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

Lookup NU author(s): Syeda Ahmed, Charlotte Alston, Sila Hopton, Dr Langping He, Gavin Falkous, Dr Monika Olahova, Professor Bobby McFarland, Professor Doug Turnbull, Dr Mariana Rocha, Professor Robert Taylor

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© 2017 The Author(s). 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.


Publication metadata

Author(s): Ahmed ST, Alston CL, Hopton S, He L, Hargreaves IP, Falkous G, Olahova M, McFarland R, Turnbull DM, Rocha MC, Taylor RW

Publication type: Article

Publication status: Published

Journal: Scientific Reports

Year: 2017

Volume: 7

Issue: 1

Online publication date: 12/11/2017

Acceptance date: 12/10/2017

ISSN (electronic): 2045-2322

Publisher: Nature Publishing Group

URL: https://doi.org/10.1038/s41598-017-14623-2

DOI: 10.1038/s41598-017-14623-2


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