Paediatrics (RCH) - Theses
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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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ItemInvestigating the biology of paediatric T cell acute lymphoblastic leukaemia to facilitate more effective individualised therapy( 2019)Acute lymphoblastic leukaemia (ALL) takes up the highest percentage of paediatric cancer. The treatment requires intensive chemotherapy for two to three years, as well as haematopoietic stem cell transplantation for poor-prognosis cases. Compared to B cell lineage ALL (B-ALL), T cell lineage ALL (T-ALL) has a lower 5-year event free rate, higher rate of relapse, and a worse outcome for relapsed cases. Individualised therapy, targeting at oncogenic changes in each patient, can make treatment more effective and less harmful. This requires understanding of the oncogenic biology of each individual leukaemia. We have attempted to develop a T-ALL model based on hiPSC-derived T cells, which will be in human origin, maintain normal genetic pattern, mimic in vivo T cell development, and can be massively produced for high throughput lab work. This model may make up for the shortcomings of conventional leukaemia cell lines and mouse models. This project investigates the biology of T-ALL by focusing on two novel fusion genes – TCF7-CSF1R and ETV6-CRX – identified by RNA sequencing of paediatric T-ALL patient samples. We have shown that TCF7-CSF1R is sufficient to immortalise mIL-3 dependent Ba/F3 cells. The ETV6-CRX fusion gene is anticipated to block differentiation. Establishing consistent expression of this fusion will require further optimization. The feasibility of setting up a hiPSC-derived T-ALL model was also assessed, with respect to protein expression in human T-ALL/lymphoma cell lines, hiPSC differentiation efficiency, hiPSC-derived T cell lentiviral infection rate, and cytokine withdrawal during differentiation. This project provides potential directions for improvement of methods for exogenous gene expression, such as the usage of CRISPR-Cas9 based techniques to introduce gene modifications for fusion genes such as ETV6-CRX that are difficult to express, particularly in hiPSC-derived T cells that have a low viral infection level. The T cell differentiation protocols also need to be optimised to make the T cell production easier and efficient. Detailed functional assay during T cell differentiation needs to be conducted in the future. In this thesis, Chapter 1 presents the background of this project; a literature review introducing human haematopoietic system, in vivo thymocyte development, paediatric TALL, novel oncogenic fusion-related genes, and in vitro T cell generation; the aims and hypothesis. Chapter 2 introduces the methods and materials used in this project. Chapter 3 presents the identification, cloning, and expression of novel fusion genes. Chapter 4 investigates the ability of these novel fusion genes to support cell survival and proliferation in conventional Ba/F3 cell line. Chapter 5 assesses the feasibility of setting up the hiPSC-derived T-ALL model. Chapter 6 makes a discussion on the results and concludes the whole project.
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ItemEnvironment versus genetics in the development of health-related problems in childhood obesity( 2017)Introduction: Childhood obesity is a major health concern affecting a quarter of all Australian youth. Obese youth are at risk of multi-systemic obesity-related comorbidities (ORC), such as Prediabetes and Type 2 Diabetes Mellitus (T2DM), Hypertension, Hyperlipidaemia, Non-alcoholic fatty liver disease (NAFLD), Polycystic ovarian syndrome (PCOS), Obstructive sleep apnoea (OSA), mental health and orthopaedic issues. However, within the obese population, the severity of obesity is a poor predictor of these comorbidities. As such, current guidelines advise screening obese youth for all comorbidities, which is both time consuming and costly. ORC, as with obesity itself, are complex polygenic disorders involving interaction between genetic and environmental risk factors. Furthermore, many of these interactions are likely mediated by epigenetic variation, linking environmental exposures with genetic predisposition. Therefore, clarification of the role of environmental exposures, genetic risk factors and epigenetic profile in obese youth may provide valuable insight into determining which obese youth are at risk of developing ORC. The aim of this study was to characterise and investigate the associations between environmental, genetic and epigenetic factors with obesity-related comorbidities in obese youth. This information obtained will allow clinicians to better predict and profile the comorbidity burden in obese youth for targeting interventions to those most likely to benefit. Materials and Methods: The Childhood Overweight BioRepository of Australia collected comprehensive clinical and anthropometric data, as well as blood samples for genetic, metabolic and hormonal profile testing from patients (age 1 to 18 years) attending the Weight Management Clinic at the Royal Children’s Hospital, Melbourne. Anthropometric measures included standard measurements of weight, height and waist circumference (converted into BMI z-score (BMI-Z) and waist-height ratio for analyses) as well as total percentage body fat (%FM) and percentage truncal body fat (%truncal) using bioimpedance. Dietary intake was assessed using the Australian Child and Adolescent Eating Survey and physical activity was measured objectively using accelerometry. Genetic typing and epigenetic profiling of single nucleotide