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Sci Rep. 2017 Nov 15;7(1):15676. doi: 10.1038/s41598-017-14623-2.

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

Author information

1
Wellcome Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle Upon Tyne, UK.
2
NHS Highly Specialised Mitochondrial Diagnostic Laboratory, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
3
The National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, London, UK.
4
School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK.
5
BHF Centre of Research Excellence, The James Black Centre, King's College London, University of London, 125 Coldharbour Lane, London, SE5 9NU, UK.
6
Wellcome Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle Upon Tyne, UK. robert.taylor@ncl.ac.uk.
7
NHS Highly Specialised Mitochondrial Diagnostic Laboratory, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK. robert.taylor@ncl.ac.uk.

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.

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