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New Fragile X Assays From NIH Could Reduce Testing Cost, Improve Sensitivity

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NEW YORK (GenomeWeb) – Researchers at the National Institutes of Health have developed a suite of PCR assays for fragile X mutations that promise improved sensitivity and reduced cost compared to currently available testing. The assays are for research use but could potentially be incorporated into a newborn screening panel.

Karen Usdin, senior author on a study describing the assays published today in the Journal of Molecular Diagnostics, told GenomeWeb in an interview that certain issues with cells in tissue culture were the original motivation for the project.

Fragile X and related disorders are caused by expansion of CGG/CCG repeat tracts in the 5' untranslated region of the FMR1 gene, located on the X chromosome.

However, AGG interruptions in the repeat tract and DNA methylation can modulate risk and disease severity, and there is a high degree of mosaicism in repeat number and methylation status.

Usdin, senior investigator on the study and chief of the Gene Structure and Disease section at NIH's National Institute of Diabetes and Digestive and Kidney Diseases, explained that she and her colleagues are interested in the mechanisms of disease pathology and the cause of the unusual expansion mutation responsible for the fragile X-related disorders, so they do a lot of work with patient stem cells.

"One of the things we noticed is that the affected gene in these cells is very unstable — the repeat tract expands and contracts, and also gains and loses DNA methylation, depending on how you're growing them," said Usdin.

This in vitro drifting can lead to "all sorts of erroneous conclusions being reached," Usdin said, for example, "that there was expansion or de novo methylation when in fact there really wasn't, it was just selection for an allele that was already present in the [in vitro cell] population."

And this is particularly important because patient cells in vitro are now used to screen for compounds that may be useful therapeutically, she said.

Unfortunately, most of the currently available tests are either too expensive for routine lab use or don't capture all the aspects of the genotype and epigenotype Usdin and her team wished to capture.

"Even in a clinical situation, commercial assays are sometimes too costly, so labs sometimes do triage and do other assays first and only as a last resort do they go to these more definitive, but quite expensive, assays," Usdin said.

But luckily, developing improved protocols is a favorite activity of first author Bruce Hayward, she said. "He tweaked existing protocols that we were using and progressively made them more and more robust and better able to amplify these long alleles, which is really challenging."

Specifically, the JMD study describes a protocol for an assay of repeat number that was improved by adjusting the polymerase and buffer conditions, and by digesting with a methylation-sensitive enzyme. This enabled amplification of an allele with approximately 940 repeats, and also compensated for preferential amplification of smaller alleles in cases of mixtures.

Interestingly, this method was sensitive enough to amplify fragile X alleles from saliva samples as well, and Usdin noted that another research group is now using it to assess tiny amounts of DNA from blastocysts and early embryos.

A second assay in the suite looks at methylation and was improved using a methylation-sensitive digestion enzyme such that it can correlate specific alleles with their methylation status. The group also worked out a method to interrogate methylation of the FMR1 promoter that does not require bisulfite modification or TaqMan primers.

Another novel assay measures the number of uninterrupted CGG repeats at the 3' end of the repeat tract in females, which, using other assays, had previously been more challenging to do than in males.

The JMD study also describes an improved assay to examine the AGG interruption pattern in the FMR1 gene, using a variant of triplet-primed PCR.

Overall, "Our assays provide an affordable solution for basic research labs, but may also offer the potential for development into assays that may be suitable for more widespread use," Usdin said. Development could potentially be done through work with an industry partner, though she said that the group is not necessarily focused on commercializing the suite of tests.

On the other hand, "We're interested in having these assays readily available to everybody, so we would be in favor of whatever would help make that happen," she said.

At a cost of around $5 per test, the assays are in line with the price point needed for population-based screening, which might benefit at-risk families as well as newborns.

Fragile X diagnosis tends to happen at age three, Usdin said. "Right now there is no cure, but there are indications that early educational intervention could be valuable, so an early diagnosis is important," she added.

For now, a few other researchers have already asked for pre-prints of the paper describing the assays, Usdin said, and she imagines they will be embraced by her colleagues.

"From anecdotal conversations that I've had with others in the field, I think [the assays] would be of interest to quite a few labs, including those working on other related diseases, like myotonic dystrophy, that pose similar testing challenges," she said. 

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