Paediatrics (RCH) - Theses
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ItemInvestigating genomic and environmental risk factors and their interactions in juvenile idiopathic arthritis( 2017)Juvenile idiopathic arthritis (JIA) is a paediatric autoimmune disease arising from an abnormal immune response to self. It is the most common childhood rheumatic disease, with a prevalence of around 1 in 1000 Caucasian children. Disease prevalence is biased towards females, with around 2–3 females affected for every male. Due to the young age of onset, JIA can have a severe effect on a child’s growing skeleton and cause serious functional disability. And though onset is in childhood, the morbidity associated with JIA can be life-long as currently there is no cure for the disease, treatments are imperfect and preventative measures aren’t available – largely due to the limited understanding of disease pathogenesis. We hypothesised that genetic and environmental risk factors contribute individually and through interaction to cause JIA, and contribute to the sex bias in disease prevalence. The first aim of this study was to replicate the association of genetic variants that had previously been associated with JIA, in our independent sample. We confirmed the association of seven risk loci in our sample, six replicated for the first time. Our findings significantly strengthen the evidence that these loci harbour true JIA risk variants. The second aim of this study was to investigate whether autosomal genetic variants confer sex-specific risk for JIA. We established that of the 68 JIA risk loci tested, eight conferred sex-specific risk for JIA. Of these, three had statistically significant evidence of sex modifying the effect of that SNP on JIA. Of note, we replicated the femalespecific association of PTPN22 rs2476601 across two independent samples. Our findings illustrate that the genetic architecture of JIA differs between the sexes. Our third aim was to investigate whether the Y chromosome contributes to JIA risk in males. We determined that genetic variation captured by Y chromosome haplogroup I was associated with JIA risk, in males over the age of 6. We also demonstrated that there was an increased risk of JIA for males that had a father with autoimmune disease. Our findings are the first to suggest that the Y chromosome may play a role in JIA risk and provide further evidence that JIA has sex-specific genetic architecture. Next we considered the role of the environment in JIA risk. The fourth aim of this study was to assess the association between factors that impact vitamin D status and JIA. We identified a protective association between increasing UVR exposure over the life course and at 12 weeks of pregnancy, and JIA. Our findings are the first to implicate insufficient UVR exposure in the development of JIA. We then considered mechanisms through which genetic and environmental risk may be mediated, such as DNA methylation and gene expression. Our fifth aim was to identify sex-specific DNA methylation differences in CD4+ T cells between oligoarticular JIA cases and healthy controls. Oligoarticular JIA cases did not have substantial sex-specific DNA methylation differences when compared to controls, but there was evidence of modest case–control differences and these were more prominent in males than females. Our findings suggest that DNA methylation is not a significant driver of the sex bias in JIA. The final aim of this study was to investigate whether CD4+ T cell gene expression profiles differed between oligoarticular JIA cases and healthy controls. Oligoarticular JIA cases had aberrant gene expression relative to controls, suggesting that disease processes are in part driven by gene regulatory differences in CD4+ T cells. In conclusion, the cumulative findings of this study improve our understanding of the aetiology of JIA by revealing sex-specific genetic architecture for the disease, establishing UVR exposure as an environmental risk factor for JIA, and characterising the DNA methylation and gene expression signatures of the active disease state.
