Paediatrics (RCH) - Theses

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End date: 2015 × Start date: 2015 × Subject: birth prevalence estimate × Subject: genetics ×

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    Using massively parallel sequencing to understand the genetic basis of mitochondrial disorders: a population-based approach
    Mountford, Hayley S. ( 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.