Extremely high levels of mutant mtDNAs co-localize with cytocohrome c oxidase-negative ragged-red fibers in patients harboring a point mutation at nt 3243

Abstract

A single mtDNA point mutation at nt 3243 has been associated with two different clinical phenotypes: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes ('MELAS3243') and progressive external ophthalmoplegia ('PEO3243'). It has been shown that there is a much higher proportion of ragged-red fibers (RRF) with cytochrome c oxidase (COX) deficiency in PEO3243 than in MELAS3243. Using PCR/RFLP analysis of isolated individual skeletal muscle fibers from patients with both syndromes, we found a direct correlation between the localized concentration of the nt 3243 mutation and impairment of COX function at the single muscle fiber level: we found relatively low levels of mutant mtDNAs (56 +/- 21%) in 'normal' fibers; high levels (90 +/- 6%) in COX-positive RRF; and an almost complete segregation of mutant mtDNAs (95 +/- 3%) in COX-negative RRF. Thus, the differential distribution of fibers with extremely high concentrations of mutant mtDNAs characterizes, and probably distinguishes, the skeletal muscle of PEO and MELAS patients harboring the same nt-3243 mutation.

Heteroplasmic ratio of the A3243G mitochondrial DNA mutation in single pancreatic beta cells

“…The amount of mutation observed was not due to the amplification of pseudogenes. The technique of singlecell lysis followed by PCR and restriction digest in a different tissue (skeletal muscle) gives results identical to those found by other authors [6]. The second possible explanation is that the patient studied was in some way atypical of patients with the A3243G mutation.…”

Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

“…For instance, in class II deletions, where only complex I and tRNAs genes are lost, the threshold for complex IV is substantially higher. Interestingly, this threshold of approximately 90% is similar to those recorded in other mtDNA defects caused by pathogenic variants in mt‐tRNA genes,37, 38, 39, 40 suggesting that complex IV deficiency in this class of deletions occurs solely due to a translational defect following removal of tRNA gene sequences. These findings are in line with a report by Hammans and colleagues, who also observed a relationship between the location of mtDNA deletion and the biochemical defect, and therefore the determinant role of mtDNA‐encoded COX genes when removed by the deletion 26.…”

23. MELAS: Clinical features, muscle biopsy and molecular genetics

“…The COX activity can be diffusely decreased or focally absent in some muscle fibers, but in these cases the COX probably is not the primary defective site of disease 9,15,22 . Thus, most of the MELAS patients have normal or increased COX activity, as in this study, possibly because the mtDNA mutation is insufficient to impair COX activity 22,25 . In our patient with COX deficiency, the number of COXdeficient fibers was greater than RRF frequency, possibly due to the greater number of muscle fiber with mitochondrial dysfunction.…”

“…Muscle biopsies from most MELAS cases, especially those associated with the most common mutation, A3243G in the tRNA Leu(UUR) gene, have RRF with normal COX activity and SSV 24 . Quantitative analysis showed 80 to 90% mutant mtDNA in muscle [25][26][27] . The total mtDNA (both normal and mutant) are extremely increased in SSV and RRF [25][26][27] .…”

“…Quantitative analysis showed 80 to 90% mutant mtDNA in muscle [25][26][27] . The total mtDNA (both normal and mutant) are extremely increased in SSV and RRF [25][26][27] . The proportion of mutant mtDNA was significantly higher in RRF than in non-RRF, as well as in SSV than in non-SSV cases 26,27 .…”