Paediatrics (RCH) - Theses
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Cellular mechanisms that spatiotemporally direct neural crest cell migration and enteric nerve system formation
The enteric nervous system (ENS) in the gastrointestinal tract is a complex nervous network. It is essential for gut secretion, absorption and peristalsis. Most of the ENS arises from vagal neural crest cells (NCCs) which migrate from the neural tube (level somite s1 to s7) into the foregut then colonize the rest of the intestine as a rostro-caudal wave and form the ENS. If this cell migration fails to be completed, the distal intestine lacks ENS, this results in Hirschsprung Disease (HSCR). My research project is going to answer: Do all levels of vagal NCC contribute to ENS equally? What is the enteric NCCs population expansion potential and how do individual initial enteric NCCs contribute to the final ENS? Does the ENS population retain its colonization capacity during development? How do enteric NC/glia cells and neurons interact in directing ENS cell invasion and axon extension? What is the mechanism that control ENS ganglion formation, especially the role of differential adhesion of enteric neurons and NCCs on gangliogenesis? This thesis using avian models combined with multiple techniques demonstrated that: The vagal NCCs of single somite levels origins migrate separately and converge at the foregut, where they all mixed. Along the whole vagal length, the mid-vagal region NC-derived cells -- that is s3 and s4 level -- arrive earliest at the foregut, and contribute the greatest numbers. During colonization along the gut, all levels of vagal NC-derived cells are mixed throughout the entire gut, but mid-vagal NC-derived cells contribute to all region of gut from foregut to distal hindgut in greatest numbers. Regarding the ENS forming potential, all levels of vagal NCC could form ENS, but mid-vagal NCCs (s3 and s4) have greatest competency. The temporal expansion of the enteric NC population in developing quail gut was investigated from E2.5 to E12, and the expansion potential was challenged by drastically reducing the starting enteric NCC numbers. By this means, the enteric NCCs showed an extremely high capacity to regulate their proliferation while forming the ENS. However, single cell lineage tracing technique developed in this thesis demonstrated that the contribution of individual enteric NCC to final ENS was unequal and unpredictabe, a few dominant ENS “superstar” cell clones emerge but most clones are small. These “superstar” clones are not predetermined, instead they achieve this status stochastically. The extremely high gut-colonization capacity of enteric NCCs from the early embryo gut was found to rapidly decline with embryonic age, and this declines was more rapid in the distal intestine-derived enteric NCCs than in more proximal enteric NCCs. This age-related loss in colonization capacity involves changes in the ENS cell population, rather than the maturity of the mesoderm. However, it is not caused by reducing the “undifferentiated” ENS cell number, since this capacity can neither be rescued with more time to catch up, nor mimicked by reducing ENS cell number from young donor. Actually, unlike their proportion in the ENS, the absolute number of apparently undifferentiated enteric NCCs does not decline with age. Therefore, this age-related loss in colonization capacity is caused by changes in qualitative aspects with age. The early development of the ENS shows two invasive events: rostral-to-caudal invasion by enteric NCCs and extension of ENS axons. Experimentally, the gut mesoderm supports these two invasive events for longer than the normal time-window. Both these events can occur bidirectionally when permitted, although normally they are unidirectional. However, a fresh invasion wave of enteric NCCs is prevent by pre-existing ENS cells. This prevention of invasion does not occur in gut with pre-existing ENS neurons and axons but without enteric NCCs. Therefore the conclusion is that this prevention is caused by the pre-existing enteric NCCs and not by other ENS components. In the other hand, pre-existing ENS neurons and axons inhibit neuronal differentiation of the invading enteric NCCs. In contrast, ENS axon invasion is not prevented by pre-existing ENS cells and axons. These invasive axons do not use pre-existing ENS axon tracts or ENS neurons to facilitate invasion of the gut, but have an absolute requirement for enteric NCC (either in situ or co-invading) in order to advance into gut mesoderm. Follow these colonizing aspects of ENS development, the ratio of enteric NCC to neuron is stabilized and ENS ganglia are formed with a core of neurons and a shell of enteric NC/glia cells. Using cell-cell aggregation assays this thesis revealed that during aggregation, both Ca+2-dependent and independent adhesion mechanisms are required. Neurons sorted to the core of aggregates, surrounded by outer enteric NCCs, showing that neurons had higher adhesion than enteric NCCs. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM (Ca+2–independent) and N-cadherin (Ca+2–dependent) on this surface of the outer non-neuronal enteric NCCs. In addition, the ganglion size is intrinsically regulated by the ratio of enteric NCCs to neurons likely by generation of an outer non-adhesive surface. Overall, my research covers almost the entire process of the ENS development from initiating of NC on its origin site to the colonization of enteric NCC along the entire gut, and the final steps of ENS ganglion formation. The results from this thesis revealed many critical issues in understanding of the cellular mechanisms that control these processes. These basic researches have important implications for understanding not only ENS development but also enteric neuropathologies and for designing NC stem cell therapies.
