Paediatrics (RCH) - Theses
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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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ItemDefining the phenotype of a cohort of children with non-syndromic Pierre Robin Sequence( 2015)Background Pierre Robin Sequence (PRS) is a common craniofacial anomaly comprising of micrognathia, cleft palate, glossoptosis and upper airway obstruction. It is a condition which affects 1 in 6000 neonates, often resulting in airway and feeding difficulties. Although it is a well-known condition, many aspects about the diagnosis, aetiology and management of Pierre Robin Sequence are contentious or unknown. Specifically, the exact phenotypic spectrum of this condition has been poorly studied. The major aim of this Master of Surgery is to provide an accurate phenotypic characterisation of a large cohort of non-syndromic Pierre Robin Sequence patients. A retrospective review of diagnosis and management of this cohort was also performed, along with a preliminary investigation into the possible genetic aetiology of a subset of patients. Methods A cohort of 141 non-syndromic PRS patients managed at the Royal Children’s Hospital in Melbourne from 1985 to 2012 was identified by cross-referencing two clinical databases. A detailed review of each patient’s medical file was performed and patients were categorised into either “Isolated PRS” or “PRS-Plus” groups. A subset of patients with a family history of cleft and/or a musculoskeletal anomaly were selected for targeted DNA sequencing of the non-coding elements of SOX9 (chromosome 17), a potential candidate gene for non-syndromic PRS. Results Our cohort comprised 83 Isolated PRS patients and 58 PRS-Plus patients. In the PRS-Plus group, the most common malformations beyond the craniofacial region involved were the musculoskeletal and ocular systems, with choanal stenosis/atresia being the single most common coexisting condition. PRS-Plus patients were found to have worse outcomes at birth as well as during the neonatal period, with a higher proportion being born small-for-gestational-age, have failure to thrive and require surgical intervention for airway and feeding. No significant genetic mutations were identified in the non-coding elements of SOX9 in the subset of patients who had DNA sequencing. A single nucleotide substitution was identified in the GATA1 transcription factor binding site, however the functional significance of this variant is yet to be determined. Summary Pierre Robin Sequence is a phenotypically diverse condition which contains a wide spectrum of features far beyond what was initially described. The results of this study supports the existence of a “PRS Spectrum” ranging from the mildest isolated PRS patients to the more complex syndromic PRS patients. Patients with additional anomalies outside of the craniofacial system had poorer outcomes at birth and during the neonatal period. Further studies are required to determine whether these differences can be explained by underlying biological causes such as genetic mutations, or whether this is a result of inadequate initial management.