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ItemThe epidemiology and aetiologies of the severe epilepsies of infancy( 2016)The severe epilepsies of infancy (SEI) are a group of infantile-onset seizure disorders characterised by frequent seizures, abnormal EEG and pharmacoresistance to anti-epileptic therapy. SEI include well-described epilepsy syndromes such as early infantile epileptic encephalopathy (EIEE), epilepsy of infancy with migrating focal seizures (EIMFS) and West syndrome. Cognitive outcome is often poor, due to effects of seizures, the underlying aetiology, and antiepileptic drugs (AEDs) on the developing brain. There is an urgent need for novel treatments. Where effective therapies are available, such as epilepsy surgery for brain malformations, treatment can be life-changing. Given that developmental outcomes may be significantly improved in the context of optimal seizure control at an early age, determining the underlying cause of SEI early in life is paramount. The aetiologies of SEI are heterogeneous; a large number of acquired and genetic brain disorders are reported. In many infants, the cause remains unknown despite investigation, and is presumed to be genetic. With the emergence of next generation sequencing (NGS) techniques such as whole exome sequencing (WES), a rapidly growing number of genetic causes of SEI is now recognised and gene discovery is ongoing. The genetic epidemiology of SEI has not been studied and the relative importance of each genetic cause is not known. Brain malformations, chromosomal abnormalities, inborn errors of metabolism and some genetic disorders can be diagnosed with technologies currently available in clinical practice. Studies of WES and other NGS techniques in epilepsy populations have shown that these techniques identify the aetiology in 10-50% of undiagnosed patients. No study has specifically looked at the yield in SEI, and no population-based studies have been reported. The yield and cost-effectiveness of NGS for SEI at a population level remains unknown, and access to genetic testing is currently poor in most regions of the world. This population-based study of SEI in Victoria, Australia aimed to study the incidence and determine the aetiologies, electroclinical phenotypes and other phenotypic characteristics of SEI. As part of a particular focus on genetic aetiologies, the study aimed to identify genetic causes in infants with SEI of unknown aetiology using WES, and determine the yield and cost of early genetic testing relative to current standard diagnostic pathways for investigation of SEI aetiology. Infants with SEI born in Victoria during 2011-2013 were identified by a comprehensive state-wide search of multiple sources. Infants with potential SEI were identified by review of all electroencephalogram (EEG) reports on children under two years old during 2011-2015 (n=4505), and search of neonatal intensive care unit (NICU) databases for neonates with seizures born 2011-2013 (n=379). Hospital records of infants with potential SEI from the three main paediatric hospitals in Victoria, The Royal Children’s Hospital (RCH), Monash Health (MMC) and The Austin Hospital, and the two NICUs not co-located with a paediatric hospital, The Royal Women’s Hospital (RWH) and The Mercy Hospital for Women (MHW), were reviewed to confirm clinical and demographic inclusion criteria were met. SEI was defined as epilepsy onset before age 18 months, frequent seizures (> daily for a week or > weekly for a month), epileptiform EEG and pharmacoresistance (failed 2 appropriate anti-epileptic therapies); infantile spasms were automatically included. In infants with confirmed SEI, medical records, EEG recordings and brain magnetic resonance imaging (MRI) were reviewed to determine each infant’s epileptic syndrome, outcome at two year old, and aetiology. Clinical assessment and WES were performed if aetiology or electroclinical phenotype was unknown. 114 infants with SEI were ascertained. The incidence of SEI in Victoria is 51/100,000 live births/year. West syndrome/infantile spasms was the most common epileptic syndrome, with an incidence of 33/100,000 live births/year. EIMFS and EIEE had incidences of 4.5 and 3.6/100,000 live births/year respectively. At two years old, 18 (16%) infants were deceased. 86/98 (90%) survivors had delayed development, and 46/98 (47%) ongoing seizures. All infants whose presenting epileptic syndrome was EIEE, early myoclonic encephalopathy (EME) or EIMFS at epilepsy onset were deceased or had severe developmental impairment. Normal development was seen in 9/64 (14%) infants who presented with West syndromes/infantile spasms or a unifocal epilepsy, and only two were deceased. The aetiology was identified in 76 (67%) and unknown in 38 (33%). Fourteen (12%) infants had an acquired brain insult such as hypoxic-ischaemic encephalopathy or perinatal stroke. The remaining infants had genetic or presumed genetic aetiologies. Brain malformations were identified in 31 (27%), including focal cortical dysplasia (FCD) in 14 (12%). Six (5%) infants had metabolic disorders and nine (8%) had chromosomal abnormalities. Sixteen (14%) had single gene disorders, including 11 (10%) with disorders of ion channel function (channelopathies). Aetiology was known from clinical testing in 61 (54%). Research MRI review identified the cause in a further 4 (4%) and research genetic testing in 11 (10%). Among 86 (75%) infants with no aetiological diagnosis prior to epilepsy onset, the highest yield investigations were MRI and genetic testing, which identified the cause in 25/85 (29%) and 16/50 (32%) respectively. 13/50 (26%) infants had a genetic variant of unknown significance (VOUS) identified on WES; these are being further investigated. Modelling of diagnostic pathways showed that performing WES early in the diagnostic pathway and reducing the amount of metabolic testing increases diagnostic yield for less cost compared with the current standard diagnostic pathway. Approximately 1:2000 infants have SEI, equating to over 150 new cases of SEI in Australia per year. Outcomes for seizure control, development and survival are poor. Brain malformations were the most common cause, were under-recognised, and should be considered in those with unknown aetiology, especially in those with West syndrome or unifocal epilepsy. Channelopathies were the most common group of single gene disorders. Next-generation genetic testing and high quality brain imaging improved diagnostic yield, with implications for treatment and reproductive counselling, and should be implemented early in the diagnostic pathway in clinical practice. Future work will focus on identifying the aetiology in the remaining infants, determining the genetic basis of brain malformations causing SEI, and studying the yield of other NGS and brain imaging techniques to improve the rate of early diagnosis. This work will inform research into development of novel and targeted treatments for these devastating disorders.