Identifying novel disease genes in genetically undiagnosed individuals with Rett syndrome and related neurodevelopmental disorders
Rett Syndrome (RTT) is a severe neurodevelopmental disorder (NDD) resulting in severe cognitive and physical impairments. Despite being predominantly caused by pathogenic variants in the methyl-CpG-binding (MECP2) gene, between 3 – 15% of classic and atypical RTT individuals do not have a genetic diagnosis. Classic RTT individuals exhibit an apparently normal development until 6 to 18 months of age after which developmental regression occurs. Atypical RTT individuals have many features of classic RTT but do not meet all the specific diagnostic criteria. Recently, the classification of RTT has been expanded to include individuals with clinical features overlapping RTT and other NDDs, often referred to as RTT-like individuals. The clinical and genetic diagnosis for RTT-like individuals is further complicated due to the complexity of NDDs and there is an unmet need to provide a precise genetic diagnosis for these individuals. Next generation sequencing (NGS) studies are continuously identifying hidden genetic links between relevant molecular pathways and RTT. Through whole genome sequencing (WGS), our lab had identified a de novo heterozygous missense variant [c.744C>A; (p.Asp248Glu)] in the motor domain of kinesin-3 family member 1A (KIF1A) in one classic RTT female. Single nucleotide heterozygous variants in KIF1A have been implicated in a number of severe neurological disorders, collectively known as KIF1A-associated neurological disorders (KANDs). KIF1A encodes a neuron-specific kinesin molecular motor protein essential for ATP-dependent anterograde axonal transport of synaptic cargos along microtubules. In order to determine additional RTT/RTT-like individuals with KIF1A variations, we collected further clinical and genetic information from our collaborators of 30 individuals with 18 different missense variants affecting the critical motor domain of KIF1A. After careful clinical assessment, we identified three additional individuals with different novel missense variants exhibiting overlapping RTT-like and KAND clinical features. In silico tools predicted all variants to affect proper protein folding and were predicted to be likely disease causing. In addition, in vitro functional analysis using the highly specific neurite tip accumulation and microtubule gliding assays, demonstrated all variants to have reduced microtubule based movement, suggesting these variants are indeed significantly pathogenic. Comparison of the clinical features of the remaining 27 KAND individuals with 16 variants in the KIF1A motor domain suggested that specific clinical features and phenotypic severity was largely dependent upon the location of the variant. Using an NGS approach, we identified pathogenic MECP2 variants, previously missed by mainstream genetic testing, in seven RTT individuals including a case of a mosaic male. In addition, we found variants in two genes that are known to be associated with NDDs and RTT-like syndromes; Structural Maintenance of Chromosomes 1A [SMC1A; c.3576delA; p.(Val1193Serfs*2)] and SH3 and multiple ankyrin repeat domains 3 (SHANK3; c.2265+1G>A). This work continues to expand the genetic basis of RTT. Through whole exome sequencing (WES), we have also identified an atypical RTT female with a heterozygous nonsense variant [c.3385C>T; p.(Arg1129*)] in Lysine Acetyltransferase 6A (KAT6A) that encodes a chromatin remodelling protein. Heterozygous protein truncating variants in this gene have been associated with KAT6A-related intellectual disability. Through various collaborations, we identified a further four individuals with KAT6A variants [c.3820G>T; p.(Glu1274*), c.3399_3400dup; p.(Lys1134Argfs*14), c.3377delC; p.(Ser1126Phefs*8) and c.3631_3632delGT; p.(Val1211*)] who had clinical symptoms overlapping with RTT/RTT-like individuals. Through systematic re-analysis of a reported cohort of 76 individuals with KAT6A-related intellectual disability we recognized two additional individuals exhibiting RTT-like clinical features with KAT6A variants [c.4254_4257del; p.(Glu1419Trpfs*12) and c.3661G>T; p.