Paediatrics (RCH) - Theses
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ItemDevelopment, characterisation and nicotinamide riboside treatment of Complex I-deficient model systems( 2018)Mitochondrial disorders are the most common inborn error of metabolism, affecting 1 in 5000 live births. Complex I (CI) de ciency is the most common cause of paediatric mitochondrial disease. Deleterious mutations in CI subunits vary in their pathological presentations and current treatments available for these disorders are limited. Investigations here focused on the CI subunits NDUFS4 and NDUFS6, specifi cally the impact of loss on different model systems and response of these models to the NAD+ precursor nicotinamide riboside (NR). NDUFS4 mutations in humans are typically associated with the progressive neurodegenerative disease Leigh Syndrome and early fatality, and Ndufs4fky=fky knockout mice have a similar progressive encephalopathy. Human NDUFS6 mutations are associated with acidosis and neonatal mortality, while the Ndufsgt=gt (gene-trap) mice have a cardiac phenotype resulting from tissue-specific c splicing of NDUFS6. Isolated mitochondria from these mice were investigated alongside a comprehensive characterisation of NDUFS4KO and NDUFS6KO HEK293T cell lines, providing a baseline for treatment investigations. As CI deficiency results in a disruption of the NAD+/NADH ratio, a range of NAD+ precursors have been investigated to attempt to ameliorate this imbalance. Dietary supplementation with NR has shown phenotypic improvement in mouse models of mitochondrial myopathy. Here, dietary supplementation of Ndufs4fky=fky mice with 0.15% NR for 4 weeks did not significantly impact phenotypic progression or biochemical parameters. However, in vitro NR treatment of NDUFS4KO and NDUFS6KO cells resulted in bene cial biochemical changes, including increased respiratory capacity. This difference in response may be due to the models investigated. While HEK293T cells are a good starting point for investigations due to their quick growth and straightforward characterisation, clinically relevant cell types provide a much more powerful tool for investigation of potential treatments and the biochemical basis of mitochondrial disease. To enable these investigations, CRISPR/Cas9 technology was used to disrupt our genes of interest in human embryonic stem cells, thus generating mutants with the potential to form clinically relevant cell types. The NDUFS6 mutant had an apparent defect in ATP synthesis and cardiac differentiation however, supercomplex disruption was not detected. Characterisation of the NDUFS4 mutants is more preliminary and clones appeared to be using an alternative start site to produce protein. More comprehensive characterisation of all mutant lines is required to provide a clear direction for future research. This work has provided additional characterisation across a range of model systems with loss of NDUFS4 or NDUFS6, providing a clear basis for future studies into potential treatments for mitochondrial disorders. While the NR treatment investigations were inconclusive, results suggested that NR can alleviate the biochemical features of CI deficiency, and the ability of NR to meaningfully impact neural cells needs to be further elucidated. The development of human embryonic stem cell mutants will facilitate more relevant models for future treatment investigations.
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ItemUsing massively parallel sequencing to determine the genetic basis of Leigh Syndrome, the most common mitochondrial disorder affecting children( 2018)Mitochondrial diseases are debilitating illnesses caused by mutations that impair mitochondrial energy generation. The most common clinical presentation of mitochondrial disease in children is Leigh syndrome. This neurodegenerative disorder can be caused by mutations in more than 85 genes, encoded by both nuclear and mitochondrial DNA (mtDNA). When this PhD commenced, massively parallel sequencing for genetic diagnosis of Leigh syndrome was transitioning into the clinic, however its diagnostic utility in a clinical setting was unknown. Furthermore, a significant number of Leigh syndrome patients remained without a genetic diagnosis, indicating that further research was required to expand our understanding of the genetic basis of disease. To identify the maximum diagnostic yield of massively parallel sequencing in patients with Leigh syndrome, and to provide insight into the genetic basis of disease, unsolved patients from a historical Leigh syndrome cohort were studied. This cohort is comprised of 67 clinically-ascertained patients diagnosed with Leigh or Leigh-like syndrome according to stringent criteria. DNA from all 33 patients lacking a genetic diagnosis underwent whole exome sequencing, with parallel sequencing of the mtDNA. A targeted analysis of 2273 genes was performed, which included known and candidate mitochondrial disease genes, and differential diagnosis genes underlying distinct disorders with phenotypic overlap. This study provided a genetic diagnosis for 11 of the 12 unsolved Leigh syndrome patients and 7 of the 21 unsolved Leigh-like patients investigated. Eight reported and eight novel pathogenic variants were identified in twelve disease genes; ALDH18A1, MT-ATP6, MT-ND3, MT-ND5, MT-ND6, MTFMT, NARS2, SCO2, SERAC1, SLC19A3, PDHA1, and PDHX. A genetic diagnosis has now been established in 78% of the total cohort, including in 34 of 35 Leigh syndrome patients and 18 of 32 Leigh-like patients. Candidate genetic diagnoses, including in differential diagnosis genes, were identified in an additional 7 Leigh-like patients, where investigation of synonymous variants, complex CNVs and heterozygous X-linked variants highlight challenges associated with variant follow-up in a diagnostic context. This thesis also describes the study of an additional patient with a firm diagnosis of Leigh syndrome. Whole exome sequencing of this patient with targeted analysis identified a homozygous splice site mutation in MRPS34, which was shown to cause abnormal splicing and loss of wild-type MRPS34 protein. MRPS34 encodes a mitochondrial ribosomal protein, and variants in this gene had not previously been described to cause disease. The data presented in this thesis demonstrate that mutation of MRPS34 causes a disorder of mitochondrial energy generation by destabilising the small mitochondrial ribosomal subunit, and therefore reducing the synthesis of mtDNA-encoded proteins. The rescue of cellular defects by lentiviral-mediated expression of wild-type MRPS34 establishes it as a bona fide disease gene. In conclusion, this thesis describes the identification of novel mutations and a new disease gene, thereby expanding our understanding of the genetic basis of Leigh syndrome. The characterisation of the aetiological basis of disease in the cohort, as well as evaluation of the outcomes of massively parallel sequencing of cohort patients, provides key insight into the diagnostic utility of this testing approach in a clinical setting.