INTRODUCTION:
UCLA researchers in the Department of Geriatric Medicine have developed a novel method for the concurrent quantification of mitochondrial DNA content and mutation frequency in both single cells and tissue homogenates.
BACKGROUND:
The human mitochondrial DNA (mtDNA) genome consists of about 37 genes, all of which are essential for mitochondrial function and oxidative phosphorylation. The development of mtDNA deletions culminates in the physiological changes of aging skeletal muscle. Specifically, an abundance of mtDNA deletions in individual cells may lead to electron transport chain-deficient fibers, ultimately driving muscle fiber loss. Even in the absence of mitochondrial disease, variations in mtDNA sequence and copy number accumulate with age. Therefore, accurate quantification of mtDNA deletions and copy number would predict heteroplasmy in single cells, while in measurement of heteroplasmy in tissue homogenates may allow for assessment of electron transport chain-deficient cells, future cell loss, and prediction of human longevity. Determinations of mtDNA deletion abundance from tissue samples are typically inferred using histological methods to count electron transport chain-deficient fibers. No current technology exists for the direct measurement and quantification of mtDNA content and deletion frequencies.
INNOVATION:
UCLA researchers have adapted a method for the absolute quantification of mtDNA deletion frequency in total cell isolates from any tissue. The assay utilizes digital PCR and direct measurement for quantification of mtDNA deletions in both individual muscle fibers and muscle homogenates. Digital PCR allows for increased quantitation sensitivity at reduced mutation frequencies, over quantitative PCR, to 2 parts per million. Mitochondrial DNA deletion frequency strongly correlates with electron transport chain deficiency determined by histochemical analysis. This method may be used to predict remaining human lifespan.
POTENTIAL APPLICATIONS:
• Quantification of temporal accumulations of mitochondrial DNA deletions to 2 parts per million
• Prediction of human longevity and remaining lifespan
ADVANTAGES:
• Digital PCR have increased sensitivity for quantification at lower mutation frequencies, and reduced error, compared to quantitative PCR
• Restriction digestion of wild-type mtDNA enriches the sample for mutant targets
• For use in single cells and tissue homogenates
• Quantification of both mtDNA content and mutation frequencies using two distinct digital PCR approaches
• Validated assay results demonstrate an underlying mechanism for aging-- electron-transport chain-deficient fiber abundance
DEVELOPMENT-TO-DATE:
This assay has been successfully demonstrated to reflect an underlying mechanistic phenomenon of aging in rat studies. Importantly, it is predictive of muscle aging and rodent lifespan.