Asuragen today announced results from a study demonstrating that its Xpansion Interpreter molecular test can improve the determination of a woman's risk of having a child with fragile X syndrome compared to existing risk measures.
The PCR-based test is designed to complement Asuragen's AmplideX FMR1 PCR assay, which detects CGG repeats in 5'-unstranslated region of the fragile X mental retardation gene, FMR1, in women to determine whether their offspring are at risk for developing fragile X syndrome.
Xpansion Interpreter, meantime, assesses the number and position of interrupting AGG sequences in the same genetic region, which are linked to instability and a higher risk of expansion to a full mutation in offspring.
Asuragen is hoping that the new data will support the combined use of AmplideX and Xpansion Interpreter to enable physicians and genetic counselors to paint a more accurate picture of their patients' risk for having children afflicted with fragile X syndrome.
"Now that we have a clinical study published, we … hope this will be incorporated into a diagnostic algorithm for physicians and genetic counselors encountering women of child-bearing age [in which] a standard fragile X assay [indicates] they have a premutation," Asuragen President and CEO Rollie Carlson told PCR Insider. "The next question would be: 'What's the risk of that mutation then expanding into your offspring?' This study really sets the foundation for clinical guidance for physicians and genetic counselors."
Fragile X syndrome is 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 PCR reagents uses gene-specific FMR1 PCR and CGG repeat primed PCR to detect full mutations up to at least 1,300 CGG, accurately size up to 200 CGG repeats, and resolve female zygosity. The company also has developed a version of this assay to detect methylation status of the FMR1 gene.
"Fragile X [risk determination] has relied on very old technology, which has mainly been Southern blots up until about three years ago, when [Asuragen vice president of research and technology development] Gary Latham and his team came up with a full PCR solution," Carlson said. "We introduced that as a kit called AmplideX, now available to the laboratory market."
Asuragen received the CE mark for an AmplideX FMR1 PCR kit in 2011 and has been offering the test commercially in Europe since that time. In addition, Asuragen offers AmplideX through its CLIA laboratory, and sells the associated reagents for research use only in the US.
Soon thereafter, Asuragen developed its Xpansion Interpreter test, which is based on the same core PCR technology as AmplideX. In November 2011, the company launched the test through its CLIA lab as a complement to the AmplideX assays it was already offering (PCR Insider, 11/3/2011). However, until this month, the company had not yet officially presented data to back up the use of Xpansion Interpreter as a tool to better determine fragile X risk in offspring.
"We made it available in our CLIA lab … primarily to support research studies," Carlson said. "It was available as a commercial offering, but really … for clinical trial support, to continue to support these studies that we're talking about now."
Asuragen scientists presented the new data today in an oral abstract at the American College of Medical Genetics and Genomics annual clinical genetics meeting in Phoenix. The data expanded upon findings presented in a scientific paper published online last month in the American Journal of Medical Genetics.
In the AJMG-published study, Asuragen — in collaboration with the New York Institute for basic Research in Developmental Disabilities, Rush University Medical Center, Emory University School of Medicine, and the MIND Institute at the University of California, Davis — used Xpansion Interpreter and AmplideX to examine 457 mother-to-child transmissions in women with intermediate or small premutation fragile X alleles (45 to 69 CGG repeats) to examine the association of CGG repeat length and AGG interruptions with repeat instability on transmission.
The results revealed that assessing the number and position of AGG interruptions — coupled with total number of CGG repeats — provided significant improvements over current risk estimates in predicting fragile X gene instability and expansion to a full fragile X mutation. All nine transmissions of the full fragile X expansion mutation in the study were from mothers with CGG repeat regions lacking AGG sequences.
In the study presented at the ACMG meeting, Asuragen expanded this to include women with 70 to 90 CGG alleles to examine risk for full mutation expansion, and combined that data with the previous analysis of mothers with 45 to 69 repeat alleles.
Overall, they examined total repeat length and AGG interruption patterns in 1,364 samples from 765 mother-to-child transmissions involving alleles of 45 to 90 repeats. "As expected, the presence of interspersed AGGs significantly modulated the risk of expansion to a full mutation," the researchers wrote in their abstract.
While the overall risk of full mutation expansion for alleles of 75 to 79 repeats was 51 percent, the risk was 77 percent without any AGG; 36 percent with one AGG; and only 8 percent with two AGGs. The number of 3' uninterrupted CGG repeats was also associated with the magnitude of expansion, which increased most dramatically for alleles exceeding a continuous stretch of 70 to 74 CGGs.
The results of the study, Asuragen said, illustrate the "critical importance of the AGG interruption pattern for estimating the risk of full mutation expansion."
Asuragen said that the methods and processes involved in its AmplideX and Xpansion Interpreter assays overcome "formidable technological challenges" and "make FMR1 genotyping efficient and AGG sequence mapping accurate" for clinical implementation.
"There is probably no more … genetic segment in the human genome more difficult to amplify than the 5' untranslated region of FMR1," Latham said. "If one wants to interpret, potentially, a subtle shift in the genotype in that region, one has to have the foundation of an efficient PCR that can contend with highly GC-rich DNA to be able to first reveal the CGG segment itself, and then to look at subtle genotypic changes within that CGG stretch, such as AGG interruptions."
Latham said that Xpansion Interpreter uses the same core technology as AmplideX to provide that additional information and help understand the AGG interruption pattern. "This then establishes a biomarker, which then we can use in a clinical study for the first time to be able to indicate how it can be useful in a clinical setting," Latham added.
Asuragen has developed its assays to run on Life Technologies' ABI family of capillary electrophoresis instruments. "This particular family of instruments has the broadest install base and, so, particularly with our AmplideX products, it's compatible with that entire family … so there is a lot of versatility in how the assay can be run and how the results can be extracted for various uses," Latham said.
The company still has designs on seeking US Food and Drug Administration clearance for AmplideX, but is still determining which version – the flagship assay or the methylation status test – to move down that path. Carlson and Latham did not provide a timeline for seeking FDA approval.
Xpansion Interpreter, meantime, will most likely remain a supplemental offering through Asuragen's CLIA lab.
"Xpansion Interpreter is a very complicated test that is not easily converted into a kit format, and requires a high degree of analytics," Carlson said. "So we see that as being a lab-developed test for the Asuragen CLIA lab, but our intent is for the AmplideX technology to seek an IVD path in the fragile X category."