JMD TIDES - Oligonucleotide and Peptide - May 18-21, 2008, Las Vegas, NV
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JMD 2005, Vol. 7, No. 5
Copyright © 2005 American Society for Investigative Pathology & Association for Molecular Pathology

Simultaneous Detection and Quantification of Mitochondrial DNA Deletion(s), Depletion, and Over-Replication in Patients with Mitochondrial Disease

Ren-Kui Bai and Lee-Jun C. Wong

From the Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, District of Columbia; and the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas

Heterogeneous clinical expression of mitochondrial DNA (mtDNA) disorders depends on both qualitative and quantitative changes in mtDNA. We developed a sensitive and effective method that simultaneously detects mtDNA deletion(s) and quantifies total mtDNA content. The percentage of deletions and mtDNA content of 19 patients with single or multiple deletions were analyzed by real-time quantitative polymerase chain reaction (real-time qPCR) using TaqMan probes specific for mtDNA (tRNA leuUUR, ND4, ATPase8, and D-loop regions) and nuclear DNA (AIB1, ß-2-microglobulin, and ß-actin). The proportion of deletion mutants determined by real-time qPCR was consistent with that determined by Southern analysis. Most patients with mtDNA deletions also demonstrated compensatory mtDNA over-replication. Multiple mtDNA deletions that were not detectable by Southern analysis due to low percentage of each deletion molecule were readily detected and quantified by real-time qPCR. Furthermore, 12 patients with clinical features and abnormal biochemical/histopathological results consistent with mitochondrial respiratory chain disorders without identified mtDNA mutations had either substantially depleted or significantly over-replicated mtDNA content, supporting the diagnosis of mitochondrial disease. Our results demonstrate that both qualitative and quantitative analyses are important in molecular diagnosis of mitochondrial diseases. The presence of deletion(s) and mtDNA depletion or compensatory over-replication can be determined simultaneously by real-time qPCR.




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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
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Copyright © 2005 by the American Society for Investigative Pathology and the Association for Molecular Pathology.