Ahmed, ST, Alston, CL, Hopton, S, He, L, Hargreaves, IP, Falkous, G, Olahova, M, McFarland, R, Turnbull, DM, Rocha, MC and Taylor, RW (2017) Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency. Scientific Reports, 7. ISSN 2045-2322

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Abstract

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.

Item Type:	Article
Uncontrolled Keywords:	Science & Technology; Multidisciplinary Sciences; Science & Technology - Other Topics; LEIGH-LIKE-SYNDROME; ASSEMBLY FACTOR; DNA MUTATION; CLINICAL PHENOTYPE; G13513A MUTATION; ND5 GENE; DISEASE; MELAS; IDENTIFICATION; SUBUNITS
Subjects:	R Medicine > RM Therapeutics. Pharmacology
Divisions:	Pharmacy & Biomolecular Sciences
Publisher:	Nature Publishing Group
Related URLs:	Author
Date Deposited:	01 Oct 2018 10:10
Last Modified:	04 Sep 2021 02:23
DOI or ID number:	10.1038/s41598-017-14623-2
URI:	https://researchonline.ljmu.ac.uk/id/eprint/9383

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