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Asuragen Research Correlates FMR1 Methylation, Symptoms in Fragile X-Associated Motor Disorder

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NEW YORK (GenomeWeb) – Women who are carriers of Fragile X mutations can have tremor and ataxia that may be associated with the number of CGG repeats in the FMR1 gene, their methylation status, and X activation ratios, but to date testing has been cumbersome, limiting understanding of the relationships.

In a study published last week in Clinical Epigenetics, researchers at Asuragen used the firm's PCR-based method of computing allele-specific methylation status in GC-rich regions to begin to characterize the epigenetic factors influencing fragile X-associated tremor and ataxia (FXTAS). The method combines both long-read PCR and methylation-specific restriction endonuclease cleavage using the HpaII enzyme, as well as two dyes, to show the fraction of methylation on each allele.

It was compared to a commonly used technique involving determining methylation with Southern blot, and Asuragen showed its test provided more detail about allelic and methylation mosaicism.  

This level of resolution has never been available before, Gary Latham, Asuragen's vice-president of research, said in an interview, and, he said, it has prompted the firm to ask, "How do we now start to connect the dots between genotype and phenotype?"

Specifically, the study looked at 39 adult women who were carriers of the fragile X premutation, meaning they had CGG expansions in the FMR1 gene of between 55 to 200 repeats. The method was not only comparable to Southern blot, but was also "unique in identifying novel and distinct patterns of methylation mosaicism in premutation carrier women, including seven sibling pairs that were assessed using FXTAS clinical rating scales," according to the study.

"Although it is small study ... it does show that there is a potential to solve a real conundrum," Latham said. Namely, Asuragen's technology may help researchers better understand the relationship between genetics and age of onset or severity of symptoms. The test can also yield insights into mosaicism of the carrier premutation repeats, and show whether, due to skewed X activation, a patient is predominantly expressing the longer premutation allele that is associated with FXTAS.

"Ultimately, this gives better information for clinicians down the road to be able to understand how they can manage their patients better," Latham said.

A 10-year-old global molecular diagnostics firm, Asuragen focuses both on neurogenetics and oncology, Latham said, and aims to address unmet needs. As such, the firm developed technology for interrogating and characterizing GC-rich regions. It homed in on a space with fragile X testing where there is both a need in the lab for streamlined and high-resolution tests as well as a need in the clinic for improved diagnostics, and, hopefully, therapeutics. 

Asuragen launched its first FMR1 detection kit in 2009. The technology relied on proprietary innovations in the gene-specific primers, the amplification buffer for CG-rich templates, and the PCR cycling conditions, as previously reported. PCR amplification of FMR1 is no small feat, considering full FMR1 mutations can sometimes have a GC content of up to 99 percent.

The company's CGG genotyping kit received CE-IVD clearance in 2011, and it followed that with a research-use only kit combining the FMR1 PCR detection with methylation detection in 2012, Latham said. The latter also comes in a variety that uses capillary electrophoresis as a readout, and is the basis for the Clinical Epigenetics study.

The method promises to "replace the low-throughput and cumbersome methodology associated with Southern blot with a much easier and more informative method," Latham said, and "leverage long-read PCR but incorporate the ability to look at the methylation status of the gene."

More recently, Asuragen has also developed bespoke reporter software, Latham said, allowing for customization of genotyping and the interface for interpreting CE output, such that it can be more easily navigated to generate genotypes.

"It is really part of a broader strategy to be able to provide end-to-end solutions around molecular profiling for the FMR1 gene and think about how we can address some of the technological gaps that have existed in this area for many years," Latham said.

In the GC-rich testing arena, the firm has also just launched a research-use-only product for detecting the hexanucleotide repeat expansion, GGGGCC, known as C9orf72. It is "another marker that was left behind by the GWAS era and limitations of sequencing technologies, including NGS, that fail to reliably detect repeats," Latham said.

The AmplideX PCR/CE C9orf72 kit can now be used by researchers interested in detailing genetic variability in this repeat, which is known to be related to familial frontotemporal dementia and amyotrophic lateral sclerosis.

"Half the human genome is repetitive, and yet this sort of 'dark matter' is rarely discussed in terms of disease associations," Latham said. Short sequence repeats, or even longer repeats, can have very demonstrative effects on gene expression, he added, including on on splicing and translation.

"We're just now starting to understand that better [and] we anticipate that the tools that we've developed will have tremendous utility going ahead as we move beyond a view of SNPs as the driving genetic element and start to think about more complex elements, like repeats, that offer more of a digital continuum in terms of their impact," said Latham.

Oncology is also a big part of Asuragen's strategy. The company has debuted a number of new products recently, including a next-generation sequencing-based lung cancer panel, and a qPCR-based BCR-ABL kit that was granted premarket clearance earlier this year.

In addition, Asuragen is active in companion diagnostic development, Latham said, although he was not at liberty to disclose any current partnerships. It has, however, announced a companion diagnostics development agreement to use Illumina's technologies in its CDx development.

Asuragen is also keeping an eye on the testing market and revenue potential for the 40 or so other triple-repeat disorders to which its technology might be applied.

"We have a unique position with the technology that we've developed, and we also appreciate that there are areas where there is a large patient need," Latham said.

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