BACKGROUND: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts. METHODS: To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue Native electrophoreses. RESULTS: Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p 50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations. CONCLUSION: Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, IV and V manifesting in peripheral blood lymphocytes. GENERAL SIGNIFICANCE: The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients.

• Klíčová slova
• AA, antimycin A, BNE, Blue Native PAGE, COX, cytochrome c oxidase, Diagnostics, FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, GP, glycerol-3-phosphate, GPDH, mitochondrial FAD-dependent glycerophosphate dehydrogenase, Lymphocytes, Mitochondrial diseases, OXPHOS, oxidative phosphorylation, Oxidative phosphorylation, PAGE, polyacrylamide gel electrophoresis, Respirometry, TMPD, tetramethylphenylenediamine, TMRM, tetramethylrhodamine methyl ester, cI–cV, respiratory chain complexes I–V, s3, state 3-ADP, s3u, state 3-uncoupled, s4o, state 4-oligomycin, ΔΨm, mitochondrial membrane potential,
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: Mitochondrial disorders (MD) may manifest in neonates, but early diagnosis is difficult. In this study, clinical and laboratory data were analyzed in 129 patients with neonatal onset of MD to identify any association between specific mitochondrial diseases and their symptoms with the aim of optimizing diagnosis. MATERIALS AND METHODS: Retrospective clinical and laboratory data were evaluated in 461 patients (331 families) with confirmed MD. RESULTS: The neonatal onset of MD was reported in 28% of the patients. Prematurity, intrauterine growth retardation and hypotonia necessitating ventilatory support were present in one-third, cardiomyopathy in 40%, neonatal seizures in 16%, Leigh syndrome in 15%, and elevated lactate level in 87%. Hyperammonemia was observed in 22 out of 52 neonates. Complex I deficiency was identified in 15, complex III in one, complex IV in 23, complex V in 31, combined deficiency of several complexes in 53, and PDH complex deficiency was identified in six patients. Molecular diagnosis was confirmed in 49 cases, including a newborn with a 9134A>G mutation in the MTATP6 gene, which has not been described previously. CONCLUSION: The most significant finding is the high incidence of neonatal cardiomyopathy and hyperammonemia. Based on our experience, we propose a diagnostic flowchart applicable to critically ill neonates suspicious for MD. This tool will allow for the use of direct molecular genetic analyses without the need for muscle biopsies in neonates with Alpers, Barth, MILS and Pearson syndromes, SCO1, SCO2, TMEM70, ATP5E, SUCLG1 gene mutations and PDH complex deficiency.

• MeSH
• hyperamonemie diagnóza   genetika   MeSH
• kardiomyopatie diagnóza   genetika   MeSH
• Leighova nemoc krev   diagnóza   genetika   MeSH
• lidé MeSH
• mitochondriální nemoci krev   diagnóza   genetika   MeSH
• mutace MeSH
• novorozenec nedonošený MeSH
• novorozenec MeSH
• retrospektivní studie MeSH
• růstová retardace plodu krev   diagnóza   genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The biogenesis of eukaryotic COX (cytochrome c oxidase) requires several accessory proteins in addition to structural subunits and prosthetic groups. We have analysed the assembly state of COX and SCO2 protein levels in various tissues of six patients with mutations in SCO2 and SURF1. SCO2 is a copper-binding protein presumably involved in formation of the Cu(A) centre of the COX2 subunit. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated that COX holoenzyme is reduced to 10-20% in skeletal muscle and brain of SCO2 and SURF1 patients and to 10-30% in heart of SCO2 patients, whereas liver of SCO2 patients' contained normal holoenzyme levels. The steady-state levels of mutant SCO2 protein ranged from 0 to 20% in different SCO2 patient tissues. In addition, eight distinct COX subcomplexes and unassembled subunits were found, some of them identical with known assembly intermediates of the human enzyme. Heart, brain and skeletal muscle of SCO2 patients contained accumulated levels of the COX1.COX4.COX5A subcomplex, three COX1-containing subcomplexes, a COX4.COX5A subcomplex and two subcomplexes composed of only COX4 or COX5A. The accumulation of COX1.COX4.COX5A subcomplex, along with the virtual absence of free COX2, suggests that the lack of the Cu(A) centre may result in decreased stability of COX2. The appearance of COX4.COX5A subcomplex indicates that association of these nucleus-encoded subunits probably precedes their addition to COX1 during the assembly process. Finally, the consequences of SCO2 and SURF1 mutations suggest the existence of tissue-specific functional differences of these proteins that may serve different tissue-specific requirements for the regulation of COX biogenesis.

• MeSH
• fibroblasty enzymologie   MeSH
• játra enzymologie   MeSH
• kojenec MeSH
• kosterní svaly enzymologie   MeSH
• lidé MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• molekulární chaperony MeSH
• mozek enzymologie   MeSH
• mutace genetika   MeSH
• myokard enzymologie   MeSH
• orgánová specificita MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• předškolní dítě MeSH
• proteiny genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• respirační komplex IV biosyntéza   chemie   metabolismus   MeSH
• transportní proteiny MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• molekulární chaperony MeSH
• podjednotky proteinů MeSH
• proteiny MeSH
• respirační komplex IV MeSH
• SCO2 protein, human MeSH Prohlížeč
• Surf-1 protein MeSH Prohlížeč
• transportní proteiny MeSH