Paediatrics (RCH) - Theses
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ItemSolving the Unsolved: Implementing new Omics and functional approaches to enable genomic diagnosis of virtually all patients with Mitochondrial disease( 2023-11)Abstract Mitochondrial disorders are the most common group of inherited metabolic disorders affecting children. Patients suffer from debilitating chronic conditions that are often life-threatening. Comprising more than 360 monogenic disorders, inherited through all modes of inheritance, this group of conditions can manifest at any age and can affect any tissue or organ system. Their high clinical and genetic heterogeneity means genomic diagnostic yields are typically only ~50%. This study aimed to extend this incomplete understanding of the genetic and mutational spectrum of mitochondrial disease through the study of a cohort of patients with a suspected mitochondrial disease who have remained undiagnosed following extensive molecular investigations, specifically exome sequencing, using new -Omics approaches. Three overlapping retrospective cohorts of paediatric mitochondrial disease patients (Leigh syndrome, Complex I deficiency or Population-based) were combined, forming a combined cohort comprising 263 patients. Among them, 77% had previously received a firm genetic diagnosis through extensive molecular and exome analyses, surpassing the typical diagnostic rate of 50% published in other studies. The remaining 61 undiagnosed individuals formed the cohort for this current study, referred to as the ‘exome-negative’ cohort, and underwent one or more of: expanded exome analysis, genome sequencing, RNA sequencing or quantitative proteomics. The analysis gene list comprised a recently updated list of genes encoding the mitochondrial proteome and the Mendeliome. At the conclusion of this PhD, 34 ‘exome-negative’ patients (56%) now have a firm genetic diagnosis; with an additional 12 requiring further functional validation of strong candidate genes. Pathogenic variants were identified in 21 different genes including 15 phenocopy genes and six mitochondrial disease genes, one of which is a novel disease gene (MRPL39). The new diagnoses consisted of structural variants that included complex rearrangements, inter– and intra-genic deletions in 33% of all the variant alleles identified, and dominant variants in 19 of 34 patients diagnosed (56%). Deep intronic variants were identified in 2 patients by genome sequencing and proteomics, and a strong splice-site altering synonymous variant was identified using RNA sequencing. Exome sequencing reanalysis directly resulted in diagnoses for 16 patients. This study has increased the overall diagnostic yield in our combined retrospective cohort by 13% to 90%. Twenty-seven patients currently remain undiagnosed, emphasising the incomplete understanding of Mitochondrial disease genetics and limitations in current technologies to detect the full mutational spectrum of disease. Our study highlights the utility of new -Omics approaches in extending diagnostic rates of ‘exome-negative’ patients. It also illustrates the benefit of including phenocopy genes in analyses and the periodic reanalysis of exome and genome sequencing to enhance diagnostic yields. The thesis is organized as follows: Chapter 1 provides an introduction to mitochondrial disease, mitochondrial genetics, and genomic diagnostic technologies to lay the foundation for subsequent discussions. Chapter 2 outlines the materials and methods utilized throughout the study. Chapter 3 offers a comprehensive overview of the study cohort, including details on biochemical, phenotypic and genetic data. Chapter 4 presents a detailed analysis of 18 patients diagnosed with phenocopy gene disorders. Chapter 5 delves into the study of 16 patients with confirmed molecular diagnoses in mitochondrial disease genes. Chapter 6 describes the identification and characterization of a novel mitochondrial disease gene, specifically one of the fifty-two proteins of the large subunit of the mitochondrial ribosome. These findings were published in the Human Molecular Genetics journal. Lastly, Chapter 7 summarise the key findings arising from this thesis and their implications for the field of mitochondrial disease research. It addresses current limitations and challenges encountered in identifying and interpreting genetic variants and offers insights into future directions for research in the field.