By Ben Butkus
Scientists from Asuragen and the University of California MIND Institute have published research demonstrating the ability of an Asuragen PCR-based assay to determine the methylation status of the FMR1 gene, a finding that the company said could improve diagnosis of FMR1-linked disorders such as fragile X syndrome.
In addition, Asuragen said that in the second half of this year it will release research-use-only reagents for evaluating FMR1 methylation status in the hopes of obtaining independent validation of the technology and, ultimately, seeking regulatory approval of the product as an in vitro diagnostic kit, Asuragen President Rollie Carlson told PCR Insider this week.
The new technology builds upon Asuragen's existing assay for detecting CGG repeats in the FMR1 gene by also assessing the gene's methylation status, which has been demonstrated to affect the severity of diseases such as fragile X syndrome.
Asuragen's existing assay, which is CE Marked and sold in Europe as the AmplideX FMR1 PCR kit, is considered an improvement upon the prior gold standard of Southern blot analysis because it similarly detects expansions in the CGG repeat region of FMR1 of anywhere from 200 to 1,000 repeats, but faster and with smaller amounts of DNA.
Detecting repeats in the FMR1 gene is only half the battle, however. "In Fragile X, if you have a full mutation, then whether or not that is actually affecting the gene is highly affected by its degree of methylation," Carlson said. "As soon as someone does a diagnosis for fragile X, then the next thing they're going to do is see if it's methylated or not."
Until now, Southern blot was also the preferred method of choice for assessing methylation status of the mutated gene. And although it is "tried and true", it is tedious, time-consuming, requires large amounts of DNA, and is often inaccurate, Carlson said.
"So the next level for us, now that we can detect full mutations with our PCR assay, was to add on this methylation technology," Carlson said. "We modified some techniques to be able to digest for and determine methylation. It's a proprietary approach, which we've filed IP for. But it is basically allows someone to amplify and determine methylation on top of the full mutation."
In the June issue of Genetics in Medicine, researchers from Asuragen and the UC-Davis MIND Institute, a long-time collaborative partner of the company, describe recent research demonstrating that the methylation PCR assay, which comprises allele-specific methylation PCR and capillary electrophoresis, could detect methlylation status as well as the gold standard of Southern blot analysis.
Specifically, the researchers surveyed eight cell lines and 80 clinical samples, including 39 full mutations, and found that not only did the new technique have a high degree of concordance to the gold standard, it revealed patterns of methylation mosaicism associated with female fragile X carriers that Southern blotting could not.
"To date, the [methylation] PCR-CE technology evaluated in this study is the only PCR-based methodology that has been reported to detect and resolve methylation status from full-length triplet-repeat-containing amplicons in both male and female samples with concordant results to [Southern blot] analysis," the researchers wrote in the paper.
"Moreover, this approach has the potential to provide new molecular information unresolvable by [Southern blot] analysis that may support a more complete understanding of fragile X disorders," the researchers added.
Asuragen said that researchers from the company and the MIND Institute are currently studying the implications of the new findings, and that the reagents described in the study are currently being evaluated at six laboratories in the US and abroad, although it did not disclose their identities.
The company also said that it will make the reagents available as RUO kits in the "second half of 2011;" even as it eyes a potential IVD kit based on the reagents down the road.
"The main reason we're putting them out as RUO reagents [that] there is a lot of skepticism that you can replace Southern blots," Carlson said. "We want to really get this into researchers' hands, although not in a clinical environment yet, to try and evaluate this and show unequivocally that it has the performance we think it does. Ultimately we'd like to try and pursue an IVD application."
Depending on how this independent evaluation period goes, and how the market shapes up for FMR1 molecular testing, Asuragen will likely pursue US regulatory approval for just one of its technologies – either the currently available AmplideX FMR1 PCR kit, which does not assess methylation status, or the new methylation PCR technique.
"I could speculate that if the [methylation] PCR plays out, then that's all anyone will need because it will detect whether or not you have a full mutation; and at the same time will give you methylation status," Carlson said. "But I don't know if that's going to happen in the clinical marketplace."
He said that customers using the AmplideX FMR1 PCR kit in countries recognizing the CE Mark already find value from that kit because "they look at it as a way to reduce the vast majority of Southern blots. All of a sudden they have eliminated it to about 2 percent of testing that has to be done on Southern blot."
Ultimately, Carlson added, "we're going to pick either one or both technologies to go down an IVD path. But we have to see how it sorts out and how we believe the molecular biology community would potentially use these."
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