Toggle Main Menu Toggle Search

Open Access padlockePrints

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 McFarlandORCiD, Emeritus Professor Doug Turnbull, Dr Mariana Rocha, Professor Robert Taylor



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


© 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

Date deposited: 12/12/2017

ISSN (electronic): 2045-2322

Publisher: Nature Publishing Group


DOI: 10.1038/s41598-017-14623-2


Altmetrics provided by Altmetric


Funder referenceFunder name
203105/Z/16/ZWellcome Trust