Paediatrics (RCH) - Theses
Permanent URI for this collection
Search Results
1 - 10 of 13
-
ItemVariation of the Microbiome in Paediatric Crohn’s Disease( 2019)Introduction: Crohn’s Disease (CD) is a chronic, relapsing condition of the human gastrointestinal system with several significant extraintestinal manifestations. It can affect any age group; 10% of patients being diagnosed prior to their 18th birthday. Pediatric CD is known to have a more severe and difficult to manage phenotype. A rising incidence has been observed in recent years although the disease phenotype has remained largely unchanged. There is a growing body of literature describing dysbiosis in patients with CD. Despite extensive research, the role and behaviour of the gut microbiome in pediatric CD is not well understood. Aims: We aimed to characterize the microbiome in patients with CD longitudinally and compare it with non-IBD patients. We also sought to explore if there were any microbiome differences between ASCA positive and ASCA negative patients. Methods: We analysed the microbiome of 345 biopsies from 204 patients, including 88 CD first diagnosis (CDFD) patients, 38 patients in relapse (CDRL), 12 patients in remission (CDRM), and 66 controls. Species identification was conducted using oligotyping in combination with ARB/SILVA taxonomic annotation. Comparison was also made between ASCA status, microbial diversity and clinical characteristics. Results: We observed 45 bacteria to be statistically different between CDFD samples and controls, with Fusobacterium being the most implicated species in CDFD patients. We also identified gender specific differences in CD. Five species showed a strong association with patients with CD in relapse and 10 species in patients with CD in remission. Three taxa showed a positive co-occurrence across the two groups. Hespellia porcina (closest taxonomic neighbor to Clostridium oroticum) had the strongest association with samples from patients with CD in relapse. Interestingly, Fusobacterium was not part of the CD relapse associated taxa group. Faecalibacterium prausnitzii was equally present in CDFD and in control samples. ASCA was highly specific but poorly sensitive for the diagnosis of CD. In patients with CD, ASCA positivity was more likely to be present in patients older than 10 years, and associated with increased likelihood of ileocolonic disease distribution and long-term risk of surgery. Microbial alpha and beta diversity were similar in patients with CD with or without ASCA, but significantly less when compared to non-IBD controls. 14 bacterial species were statistically associated with ASCA positive patients with CD and 14 species with ASCA negative patients (p< 0.05). By using a false discovery rate corrected P value, two species remained statistically associated with both the groups. Ruminococcus torques and bacterium Yersinia enterocolitica were statistically associated with CD ASCA positive patients (p = 0.0178). Enterobacter cloacae and Faecalibacterium prausnitzii were statistically associated with CD ASCA negative patients (p = 0.0178 and 0.0342, respectively). Conclusions: This is the first study to investigate gut mucosal microbiome in a pediatric CD cohort with longitudinal sampling. Significant differences in microbiome were observed between treatment naïve patients with CD, patients with CD in relapse, patients with CD in remission and non-IBD patients. We also identified differences in the gut microbiome between patients with CD depending on presence of ASCA.
