Unravelling the molecular mechanisms underlying single, large-scale mtDNA deletions

Tuppen HA, Rocha M, Rosa H, Taylor RW and Turnbull DM.

Wellcome Trust Centre for Mitochondrial Research, Newcastle University, Newcastle-upon-Tyne, NE2 4HH, UK

The underlying genetic causes of mitochondrial diseases are now well-defined and focus has shifted to elucidating the pathogenic mechanisms of disease. Due to their genetic heterogeneity, the study of pathomechanisms associated with single, large-scale mtDNA deletions is particularly challenging. To address this we are pursuing a two-staged approach, investigating single muscle fibres at both the protein and RNA level. First, we combined our recently developed quantitative single cell immunofluorescence assay, which accurately measures key complex I and IV protein levels, with single cell measurements of mtDNA deletion level and copy number, to analyse individual fibres from 23 adult patients with a single, large-scale mtDNA deletion. This revealed that both the expression and severity of the ensuing mitochondrial respiratory chain deficiency are affected by the specific location of the mtDNA deletion, with the loss of protein-encoding genes being the determining factor in the pathogenesis of single, large-scale deletions. Furthermore, the removal of mt-tRNA genes impacts specific complexes only at very high levels of deletion in the absence of affected protein-encoding genes. Second, we are developing an mRNA-seq workflow for the analysis of gene expression profiles in individual muscle fibres. Optimisation of the processing, staining and handling of the skeletal muscle sections for laser capture microdissection of individual fibres will be described. We will also discuss initial findings from the transcriptomic analysis on a Next-Seq 500 of functionally wild-type and deficient fibres from single deletion patients, identified as such by the immunofluorescent assay. Direct correlations between genomic, transcriptomic and proteomic profiles will improve our understanding of the molecular mechanisms underlying single, large-scale mtDNA deletion disease and may help in the continuing search for therapeutic targets.