(Glu1221*)] . All the identified variants in the seven RTT-like individuals were clustered in the last exon of KAT6A and in silico analysis predicted the variants to cause a dominant-negative effect. These seven individuals exhibited clinical features overlapping between RTT and KAT6A-related intellectual disability that was previously unrecognized. Using singleton WGS, a novel heterozygous nonsense variant in another chromatin regulator gene, Chromodomain helicase DNA-binding protein 8 [CHD8; c.5017C>T; p.(Arg1673*)] was identified in an atypical RTT individual. Heterozygous protein truncating variants in CHD8 have been implicated in NDDs including Autism Spectrum Disorders (ASDs). Functional analysis confirmed reduction in CHD8 protein levels in her fibroblasts, confirming the pathogenicity of the identified variant. In another RTT-like female, a de novo heterozygous missense variant [c.271G>A; p.(Asp91Asn)] in the Eukaryotic Translation Elongation Factor 1 Alpha 2 (EEF1A2), involved in protein translation, was identified through singleton WES. This variant has been previously reported in a female with NDD. Interestingly, a single case with the same EEF1A2 variant [c.274G>A, p.(Ala92Thr)] has also been reported in a RTT-like female. Thus, our findings further established the casual association between EEF1A2 and a RTT-like phenotype. In a RTT-like individual, a de novo large deletion at chromosome 9q34.11 (hg19:131,455,942-131,743,585) resulted in the loss of 13 genes, including the 3’ end of the coding sequence of SET Nuclear Proto-Oncogene (SET) and the 5’ end of the coding sequence of Nucleoporin-188 kDa (NUP188). The truncation of SET resulted in the loss of a highly conserved critical nucleosome assembly protein (NAP) domain crucial for assembly of nucleosomes and chromatin fluidity. Subsequent WES in the same individual identified a missense variant in NUP188 [c.3922C>T; p.(Arg1308Cys)] on the other allele. Preliminary functional studies in individual’s fibroblasts showed reduced NUP188 protein levels and enlarged nuclei, suggestive of perturbed NUP188 function. In this individual two genes, SET and NUP188, may be contributing to the affected individual’s phenotype. Interestingly, a homozygous variant in a novel candidate disease gene, Potassium Channel Tetramerization Domain Containing 16 [KCTD16; c.937T>A; p.(Ser313Thr)], was also revealed in a classic RTT female. KCTD16 encodes an auxiliary subunit that associates with GABA-B receptor and regulates receptor response in an agonist concentration dependent manner. Although variants in the KCTD family of proteins have previously been reported in individuals with NDDs, defects specifically in KCTD16 are yet to be linked with any human disease. Our preliminary electrophysiological studies in Xenopus oocytes investigating perturbed GABA-B receptor kinetics in response to the KCTD16 variant [p.(Ser313Thr)] did not reveal any significant differences when compared to wild type, as well as a variant commonly found in the healthy population [p.(Asp160Asn)]. Despite this, we plan to continue these investigations in a mammalian Chinese Hamster Ovary (CHO) cell-based model to further evaluate the variant’s pathogenicity. Overall, this study has provided functional evidence of variations affecting the motor domain of KIF1A in the pathophysiology of RTT/RTT-like disorders. In addition to identifying pathogenic variations in four known RTT-related genes (MECP2, SHANK3, SMC1A and EEF1A2), in this project we have further expanded the genetic landscape of RTT/RTT-like disorders to include variations in five additional genes (KAT6A, CHD8, SET, NUP188 and KCTD16). We recommend that the testing of these genes should be considered routinely while analysing NGS data in mutation negative RTT/ RTT-like individuals. The identification of additional members of key molecular pathways perturbed in RTT has further expanded our understanding of the underlying biology behind RTT, and this may pave the way for future targeted therapeutic options for RTT.
Investigating the DNA methylation profiles of children with oligoarticular juvenile idiopathic arthritis (JIA)
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.
