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University of Utah's ARUP Labs, Celera Develop PCR-Based Assay for Fragile X Expanded Alleles

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By Ben Butkus

This story has been updated from a version posted May 5 to clarify information regarding the availability of Quest's fragile X test.

Scientists from Celera and a national reference laboratory at the University of Utah have developed a PCR-based assay to detect the presence or absence of an expanded allele in the FMR1 gene on the X chromosome that is associated with fragile X syndrome and other developmental and degenerative disorders.

The assay, which combines triplet repeat-primed PCR with high-throughput automated capillary electrophoresis, is fast enough and sensitive enough to serve as the basis for a first-tier routine population screen for fragile X and associated conditions, according to a research paper published online last week in the Journal of Molecular Diagnostics.

However, while the cost of such a screen makes it feasible to be immediately adopted for pre-mutation carriers, it may still be too expensive to use routinely for newborn testing. In addition, more trials are needed to assess the efficacy of the test in a clinical setting, the researchers said.

The assay was developed in the laboratory of Elaine Lyon, an associate professor of clinical pathology at the University of Utah; division medical director of genetics at the Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology; and corresponding author on the JMD paper.

Scientists have developed a variety of tests to diagnose fragile X syndrome in patients or identify pre-mutation allele carriers. Of these, the most commonly used is PCR amplification of CGG repeat regions in the FMR1 gene followed by Southern blot of the genomic DNA.

Although this method is relatively effective, it can miss certain large pre-mutations, especially in females, and the use of Southern blot makes it expensive and time-consuming. Thus, it is not ideal for use in large-scale population screening.

Currently, in order to perform fragile X population screening, either as a newborn screen or carrier screen, "we could not use Southern blot for every patient; the workload would be too great," Lyon told PCR Insider this week. "We'd basically be performing PCR and a Southern blot on every patient."

Instead, reference laboratories such as ARUP Laboratories, along with diagnostic test manufacturers, have been trying to develop a PCR-based test that can positively identify a certain number of CGG repeats in the FMR1 gene with high sensitivity and specificity. Individuals that test positive for an expanded allele — a small percentage of the general population — could then undergo further genetic testing using traditional methods.

To wit, researchers from the MIND Institute at the University of California, Davis, recently published a study demonstrating how a PCR-based method developed by Asuragen for detecting fragile X mutations was faster, more accurate, and more sensitive than existing methods (PCR Insider, 3/4/2010); and Quest Diagnostics in February launched a fragile X test called XSense, which also combines triplet-primed PCR with capillary electrophoresis.

"I needed a PCR test that I could be confident would not miss expanded alleles," Lyon said. "I just wanted a quick, easy test to tell me if there was an expanded allele or not. If not, I stopped right there. And about 98 percent of the samples will stop as a negative test. Those that were positive and showed an expanded allele, I would then take on to the diagnostic test."

Lyon had begun developing such a "pre-screen" test several years ago using a product previously sold by Applied Biosystems but no longer available, Mike Zoccoli, head of the products business at Celera and a co-author on the paper, told PCR Insider this week.

The kit enabled her to amplify the expanded CGG repeat using primers that bound internally to the repeat and generated so-called trinucleotide "ladders" that could easily be detected using Southern blot.

In developing a higher throughput version of the assay, Lyon began working with reagents manufactured by Celera and distributed by Abbott to try and recreate the previous assay, but couldn't. So Lyon began working with Celera to optimize its reagents for the CGG-repeat expansion assay, Zoccoli said. The collaborators rejiggered the "molecular biology of the PCR reagent, and the design of the molecules in order to get them to work with the other reagents that we sell," he added. "It was partly computer design, partly experience, and partly empirical testing."

Zoccoli also stressed that the reagents Celera supplied to Lyon are analyte-specific and general-purpose reagents "that enable a laboratory to develop a laboratory-developed test."

In the JMD paper, Lyon, Zoccoli, and colleagues describe the assay in more detail. Specifically, they combined a triple repeat-primed PCR-based assay using a fluorescently labeled primer with fragment analysis on an Applied Biosystems PRISM 3100 genetic analyzer. Zoccoli noted that the reagents could also be run on ABI's 3130xl Genetic Analyzer; and that Celera has collaborations in place to look at fragment equipment made by other undisclosed manufactures. "We don't have results on those, but our hope is to have it be run on a variety of platforms," he said.

The researchers used their assay to analyze 205 previously genotyped DNA samples. Of those samples, 132 had expanded alleles and 73 had normal or intermediate samples. All 205 samples showed 100 percent concordance with previous results obtained with sizing PCR and Southern blot analysis.

What's more, the assay demonstrated a high degree of sensitivity and specificity; and the researchers showed that it could be used to analyze DNA obtained from dried blood spots, which are routinely collected from newborns for various genetic tests.

Overall, the researchers surmised that using the new assay would reduce the number of samples requiring Southern analysis by 98 percent, and that of those samples reflexed to further testing, approximately five to seven percent would be affected with full mutations.

Lyon said that the new assay "could replace our current test and reduce the number of Southern blots we do. It can be used as a first-tier test for diagnostic purposes, but the real power would be if it becomes a test for population screening."

Currently, there is no routine population testing in the US for fragile X. The decision to do so would be up to individual states, Lyon said. However, professional organizations such as the American College of Medical Genetics and the American Congress of Obstetricians and Gynecologists are currently debating the creation of guidelines for such routine screens, which would greatly influence those charged with implementing routine screening.

"Up until now, there haven't really been Fragile X assays that are good enough to drop the Southern blot for every testing scenario," Lyon said. "But now that the technical part is solved, we can begin discussing the ethical and counseling issues for screening. Screening hasn't seriously been discussed because the technical issues have always been in the way of moving forward."

Lyon said that the current iteration of the screen, if used for carrier testing, "is really doable. It's very similar to cystic fibrosis carrier screening in terms of the cost." However, for newborn screening, "the costs need to come down a bit more, but that could happen with volume. The next step would be clinical trials."

Zoccoli agreed that by selling the reagents in large volumes, "it has the potential to meet the price point that is necessary for newborn screening on the order of a couple million tests per year.

"It's a commercial opportunity for us; it's an availability of high-quality commercial reagents for labs to get a test running; and then there is the unmet medical need that fragile X expansion is relatively frequent in males, and the major cause of mental retardation," he added.

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