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ItemThe identification and characterisation of novel genes associated with cardiomyopathy( 2017)Cardiomyopathies are heart muscle disorders that have a diverse clinical presentation, spectrum of severity and underlying aetiology. The aim of this PhD project was to utilise new genetic technologies to identify and subsequently characterise novel genes associated with cardiomyopathy. Using this approach a novel cardiomyopathy associated gene called ALPK3 was identified. Utilising heart cells generated from patient induced pluripotent stem cells (iPSCs) we discovered that ALPK3 provides internal structure for heart cells and assists in the connection between cells.
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ItemBioinformatic challenges in translating high throughput sequencing into clinical settings( 2016)In the last ten years, genomic sequencing has undergone a technological revolution, enabling large numbers of genes, or even entire exomes or genomes to be sequenced economically. These capabilities, based around the new technology of high throughput sequencing (HTS), have had a transformational impact on biomedical research. The impact in clinical settings is likely to be equally as profound due to the potential to enhance the diagnosis, treatment and management of disease. These impacts, however, are only beginning to be explored. Clinical genetics is in a transitional phase, as the new technologies are being validated and tailored to the clinical setting. In that process, significant new challenges are being faced. This thesis focuses on one particular aspect: the essential role of bioinformatics in the translational process. The thesis discusses the bioinformatics of clinical translation from a variety of points of view. In the first chapter, an overview of the current clinical setting is presented. This includes a description of the general challenges of clinical translation, background about the history and context of modern clinical genetics, and the key bioinformatic methods that are applied to derive clinical results. The second chapter consists of a published paper describing an analysis pipeline, Cpipe. This analysis pipeline was developed as part of the Melbourne Genomics Health Alliance demonstration project, a prototype for deployment of clinical exome sequencing. It illustrates many of the key issues associated with moving high throughput sequencing into a clinical setting. In the third chapter, a specialised sequencing technology, HaloPlex, is described in detail. HaloPlex is in use at the Murdoch Childrens Research Institute as part of a sequencing project that aims to diagnose disorders of sexual development. HaloPlex data has many unique characteristics, and these form the basis of several subsequent chapters of the thesis. The fourth chapter extends the discussion of HaloPlex into an evaluation of the accuracy of variant detection from HaloPlex data. The key problems in analysing HaloPlex data are identified, and one particular issue, adapter contamination, is discussed in depth. A novel method is described for removal of adapter contamination in HaloPlex data. The fifth chapter discusses the difficulty of detecting some clinically relevant types of mutation from HTS data. Exon and larger size deletions are identified as particularly important. The chapter introduces a novel simulation method, Ximmer, which is specifically designed to simulate deletions in targeted sequencing data. Using the simulation method, we present an evaluation of performance of existing deletion detection methods. Lastly, the thesis returns to HaloPlex to consider a dedicated HaloPlex algorithm for identifying single copy deletions. An evaluation of current methods is presented and the statistical basis of a method, called Angel, is described and evaluated to show that significantly improved performance is attained by leveraging the unique characteristics the HaloPlex technology. In summary, this thesis describes the bioinformatic context of clinical genomics, and details several related novel methods to address key challenges that are identified. These include a clinical exome sequencing pipeline, a method for simulating deletions in exome sequencing data, and a method for detection of deletions in HaloPlex sequencing data.
