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New Multiplex qPCR Assay Measures mtDNA Copy Number, Aging-related Deletions

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Researchers at the University of North Texas Health Science Center recently developed an assay capable of quantifying nuclear DNA, mitochondrial DNA copy number, and the ratio of age-related deletions in the mitochondrial genome, in a single qPCR. This triplex assay could help standardize mitochondrial DNA analysis, and has potential applications in age-related disease research and forensic identification of human remains.

The work, published in Scientific Reports, describes a probe-based single-tube multiplex assay which aims to eliminate the well-to-well variability of assaying nuclear and mitochondrial targets separately. The assay measures a rarely mutated mtDNA region and compares that to nuclear DNA. It also measures proportions of mitochondrial genomes with common large deletions in the major arc, where age-related deletions often accumulate. These deletions may be a proxy for biological aging — recent studies have suggested that some people age more quickly, and thus may be more susceptible to aging-related diseases, the researchers said.

Developing the assay was part of a larger project spearheaded by postdoc Nicole Phillips in the laboratory of Rhonda Roby, an associate professor in the department of molecular and medical genetics who is also affiliated with the center for human identification at UNTHSC. In an interview with PCR Insider, Roby said the assay was perfected by Phillips, who was interested in tracking changes that may accumulate in the mitochondrial genome with age-related diseases, specifically Alzheimer's disease. Sporadic Alzheimer's ─ which is not as obviously heritable and not as associated with specific mutations as is the familial form of the disease ─ may be related to an underlying mitochondrial dysfunction. This mitochondrial hypothesis is gaining traction. As reported in PCR Insider, a Spanish group recently showed diminished levels of cell-free mtDNA in cerebrospinal fluid of patients with, or at risk of developing, Alzheimer's disease,.

Phillips suggested that tracking integrity of mtDNA over time could indicate susceptibilities early, and help increase the precision of peripheral blood-based panels of differential protein expression, or even common neurocognitive tests used to diagnose Alzheimer's.

However, the Roby lab is also intrigued by recent studies suggesting an inverse relationship between Alzheimer's or dementia and prostate cancer. Mitochondrial dysfunction is a commonality between those conditions, and, Roby said, "It's important for us to see if there is anything we can learn by looking at these two very different age-related diseases."

Roby also has been instrumental in developing mtDNA assays for forensic applications. She worked for three and a half years to help indentify human remains at the World Trade Center site in New York City, she said. In fact, she was one of the first forensic geneticists on the scene. According to J. Craig Venter's autobiography, as the disaster was unfolding, he volunteered his company's resources to identify victims' remains. He wrote that his private flight to NYC was the first non-military flight allowed after the attack, and they risked being shot down if they did not contact NORAD every 30 minutes. Roby, then head of the forensics division at Applied Biosciences, accompanied Venter on that flight. Because of the state of the remains, mitochondrial DNA was used for identification. Roby's laboratory has since worked to optimize an mtDNA quantification assay developed by the FBI.

The triplex qPCR assay could assist forensic analyses, particularly ones on skeletal remains, Roby said. "There's so many copies of mitochondrial compared to the nuclear DNA, we can oftentimes detect mitochondrial DNA out of an old bone sample where we can't get the nuclear DNA. Since mitochondrial DNA is maternally inherited, we can make identifications or associations with that," she said. DNA from evidentiary material must be quantified prior to DNA testing, Roby said. This is a standard in the field of forensics, but "the standard is written for nuclear DNA because there is no [standardized] mitochondrial DNA qPCR assay," she said.

Roby said she was aware of only seven publications that have ever tried to analyze mitochondrial and nuclear DNA simultaneously. "Some of the naysayers to it will say that you can't accurately measure the nuclear and the mito together because of the disparity in copy number of mito versus the nuclear," she said. However, Roby and her colleagues are extremely confident in the sensitivity and reproducibility of their assay.

In fact, Roby and Phillips believe the field of mtDNA analysis in general could greatly benefit from their enhanced technique. "We also think that it could be good for [other] metabolic diseases such as diabetes and cardiovascular diseases. … We definitely want to have it available in some kind of kit format," Roby said. "If a company could develop it into a kit that would be easy to use … I think that would be fantastic." In the past, Roby has collaborated with TriLink Biotechnologies to develop primers for mitochondrial DNA PCR amplification and sequencing for forensic identification.

The researchers also pointed out that the mitochondrial DNA field could use guidelines similar to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments, too. The MIQE guidelines, a set of 15 criteria aimed at improving qPCR and making results more reliable and reproducible between laboratories, debuted in 2009. But these guidelines are for nuclear DNA. For mtDNA, Phillips said, "Everyone has their home-brewed qPCR approach to assessing copy number and deletion ratio, and I learned through the design of this [assay] that you could get vastly different results just with slight differences in protocol." She added, "If this assay could help move the field in the direction of standardization, then I think it would definitely be beneficial."

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