-
ItemStructural and Regulatory Changes to the Mouse Y Chromosome( 2020)The X chromosome and the Y chromosome are the sex chromosomes of mammals. While the X chromosome carries many genes with functions beyond sex, the Y chromosome is relatively gene poor. It serves as the trigger for development of a male embryo, with its genes having roles in testis determination and function. The sex chromosomes exist as a pair; in females, this pair are XX, and in males, XY. Two different mouse Y chromosomes were the focus of this project. The first was a transgenic fluorescent reporter Y chromosome, Ddx3y^mKate2, which was created to be used as a tool in chromosome instability research. The second was the naturally occurring Y chromosome from the A/HeJ inbred mouse strain, Y^A/HeJ. The work associated with these two Y chromosomes comprised the two studies in this project. The Ddx3y^mKate2 Y chromosome was generated by targeted insertion of a transgene cassette containing the red fluorescent protein gene, mKate2, into the mouse Y chromosome adjacent to the Ddx3y locus. This was selected as the relatively broad expression of Ddx3y indicated a permissive environment for expression of the mKate2 transgene. As male mice are XY, all male Ddx3y^mKate2 mice carried the fluorescent reporter. The mKate2 fluorescence in this strain has been previously assessed, revealing that the reporter was detectable from the preimplantation embryo, through to sexual maturity. To finalise the characterisation of the Ddx3y^mKate2 mouse, this study assessed the fluorescence in the Ddx3y^mKate2 male in advanced age. Additionally, this study characterised another transgenic reporter strain, Hprt^DsRed-Express, to serve as a comparison. To demonstrate the utility of the Ddx3y^mKate2 reporter, the Ddx3y^mKate2 Y chromosome was crossed onto two known chromosome instability backgrounds: the Trp53 knockout and Cenpagfp fusion knock-in backgrounds. Following in vivo and in vitro experiments, it was determined that the Cenpagfp background was the better background to model the Ddx3y^mKate2 reporter Y chromosome; however, more work using this genetic background in embryonic stem cells is advised for assessing the Ddx3y^mKate2 reporter Y chromosome. The Y^A/HeJ chromosome from the inbred A/HeJ strain was causally associated with a disturbance in the testis determination pathway. The Y^A/HeJ chromosome resulted in a subset of male mice having developed either ovotestes (gonads containing ovarian and testicular tissue) or abnormally small testes without epididymal sperm. It was confirmed that the Y^A/HeJ chromosome still carried the testis triggering gene, Sry. The gonadal and cytological abnormalities associated with this Y chromosome lead to the speculation that there was a structural change at or near its centromere. This study extended the characterisation of the A/HeJ phenotype, including identification of an age-related decline in male gonad mass, as well as assessment of the Y^A/HeJ chromosome. It was confirmed that the structural change to the Y^A/HeJ chromosome was a halving of the Rbmy tandem repeat array. This was predicted to adversely affect Sry gene expression during testis determination, resulting in the A/HeJ gonadal phenotype. Assessment of Sry regulation and gene expression from the Y^A/HeJ chromosome is recommended to complete the work associated with this Y chromosome.
-
ItemCombined genetic and epigenetic analysis to identify early life determinants of complex phenotype( 2019)There is now considerable evidence indicating that risk of many complex diseases in adulthood may be influenced by exposure to environmental exposures in utero. A growing number of studies suggest epigenetic markers, including DNA methylation, are involved in this process. Understanding how DNA methylation is impacted by pregnancy exposures, and related to later health, may both contribute to unravelling the aetiology of complex disease risk in later life and provide a potential early-life biomarker for risk prediction. However, current evidence is limited. There has been a predominance of small, poorly powered studies, failure to consider the effects of genetic variation, and limited replication of previous findings. In addition, previous studies investigating the relationship between DNA methylation and offspring health have been primarily cross-sectional. For these reasons, I investigated the associations between pregnancy exposures (in particular, maternal smoking, nutrition and metabolic health, psychosocial stress, and adverse pregnancy conditions), birth outcomes, and offspring blood DNA methylation of the insulin-like growth factor 2 (IGF2) and H19, hypoxia-inducible factor 3A (HIF3A), leptin (LEP) genes. I also considered how genetic variation impacted on these associations. I then investigated the longitudinal relationship between early life methylation and anthropometry, as well as the association between early life methylation and later childhood measures of weight, adiposity, and cardiovascular health. To do this, the large, population-based longitudinal Barwon Infant Study pre-birth cohort (n=1,074) was used, with clinical and questionnaire measures from 28 weeks pregnancy, birth, 12 months post-birth and 4 years post-birth time points. DNA methylation of candidate regions was measured using the Sequenom EpiTyper mass-spectrometry platform in cord (birth) and peripheral (12-month) blood. Infant genetic variation in and near the candidate genes was considered. Infant adiposity was assessed as sum of triceps and subscapular skinfold thicknesses in infancy, and with DEXA scanning at 4 years of age. We found evidence that exposure to maternal psychosocial stress, gestational diabetes, and pre-eclampsia was associated with differences in offspring methylation at the candidate regions, as was infant sex. Genetic variation showed strong effects on DNA methylation levels, with some evidence for the associations of pre-eclampsia and infant adiposity with LEP methylation differing by infant genotype. Early life methylation of HIF3A and LEP showed modest associations with four-year blood pressure and BMI, respectively. While these associations persisted with adjustment for potential confounding factors, they explained relatively little variance in the four-year phenotypes compared to traditional predictors, such as weight. These findings suggest that offspring DNA methylation of these candidate genes involved in regulation of growth and metabolism are sensitive to several environmental exposures and genetic factors. While there is modest evidence for methylation in infant blood associating with later phenotypes, methylation of these genes appears unlikely to have useful predictive utility in isolation. This study is the first to perform early life longitudinal analysis to investigate the association between anthropometry and methylation in infancy. It is also the first to report evidence of earlier methylation associating with later cardiovascular phenotypes. However, as gene expression data was not available, the functional consequences of the altered methylation observed in blood is unclear. Further work is required to replicate these findings in independent cohorts, to determine the nature of expression of these genes in blood, and to investigate if the relationship between early life methylation and later health persists into adulthood.
