To correlate mitochondrial genetics and respiratory chain deficiency at a single cell level in patients with mitochondrial DNA (mtDNA) maintenance disorders.

mtDNA deletions are an important pathological mechanism in adults with mtDNA maintenance disorders. The threshold level of a single mtDNA deletion required to cause OXPHOS deficiency is different for complex I (CI) and complex IV (CIV). Different thresholds were observed depending on the size and location of the resulting mtDNA deletion that together determine the number of CI, CIV and tRNA genes deleted. Whilst these relationships appear straightforward in patients with single, large-scale mtDNA deletions, they are likely to be more complex in patients with multiple mtDNA deletions. 

We used immunofluorescence to quantify mitochondrial respiratory chain deficiency in muscle biopsies from patients with mtDNA maintenance disorders (n=16). Further studies on 6/16 patients (2 POLG, 2 RRM2B,1 TWNK, 1 SLC25A4(ANT1)) included the correlation of the biochemical deficiency with the mtDNA abnormality in individual cells, following lasercapture microdissection and determination of the size and level of clonally-expanded mtDNA deletion within fibres by real-time PCR, long-range PCR and sequencing of breakpoints.

Quadruple immunocytochemical studies show that the muscle biochemical phenotype is different in patients with multiple mtDNA deletions compared to other mtDNA mutations, but showed no difference between genotypes. Genetic analysis demonstrated major arc deletions to be more common and showed a clear correlation between deletion level and respiratory chain deficiency. We find that 62.8% of respiratory chain deficient muscle fibres contained a single deletion, 34.6% two deletions and 2.6% three deletions.

Patients harbouring multiple deletions have a distinct muscle respiratory chain profile, however, no genotype based pattern of respiratory chain deficiency has been found. A clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell was demonstrated.