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Nucleic Acids Research, 2001, Vol. 29, No. 3 e13
© 2001 Oxford University Press

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

Károly Szuhai, Jody M. van den Ouweland, Roeland W. Dirks, Marc Lemaître1, Jean-Christophe Truffert1, George M. Janssen, Hans J. Tanke, Elisabeth Holme2, J. Antonie Maassen and Anton K. Raap*

Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands, 1Eurogentec S.A., Department of Production, Parc Scientifique, B-4102-Seraing, Belgium and 2Department of Clinical Chemistry, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden

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 tRNALys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

* To whom correspondence should be addressed at: Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333AL Leiden, The Netherlands. Tel: +31 71 5276187; Fax: +31 71 5276180; Email: a.k.raap{at}lumc.nl Present address:Jody M. van den Ouweland, Isala Clinics, Clinical Chemistry Laboratory, PO Box 10500, 8000 GM Zwolle, The Netherlands


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