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Quantification of Mitochondrial DNA Deletion, Depletion, and Overreplication: Application to Diagnosis

¹ Unité du Métabolisme Protéino Energétique, UMR INRA 1019, 63009 Clermont-Ferrand, France.

² Laboratoire de Biochimie, Hôpital Debrousse, Hospices Civils de Lyon, 69322 Lyon, France.

³ Laboratoire de Biolphysique Moléculaire et Cellulaire, UMR CNRS 5090, CEA Grenoble, 38054 Grenoble, France.

^aAddress correspondence to this author at: UMPE, UMR INRA 1019, 58, rue Montalembert, 63009 Clermont-Ferrand, France. Fax 33-4-7360-8255; e-mail stepien{at}clermont.inra.fr.

Background: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations.

Methods: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication.

Results: Both methods quantified 5–76% of deleted mtDNA in muscle, 59–97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r²) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis.

Conclusions: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis.

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