Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency

• Published in: Scientific reports 2017-11, Vol.7 (1), p.15676-13, Article 15676
• Main Authors: Ahmed, Syeda T., Alston, Charlotte L., Hopton, Sila, He, Langping, Hargreaves, Iain P., Falkous, Gavin, Oláhová, Monika, McFarland, Robert, Turnbull, Doug M., Rocha, Mariana C., Taylor, Robert W.
• Format: Article
• Language: English
• Subjects: 13/1; 13/51; 45; 45/23; 631/208/1516; 631/45/881; 82; 82/29; Biopsy; Cell Nucleus - genetics; Child; Child, Preschool; DNA, Mitochondrial - genetics; Electron transport; Electron transport chain; Electron Transport Complex I - deficiency; Electron Transport Complex I - genetics; Enzymatic activity; Female; Fluorescent Antibody Technique; Fluoroimmunoassay - methods; Genetic Heterogeneity; Genomes; Humanities and Social Sciences; Humans; Immunoreactivity; Male; Mitochondria - genetics; Mitochondrial Diseases - diagnosis; Mitochondrial Diseases - genetics; Mitochondrial Diseases - pathology; Mitochondrial DNA; multidisciplinary; Muscle, Skeletal - metabolism; Muscle, Skeletal - pathology; Mutation; NADH Dehydrogenase - genetics; NADH-ubiquinone oxidoreductase; Oxidative Phosphorylation; Science; Science (multidisciplinary); Skeletal muscle; Spectrophotometry
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-14623-2

Isolated Complex I (CI) deficiency is the most commonly observed mitochondrial respiratory chain biochemical defect, affecting the largest OXPHOS component. CI is genetically heterogeneous; pathogenic variants affect one of 38 nuclear-encoded subunits, 7 mitochondrial DNA (mtDNA)-encoded subunits or 14 known CI assembly factors. The laboratory diagnosis relies on the spectrophotometric assay of enzyme activity in mitochondrially-enriched tissue homogenates, requiring at least 50 mg skeletal muscle, as there is no reliable histochemical method for assessing CI activity directly in tissue cryosections. We have assessed a validated quadruple immunofluorescent OXPHOS (IHC) assay to detect CI deficiency in the diagnostic setting, using 10 µm transverse muscle sections from 25 patients with genetically-proven pathogenic CI variants. We observed loss of NDUFB8 immunoreactivity in all patients with mutations affecting nuclear-encoding structural subunits and assembly factors, whilst only 3 of the 10 patients with mutations affecting mtDNA-encoded structural subunits showed loss of NDUFB8, confirmed by BN-PAGE analysis of CI assembly and IHC using an alternative, commercially-available CI (NDUFS3) antibody. The IHC assay has clear diagnostic potential to identify patients with a CI defect of Mendelian origins, whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work-up of patients with suspected mitochondrial disease.