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3rd International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV23-26 October 2001, Athens, Greece |
QUANTIFICATION OF MITOCHONDRIAL DNA RELATIVE TO NUCLEAR DNA IN PERIPHERAL BLOOD MONONUCLEAR CELLS: VALIDATION OF AN ACCURATE DUPLEX ASSAY FOR USE IN PATIENTS UNDERGOING ANTIRETROVIRAL THERAPY
Antiviral Therapy 2001; 6(Suppl. 4):14 (abstract no. 19)
B van Gemen1, A de Ronde1, M Casula2, E Timmermans1, M van Dooren1, M Westrop1 I Dobbelaar1, M Bakker2, GJ Weverling2, J Lange2, J Goudsmit2 and P Reiss2
1 PrimaGen, Amsterdam, The Netherlands; and 2 Department of Human Retrovirology, AMC, Amsterdam, The Netherlands
BACKGROUND: Several of the adverse effects of antiretroviral therapy may result from inhibition of mitochondrial (mt)DNA polymerase γ by nucleoside analogue reverse transcriptase inhibitors (NRTIs). Evidence has been presented that mtDNA content in tissue like muscle, and fluids like sperm may be an indicator of integrity of mitochondrial function. Less data are available on mtDNA content of more accessible body fluids like blood as an indicator of mitochondrial dysfunction.
OBJECTIVE: To validate a new accurate quantitative duplex assay designed specifically to measure mtDNA content, without the need for first having to perform cell counts.
METHODS: We developed a duplex real time NASBAbased assay using conditions favouring accurate DNA quantification of two targets. The assay reliably and robustly detects and quantifies simultaneously mtDNA and nuclear DNA, resulting in a ratio of mtDNA over nuclear DNA. Reconstruction experiments indicate that the dynamic range of the assay is 10-500 copies of mtDNA per cell using a total input DNA amount equivalent to 105 cells or leI's. To validate the ability of the assay to sensitively monitor fluctuations in mtDNA content in peripheral blood mononuclear cells (PBMe), we tested PBMCs of 48 treatment-naïve individuals from the Delta trial randomly allocated to zidovudine alone, zidovudine plus didanosine, or zidovudine plus zalcitabine, prior to and at start of therapy, as well as after 48 weeks. As input the DNA equivalent of 105 cells was used.
RESULTS: The duplex NASBA was able to discriminate and quantify small (less than a factor 2) changes in the mtDNA content of PBMC. Before, and at start of therapy the three patient groups (zidovudine, zidovudine plus didanosine, zidovudine plus zalcitabine) did not differ significantly in the mtDNA content of their PBMC. However, after 48 weeks of therapy the mtDNA content per cell in the PBMC was significantly reduced in the patients on zidovudine plus didanosine or zidovdine plus zalcitabine, but not in those on zidovudine monotherapy.
CONCLUSIONS: (1) The duplex assay we developed is able to quantify small changes in the amounts of mtDNA using nuclear DNA content as reference. (2) PBMC DNA can be used as input material to measure changes in mtDNA content in patients receiving treatment containing NRTIs. (3) Therapies containing standard dosages of NRTIs may result in reductions of mtDNA content detectable in PBMC using this duplex NASBA assay. Taken together these preliminary results indicate that this assay is suitable for monitoring mtDNA reductions resulting from different antiretroviral therapy regimens.
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