Identification and functional validation of new mtDNA and nuclear gene variants responsible for mitochondrial disorders

My project has been focused on the identification and validation of new mitochondrial gene variants and new mitochondria-related genes. The first report is about a woman presenting with a stroke-like episode and an history of severe hearing loss, frequent migraines, exercise intolerance, myalgias and limb-girdle muscle weakness indicating a slowly progressive myopathy and secondary amenorrhea with low gonadotropin levels. A muscle biopsy showed ragged-red, cytochrome c oxidase-negative fibers, and an isolated defect of cytochrome c oxidase activity in muscle mitochondria and sequence analysis of muscle mtDNA revealed a new heteroplasmic m.6597C>A transversion in the MTCOI gene, encoding subunit I of cytochrome c oxidase. Analysis on transmitochondrial cybrids demonstrated that the mutation is indeed associated with COX deficiency, i.e. pathogenic. The second report is about a new phenotype associated to mutations in the AARS2 gene encoding for the mitochondrial aminoacyl tRNA synthetase, identified in six patients presenting with primary ovarian failure, cerebellar and pyramidal signs and cognitive or behavioural disturbances. Two patients underlined a muscle biopsy which showed a severe complex IV defect at histochemical and biochemical analyses. The third report is about the clinical and biochemical phenotypes associated with mutations in two new mitochondrial aminoacyl tRNA synthetases (ARSs2) genes. In the first patient, an 8 years old child presenting with psychomotor delay, seizures, facial dysmorphisms and hyperlactacidemia and a brain MRI showing hyperintense lesions in the insula and fronto-temporal right cortex, whole exome sequencing (WES) identified a homozygous missense mutation in VARS2, encoding the mitochondrial valyl tRNA-synthetase. In two siblings presenting with a phenotype characterized by hypotonia and psychomotor retardation, high plasma and liquor lactate, both died at few months of age WES revealed two variants in TARS2, encoding the mitochondrial threonyl tRNA-synthetase: a missense and a splice site mutation. VARS2 and TARS2 mutations segregate within patients families. Patients’ clinical- biochemical phenotype and in silico and in vitro analyses of VARS2 and TARS2 mutations clearly indicate these genes as disease-causative. Expression of the corresponding wild-type enzymes led to recovery of the biochemical impairment of mitochondrial respiration in immortalized mutant fibroblasts; yeast modelling of the VARS2 mutation confirmed its pathogenic role.