Two pathogenic point mutations exist in the authentic mitochondrial genome, not in the nuclear pseudogene

Abstract

Technical advancements in molecular genetics have shown various mitochondrial DNA (mtDNA) abnormalities in patients with mitochondrial myopathies. Recently, it has been revealed that, in these patients, the nuclear DNA carries sequences similar to those of the mtDNA (nuclear pseudogene) and it has several point mutations previously reported to be pathogenic. We verified the existence of the T3250C and T3291C mutations, which we have found in patients with mitochondrial myopathy, in the authentic mitochondrial genome. A long polymerase chain reaction provides a powerful tool for avoiding nuclear pseudogene amplification and for ruling out ambiguity in the detection of the mutation for diagnosis.

Mutations in mitochondrially encoded complex I enzyme as the second common cause in a cohort of Chinese patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes

“…18 For mutation analysis, first, the DNA samples were analyzed for the presence of large deletions and five common point mutations (A3243G, T3271C, A8344G, T8993G and T8993C) by southern blot/long-range PCR and PCR-restriction fragment-length polymorphism, respectively, according to published procedures. 15,19 Second, for those patients without common mutations, the entire mitochondrial genome extracted from muscle was sequenced by direct DNA sequencing of the PCR product using a BigDye terminator cycle sequencing kit (Applied Biosystem, Foster City, CA, USA) and an ABI 3730XL (Applied Biosystem) automated sequencer. We applied the long PCR-based sequencing method to avoid nuclear pseudogene amplification.…”

Leigh syndrome caused by mitochondrial DNA G13513A mutation: frequency and clinical features in Japan

“…A mixture-template standard curve was used to revise the percentage of mutant mtDNA. We performed total mtDNA sequencing for selected cases, such as those with diffuse COX deficiency in their muscle biopsy, by the method previously described (Akanuma et al 2000).…”

Co‐existing point mutations of mitochondrial DNA in a patient with a heart abnormality and Pearson syndrome‐like symptoms

“…No deleted band was amplified by long PCR (data not shown). Next, we sequenced the entire mtDNA of the patient as previously described [Akanuma et al, 2000].…”