title: Low frequency of mtDNA point mutations in patients with PEO associated with POLG1 mutations.
abstract: Mitochondrial myopathy in progressive external ophthalmoplegia (PEO) has been associated with POLG1 mutations. POLG1 encodes the catalytic alpha subunit of polymerase gamma 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.
authors.affiliation: Department of Pathology, Sahlgrenska University Hospital, Göteborg, Sweden.
authors.forename: Gittan
authors.initials: G
authors.lastname: Kollberg
authors.forename: Monica
authors.initials: M
authors.lastname: Jansson
authors.forename: Asa
authors.initials: A
authors.lastname: Pérez-Bercoff
authors.forename: Atle
authors.initials: A
authors.lastname: Melberg
authors.forename: Christopher
authors.initials: C
authors.lastname: Lindberg
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Holme
authors.forename: Ali-Reza
authors.initials: AR
authors.lastname: Moslemi
authors.forename: Anders
authors.initials: A
authors.lastname: Oldfors
mesh_terms: D004252:DNA Mutational Analysis; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005260:Female; D005787:Gene Frequency; D006801:Humans; D008297:Male; D008931:Mitochondria, Muscle; D018485:Muscle Fibers, Skeletal; D017246:Ophthalmoplegia, Chronic Progressive External / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: D004272:DNA, Mitochondrial; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human