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ItemUsing massively parallel sequencing to understand the genetic basis of mitochondrial disorders: a population-based approach( 2015)Inherited defects in mitochondrial oxidative phosphorylation (OXPHOS) are the most common inborn error of metabolism, affecting at least 1 in 5000 live births (Skladal, Halliday et al. 2003), and predominantly affect organs with high-energy consumption such as the brain, skeletal muscle, cardiac muscle and liver. Mitochondrial diseases are notoriously difficult to diagnose, as they show extreme clinical heterogeneity, presenting at any age and with any level of severity, and typically impact on multiple organ systems (Munnich and Rustin 2001). They are also genetically heterogeneous with over 200 mitochondrial DNA and nuclear DNA encoding genes associated with OXPHOS disease. Despite the large number of disease genes being identified, many patients with OXPHOS disease remain without a molecular diagnosis. We developed a targeted DNA capture and massively parallel sequencing method to detect variants within 1,034 genes encoding proteins known or implicated as having a mitochondrial function, known as the MitoExome. My PhD studies have focused on the characterisation of two novel genes identified by MitoExome sequencing; UQCC2 and UQCR10. Both UQCC2 and UQCR10 are components of mitochondrial complex III. By using a panel of patients with causative mutations in a range of different complex III subunits and assembly factors, we have further characterised the assembly pathway of complex III. Of the 45 patients who underwent MitoExome sequencing, a third remain without a molecular cause identified. To address this, I utilised several alternative analysis strategies to pursue molecular diagnoses in patients where a causative mutation had not been easily identified. Reanalysis of the MitoExome data using two different analysis pipelines (Cpipe and xBrowse) identified an additional patient diagnosis in RMND1. Comparison between the two pipelines highlighted some key differences between analyses for research compared to a clinical setting. The Birth Prevalence cohort is a 12 year follow up study to revisit the original cohort reported by Skladal and colleagues in 2003 (Skladal, Halliday et al. 2003). This study identified 86 patients with a confirmed diagnosis of mitochondrial disease who were born in South Eastern Australia between 1987 and 1996. This cohort was used to calculate the birth prevalence of mitochondrial disorders as 1 in 5000 live births. At the time of publication 23% (n=20/86) of patients had a molecular diagnosis identified. The 2015 review of this cohort found an additional 19 patients who fit the inclusion criteria, bringing the total number of patients to 105. Currently, 70% (n=73/105) of Birth Prevalence cohort patients have a molecular cause identified. This PhD describes some preliminary molecular investigation of 19 patients, who are as yet without a molecular diagnosis.
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ItemVery often the answer's not black or white: current practice, clinician and parental experiences of genomic testing in paediatric genetics clinics( 2015)Developmental delay (DD) has been estimated to affect 1 – 3% of the population and the underlying cause often remains unknown. For parents, receiving a diagnosis for their child’s DD can be a source of validation, and may impact upon their child’s prognosis, treatment and access to available supports. New genetic technologies, chromosomal microarrays (CMA), are now used across Australia to help end the ‘diagnostic odyssey’ families often experience. CMA tests have the capacity to identify genetic changes at much higher resolutions than was previously possible, but may increase the complexity and uncertainty of results. There is no research into the process of these consultations and the way in which this information is communicated to patients attending a genetics clinic. Using qualitative techniques and guided by the frameworks of symbolic interactionism and interactional sociolinguistics, this study provides a rich and nuanced analysis of paediatric genetic consultations. A multi-layered approach was used, enabling investigations of both the ‘front stage’ (consultations) and ‘backstage’ (interviews with parents and clinicians) to facilitate and further understand emerging patterns. Four data sources were used: (1) pre-consultation surveys with parents (n=32); (2) audio-recordings from consultations (n=32); (3) post-consultation telephone interviews with parents (n=32); and (4) post-consultation interviews with clinical geneticists (n=10). Overwhelmingly, parents were complimentary regarding consultations and described feeling reassured. Those who were disappointed were largely parents who were desperate for a diagnosis. The vast majority of parents reported a positive relationship with the clinician and felt that the genetic information had been explained in a very useful manner. Clinicians described consultations as varied and dependent on many factors including parents’ hopes and diagnostic expectations. They