1. ¹Department of Pathology, Sahlgrenska University Hospital, Göteborg, Sweden
2. ²Department of Neuroscience, Uppsala University Hospital, Uppsala, Sweden
3. ³Department of Neurology, Sahlgrenska University Hospital, Göteborg, Sweden
4. ⁴Department of Clinical Chemistry, Sahlgrenska University Hospital, Göteborg, Sweden

Correspondence: Dr A-R Moslemi, Department of Pathology, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. Tel: +46 313 423335; Fax: +46 31 417283; E-mail: ali-reza.moslemi@pathology.gu.se

Received 27 May 2004; Revised 12 October 2004; Accepted 13 October 2004; Published online 9 February 2005.

Abstract

Mitochondrial myopathy in progressive external ophthalmoplegia (PEO) has been associated with POLG1 mutations. POLG1 encodes the catalytic subunit of polymerase and is the only polymerase known to be involved in mtDNA replication. It has two functionally different domains, one polymerase domain and one exonuclease domain with proofreading activity. In this study we have investigated whether mtDNA point mutations are involved, directly or indirectly, in the pathogenesis of PEO. Muscle biopsy specimens from patients with POLG1 mutations, affecting either the exonuclease or the polymerase domain, were investigated. Single cytochrome c oxidase (COX)-deficient muscle fibers were dissected and screened for clonally expanded mtDNA point mutations using a sensitive denaturing gradient gel electrophoresis analysis, in which three different regions of mtDNA, including five different tRNA genes, were investigated. To screen for randomly distributed mtDNA point mutations in muscle, two regions of mtDNA including deletion breakpoints were investigated by high-fidelity PCR, followed by cloning and sequencing. Long-range PCR revealed multiple mtDNA deletions in all the patients but not the controls. No point mutations were identified in single COX-deficient muscle fibers. Cloning and sequencing of muscle homogenate identified randomly distributed point mutations at very low frequency in patients and controls (<1:50 000). We conclude that mtDNA point mutations do not appear to be directly or indirectly involved in the pathogenesis of mitochondrial disease in patients with different POLG1 mutations.