Clonal expansion of mtDNA deletions: different disease models assessed by digital droplet PCR in single muscle cells

Selena Trifunov,Patrick Yu-Wai-Man,Michela Rugolo,Stephanie Kleinle,Rita Horvath,Isabel Diebold,Maria Lucia Valentino,Florence Burté,Valerio Carelli,Angela Pyle,Jennifer Duff,Rocco Liguori

doi:10.1038/s41598-018-30143-z

Abstract

Deletions in mitochondrial DNA (mtDNA) are an important cause of human disease and their accumulation has been implicated in the ageing process. As mtDNA is a high copy number genome, the coexistence of deleted and wild-type mtDNA molecules within a single cell defines heteroplasmy. When deleted mtDNA molecules, driven by intracellular clonal expansion, reach a sufficiently high level, a biochemical defect emerges, contributing to the appearance and progression of clinical pathology. Consequently, it is relevant to determine the heteroplasmy levels within individual cells to understand the mechanism of clonal expansion. Heteroplasmy is reflected in a mosaic distribution of cytochrome c oxidase (COX)-deficient muscle fibers. We applied droplet digital PCR (ddPCR) to single muscle fibers collected by laser-capture microdissection (LCM) from muscle biopsies of patients with different paradigms of mitochondrial disease, characterized by the accumulation of single or multiple mtDNA deletions. By combining these two sensitive approaches, ddPCR and LCM, we document different models of clonal expansion in patients with single and multiple mtDNA deletions, implicating different mechanisms and time points for the development of COX deficiency in these molecularly distinct mitochondrial cytopathies.

Highlights

Mitochondria are cytoplasmic organelles crucially involved in respiration and energy production of eukaryotic cells, unique for containing their own maternally inherited genome[1]
As we found that droplet digital PCR (ddPCR) is much more sensitive than Southern blot in detecting and quantifying mitochondrial DNA (mtDNA) deletions, we tested whether it detects single mtDNA deletions in blood cells from patients with the Pearson and Kearns Sayre syndromes or chronic progressive external ophthalmoplegia (CPEO) (n = 16)
The patients affected with dominant optic atrophy (DOA) “plus” and carrying dominant OPA1 mutations leading to defective mitochondrial fusion had extremely low amounts of deleted mtDNA molecules in the c oxidase (COX) positive fibers, comparable to controls

Summary

Introduction

Mitochondria are cytoplasmic organelles crucially involved in respiration and energy production of eukaryotic cells, unique for containing their own maternally inherited genome[1]. MtDNA is a high copy number genome and mitochondrial mass varies amongst different cell and tissue types depending on the metabolic demands[2]. The mtDNA deletions remove various portions of the genome, resulting in shorter molecules, and they may occur either as single or multiple deletions[5,6]. Single mtDNA deletions are considered sporadic, somatic mutational events occurring early during embryonic development in the majority of cases. A few cases of maternally inherited single mtDNA deletions have been reported and considered rare exceptions[10,11,12,13,14]. Multiple mtDNA deletions may accumulate somatically in post-mitotic tissues secondary to mutations in nuclear genes inherited as Mendelian traits, directly involved in mtDNA replication, or in the nucleotide pool balance or, more recently, in mitochondrial dynamics[15]. MtDNA deletions generated by different genetic defects may propagate within muscle fibers with different modes and timing

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