Identifying new genes and molecular mechanisms in mitochondrial disease

Ph. D. ThesisAn estimated 1158 nuclear encoded proteins function within mammalian mitochondria. Over the past ten years the increased implementation of next generation sequencingdriven diagnostics, particularly whole exome sequencing (WES), has resulted in the identification of over 150 nuclear genes not previously implicated in mitochondrial disease, necessitating extensive functional studies to determine and confirm pathogenicity. Over one third of reported mitochondrial disease-causing nuclear genetic variants reside in genes encoding proteins with a role in mtDNA gene expression. The overarching aim of my research project was to functionally validate novel candidate mitochondrial disease variants, focusing on those identified in genes encoding proteins with roles in mitochondrial translation, in order to achieve genetic diagnoses for affected patients. WES of two unrelated patients with early-onset neurological disease presentations identified different and previously unreported variants in the GFM2 gene encoding ribosome recycling factor mtEFG2. Differential and tissue-specific patterns of combined OXPHOS defects were identified in each patient. Novel compound heterozygous variants in the TSFM gene, encoding the elongation factor mtEF-Ts, were identified in a single patient presenting with adult-onset hypertrophic cardiomyopathy. The abundance of mtEF-Ts was significantly decreased in patient fibroblasts and cardiac tissue. The resulting OXPHOS deficiency was most severe in cardiac tissue, accompanied by decreased levels of elongation factor mtEF-Tu. A homozygous nonsense mutation in the MRPL47 gene, encoding a mitoribosomal large subunit (LSU) protein, was identified in three unrelated paediatric patients presenting with metabolic acidosis, epilepsy and liver involvement. Patient fibroblasts exhibited disorders of translation and combined OXPHOS defects. Some truncated MRPL47 protein was visualised within the destabilised LSU. Variants in MRPL65, a second LSU gene, were identified in five paediatric patients presenting with overlapping clinical features including developmental delay, ataxia and nystagmus. Attempts to generate a CRISPR/Cas9 MRPL65 knock-out cell line were unsuccessful due to polyploidy of the target genomic region, highlighting the importance of ii karyotyping in gene editing studies. MRPL65 cDNA analysis in a single patient identified retention of intronic sequence due to a +5 splice variant. Patient fibroblasts displayed normal levels of MRPL65 and OXPHOS proteins, but appeared to exhibit a defect in mitochondrial translation alongside some abnormal assembly of the LSU. This work expands upon our current knowledge of Mendelian mitochondrial disorders, adding to a growing list of nuclear genes implicated in disorders of translation and achieving genetic diagnosis in the presented families. Tissue-specific defects illustrate the complexity of the pathomechanisms underlying these disorders and highlight the gaps in our understanding of adaptive compensatory mechanisms employed in different tissues. This research also emphasises the importance of identifying and characterising multiple families when assigning pathogenicity to novel disease-causing variants.Lily Foundatio