NEW YORK (GenomeWeb) – An assay from Asuragen to detect the most common genetic mutation associated with amyotropic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has been shown to have significant advantages over a lab-developed test in an evaluation by researchers at the University of Pennsylvania.
Asuragen's research-use-only PCR test for GC-rich expansion repeats in the gene C9orf72 is part of a growing menu for genotyping extremely hard-to-amplify regions that the firm believes will be valuable to both researchers and pharmaceutical developers.
The evaluation, published this week in the Journal of Molecular Diagnostics, was conducted in the lab of Vivianna Van Deerlin, a professor of pathology and laboratory medicine at UPenn.
Van Deerlin's lab studies neurodegenerative diseases like ALS, FTD, and Alzheimer's and Parkinson's diseases. She collaborates with neuropathologists and clinical neurologists to identify and study genetic factors associated with risk for disease, clinical and pathological phenotype, disease course, and prognosis, she said in an interview.
The group uses genome-wide genotyping and next-generation sequencing to identify risk factors and new mutations, then integrates information from these genetic studies, RNA expression analysis, clinical phenotype, biomarkers, imaging, and neuropathology to help better understand the pathogenesis of disease.
"The ultimate goal is to be able to predict who is at risk of disease before onset or progression, and, in cases of disease, predict the underlying pathological protein for targeting therapeutics," she said.
As part of this research, Van Deerlin and her team need to screen patients for known pathogenic genetic variations, including C9orf72, to determine eligibility of patients and family members for clinical studies and future therapeutic trials, she said.
In the course of this work she developed an LDT for C9orf72 — a GGGGCC repeat discovered by two independent research groups in 2011 that can be repeated hundreds of times to cause ALS and FTD pathologies and that typically has greater than 98 percent GC content.
However, her team reported in the JMD study that the Asuragen Amplidex PCR/CE C9orf72 kit performed almost as well as the LDT, but also had significant advantages.
"The AmplideX-C9 test performs both the gene-specific amplification and repeat-primed assay in a single reaction, requires less DNA input to achieve a higher peak intensity, and can provide expansion size up to 140 repeats compared to around 70 for the LDT," Van Deerlin said.
Also, because of its greater analytic sensitivity, the AmplideX-C9 assay has enabled her team to detect mosaicism, assess sequence variations with greater specificity, and amplify expansions from some formalin-fixed paraffin-embedded tissue DNA samples.
However, the LDT still has a slightly higher clinical sensitivity, she said, "because a reverse direction repeat-primed assay is included in the design to detect situations where a sequence variation prevents the standard repeat-primed assay from working."
Van Deerlin and lab have worked with Asuragen for a number of years on several molecular diagnostic projects. "They contacted me to collaborate on the C9orf72 test development because I am in a unique position of having both a research and a clinical laboratory that does testing for C9orf72," she said. In particular, Van Deerlin and her colleagues have access to a large cohort of samples that have been evaluated by different methods, and they have experience with clinical test validation.
"As soon as the C9orf72 reagents became available I wanted to see how they compared to my optimized LDT for C9orf72," she said, adding, "I came away very impressed with the performance."
Gary Latham, Asuragen's senior VP of research and development, said in an interview that development of the C9orf72 kit was initially spurred by scientists such as Van Deerlin who reached out to the firm searching for PCR-based technologies to genotype the challenging hexanucleotide repeat, "which at that time had resisted amplification beyond maybe 30 to 60 repeats by known methods."
Asuragen's technology was originally developed to navigate a CGG repeat region in FMR1, a Fragile X-associated expansion. "It was immediately obvious to us that this technology would have broader applications across other disorders linked to repetitive DNA," Latham said.
The method combines specialized reagents, optimal thermal cycling conditions, and software, he said. "We have some proprietary components within the PCR buffer that are important to help drive the capability, but we also rely on how we combine that with other components," he explained.