Autism Spectrum Disorder (ASD) and Anaesthesia
This thesis addresses the question of what is the best evidence-based management for children with ASD (autism spectrum disorder) coming under anaesthetic care in the hospital setting. The increasing prevalence of ASD (1) has meant that most anaesthetists need to become proficient in understanding and managing children with ASD. Children with ASD have higher hospital contact than their neurotypical peers.(2) Behavioural problems, sensory sensitivities, language deficit, and inflexibility with change contribute to the difficulties experienced by children with ASD in the hospital setting. (3)Hospitals may be inherently challenging to children with ASD: being inflexible places, with the sound of crying children, with invasive monitoring techniques and bright lights.(4) One unpleasant anaesthetic experience can lead to heightened anxiety and future refusal to attend hospital. In order to find the context for best anaesthetic care, we have reviewed the existing literature about ASD and its management in chapter one. The first part of chapter two is a review of anxiety and premedication in general terms. The evidence for current best practices in managing children with ASD in the perioperative period is outlined in the second part of chapter two. To further understand the family and staff perspective of optimal care, we conducted a qualitative study of 29 individuals including 15 parents of children with ASD who had had a recent anaesthetic and 14 staff members that had looked after them in different capacities at two hospitals in Melbourne, Australia in chapter three. Chapter four contains discussion and conclusion. It includes discussion about the discontinued preparation/premedication trial (CLOMID). The flaws in the design and obstacles in its execution are examined. Our data showed important organisational, educational and resource matters. Problems such as prolong waiting for an operation date, lack of training of staff including anaesthetists and nurses, lack of availability of simple equipment and private spaces in the recovery rooms- were to be addressed. Good communication, clear explanation, and friendly attitudes as well as flexibility and individualised care of patients were considered useful. The supplementary material includes a protocol for a preparation /premedication study that has not been concluded as well as two social stories that I have designed.
An epigenomic and omics approach to neurodevelopmental disorders
Neurodevelopmental disorders such as cerebral palsy (CP) and epilepsy are some of the most prevalent childhood neurological disorders caused by damage to the growth and development of the brain. Early life environments predispose children to later health outcomes evidenced by the developmental origins of health and disease (DOHaD) phenomenon. Epigenetics, which refers to modifications of DNA without change in DNA sequence, is one way by which environmental exposures may contribute to development of disease. DNA methylation, arguably the most highly studied epigenetic mark, has been correlated with early life environmental exposures and have implications in both disease mechanisms as well as clinical biomarkers of neurodevelopmental diseases. These modifications most likely originate in utero, in line with the DOHaD hypothesis. The study of monozygotic (MZ) twins, in which genetics, age, sex, parental factors and shared environment are controlled for, helps in distinguishing the extent of effect of genetics and environment. Discordance for neurodevelopmental disorders has been recorded in MZ twins indicating a potential role of non-shared factors in disease risk. The aim of this PhD was to utilise the discordant MZ twin model to understand epigenetic changes associated with neurodevelopmental disorders. Genome-wide DNA methylation was measured within MZ twin cohorts discordant for CP or epilepsy using Illumina’s Infinium HumanMethylation450 and EPIC arrays. Statistical and bioinformatics pipelines were applied to evaluate the association of DNA methylation data to disease phenotypes. As detailed in Chapter Three of this thesis, DNA methylation analysis of CP-discordant twin pairs provides the first evidence that environmentally mediated differential methylation in genes involved in known processes such as hypoxia and inflammation, and processes such as cell adhesion, may contribute to the development of CP. As detailed in Chapter Four, an epigenome-wide analysis of epilepsy discordant MZ twin pairs revealed distinct patterns of DNA methylation within subtypes of epilepsies of unknown cause. Differentially methylated genes within epilepsy subtypes included those with a role in metabolic pathways, voltage-gated channel signalling and neurotransmitter processes. This research paves the way for future larger studies, as understanding DNA methylation profiles associated with neurodevelopmental disorders, may facilitate biomarkers for earlier diagnosis. This could lead to possible intervention strategies for patients suffering from a broad spectrum of disorders. Analysing epigenetic data from disease discordant twins provides an elegant study design and has the power to explore non-shared environmental factors that further refine models of disease mechanisms and biomarkers. The findings of this thesis suggest that epigenetic factors may play a role regulating biological pathways that underlie neurodevelopmental disorders, some of which arise as early as the prenatal period. Replication in other larger and similar cohorts of discordant twin pairs may provide novel targets for biomarker development, thereby allowing for early interventions and helping the health of children.
