By Ben Butkus
Clinical researchers from the National Heart Lung and Blood Institute, in collaboration with Qiagen, have developed a simplified, automated real-time PCR assay to measure telomere length in less than half the time of other PCR-based methods while using 20-fold less genetic material, according to research being presented at a conference next week.
The researchers are now deploying the assay as part of a clinical study at the National Institutes of Health Clinical Center designed to monitor changes in telomere length in response to certain therapies in patients with diseases in which telomere length is implicated, such as aplastic anemia and pulmonary fibrosis, one of the assay's developers said this week.
In addition, the group is hoping that the automated, optimized assay will eventually meet the requirements necessary to receive a CLIA waiver so it can eventually be used to help diagnose diseases in which telomere length is implicated; such as leukemia or cirrhosis of the liver.
The assay seeks to build on the growing body of evidence that telomeres, the tandem hexameric nucleotide repeats at the ends of chromosomes, are part of a complex molecular clock associated with aging, cancer progression, and a host of other diseases.
As such, research groups are increasingly becoming aware that measuring telomere length from patient samples may help in monitoring a patient's health status, mortality risk, and response to certain drugs; and may also provide valuable information for drug development.
To wit, last week one-year-old startup Telome Health came out of stealth mode by disclosing details about its core technology for measuring telomere length and its plans to work with others to use the method in various research, drug-development, and health-monitoring applications (PCR Insider, 3/24/11).
Telome Health's core technology, a qPCR-based method developed by University of Utah researcher Richard Cawthon for measuring telomere length, also serves as the basis for the new assay developed by NHLBI and Qiagen, who used Cawthon's published primers but developed an automated version of the assay, NHLBI researcher Rodrigo Calado told PCR Insider this week.
The NHLBI/Qiagen assay will be the subject of a poster presentation at next week's American Association for Cancer Research meeting in Orlando, Fla.
According to Calado, the Cawthon method, originally published in Nucleic Acids Research in 2002, "is a very homemade method, because you have to add a variety of reagents to the master mix, and there is a lot of variability also, inherent to having to mix all that together."
Further, the method requires "multiple controls" and has to be repeated multiple times due to the inherent variability; requires a fairly substantial amount of sample genetic materials, about 20 nanograms; and takes about three hours to complete — about an hour and a half each for the qPCR measurements of telomere length and a housekeeping gene, according to Calado.
All of these limitations make the originally published assay unsuitable for a clinical test, Calado said.
"We wanted to make this a high-throughput … and more accurate method that we can eventually use for CLIA purposes, for testing patients," Calado said. "The reproducibility and throughput of the method is much better than we used to get, so we can really [use this] for clinical purposes and to measure telomere length in clinical samples."
Officials from Telome Health could not be reached for comment before press time.
To optimize an assay based on Cawthon's published primers, Calado's lab worked with Qiagen scientists led by James Qin, who, after analyzing NHLBI's criteria for a fast and reproducible assay, ported it to an existing Qiagen product, the one-step Rotor-Gene SYBR Green PCR kit.
"What's unique about this kit is that we put in specific compounds, Q-Bond and Synthetic Factor MP," Qin told PCR Insider. "They actually enable fast cycling by promoting annealing of the Taq polymerase," and also promote primer-probe binding to the specific target and enable equal amplification the tandem GC-rich hexamer repeats characteristic of telomeres, he added.
In addition, Qiagen optimized the NHLBI assay for use on the Rotor-Gene GQ thermal cycler, the rotary design of which enables higher throughput and reduces variation; and incorporated the QIAgility benchtop PCR setup instrument for increased automation.
All told, the modifications reduced the amount of time it took to run the assay from three hours to about 47 minutes, according to the researchers; and reduced the amount of starting material from 20 nanograms to about 1 nanogram.
Qin said that the necessary chemistry "was already optimized in the Rotor-Gene SYBR Green master mix. You just mix your primer into the master mix and then load it on the QIAgility and RGQ. You don't even need to use a different tube."
In their AACR poster, Qin and Calado detail how they used the optimized assay to evaluate 299 healthy human subjects of varying ages ranging from 0 (cord blood) to 99 years, and demonstrated an inverse correlation between telomere length and age. In 13 samples, they measured telomere length using the gold standard Southern blot method and the new method, resulting in a correlation higher than previously described in a 2009 publication by Cawthon's group.
Calado said that NHLBI will now incorporate the optimized assay into a clinical trial that will recruit patients with telomere diseases — for instance, mutations in telomerase genes and associated clinical phenotype, aplastic anemia, and pulmonary fibrosis — and will treat the subjects with androgen therapy.
"Telomere length will be monitored while patients will be in the protocol under treatment and response rate will be correlated with telomere length behavior," Calado said.
In addition, the group plans to eventually seek a CLIA waiver for "using this in the clinic as a diagnostic procedure for patients; as well as for large trials of bone marrow failure, or liver cirrhosis, or pulmonary fibrosis," Calado added.
"We have also found for several patients that when they have shorter telomeres they are at higher risk of developing leukemia," he added. "So this can all help us with therapeutic options; or help us decide on transplant or other therapies."
As for Qiagen, Qin was unsure as to whether the company would seek to add the assay to its stable of qPCR-based tests. He said the company "realizes the potential" of the method, but has made no commercialization overtures thus far.
"We are thinking about, from a marketing standpoint, having a series of webinars this year on this topic," Qin said. "Now we are starting to send out questionnaires to our customer base to see who is interested in topics related to this work. This telomere detection area has become a hot topic."
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