Study of mitochondrial DNA depletion in muscle by single-fiber polymerase chain reaction.

We studied muscle biopsies from 3 children with a mitochondrial myopathy characterized histochemically by the presence of ragged-red fibers (RRF) and various numbers of cytochrome c oxidase (COX)-negative fibers. We quantitated the absolute amounts of total mitochondrial DNA (mtDNA) in isolated normal COX-positive muscle fibers and in COX-negative RRF. There was severe mtDNA depletion in all fibers from the two most severe cases. In the third case mtDNA depletion could not be established with conventional diagnostic tools, but it was documented in single COX-negative fibers; COX-positive fibers showed the same amounts of mtDNA as fibers from aged-matched controls. Our observations indicate that mtDNA single-fiber PCR quantitation is a highly sensitive and specific method for diagnosing cases with focal mtDNA depletion. This method also allows one to correlate amounts of mtDNA with histochemical phenotypes in individual fibers from patients and age-matched controls, thereby providing important information about the functional role of residual mtDNA.