Improving the diagnosis of scabies in low-resource settings
Scabies is a parasitic disease and a global health problem that predominantly affects disadvantaged communities in low-resource settings. Scabies significantly impacts the health and quality of life of those with the disease. To accurately assess the global burden of disease and to compare data across regions, standardised diagnosis with consistent disease definitions is necessary. In low-resource settings, diagnosis by clinical assessment is the principal diagnostic method. In the absence of available experts, non-expert health workers are likely to play critical roles in diagnosis, including for prevalence mapping. Currently, standardised processes for clinical diagnosis for scabies do not exist. This thesis explores the diagnosis of scabies in low-resource settings and the role and utility of non-expert health workers in the diagnosis of scabies. Chapter 3 describes the evaluation of non-expert health workers in the diagnosis of scabies and impetigo using clinical criteria. The diagnosis of four briefly-trained nurses was compared to the consensus diagnosis of two experienced doctors. The sensitivity of the nurses’ diagnosis compared to the reference standard was 55.3% for scabies with a specificity of 89.9% Sensitivity for moderate to severe scabies was 93.5% with a specificity of 74%. The accuracy of diagnosis by non-expert health workers is promising and may be acceptable for scabies and impetigo disease mapping in low-resource settings. Chapter 4 describes the development and evaluation of a training protocol for the diagnosis of scabies and impetigo for non-expert health workers. The aim of this study was to measure the change in knowledge and confidence of the participants and explore their experience and perceptions of the training. Training was evaluated using a case-based test, a questionnaire and semi-structured interviews. The overall results of the case-based test were 90% for scabies and 75.5% for impetigo. The mean score for both self-reported knowledge and confidence increased from 2.5 to 4.5 following training and scores increased for all nurses (mean difference 2, 95%CI 1.1-2.9, P=0.005). The study showed that training local health staff in scabies diagnosis was enjoyable for participants and led to improvements in self-reported knowledge and confidence. Chapter 5 investigates the prevalence of scabies and impetigo using a cross-sectional study in a primary school in Gizo in the Solomon Islands. Using the International Alliance for the Control of Scabies (IACS) diagnostic criteria the classified the diagnosis of scabies. The prevalence of scabies was 54.3% and prevalence of impetigo was 32.1%. 63.5% of those with impetigo had scabies, corresponding to a population attributable risk of 11.8%. The study highlighted the extremely high burden of these diseases supporting the need for interventions for scabies in this community. Chapter 6 evaluates the methods of data collection, analysis and display for describing in detail the distribution of scabies lesions in a pilot study. The study used a novel technique of representation of dermatological lesions in the form of a choropleth map. The study found that the methods used were feasible for a larger population and would describe valuable detailed information on specific lesion location in scabies. The study will provide information on lesions at specific body sites to determine if simplified examinations are appropriate for prevalence surveys. The public health control of scabies requires identification of high-prevalence communities to target interventions, as well as methods to monitor the effectiveness of interventions. Such programs would be dependent on accurate and standardised diagnosis for population mapping. This thesis suggests methods to improve the diagnosis of scabies in low-resource settings. Modifications to training and diagnostic methods are likely to improve diagnostic accuracy. Improvements to scabies diagnosis will contribute to efficient collection methods and reliability of prevalence data.
Improving the quality of Essential Newborn Care in Solomon Islands
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.
