In this study, we report on a novel heteroplasmic pathogenic variant in mitochondrial DNA (mtDNA). The studied patient had myoclonus, epilepsy, muscle weakness, and hearing impairment and harbored a heteroplasmic m.8315A>C variant in the MTTK gene with a mutation load ranging from 71% to >96% in tested tissues. In muscle mitochondria, markedly decreased activities of respiratory chain complex I + III and complex IV were observed together with mildly reduced amounts of complex I and complex V (with the detection of V*- and free F1-subcomplexes) and a diminished level of complex IV holoenzyme. This pattern was previously seen in other MTTK pathogenic variants. The novel variant was not present in internal and publicly available control databases. Our report further expands the spectrum of MTTK variants associated with mitochondrial encephalopathies in adults.

• Keywords
• MTTK gene, OXPHOS, heteroplasmy, m.8315A>C, mtDNA,
• MeSH
• Adult MeSH
• Humans MeSH
• DNA, Mitochondrial genetics   MeSH
• Mitochondrial Encephalomyopathies * pathology   MeSH
• Electron Transport Complex IV MeSH
• Mitochondria, Muscle metabolism   MeSH
• MERRF Syndrome * genetics   pathology   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Mitochondrial MeSH
• Electron Transport Complex IV MeSH

BACKGROUND: Maternally inherited complex I deficiencies due to mutations in MT-ND genes represent a heterogeneous group of multisystem mitochondrial disorders (MD) with a unfavourable prognosis. The aim of the study was to characterize the impact of the mutations in MT-ND genes, including the novel m.13091 T > C variant, on the course of the disease, and to analyse the activities of respiratory chain complexes, the amount of protein subunits, and the mitochondrial energy-generating system (MEGS) in available muscle biopsies and cultivated fibroblasts. METHODS: The respiratory chain complex activities were measured by spectrophotometry, MEGS were analysed using radiolabelled substrates, and protein amount by SDS-PAGE or BN-PAGE in muscle or fibroblasts. RESULTS: In our cohort of 106 unrelated families carrying different mtDNA mutations, we found heteroplasmic mutations in the genes MT-ND1, MT-ND3, and MT-ND5, including the novel variant m.13091 T > C, in 13 patients with MD from 12 families. First symptoms developed between early childhood and adolescence and progressed to multisystem disease with a phenotype of Leigh or MELAS syndromes. MRI revealed bilateral symmetrical involvement of deep grey matter typical of Leigh syndrome in 6 children, cortical/white matter stroke-like lesions suggesting MELAS syndrome in 3 patients, and a combination of cortico-subcortical lesions and grey matter involvement in 4 patients. MEGS indicated mitochondrial disturbances in all available muscle samples, as well as a significantly decreased oxidation of [1-14C] pyruvate in fibroblasts. Spectrophotometric analyses revealed a low activity of complex I and/or complex I + III in all muscle samples except one, but the activities in fibroblasts were mostly normal. No correlation was found between complex I activities and mtDNA mutation load, but higher levels of heteroplasmy were generally found in more severely affected patients. CONCLUSIONS: Maternally inherited complex I deficiencies were found in 11% of families with mitochondrial diseases in our region. Six patients manifested with Leigh, three with MELAS. The remaining four patients presented with an overlap between these two syndromes. MEGS, especially the oxidation of [1-14C] pyruvate in fibroblasts might serve as a sensitive indicator of functional impairment due to MT-ND mutations. Early onset of the disease and higher level of mtDNA heteroplasmy were associated with a worse prognosis.

• Keywords
• Complex I, Leigh syndrome, MEGS, MELAS syndrome, MT-ND genes, Mitochondria, mtDNA,
• MeSH
• Biopsy MeSH
• Child MeSH
• Adult MeSH
• Fibroblasts metabolism   MeSH
• Infant MeSH
• Muscle, Skeletal metabolism   MeSH
• Cells, Cultured MeSH
• Leigh Disease genetics   MeSH
• Humans MeSH
• Magnetic Resonance Imaging MeSH
• DNA, Mitochondrial * MeSH
• Mitochondrial Diseases genetics   MeSH
• Adolescent MeSH
• Brain diagnostic imaging   pathology   MeSH
• Mutation * MeSH
• Infant, Newborn MeSH
• Electron Transport Complex I deficiency   genetics   metabolism   MeSH
• MELAS Syndrome genetics   MeSH
• Age of Onset MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Infant, Newborn MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Mitochondrial * MeSH
• Electron Transport Complex I MeSH

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

• MeSH
• Apoptosis MeSH
• ATPases Associated with Diverse Cellular Activities MeSH
• Gene Knockdown Techniques MeSH
• GTP Phosphohydrolases metabolism   MeSH
• Humans MeSH
• Metalloendopeptidases metabolism   MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondrial Proteins MeSH
• Mitochondria metabolism   MeSH
• NADH, NADPH Oxidoreductases metabolism   MeSH
• Cell Proliferation * MeSH
• ATP-Dependent Proteases metabolism   MeSH
• Peptide Hydrolases metabolism   MeSH
• Protein Isoforms metabolism   MeSH
• Electron Transport Complex I MeSH
• Electron Transport Complex IV metabolism   MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Saccharomyces cerevisiae cytology   metabolism   MeSH
• Electron Transport * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATPases Associated with Diverse Cellular Activities MeSH
• GTP Phosphohydrolases MeSH
• Metalloendopeptidases MeSH
• Mitochondrial Proteins MeSH
• NADH, NADPH Oxidoreductases MeSH
• NDUFB6 protein, human MeSH Browser
• OPA1 protein, human MeSH Browser
• ATP-Dependent Proteases MeSH
• Peptide Hydrolases MeSH
• Protein Isoforms MeSH
• Electron Transport Complex I MeSH
• Electron Transport Complex IV MeSH
• Saccharomyces cerevisiae Proteins MeSH
• YME1 protein, S cerevisiae MeSH Browser
• YME1L1 protein, human MeSH Browser