projects demonstrate the strengths and challenges of using kidney organoids as models of inherited renal disease. Kidney organoids stand to complement animal and 2D unicellular disease models rather than replace them. We proposed that patient-derived kidney organoids are best placed to model paediatric onset kidney diseases with the future potential of providing personalised therapeutic screening.
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ItemThe Use of Pluripotent Stem Cells (PSCs) and CRISPR Genome Editing to Study the Roles of TRPV4 Ion Channels in Skeletal Malformation( 2019)Transient Receptor Potential Vanilloid 4 (TRPV4) is a non-selective calcium channel that plays an important role in the mechanotransduction system in chondrocytes. Heterozygous TRPV4 mutations cause skeletal disorders with varying severity. Heterologous cells such as fibroblasts and HEK-293 cells are commonly used to model TRPV4-inherited skeletal diseases in vitro. Studies using human chondrocytes are limited because cartilage is rarely available from patients and controls. Although heterologous cells cannot completely recapitulate the biological processes occurring in human chondrocytes, the studies show that two distinct disease phenotypes, TRPV4 skeletal dysplasia and arthropathy, might be caused by differences in the way the mutations change TRPV4 channel behaviour. TRPV4 skeletal dysplasia causing mutations show channel over-activity whereas arthropathy causing mutations show reduced channel activity upon channel stimulation. However, the downstream pathogenic mechanisms responsible for the distinct skeletal phenotypes remain undefined. Recognising the limitations of previous studies, human-induced pluripotent stem cells (hiPSCs) offer a new approach for inherited disease modeling. Their ability to differentiate into disease-relevant cells such as chondrocytes creates new opportunities for TRPV4-inherited skeletal disease modelling. Therefore, this PhD project aims to model the disorders caused by two TRPV4 mutations using hiPSCs and identify the pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal disease phenotypes. To obtain disease-relevant cells, establishing a robust and reproducible chondrocyte differentiation protocol is required. To do this, a reporter hiPSC line, SOX9-T2A-tdTom, was generated from MCRIi001-A (PB001.1) (1) using CRISPR/Cas9 genome editing. Thus in vitro chondrocyte differentiation could be monitored in real-time. The T2A linker and tdTomato fluorescent reporter gene were inserted downstream of the SOX9 coding sequence through homology-directed repair. The targeted allele was designed to produce SOX9 with the T2A sequence at the C-terminal end and a separate tdTom fluorescent protein. Genomic DNA sequencing of the SOX9-T2A-tdTom hiPSC line confirmed that the hiPSC line had one SOX9 allele with the T2A tdTom gene fusion and one wild type allele. Pluripotency was maintained as indicated by expression of pluripotency markers OCT4 and NANOG (immunostaining); CD9, CD326, and SSEA-4 (flowcytometry); and the ability to form tissues derived from three germ layers. SNP array showed there were no aneuploidies. The SOX9-T2A-tdTom hiPSC line had a similar capability to the parental line, MCRIi001-A, to form sclerotome. Western blotting showed that SOX9 protein expression was similar between SOX9-2A-tdTom and its parental line suggesting that adding tdTom gene sequence downstream of SOX9 gene did not disrupt the SOX9 expression and stability. The chondrocyte differentiation protocol was established using the SOX9-T2A-tdTom hiPSC line. Two stages of differentiation were performed. First, sclerotome induction was achieved by culturing hiPSCs in a 6-day multiple-step chemically defined culture mimicking embryonic development with pellet culture format was established on day 4. Secondly, chondrocyte differentiation was performed by transferring day-6 pellets into chondrogenic media in swirling culture format up to 10 weeks. A 4-week course of FGF2 treatment followed by an optional TGFB3 and GDF5 treatment until week 10 was performed during chondrocyte differentiation. RNA was collected every day during a 6-day sclerotome induction and at different time points during chondrocyte differentiation. The optimised protocol that involved a multiple-step chemically-defined 3-dimensional (3D) culture with swirling in an extended culture that included a 4-week FGF2 supplementation and optional subsequent TGFB3 and GDF5 treatment was able to generate cartilage that closely resembles fetal cartilage. CRISPR/Cas9 genome editing was also used to introduce two human TRPV4 mutations, a TRPV4 c.819C>G (p.F273L) mutation causing familial digital arthropathy with brachydactyly (FDAB) and a TRPV4 c.2396C>T (p.P799L) mutation causing metatropic dysplasia, into the SOX9-T2A-tdTom hiPSC line. For in vitro disease modelling, the mutant and their isogenic wild-type control (SOX9-T2A-tdTom) hiPSC lines were differentiated towards chondrocytes using optimised chondrocyte differentiation. The phenotypic differences between mutants and wild-type were assessed using various techniques including gene (RNA sequencing) and protein expression analysis. The two mutant cell lines and their isogenic wild-type control (SOX9-T2A-tdTom) were able to form cartilage. The pellet cartilage histology did not show any striking differences between the two mutants and their isogenic control. COL2A1 and TRPV4 protein expression was similar between mutants and control and this was consistent with the RNA sequencing data. RNA sequencing suggested that the pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal diseases were different. Compared to the isogenic control, F273L mutant cartilage had 263 differentially expressed genes. F273L cartilage showed a slight reduction in cartilage related gene expression including COL2A1, CSPG4, BGN, and CILP2. The F273L cartilage tissue was also less mature than the wild-type as indicated by increased SHH expression. On the other hand, P799L cartilage had more differentially expressed genes (655 genes) than F273L. MEF2C, the main regulator of chondrocyte hypertrophy, was upregulated in P799L. The hypertrophic chondrocyte markers such as RUNX2, SPP1 or osteopontin, and PTH1R, were also upregulated in P799L suggesting increased chondrocyte hypertrophy of P799L chondrocytes. The other characteristics of hypertrophic chondrocytes such as a reduction in cell proliferation and increased apoptosis were also observed in P799L cartilage. In conclusion, this study is the first study that conducts global gene expression analysis using RNA sequencing to characterise gene expression changes downstream of TRPV4 mutations in hiPSC-derived chondrocytes. The pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal diseases are different. The fewer differentially expressed genes in the F273L cartilage than in P799L suggests a milder disease phenotype. The slight reduction in cartilage marker expression in F273L cartilage might cause the cartilage tissues less resilient to physical forces thus leading to FDAB. In contrast, accelerated chondrocyte hypertrophic maturation can be the pathogenic mechanism underlying TRPV4 skeletal dysplasia phenotype. Accelerated chondrocyte hypertrophic maturation can disrupt growth plate development and cause systemic skeletal defects seen in patients. This thesis demonstrates that hiPSCs are a powerful tool to model inherited skeletal disease in vitro.
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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.