Long-term outcomes of truncus arteriosus repair
Truncus arteriosus is a rare congenital cardiac defect which results in a single common arterial trunk exiting the heart which supplies the systemic, pulmonary, and coronary circulations. The truncus itself is guarded by a single, often large common valve – the truncal valve – which separates the truncus from both the left and right ventricle. Truncus arteriosus has an incidence of 3 to 10 per 100,000 live births. Although only 0.7% – 3% of all congenital cardiac anomalies are due to truncus arteriosus, it accounts for 4% of all critical congenital cardiac anomalies. Patients typically present early in life with symptoms of cyanosis and congestive cardiac failure. Nowadays, surgery is undertaken early in life prior to the development of irreversible pulmonary hypertension. Improvements in surgical techniques and perioperative management has drastically reduced early mortality to 3 – 20%. Therefore, many children who have undergone truncus arteriosus repair are living well into adulthood. Despite this, there are few large studies addressing the long-term outcomes of truncus arteriosus repair. Furthermore, the impact of concomitant anomalies and the truncal valve are insufficiently described. This Doctor of Philosophy focuses on the long-term outcomes of truncus arteriosus repair in order to determine the current results and risk factors for poor outcomes. This research constitutes the largest single-institutional and multi-institutional experience assessing the long-term outcomes of truncus arteriosus repair with the longest follow-up time. I demonstrated that the majority of mortality following truncus arteriosus repair occurs within the first year after repair, and survival beyond the first year is excellent. I found that the presence of a coronary artery anomaly was associated with both early and late mortality and suggest that the coronary anatomy be clearly identified intraoperatively. Furthermore, patients with DiGeorge syndrome are at risk of late mortality, most commonly due to infection. Interestingly, I found that patients with mild or less truncal valve insufficiency are free from truncal valve surgery for up to 25 years, despite their truncal valve anatomy. In contrast, most patients with moderate or greater truncal valve insufficiency – particularly those with a quadricuspid truncal valve – will require truncal valve surgery at some stage in their lifetime. Of note however, the durability of truncal valve repair as a whole is poor, with most patients requiring reoperation on the truncal valve. In those with a quadricuspid truncal valve, repair by tricuspidization appears to be the most durable option with good long-term outcomes. Tricuspidization provided better long-term outcomes even if the non-tricuspidization group included younger children (less than 6 years of age), in whom truncal valve replacement was performed. This is an important finding as it suggests that younger children may benefit from truncal valve repair rather than a replacement with a smaller (non-adult sized) mechanical prosthesis which may require repeat replacement. Furthermore, if repair of the truncal valve is possible, this would avoid life-long anti-coagulation and the associated risks. In the long-term, patients had an excellent functional status following truncus arteriosus repair but had a high rate of reoperation due to the use of a conduit for reconstruction of the right ventricular outflow tract. Despite the high reoperation rate, patients have similar quality of life compared to age-matched Australian controls. This Doctor of Philosophy has redefined our understanding of the long-term outcomes of truncus arteriosus repair. The findings presented will impact clinical decision making, and I envision an improvement in the outcomes of these rare and complex patients.
INVESTIGATING THE ROLE OF INNATE IMMUNITY IN MEDIATING THE NON-SPECIFIC EFFECTS OF BACILLE CALMETTE-GUÉRIN VACCINE
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.
Outcomes of the arterial switch operation
The last few decades have seen great advances in the world of paediatric cardiac surgery. In particular, the arterial switch operation (ASO) is one of the specialty’s most impressive achievements. It is the procedure of choice for most children born with transposition of the great arteries (TGA). This project aimed to expand our understanding of the long-term outcomes and prognosis of patients who have had an ASO for TGA. The ultimate goal of this work was to not only increase clinicians’ knowledge of the long-term consequences of the ASO but also to allow cardiologists, cardiac surgeons and paediatricians to give the parents of patients, and patients themselves, more information about their future cardiac health. We aimed to achieve these goals by retrospectively reviewing patients who underwent an ASO at The Royal Children’s Hospital in Melbourne. This unit has performed the ASO for more than 30 years with a very low hospital mortality from the beginning. This allows for review of a large population of surviving patients from which long-term outcomes can be investigated, and makes possible the analysis of rarer subgroups of patients undergoing the ASO. We showed that the long-term outcomes of patients undergoing the ASO are generally good extending into young adulthood. However, reintervention is common and the incidence of clinically significant neo-aortic regurgitation or the need for neo-aortic root reoperation remains an issue for survivors with more than 25 years of follow-up. In regards to the subgroups examined, we showed that patients with intramural coronaries can be operated with excellent long-term outcomes using a simple, reproducible technique. Children who are less than 2.0 kilograms at time of ASO represent one of the highest mortality risk subgroups. Female adult survivors can undergo pregnancy with a low risk of maternal cardiac complications. Patients with associated aortic arch obstruction have a higher rate of reintervention for right sided obstruction and have a low rate of recurrent arch obstruction when avoiding the use of patch material. We also demonstrated that quality of life in adult ASO survivors was comparable to the general population using a validated quality of life questionnaire.
