Figure 1.

Molecular Findings in Muscle Tissue of Probands with DNA2 Mutations

(A) Southern-blot analysis of muscle mtDNA derived from probands 1–4 revealed the presence of multiple bands. “C” indicates control samples that display only a single band corresponding to WT mtDNA, and “+” indicates a positive control (POLG-mutated subject).

(B) Long PCR. This panel shows an agarose-electrophoresis separation of the WT mtDNA long-PCR amplified fragment (approximately 8,520 bp). Variably abundant extrabands due to mtDNA-deleted molecules from muscle DNA are observed in all the probands, but not in age-matched controls (“C”).

(C) Quantitative PCR analysis of mtDNA content in patients’ muscle. Real-time quantitative PCR analysis was performed with primers and probes for human MTCYB (MIM 516020, mtDNA) and APP (MIM 104760, nDNA). Primer sequences and PCR conditions are available upon request. Results were analyzed with a Student’s t test. The mtDNA copy number was determined by quantitative real-time PCR in skeletal-muscle samples of our probands (P1–P4, n = 4), age-matched control individuals (n = 4), and three groups of individuals with autosomal-dominant or -recessive PEO and mutations in POLG (n = 4), PEO1 (n = 4), or DGUOK (n = 4); no significant difference was observed. All determinations were performed in triplicate. Values are normalized to a control sample. Horizontal bars indicate mean values.