Published online before print July 25, 2007

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Journal of Molecular Diagnostics 2007, Vol. 9, No. 4
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2007.060183

Assessing Heteroplasmic Load in Leber’s Hereditary Optic Neuropathy Mutation 3460GA/MT-ND1 with A Real-Time PCR Quantitative Approach

Anna Genasetti^*, Maria L. Valentino, Valerio Carelli, Davide Vigetti^*, Manuela Viola^*, Evgenia G. Karousou^*, Gian Vico Melzi d’Eril, Giancarlo De Luca^*, Alberto Passi^* and Francesco Pallotti^*

From the Dipartimento di Scienze Biomediche Sperimentali e Cliniche, * Università degli Studi dell’Insubria, Varese; the Dipartimento di Scienze Neurologiche, Università degli Studi di Bologna, Bologna; and the Dipartimento di Medicina, Chirurgia e Odontoiatria, Università degli Studi di Milano, Milano, Italy

To quantify the amount of the 3460GA/ND1 point mutation responsible for Leber’s hereditary optic neuropathy, we developed a quantitative real-time polymerase chain reaction method based on the SYBR Green assay and a new approach using the TaqMan assay. Both methods were based on the amplification refractory mutation system, comparing the heteroplasmic load quantified by restriction fragment length polymorphism in 15 Leber’s hereditary optic neuropathy family members, with the results obtained using quantitative real-time polymerase chain reaction methods. The comparative evaluation of mitochondrial DNA (mtDNA) heteroplasmy from blood samples showed significant correlation between restriction fragment length polymorphism analysis, real-time SYBR Green assay, and TaqMan assay. We validated the last method by measuring experimental samples composed by a known proportion of cloned plasmids containing either the wild-type or mutant sequence, giving a correlation coefficient of 0.999 (P < 0.0001). The real-time amplification refractory mutation system polymerase chain reaction by TaqMan assay provides a rapid, reliable, sensitive, reproducible, and one-step quantitative method to detect heteroplasmic mutant mtDNA. This method allows the quantitation of a broad range of mutational load (up to 100%, down to 0.01%) on the basis of in vitro calibration, thus rendering the TaqMan assay suitable for the diagnostic analysis of heteroplasmic load in mtDNA-related disorders.