The epigenetic landscape of paediatric acute myeloid leukaemia
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.
Saving the hair cell: Investigating Apoptosis signal-regulating kinase 1 as a molecular target for preventing aminoglycoside induced hearing loss
Aminoglycoside antibiotics are lifesaving medicines, crucial for the treatment of chronic or drug resistant infections. However, aminoglycosides can destroy the sensory hair cells of the ear. As a result, aminoglycoside treated individuals frequently experience a reduced quality of life, stemming from permanent high-frequency hearing loss and vestibular impairment. Currently, no alternate antibiotic substance has the same bactericidal profile or efficacy as aminoglycosides, and an adjuvant therapy capable of mitigating ototoxic outcomes does not exist. In order to develop an otoprotective therapy, the mechanisms of aminoglycoside-induced hair cell death must be elucidated. With respect to this goal, the production of reactive oxygen species and subsequent c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) phosphorylation has been extensively documented in aminoglycoside treated hair cells. However, strategies directly targeting ROS, JNK or P38 are limited by the importance of these molecules for normal cellular function. Notably, the upstream regulators of JNK or P38 have not been well studied in the ear. Therefore, this project aimed to elucidate the role of upstream regulator Apoptosis signal-regulating kinase 1 (ASK1) within the auditory system. ASK1 is a key mediator of ROS induced JNK and P38 mediated disease. Importantly, ASK1 inhibition has previously been shown to reduce the pathological consequences of ROS, P38 and JNK signalling in varied disease models; without impeding the normal homeostatic cell function of these molecules. Therefore, ASK1 inhibition may represent a novel strategy for preventing aminoglycoside ototoxicity. However, the role of ASK1 as a mediator of drug-induced hair cell death has not been investigated. Moreover, the role of ASK1 in the development and function of auditory structures has not been explored. This project first aimed to elucidate the importance of ASK1 in the inner ear by characterising the auditory phenotype of Ask1 knockout mice. Histology indicated normal development of cochlear structures and the auditory brainstem response (ABR) demonstrated that Ask1 knockout mice had hearing thresholds comparable to C57BL/6 controls. However, ABR peak analysis indicated that auditory signal transduction is faster in Ask1 knockout mice. In addition, the acoustic startle response showed Ask1 knockout mice to be hypersensitive to auditory stimuli. Combined, this data indicates that ASK1 is important for neuronal function and that Ask1 knockout mice have an auditory processing disorder. This project then evaluated the importance of ASK1 as a mediator of drug-induced hair cell death. To test the hypothesis that ASK1 deficiency is protective against aminoglycoside-induced hair cell death, cochlear explants from Ask1 knockout and C57BL/6 mice were treated with neomycin in vitro. After six hours of 1 mM neomycin treatment, immunohistochemistry demonstrated that p-JNK staining was reduced in Ask1 knockout explants when compared to C57BL/6 explants. Moreover, hair cell death was significantly attenuated in Ask1 knockout explants. This data provides robust evidence that ASK1 has an important role in the process of hair cell death, and that ASK1 inhibition could limit aminoglycoside-induced hair cell death. Therefore, the final aim of this project was to test ASK1 inhibition as an otoprotective strategy. ASK1 inhibition significantly attenuated neomycin-induced outer hair cell death in C57BL/6 cochlear explants. Immunohistochemistry and western blot analysis suggested that ASK1 inhibition attenuated both P38 and JNK phosphorylation in neomycin-treated explants. Importantly, ASK1 inhibition did not impact the efficacy of the aminoglycoside antibiotics against P. aeruginosa in the broth dilution test. This indicates that ASK1 inhibition may mitigate ototoxic outcomes without impairing the primary aminoglycoside treatment. Overall, this thesis represents the first aural characterisation of the Ask1 knockout mouse strain, providing valuable insight regarding the role of ASK1 in the auditory pathway. Moreover, this work has provided compelling evidence that ASK1 inhibition may be a useful strategy for the prevention of aminoglycoside induced hearing loss.