polymorphisms (SNP) and measures of DNA methylation were performed using the SEQUENOM MassARRAY platform. Results: 349 participants were included in the study. Both metabolic and non-metabolic ORC are common in obese youth. The most commonly used measure of adiposity, BMI-Z, was the anthropometric marker least associated with ORC, while important ORCs such as T2DM and hyperlipidaemia were not significantly associated with any of the anthropometric measures. The presence of two or more significantly elevated anthropometric markers was associated with the presence of a higher number of ORCs. Almost all obese youth reported poor vegetable intake and high sedentary behaviour (TV-watching and total media) time. Although initial analyses observed associations between sedentary behaviour (TV-watching and total media use time) with prediabetes and hyperlipidaemia, unhealthy food options with mental health issues, and high fruit intake with NAFLD, these associations lost significance after correction for multiple testing by false discovery procedure (FDR). However, low vitamin D levels were significantly associated with hypertension, independent of adiposity and season. The Fat Mass and Obesity (FTO) gene has the largest influence on adiposity amongst obesity-risk genes to date and has been associated with ORCs. In our cohort, the FTO risk allele was associated with poorer longitudinal weight outcomes in pre-pubertal children, but not pubertal adolescents. The FTO SNP however was not associated with any ORCs. We also examined the association between the top 22 T2DM risk SNPs identified from previous studies and Prediabetes or T2DM status. The SLC30A8 rs13266634 SNP appeared to be protective of prediabetes/T2DM status in unadjusted analysis but lost significant on FDR testing. The addition of T2DM SNPs profile to TV-watching time improved the predictive model for Prediabetes/T2DM status. We were not able to replicate the previously reported association of FTO CpG hypomethylation with prediabetes/T2DM in obese children and adolescents. However FTO hypomethylation was seen in obese youth with NAFLD, independent of age, sex and total body adiposity. FTO methylation was independent of FTO expression, which was not associated with any ORC. Conclusion: Anthropometry is not associated with comorbidities in childhood obesity. Environmental factors do not appear to be associated with comorbidities. Low vitamin D level is associated with hypertension. Genetic factors impact on weight gain but not comorbidities, and epigenetic factors may contribute to NAFLD. Combining these environmental and genetic elements with established clinical risk factors in the assessment of each individual ORC may assist in enhancing current clinical models of care for obese children and adolescents.
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ItemA tool for risk profiling and accurate prognostication in paediatric glioma integrating clinical features with epigenetics: it is time to move on from the binary classification( 2016)Paediatric glioma, the most common group of brain tumours in children, encompasses a wide range of entities with highly variable prognoses. Gliomas are grouped by histopathological features into high and low grade glioma but this classification does not take into account many established and emerging risk factors in this disease. Research into the molecular features of these lesions has shown that histology does not always correlate with biology and, where they differ, molecular features are usually superior at predicting outcome. Risk classifications have been developed for other paediatric malignancies which combine clinical, radiological, pathological and molecular factors to predict disease risk and prognosis. A comprehensive risk classification has not been published for paediatric glioma despite many risk factors being established in this disease. Using a clinical cohort of all paediatric glioma treated at a single institution (Royal Children’s Hospital, Melbourne, Australia) over an 18 year period a database was developed incorporating clinical, radiological, pathological and treatment factors. Where sufficient tumour tissue was available genome-wide methylation analysis was performed. The results of this were processed and evaluated by an established cluster analysis algorithm. Breaking the cohort into clinically appropriate subgroups, risk factors for disease progression and death were determined and prognosis estimated for distinct groups. A tool for robust risk profiling and prognostication was established with 5 main risk groups and 10 subgroups. Patients in the very low risk group have a predicted 100% overall survival and the majority require no treatment. In contrast there was a group of patients with 100% mortality within a short space of time where attempts at curative therapy are futile and may be deleterious to quality of life. The intermediate risk group contained a number of entities with a prognosis genuinely falling between that of low grade glioma and that of high grade glioma. Histopathological diagnosis retained prognostic importance for some, but not all, groups and methylation analysis was shown to have a significant role to play, particularly in high grade lesions or where diagnosis was unclear. This is likely to become part of routine care in the future and its place is strongly supported by the analysis presented here. Risk profiling is possible in paediatric glioma with far more accuracy than the current binary classification provides. A combination of established clinical factors and emerging molecular features provides an accurate and nuanced assessment of risk and prognosis.