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

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.

Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase

“…Taken together, these values suggest wild type Pol g and Twinkle also remain coupled at the mtDNA replication fork. Missense disease variants of TWNK and POLG disrupt the function and stability of the enzymes, which interferes with coordinated action of the enzymes and likely promotes the formation of mtDNA deletions during replication and repair of mtDNA in vivo (35,64,65). For example, persistent pausing of the helicase, such as at DNA secondary structures, would stall the mtDNA replication fork and promote disassociation of Twinkle from the unwinding site.…”

Roles of the mitochondrial replisome in mitochondrial DNA deletion formation

“…In addition, a recent comparative study demonstrated that DmtDNA molecules generated in patients with POLG1 mutations undergo clonal expansion similarly to the single large deletions sporadically generated in patients. In contrast, DmtDNA generated in patients with a mutation in OPA1, which encodes a protein involved in mitochondrial fusion, did not expand clonally (Trifunov et al, 2018). In both POLG1-related and sporadic deletions (but not in OPA1-related deletions), the authors observed an increase in the overall mtDNA copy number, which may link the phenomenon of clonal expansion to a compensatory upregulation of replication as a response to inefficient OXPHOS.…”

“…affect its development, whereas the homozygous POLG1 knockout is lethal at the early embryonic stage (Hance et al, 2005). This lack of an`intermediate' phenotype implies significant tolerance to the limited level/activity of Pol g. Also, the phenomenon of clonal expansion seems to be consistent with the concept of threshold activity, as the lack of replication capacity would also exclude any genome expansion, and yet it has recently been documented for deletions resulting from defective Pol g (Trifunov et al, 2018). Therefore, each individual who develops beyond the embryonic stage must possess the capacity to replicate mtDNA.…”

High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases

“…Single-cell ddPCR (sc-ddPCR), which takes advantage of an intact cell instead of lysed cells or eluted nucleotides and is executed in a single droplet enclosing a single cell, has been developed by using a droplet-to-digital device. To examine a deletion in mtDNA, this technology was applied to a single skeletal muscle cell collected by laser-capture microdissection (LCM) from muscle biopsies 26 . In ex vivo gene therapy using hematopoietic stem cells, sc-ddPCR enabled the tracing of gene-modified donor cells 27 .…”