Mitochondrial disease associated with the pathogenic m.3243A>G variant is a common, clinically heterogeneous, neurogenetic disorder. Using multiple linear regression and linear mixed modelling, we evaluated which commonly assayed tissue (blood N = 231, urine N = 235, skeletal muscle N = 77) represents the m.3243A>G mutation load and mitochondrial DNA (mtDNA) copy number most strongly associated with disease burden and progression. m.3243A>G levels are correlated in blood, muscle and urine (R 2 = 0.61–0.73). Blood heteroplasmy declines by ~2.3%/year; we have extended previously published methodology to adjust for age. In urine, males have higher mtDNA copy number and ~20% higher m.3243A>G mutation load; we present formulas to adjust for this. Blood is the most highly correlated mutation measure for disease burden and progression in m.3243A>G‐harbouring individuals; increasing age and heteroplasmy contribute (R 2 = 0.27, P < 0.001). In muscle, heteroplasmy, age and mtDNA copy number explain a higher proportion of variability in disease burden (R 2 = 0.40, P < 0.001), although activity level and disease severity are likely to affect copy number. Whilst our data indicate that age‐corrected blood m.3243A>G heteroplasmy is the most convenient and reliable measure for routine clinical assessment, additional factors such as mtDNA copy number may also influence disease severity.

ObjectiveThe pathogenic mitochondrial DNA m.3243A>G mutation is associated with a wide range of clinical features, making disease prognosis extremely difficult to predict. We aimed to understand the cause of this heterogeneity.MethodsWe examined the phenotypic profile of 238 adult m.3243A>G carriers (patients and asymptomatic carriers) from the UK MRC Mitochondrial Disease Patient Cohort using the Newcastle Mitochondrial Disease Adult Scale. We modeled the role of risk factors for the development of specific phenotypes using proportional odds logistic regression. As mitochondria are under the dual control of their own and the nuclear genome, we examined the role of additive nuclear genetic factors in the development of these phenotypes within 46 pedigrees from the cohort.ResultsSeizures and stroke‐like episodes affect 25% and 17% of patients, respectively; more common features include hearing impairment, gastrointestinal disturbance, psychiatric involvement, and ataxia. Age, age‐adjusted blood heteroplasmy levels, and sex are poor predictors of phenotypic severity. Hearing impairment, diabetes, and encephalopathy show the strongest associations, but pseudo‐R 2 values are low (0.14–0.17). We found a high heritability estimate for psychiatric involvement (h 2=0.76, P = 0.0003) and moderate estimates for cognition (h 2=0.46, P = 0.0021), ataxia (h 2 = 0.45, P = 0.0011), migraine (h 2 = 0.41, P = 0.0138), and hearing impairment (h2 = 0.40, P = 0.0050).InterpretationOur results provide good evidence for the presence of nuclear genetic factors influencing clinical outcomes in m.3234A>G‐related disease, paving the way for future work identifying these through large‐scale genetic linkage and association studies, increasing our understanding of the pathogenicity of m.3243A>G and providing improved estimates of prognosis.

Here we report the application of a mass spectrometry-based technology, imaging mass cytometry, to perform in-depth proteomic profiling of mitochondrial complexes in single neurons, using metal-conjugated antibodies to label post-mortem human midbrain sections. Mitochondrial dysfunction, particularly deficiency in complex I has previously been associated with the degeneration of dopaminergic neurons in Parkinson’s disease. To further our understanding of the nature of this dysfunction, and to identify Parkinson’s disease specific changes, we validated a panel of antibodies targeting subunits of all five mitochondrial oxidative phosphorylation complexes in dopaminergic neurons from Parkinson’s disease, mitochondrial disease, and control cases. Detailed analysis of the expression profile of these proteins, highlighted heterogeneity between individuals. There is a widespread decrease in expression of all complexes in Parkinson’s neurons, although more severe in mitochondrial disease neurons, however, the combination of affected complexes varies between the two groups. We also provide evidence of a potential neuronal response to mitochondrial dysfunction through a compensatory increase in mitochondrial mass. This study highlights the use of imaging mass cytometry in the assessment and analysis of expression of oxidative phosphorylation proteins, revealing the complexity of deficiencies of these proteins within individual neurons which may contribute to and drive neurodegeneration in Parkinson’s disease.