P39. Assigning pathogenicity to three novel mt-tRNA mutations associated with different mitochondrial disease presentations

Abstract

Mitochondrial transfer RNA (mt-tRNA) mutations are the commonest mitochondrial (mtDNA) mutations associated with human disease. Assigning pathogenicity to novel mt-tRNA variants is very important particularly regarding the polymorphic nature of mtDNA. We report three patients in whom we have identified novel mt-tRNA point mutations. Clinical presentation of patients 1 and 2 was isolated myopathy. Patient 3 showed multisytemic disease with myopathy, spinal ataxia, deafness, cataract and cognitive impairment. Muscle biopsies revealed extensive histopathological findings (up to 40% COX-deficient fibres). mtDNA sequencing in muscle identified variants in the mt-tRNA Ala gene (m.5631G>A and m.5610G>A) in patients 1 and 2, and a m.7539C>T mt-tRNA Asp transition in patient 3. Quantitative restriction fragment length polymorphism analysis and pyrosequencing confirmed mtDNA heteroplasmy for all mutations, with highest levels being demonstrated in muscle and lower levels observed in blood, buccal epithelium, urinary sediments and hair shafts. Family analysis in patients 1 and 2 showed maternal transmission of the mutations. Single muscle fibre segregation studies in individual, laser-microcaptured COX-positive and COX-deficient fibres detected statistically-significant higher mutation loads in COX-deficient fibres than in COX-positive fibres (patient 1: 95.1% + 0.45 vs. 83.8% + 3.38; patient 2: 98.0% + 0.46 vs. 78.9% + 4.81; patient 3: 96.1% + 0.38 vs. 69.1% + 2.98). Absence from control databases, presence of mtDNA heteroplasmy, hierarchical mt-tRNA mutation segregation within tissues, occurrence at conserved sequence positions, and single-fibre studies confirm these novel mt-tRNA mutations to be pathogenic. Interestingly, patients 1 and 2 showed very high mutation levels in all tissues, as described for other mt-tRNA mutations (e.g. m.14709T>C); moreover, the threshold for impairment of oxidative phosphorylation, as evidenced by COX deficiency, appears to be extremely high for both novel mt-tRNA Ala mutations. Previously-described cases of mt-tRNA Ala mutations (m.5591G>A, m.5650G>A) are also associated with a pure myopathic phenotype and demonstrate very high mutation thresholds, inferring at least some genotype:phenotype correlation for mutations within this tRNA gene.