Figure 4. Respiratory deficient CP epithelial cells in multiple sclerosis harbored clonally expanded Δ-mtDNA.

A: Multiple Δ-mtDNA were detected when DNA was extracted from multiple respiratory deficient cells (a, 20 respiratory deficient cells pooled per lane) from MS cases (lanes 1–4). In contrast, Δ-mtDNA were infrequent in pooled respiratory deficient cells (n=20 per lane) from PD (lanes 5–6), AD (lanes 7–8) and controls (lanes 9–10). Negative control (meninges from control case) showed full-length amplified product (+).

B: When long-range PCR was performed using single cells (DNA extracted from one respiratory deficient cell per lane) more than one Δ-mtDNA was rarely detected in controls (lanes 1–5) or MS cases (lanes 6–10).

C–D: Sequencing of Δ-mtDNA extracted from gel following long-range PCR of single respiratory deficient cells confirmed the Δ-mtDNA by identifying the breakpoints (c–d). In a proportion of Δ-mtDNA, the sequence that flanked the breakpoints showed perfect or imperfect repeats (see Table 2). The repeat sequence of an imperfect repeat is underlined on the 3’ end from the breakpoint, with the imperfect base shown above the sequence in bold (c). Δ-mtDNA without repeat sequences were also detected within respiratory deficient cells in MS (d).

E: Clonality of Δ-mtDNA in respiratory deficient CP cells was confirmed using single molecule PCR. Long range PCR was performed multiple times from diluted DNA of six individual cells. Three representative positive reactions are presented for each of six respiratory deficient cells from MS. Note that each cell contained only one species of mtDNA molecule: either full length wild type (cells 1 and 6), or mtDNA deletions (cells 2–5). Single species of Δ-mtDNA by single molecule PCR in cells 2–5 indicated a hallmark of clonal expansion of Δ-mtDNA.