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Asuragen PCR Assay Provides More Evidence of Risk Factors for Mutation Expansion in Fragile X

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NEW YORK (GenomeWeb) — Scientists from Asuragen and the New York Institute for Basic Research in Development Disabilities have published research that they claim provides the most compelling evidence to date for a relationship between AGG sequence interruptions and the risk of triplet repeat expansion in the fragile X gene, FMR1.

To arrive at their findings, the scientists used Asuragen's PCR-based tests for fragile X carriers that determine the total number of CGG repeats followed by the number of AGG interruptions in those repeats to help determine parental risk of having a child with the syndrome.

Fragile X syndrome and associated disorders are caused by a mutation in the FMR1 gene that alters the production of a protein required for normal brain development. The mutation is the result of repeat CGG sequences on a fragile area of the X chromosome. These sequences are categorized into four classes based on repeat length: normal (fewer than 45 repeats); intermediate (45-54 repeats); premutation (55-200 repeats); and full mutation (more than 200 repeats).

Asuragen's AmplideX FMR1 assay uses gene-specific FMR1 PCR and CGG repeat primed PCR to detect full mutations up to at least 1,300 CGGs, accurately size up to 200 CGG repeats, and resolve female zygosity.

However, researchers have also found that AGG interruptions in CGG pre-mutation carriers can act as "anchors" of sorts that help keep the pre-mutations from expanding to the full mutations associated with fragile X in offspring. In other words, fewer AGG interruptions in CGG repeats are associated with a higher probability of full mutation expansion.

Building on this knowledge, Asuragen developed Xpansion Interpreter, which uses the same core technology as AmplideX to add valuable information about the number and location of AGG interruptions present in CGG repeat areas.

In a study published in the American Journal of Medical Genetics in February 2013, researchers from the company and partnering institutions used Asuragen's assays to demonstrate in 538 parental transmissions that the number of AGG interruptions and the length of uninterrupted CGG repeats at the 3' end of the FMR1 repeat structure correlated with repeat instability on transmission. They also found that maternal alleles with no AGGs conferred the greatest risk for unstable transmissions, and that all nine full mutation expansions studied were inherited from maternal alleles with no AGGs.

In the study published this week in Genetics in Medicine, the team essentially doubled the previous cohort, using Asuragen's assays to determine CGG repeat length and AGG interruptions for 1,040 parent-to-child transmissions of intermediate and premutation-sized fragile X alleles from 705 families.

They grouped transmissions into nine categories of five CGG repeats by parental size, and found that in every size category, alleles without AGG interruptions had the greatest risk for instability. For maternal allele repeats, 89 percent, or 24 of 27 cases that expanded to a full mutation had no AGG interruptions.

Furthermore, two contractions in maternal transmission were accompanied by a loss of AGG interruptions, suggesting a mechanism for generating alleles that lack AGG interruptions.

"Our study demonstrates that AGG sequences are critical 'stability anchors' in the fragile X CGG repeat tract," Sally Nolin, director of the Fragile X Laboratory at the New York Institute for Basic Research in Developmental Disabilities and lead author on the latest publication, said in a statement.

"By extending our previous work to include parent repeat sizes up to 90 CGG, we were able to show that AGG interruptions can alter the risk of full mutation expansion by more than 10-fold for individuals with the same number of CGG repeats," she added. "In addition, since more than half of our study cohort was drawn from patients identified by population screening, rather than families with a history of fragile X, our results should be generally applicable to fragile X carrier screening."

Regarding the latter point, such a scenario would stand to greatly benefit Asuragen, which currently sells AmplideX FMR1 PCR as a research-use only kit, and offers its Xpansion Interpreter test out of its CAP-accredited CLIA laboratory. In addition, in July the company received a clinical laboratory permit from New York State to offer Xpansion Interpreter (as well as its miRInform thyroid tests) in the state.

The company has previously stated that it is pursuing US Food and Drug Administration Clearance for an AmplideX FMR1 test kit. A company official told PCR Insider that Asuragen continues to work on its FDA registration strategy but could not provide an updated timeline.

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