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Molecular Diagnostics and Genetics |
1 Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC.
aAddress correspondence to this author at: Institute for Molecular and Human Genetics, Georgetown University Medical Center, M4000, 3800 Reservoir Rd. NW, Washington, DC 20007. Fax 202-444-1770; e-mail wonglj{at}georgetown.edu.
Background: The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation.
Methods: To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences.
Results: A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR–restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations.
Conclusions: The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA.
The following articles in journals at HighWire Press have cited this article:
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  S M Ware, N El-Hassan, S G Kahler, Q Zhang, Y-W, E Miller, B Wong, R L Spicer, W J Craigen, B A Kozel, et al. Infantile cardiomyopathy caused by a mutation in the overlapping region of mitochondrial ATPase 6 and 8 genes J. Med. Genet., May 1, 2009; 46(5): 308 - 314. [Abstract] [Full Text] [PDF]
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 R. S. Jahangir Tafrechi, F. M. van de Rijke, A. Allallou, C. Larsson, W. C.R. Sloos, M. van de Sande, C. Wahlby, G. M.C. Janssen, and A. K. Raap Single-cell A3243G Mitochondrial DNA Mutation Load Assays for Segregation Analysis J. Histochem. Cytochem., November 1, 2007; 55(11): 1159 - 1166. [Abstract] [Full Text] [PDF]
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 A. Genasetti, M. L. Valentino, V. Carelli, D. Vigetti, M. Viola, E. G. Karousou, G. V. Melzi d'Eril, G. De Luca, A. Passi, and F. Pallotti Assessing Heteroplasmic Load in Leber's Hereditary Optic Neuropathy Mutation 3460G->A/MT-ND1 with A Real-Time PCR Quantitative Approach J. Mol. Diagn., September 1, 2007; 9(4): 538 - 545. [Abstract] [Full Text] [PDF]
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 K. S. Lim, R. K. Naviaux, and R. H. Haas Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC Clin. Chem., June 1, 2007; 53(6): 1046 - 1052. [Abstract] [Full Text] [PDF]
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L-J C Wong, D Yim, R-K Bai, H Kwon, M M Vacek, J Zane, C L Hoppel, and D S Kerr A novel mutation in the mitochondrial tRNASer(AGY) gene associated with mitochondrial myopathy, encephalopathy, and complex I deficiency. J. Med. Genet., September 1, 2006; 43(9): e46 - e46. [Abstract] [Full Text] [PDF]
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H. Fan, C. Civalier, J. K. Booker, M. L. Gulley, T. W. Prior, and R. A. Farber Detection of Common Disease-Causing Mutations in Mitochondrial DNA (Mitochondrial Encephalomyopathy, Lactic Acidosis with Stroke-Like Episodes MTTL1 3243 A>G and Myoclonic Epilepsy Associated with Ragged-Red Fibers MTTK 8344A>G) by Real-Time Polymerase Chain Reaction J. Mol. Diagn., May 1, 2006; 8(2): 277 - 281. [Abstract] [Full Text] [PDF]
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 D. Waku-Kouomou, A. Alla, B. Blanquier, D. Jeantet, H. Caidi, A. Rguig, F. Freymuth, and F. T. Wild Genotyping Measles Virus by Real-Time Amplification Refractory Mutation System PCR Represents a Rapid Approach for Measles Outbreak Investigations J. Clin. Microbiol., February 1, 2006; 44(2): 487 - 494. [Abstract] [Full Text] [PDF]
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R.-K. Bai and L.-J. C. Wong Simultaneous Detection and Quantification of Mitochondrial DNA Deletion(s), Depletion, and Over-Replication in Patients with Mitochondrial Disease J. Mol. Diagn., November 1, 2005; 7(5): 613 - 622. [Abstract] [Full Text] [PDF]
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