A team led by clinical researchers from the Emory University School of Medicine has published a paper validating the use of RainDance Technologies' multiplexed PCR-based target enrichment technology upstream of a next-generation sequencing panel for comprehensive mutational analysis in congenital muscular dystrophy.
According to the researchers, the study, published this month in PLoS One, demonstrates how such an approach results in higher diagnostic yields than individual gene testing by Sanger sequencing, which traditionally has been the diagnostic method of choice for such multi-gene mutational analyses.
In addition, the researchers believe that the CMD example serves as model disorder group for developing and validating NGS-based tests for multiple diagnostic applications. As such, Emory Genetics Laboratory already offers tests based on the workflow for CMD, X-linked intellectual disability, and congenital disorders of glycosylation, and has other tests in the works.
"We expect the diagnostic yield to be higher for these panel tests," Alexander Valencia, lead author on the paper and a former clinical molecular genetics fellow at Emory, told PCR Insider this week. "However, we're just starting to see papers coming out, like this one, proving that point. So [the field] is definitely changing, moving toward more comprehensive testing, either panel testing or, down the line, exome testing, and it is no longer the one-gene approach."
Highly multiplexed and reliable target-enrichment methods are a crucial aspect of this sea change, and although several commercial solutions exist for this, the Emory Genetics Lab has in recent months begun favoring RainDance's technology, the RDT 1000, which uses the company's picodroplet-based technology and single-plex PCR to amplify hundreds to thousands of genomic loci with high specificity and uniformity.
In May of last year, many of the same authors of the recently published PLoS One paper published a study in the Journal of Molecular Diagnostics comparing the RainDance target-enrichment technology with that of Agilent's SureSelect solution-based hybridization technology to identify mutations in 321 exons representing 12 different genes involved with CMDs.
In that study, the group showed that both enrichment technologies produced suitable calls for use in clinical laboratories, but that microdroplet-based PCR target enrichment was more appropriate for such applications due to excellent sequence specificity and uniformity, reproducibility, high coverage of target exons, and the ability to distinguish the active gene versus known pseudogenes.
"Now that we've done the validation, our focus in [the PLoS One paper] was now: How many samples are actually giving us a positive or increased diagnostic yield?" said Valencia, who recently left Emory to become assistant director of the molecular genetics laboratory at the Cincinnati Children's Hospital Medical Center.
In their most recently published study, the researchers used the same RainDance target enrichment and NGS-based panel designed to amplify all exons of the 12 known CMD-associated genes. To assess the clinical utility of the panel, they analyzed 20 samples from patients with clinically suspected CMD, diagnosed and analyzed with Sanger sequencing by co-author Carsten Bonnemann, currently of the National Institutes of Health and formerly of Children's Hospital Philadelphia.
The Emory Genetics Lab employees were blinded to the identity, clinical phenotype, and prior genetic testing results of the 20 samples. They found that the targeted NGS approach was able to identify all disease-causing mutations that had been previously identified in the Bonnemann laboratory.
As reported in 2010 by PCR Insider sister publication In Sequence, based on this successful validation the Emory Genetics Laboratory launched a clinical CMD NGS panel, as well as a panel for X-linked intellectual disability and congenital disorders of glycosylation.
Since launching the CMD panel, the researchers wrote in their most recent paper, hundreds of the tests have been ordered and have, on average, yielded a higher call percentage — the identification and report of pathogenic mutations in the respective gene — than corresponding single-gene tests using Sanger sequencing.
For example, the researchers reported, the LAMA2 and FKTN genes yielded the highest percentages for single-gene tests, with 64 percent and 35 percent call percentages. In contrast, two panels that contained the same two genes had call percentages of 54 percent and 94 percent, respectively. In addition, the total percentage call for the gene-by-gene approach was 17 percent compared to 41 percent obtained by the use of four different NGS panels.
"This indicates that the diagnostic yield is increasing, it is more cost-effective to the patient, and you get more rapid results," Valencia said. "This is what we wanted to emphasize … from about 300 cases that we've analyzed in the past year or so."
Valencia added that the RainDance target enrichment was a primary reason for these increased call percentages.
"It's because of that … and also simply because we include all of the genes, so it's a more comprehensive test, and we're not doing the gene-by-gene approach anymore," he said.
Regarding the cost effectiveness point, the researchers wrote in their paper that the new "expedited approach to molecular diagnostics avoids the diagnostic odyssey and cost associated with a serial gene testing approach."
For example, they noted that a single-gene approach would cost around $2,500 per gene for molecular analysis, clinical interpretation, and report issuance. On the other hand, the NGS-based sequencing panel costs around $5,000 and includes "comprehensive analysis of the current 12 disease-associated genes. In addition, as more and more disease-causing genes are identified, they can be added to the panel without a significant increase in the overall cost, which is very unlikely to be the case for a gene-by-gene approach."
This last point is also a minor Achilles' heel for the RainDance platform, Valencia said, adding that it was really the only drawback to the technology — at least in a CLIA-validated setting.
"From a technical standpoint, once you validate a test, it's difficult to change it," he said. "Let's say you have 50 genes that you want to amplify, and then somebody discovers a new gene and you want to add this to it. You have to revalidate it. There are some limitations … because you have all of these mixed primers in the library … and if you were to add another primer set for another gene, it means you kind of have to test the library again … to make sure it still amplifies the genes that you want in addition to the next gene that you've added."
The bottom line, the researchers noted, is that the "flexibility of both batch processing and single-sample processing offered by RainDance minimizes reagent wastage and maintains rapid turnaround time, even when processing is less frequently ordered for samples of rare disorders in diagnostic laboratories."
This, in addition to the increased call percentage offered by the workflow compared to single-gene sequencing tests, means that the Emory Genetics Laboratory and others may adapt it to similar mutational analysis panels. Valencia said that EGL was currently developing other panels in addition to the three it has already launched, but was not able to provide additional details on those at this time.