-
ItemEnvironmental and genetic risk factors for food allergy in children with Asian ancestry( 2019)Epidemiological and genetic studies on food allergy to date have focused primarily on the Caucasian population. This is despite emerging evidence that food allergy appears to be rising in Asian countries, alongside the increasing Westernisation and urbanisation in these countries. Even less is known about Asian migrants living in Western countries. A population-based study on food allergy found the risk of food allergy to be three times higher in infants of East Asian ancestry than infants of Caucasian ancestry. It is thought that infants growing up in Australia are exposed to environmental agents that have a differential effect on the immune system depending on their genetic background. However, the influence of specific genetic and environmental risk factors is currently unknown. It is also unclear if the increased risk of food allergy translates to higher risk of other allergic diseases later in childhood. The main objective of this thesis is to identify specific environmental and genetic factors on the risk of food allergy in the Asian population living in Australia. This thesis aims to quantify the prevalence of, and identify risk factors for food allergy and allergic comorbidities in the Asian population. An additional aim is to identify genetic variants that increase the risk of food allergy in the East Asian population and compare this to the Caucasian population living in Australia. This thesis primarily used data and samples from the longitudinal HealthNuts study where 5,276 1-year-old infants attending council run vaccination sessions across Melbourne were recruited. Skin prick tests to a range of food were carried out on infants and those with a wheal size less than 1 mm underwent an oral food challenge. The 1-year-old infants were followed up again at age 6 years and data collected at this follow-up visit were also used for analyses in this thesis. Additionally, data collected from a Growing Up in Singapore Towards Healthy Outcomes (GUSTO) birth cohort was also used to compare the risk factors and prevalence of allergic diseases between East Asians living in Melbourne and East Asians living in Singapore. This thesis reports that Australian-born children with East Asian parents have a higher burden of allergic rhinitis, eczema and aeroallergen sensitisation but not asthma, in the first six years of life compared to children of Caucasian ancestry. Moreover, children with IgE-mediated food allergy and eczema in infancy were 3 times more likely to have asthma and 2 times more likely to have allergic rhinitis at age 6 years, irrespective of ancestry. Additionally, East Asian children living in Melbourne have a higher risk of food allergy compared to East Asian children living in Singapore. Despite delayed introduction of allergens into the diet compared to the Asian population in Melbourne, Asian children in Singapore had less food allergy. While eczema rates were lower in Singapore than in Melbourne, early onset eczema was associated with an increased risk of food allergy in both Singapore and Melbourne. In terms of genetic risk factors, a systematic review conducted as part of this thesis identified several genes of interest known to be involved in immune regulation, cell function and epidermal barrier function. However, studies were of varied quality and the reproducibility of findings for the same SNPs were minimal. Some of the highly re-producible genes identified from the literature include HLA, FLG and IL13. Additionally, there was also a paucity of studies carried out in the Asian population that were able to elucidate underlying mechanisms for the differential food allergy risks observed in the population. This highlighted the need for genetic studies focused in this population. This thesis found that HLA rs7192 minor allele was associated with increased risk of peanut allergy in the Caucasian population but not East Asian population. Among sensitised children with two East Asian born parents, those with the minor allele for rs231735, rs231804 or rs11571291 (all CTLA4) have a reduced risk of egg allergy. The findings of this thesis identify Asian children living in Australia as a high risk allergic group not just in infancy but throughout early childhood. As a multifactorial disease, both environmental and genetic factors are known to contribute to the pathogenesis of food allergy. Therefore, it may be that the increased risk of food allergy observed in genetically predisposed East Asian children living in Melbourne unmasked upon exposure to environmental risk factors.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.