identified their primary role as a diagnostician but also acknowledged the therapeutic benefits these consultations could provide to families. Clinicians displayed mixed opinions regarding new tests, appreciating the benefits while also wary of inherent complexities and uncertainties that more detailed genetic testing would bring. Seven phases were identified within consultations, and on average clinicians contributed more words than parents, however there was variation across and within consultations. Although content of consultations was extremely similar, clinicians appeared to have varied approaches, and different styles were described including the clinical assessment approach and conversational style. Some parents were able to describe their experiences of their child’s development as a ‘narrative’, while during other consultations clinicians seemingly had a checklist of closed questions. Most clinicians used an historical narrative to describe the evolution of genetic testing; comparing past and current limitations with the promise of future genomic technologies. Findings enabled the development of an ‘ideal’ consultation in this setting, which highlights the importance of a positive clinician-parent relationship, especially in light of diagnostic uncertainty and advancing genomic testing capabilities. Narrative medicine could play a valuable role both for parents and clinicians as they make sense of genomic testing and diagnostic uncertainty. With the impending introduction of whole genome sequencing into a clinical setting, now is an ideal time to reflect and learn from past and present experiences, in order to maximise the therapeutic potential of such scientific discoveries.
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ItemExploration of antenatal β-thalassaemia carrier screening in Victoria, Australia( 2012)Currently there is no coordinated β-thalassaemia carrier screening program carried out in Australia. Unlike other genetic screening programs, this screening has been incorporated into routine healthcare practice. This is attributable to most women undergoing a full blood examination (FBE) at their initial prenatal visit, with a low mean corpuscular volume (MCV) or mean corpuscular haemoglobin (MCH) indicating that they may be a carrier, therefore triggering further β-thalassaemia diagnostic testing. Little is known about the processes currently applied across the different hospitals and practices within Australia to screen women for β-thalassaemia, as well as both women’s and healthcare professionals’ attitudes towards this screening process. To further understand the β-thalassaemia carrier screening process(es) undertaken within Victoria as well as the acceptability of this different approach to carrier screening, a qualitative study was carried out. This aimed to explore carriers’ and health professionals’ experiences of and attitudes towards the β-thalassaemia carrier screening process in Victoria. Semi-structured interviews were carried out with 26 female carriers of β-thalassaemia who had been pregnant within 12 months prior to being interviewed, 10 carrier couples of β-thalassaemia, as well as 23 healthcare professionals who attend to women during the antenatal β-thalassaemia screening process. Data were analysed using inductive content analysis and process mapping. Findings revealed that women had undergone variable experiences while being identified as a carrier, with surprisingly more than half of the participants being made aware of their carrier status prior to pregnancy, at various ages. This was due to women having undergone FBEs for numerous reasons, other than thalassaemia screening specifically. Pre-pregnancy screening was seen to be preferable due to both women and healthcare professionals believing this to be the most suitable time for testing. Other women had only become aware of their carrier status during pregnancy. Variation was also seen amongst the screening processes carried out by the different healthcare professionals, with often little use of guidelines and lack of knowledge, which was often not believed to be ideal by healthcare professionals. This highlights a need for further education of healthcare professionals. Most of the women did not recall being informed about β-thalassemia before notification of their carrier status and therefore did not make a decision about being screened. They were generally satisfied, however, for doctors to make the decisions regarding tests conducted. The women however would have preferred to have been informed about the screening test before it was carried out. Insufficient information was also reported being provided to women after they were notified of their carrier status, leading to misconceptions and confusion. Even though most women did not provide informed consent, this variable thalassaemia carrier screening process incorporated into routine care was seen to be acceptable amongst this community who overall had positive attitudes. A greater emphasis, however, should be placed on information provision, both pre and post testing, as well as further education for healthcare professionals.