title: Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR.
abstract: The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype-phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA(Lys) mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: K
authors.initials: K
authors.lastname: Szuhai
authors.forename: J
authors.initials: J
authors.lastname: Ouweland
authors.forename: R
authors.initials: R
authors.lastname: Dirks
authors.forename: M
authors.initials: M
authors.lastname: Lemaître
authors.forename: J
authors.initials: J
authors.lastname: Truffert
authors.forename: G
authors.initials: G
authors.lastname: Janssen
authors.forename: H
authors.initials: H
authors.lastname: Tanke
authors.forename: E
authors.initials: E
authors.lastname: Holme
authors.forename: J
authors.initials: J
authors.lastname: Maassen
authors.forename: A
authors.initials: A
authors.lastname: Raap
mesh_terms: D002460:Cell Line; D004272:DNA, Mitochondrial / Q000235:genetics*; D005453:Fluorescence; D018628:Gene Dosage; D006801:Humans; D017243:MERRF Syndrome / Q000235:genetics* / Q000473:pathology; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004272:DNA, Mitochondrial