Van Deerlin and her colleagues plan to continue using the Asuragen AmplideX-C9 assay to study mosaicism and sequence variations in the C9orf72 gene as well as its relevance to ALS and FTD clinical phenotypes and disease progression, she said. There are cohorts of C9orf72-expanded patients and at-risk family members, and C9orf72-targeted clinical trials are starting, she said, "so having a clinically validated test for C9orf72 is important," as is "having the ability to easily start up a test and to standardize testing across laboratories."
Having the test evaluated by a key opinion leader such as Van Deerlin may now help with uptake in research as well as pharmaceutical spaces. Furthermore, the fallback method for the C9orf72 region is Southern blot, which has limitations in terms of input, workflow, and resolution, Latham said, but the firm's kit could now potentially enable labs to switch to a PCR-only workflow.
Latham noted that running the assay on capillary electrophoresis platforms sets an upper limit on the length of the expansions that can be genotyped — around 900 or so basepairs — but that the kit can be modified slightly by users and run on an agarose gel to detect much longer products. Work describing detection of up to 950 hexanucleotide repeats is currently being submitted for publication, Latham said.
Still, the firm continues to be very invested in the PCR/CE technology, he said. "We've found that it is much more extensible than perhaps some have thought and it is rapidly growing our menu to other repeat expansions." Asuragen is now using PCR/CE to develop a kit for CAG expansions associated with Huntington's disease and hopes to have that product launched by the end of the year.
"We're excited about the applications for this particular PCR technology because it addresses a real niche and emerging biomarkers in the space," Latham said. There are more than 30 known repeat-type markers linked to Mendelian disorders, "but they've evaded detection with conventional methods such as PCR and next-generation sequencing, because it is just such rough sledding to be able to successfully amplify and detect them."
Since the firm began developing kits for tough-to-PCR regions, Latham said technologies like long-read NGS have come online to enable researchers to mine the "dark matter" of the genome that had been left behind in the GWAS era, when methods used weren't able to pick up repetitive DNA. "We're learning more about these types of markers, in addition to the ones that are already established," he said, noting that this provides opportunities for more repetitive markers to be useful in diagnostic contexts in the future.
The overall process of developing the C9orf72 kit has led to commercial benefits for Asuragen, Latham said. "It has demonstrated how we can extend from FMR1 to new and emerging markers that may be important in neurodegenerative disease," he said. The test has become a clinical research tool and Latham said the firm has seen uptake both in the US and abroad, adding that "biopharma has shown interest in this type of technology for use in areas such as animal or cell-based models" to foster drug development programs.
Asuragen has launched an RUO kit for the DMPK gene which can have more that 1,000 CTG repeats and is associated with a type of muscular dystrophy, a kit for FMR1 expansion repeats which has been used to demonstrate a correlation between number of repeats and severity of disease, as well as kits for poly-T polymorphisms associated with Alzheimer's disease.
The firm is still considering a US regulatory strategy for these tests, Latham said. "We're monitoring the market very actively," he said, with the possibility of seeking US Food and Drug Administration clearance "once facts and circumstances justify the investment."
The FMR1 kit is CE-IVD marked and has obtained regulatory clearances in Canada, Australia, and South Korea, Latham said. The firm is also in the process of obtaining CE-IVD on its DMPK kit.
It also recently launched an SMN1 kit as part of a collaboration to develop and commercialize capillary electrophoresis-based tests on the Thermo Fisher Scientific 3500 Dx Series Genetic Analyzer CS2 instrument. That test measures copy number of a gene linked with spinomuscular atrophy and is a common carrier assay performed on would-be parents.
Asuragen is "working on an assay that can combine SMN1 and SMN2 copy number, reading out on a CE box with very accurate quantification," Latham said. It also is developing assays that can combine CNVs, repeat sequences, SNVs, and indels into "perhaps the first truly unified workflow outside of NGS" that can look at common markers associated with carrier screening and reproductive health.
"You can imagine in the future having a FMR1 kit combined in a single workflow with SMN1 and with SNVs and indels — those are among the most common carrier gene screenings used and can create a very facile approach for labs to run those types of assays and achieve great economy and operational scale, using a method that is far less complex, and, for these targets, far more reliable than NGS," said Latham.