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Asuragen, UC-Davis MIND Institute Publish Study Vetting PCR-Based Assay for Fragile X Mutations

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By Ben Butkus

Researchers from Asuragen and the MIND Institute at the University of California, Davis, have published a study demonstrating how Asuragen's PCR-based method for detecting mutations associated with fragile X syndrome is faster, more accurate, and more sensitive than existing methods.

The method described in the study is currently available from Asuragen as a research-use-only PCR kit, but the company is hoping that the publication is an important step toward developing a routine PCR-based diagnostic for the disorder, Asuragen President Rollie Carlson told PCR Insider this week.

The research paper, published in the March issue of Clinical Chemistry, is the culmination of a collaboration begun between Asuragen and the MIND Institute more than two years ago, Carlson said.

Asuragen, based in Austin, Texas, had been developing chemistry and methods to improve on current PCR-based methods for detecting mutations in the FMR1 gene, which play a large role in the development of fragile X and other related disorders, such as fragile X-associated tremor/ataxia syndrome and primary ovarian insufficiency.

Specifically, fragile X is a trinucleotide-repeat disease caused predominantly by the expansion of CGG sequences in the 5' untranslated region of FMR1. Expansions of more than 200 CGG sequences are associated with fragile X, while smaller expansions can contribute to FXTAS and FXPOI.

"There are these very large allele expansions, and available PCR tests … can really only see up to 100 to a little less than 200 repeats," Carlson said. Currently, the gold standard for testing for fragile X syndrome involves using PCR with size resolution by electrophoresis to detect up to 150 CGG repeats.

In addition, labs routinely use Southern blot analysis to characterize samples with CGG repeat numbers too large to amplify by PCR and to determine the methylation status of the gene, which can provide additional information about disease phenotype.

"Unfortunately, this workflow is costly, is time- and labor-intensive, and requires large amounts of genomic DNA, making it unsuitable for higher testing volumes or population screening," the researchers wrote in their paper. "PCR has the potential to address each of these limitations, yet the highly GC-rich character of the fragile X triplet-repeat sequence historically has been refractory to amplification."

To solve these issues, Asuragen scientists set out to develop optimized PCR reagents and methods to enable highly efficient amplification of GC-rich DNA. Specifically, they developed proprietary innovations in the gene-specific primers used; the amplification buffer for CG-rich templates; and the PCR cycling conditions, according to the paper.

Carlson told PCR Insider that Asuragen teamed up with scientists at the MIND Institute primarily because it is one of the foremost research institutions for fragile X studies and has access to a large number of patients for genomic samples.

"We were doing development on our side, and we wanted to be able to test for unique and novel types of fragile X samples, and certainly the MIND Institute had that diversity in its sample collection," Carlson said.

Scientists from Asuragen and the MIND Institute exchanged reagents and specimens – 146, according to the paper – "a number that would normally take years to accumulate, because the instance of fragile X is very low," Carlson said. "And we were able to use those samples to challenge our reagent designs that we were putting together. The culmination of that work over the past couple of years is in this publication."

In the study, the researchers showed that Asuragen's PCR reagents were able to amplify fragile X alleles with as many as 1,300 repeats and detected every one of 66 full mutations that were co-detected using Southern blot analysis.

Across the 146 clinical samples, which included both expanded and normal alleles, Asuragen's fragile X PCR detection method produced results consistent with the reference method, but with more accurate repeat quantification and greater detection sensitivity, and did so in about a tenth of the time using 175-fold less DNA sample, according to the researchers.

"This innovative PCR approach has tremendous potential for clinical research into fragile X biology, and could shift the paradigm for routine fragile X testing," Paul Hagerman, a professor of biochemistry and molecular medicine, and senior co-author of the study, said in a statement.

Furthermore, as the researchers concluded in their paper, "the capability to reproducibly amplify expanded alleles represents a critical first step toward the development of a PCR-only workflow that can support routine FMR1 analysis."

According to Carlson, Asuragen has introduced the PCR test in research-use-only kits to allow more researchers to evaluate the technology.

"While we might have a preconceived notion of what the potential clinical utility of the test would be, we want to make sure that it gets out and evaluated," Carlson said. "Our intent is to certainly go down the path toward a diagnostic test in the future, after we've gotten more experience with it in the research community." He did not provide a timeline for the potential development of a diagnostic, however.

In addition, the test might be applicable to other are some other rare diseases in which repeat expansion plays a role, Carlson said, but didn't specify which ones. Other notable examples include Huntington's disease and Friedrich ataxia, according to the National Fragile X Foundation.

Carlson said that Asuragen has filed for patents that would cover Asuragen's chemistry and testing methods. "The core PCR component is something that is pretty standard, but we've been able to optimize and bring in other technology," he said.

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