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

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.

analysis revealed the presence of multiple mtDNA deletions by long-range PCR. The patient belonged to a large family with dominant transmission of the same phenotype. Screening of candidate genes involved with multiple mtDNA deletions revealed a heterozygous missense mutation in OPA1, affecting nucleotide c.1462G>A. This mutation affects the conserved amino acid p.G488R in the GTPase domain of OPA1. This family has been previously reported (Carelli, 2015).

Patient 2:
This is a 49-year-old man who had poor vision since he was 4-year-old that progressed to complete blindness later in life. Since 9 years of age he also suffered a progressive hearing loss needing acoustic prosthesis. At 30 years of age he developed gait difficulties with frequent falls due to peripheral neuropathy. At the time of muscle biopsy he was 42, and biopsy was positive for COX negative fibres. Electron microscopy of skeletal muscle showed mitochondria with morphologically abnormal cristae and accumulation of lipid droplets. Long-range PCR analysis of mtDNA revealed the presence of mtDNA multiple deletions. Screening of candidate genes was positive for the mutation c.1316 G>T affecting a conserved amino acid p.G439V in the GTPase domain of OPA1. The patient's daughter developed the same phenotype and this case has been previously reported (Amati-Bonneau, 2008).

Patient 3:
This is a 63-year-old man with a multisystemic syndrome known with the acronym of SANDO.
He suffered with ptosis and ophthalmoplegia by the age of 30; ten years later he started to develop a progressive skeletal muscle weakness and a sensitive polyneuropathy causing an ataxic gait. By the age of 50 he had dysarthria. His muscle biopsy showed a mitochondrial myopathy with Ragged Red Fibres and COX negative fibres. He carried two heterozygous mutations in the POLG1 gene (a missense mutation c.934T>C causing the amino acid change p.W312R and an insertion, c.3629insA, causing a stop codon p.Y1210X). mtDNA multiple deletions were evident with long range PCR of muscle DNA.

Patient 4:
This 67-year-old woman suffered from ptosis and ophthalmoplegia from the age of 30 years.
By the age of 56 years she clinically presented with ptosis, ophthalmoplegia, skeletal muscle weakness, peripheral polyneuropathy. A few years later she also developed dysphagia and a Parkinsonian syndrome with hypomimia, hypophonia, camptocormia and bradykinesia. Her muscle biopsy showed a mitochondrial myopathy with Ragged Red Fibres and COX negative fibres. She carried a homozygous missense mutation c.1943C>G in POLG1 gene leading to the amino acid change p.P648R. Multiple mtDNA deletions were evident with long range PCR.

Patient 5:
This 50-year-old man started to develop a progressive bilateral ptosis and ophthalmoplegia by the age of 17 years. By the age of 45 years he suffered from diabetes mellitus, cardiomyopathy and skeletal muscle weakness. His muscle biopsy revealed numerous COX negative fibres. He carried a single mtDNA deletion of 4977 base pairs (nt. 8469-nt.13447).

Patient 6:
This 23-year-old girl suffered from monolateral ptosis by the age of 15 years without ophthalmoparesis. Her neurological examination was negative except for monolateral ptosis and electromyography didn't show signs of myopathy and/or polyneuropathy. Audiometry was normal. She had lactic acidosis after aerobic effort and muscle biopsy showed a mitochondrial myopathy with COX negative fibres. She carried a single mtDNA deletion of 4977 base pairs (nt. 8469-nt.13447). A-biopsy belonging to patient 1; B-biopsy belonging to patient 2; C-biopsy belonging to patient 3; D-biopsy belonging to patient 4; E-biopsy belonging to patient 5; F-biopsy belonging to patient 6; G-biopsy belonging to patient 7; H-biopsy belonging to control 1; Ibiopsy belonging to control 2; J-biopsy belonging to control 3.