By Julia Karow
In an effort to increase the content and decrease the cost of genetic testing, the Laboratory for Molecular Medicine at the Partners Healthcare Center for Personalized Genetic Medicine is getting ready to switch its gene tests for inherited cardiomyopathies from a chip-based platform to next-generation sequencing.
Next month, the LMM plans to launch the Pan Cardiomyopathy Panel, a comprehensive sequencing-based genetic test that will comprise 46 genes involved in inherited cardiomyopathies. The test, the lab's first on a next-gen sequencing platform, will run on the Illumina HiSeq, supplemented by some Sanger sequencing for regions of low coverage and to confirm mutations. In addition, the lab is working on next-gen sequencing panels for other disease areas.
Although the price of the new test has not been finalized, it will likely be similar to that of the current chip-based test for dilated cardiomyopathy, which covers 19 genes and costs $3,700.
Launched in 2003, the LMM is a CLIA-certified molecular diagnostic lab that performs about 5,000 tests per year, the vast majority of them large and complex DNA sequencing tests that comprise panels of genes.
The Cambridge, Mass.-based lab focuses primarily on tests for cardiovascular disease — in particular, cardiomyopathy — as well as for inherited cancer syndromes, hearing loss and related syndromes, other genetic syndromes, and cancer somatic mutation testing. It also offers some pharmacogenetics testing for drug metabolism.
Four years ago, the lab started transferring its largest DNA sequencing test onto Affymetrix GeneChip custom resequencing arrays and currently offers four array-based testing panels: the dilated cardiomyopathy CardioChip, which covers 19 genes; the hypertrophic cardiomyopathy CardioChip, which covers 11 genes; the OtoChip test for hearing loss and Usher syndrome, which assays 19 genes; and the Noonan Spectrum Chip, which analyzes 10 genes. Chip-based results are validated by Sanger sequencing.
Switching to the array platform allowed the lab to cut DNA sequencing costs in half, so it could offer more affordable tests with more content, Heidi Rehm, LMM's director, told Clinical Sequencing News. But the platform has limitations, she said, for example in detecting insertions and deletions, and because it generates false-positive results that requires Sanger validation.
To increase the content and decrease the cost of its tests even further, the lab has been working for the past year on a next-generation sequencing pan-cardiomyopathy test. The new test will combine the existing panels for DCM and HCM and add a number of genes for arrhythmogenic right ventricular cardiomyopathy and several other forms of the disease, for a total of 46 genes. For several of the genes, testing has not been available before.
Physicians will be able to order testing for only a subset of genes that match their patient's phenotype, which will reduce the risk of discovering variants of unknown significance. In that case, the lab will still run the entire test but mask the unwanted data, and will only confirm and report variants in the genes ordered. Pricing for these subsets of genes will be lower than for the entire test.
According to Calum MacRae, an associate physician in the cardiovascular division at Brigham and Women's Hospital who orders tests from the LMM, the new test will "move us closer to a molecular classification of cardiomyopathies." In particular, it will help diagnose patients with clinical phenotypes that differ from traditional phenotypes.
The inclusion of new genes in the panel will likely increase the number of patients with dilated cardiomyopathy that can be diagnosed, according to Neal Lakwadala, another cardiologist at Brigham and Women's. Only about 40 percent of patients with DCM have a hereditary form of the disease, he said, and genetic testing detects a pathogenic mutation in only about 20 percent of them. The new test could increase that number substantially, he said.
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Identifying a mutation has "clinically meaningful implications," Lakwadala said. "In many of these patients, we are entertaining alternative diagnoses, which have different, very specific therapies … and so simply by understanding exactly why a patient has dilated cardiomyopathy is in and of itself a very important contribution to clinical care."
A molecular diagnosis is also clinically useful because it allows doctors to identify family members at risk of developing cardiomyopathy — which is inherited in an autosomal dominant fashion — and to rule out family members not at risk.
Clinical trials to test medications to prevent the disease from developing in at-risk patients are ongoing. If they are found to be effective, "knowledge of genotype will be critical because it will allow us to realize the promise of preventative therapy through genetic testing," Lakwadala said.
The new test will use Agilent's SureSelect in-solution capture — a method the LMM chose after testing several others — to enrich the genes and will sequence them on the Illumina HiSeq platform. The main reason the lab chose the HiSeq is that the center's research core already had that platform installed, and it was easier to transition the HiSeq into a CLIA environment than an entirely new platform, according to Rehm. "One of our strategies in our center is to support core facilities in the same physical space as the CLIA lab, so that those instruments and the staff to run them and the infrastructure are already set up when it's time to transition those technologies into clinical use," she explained.
In addition, she said, other sequencing platforms — like the 454 FLX or the PacBio RS — currently do not produce enough reads per run for the large sequencing panels the lab is developing.
Like the Affymetrix chip-based tests, the pan-cardiomyopathy test will still require some Sanger sequencing, which is included in the price. Areas of low coverage will need to be supplemented by Sanger so that all bases are covered sufficiently for variant calling. Also, potentially clinically relevant variants need to be confirmed by Sanger since it is "not yet accepted in the field to not confirm variants from next-gen," Rehm said.
Also, for a molecular diagnostic test, "you just need to have that extra level of confidence, which is very different from running a whole-genome sequencing test, where any one variant found is not really influencing that patient's life in a substantial way," she said. "It's a different scenario when you are talking about targeted testing for a patient with a disease."
Another reason for confirming mutations by Sanger sequencing is to ensure that no samples were mixed up. "It's not just [whether] you do or don't trust the original variant call, it's also that you ensure it came from the correct sample," Rehm said.
Of the more than 1,000 exons sequenced in the test, about 30 to 50 will need to be Sanger-sequenced initially, she estimated. That number will likely decrease over time as researchers learn more about the clinical significance of certain variants and may classify some as benign, so they do not need to confirm them. Also, as variant calling improves, there will likely be fewer false positives in the future, and as DNA capture improves, the exons will be covered more equally.
Compared to the chip-based tests, the new next-gen sequencing test will have improved indel detection, though Rehm said it will still not be perfect. The ability to detect indels depends on the alignment software, read length, and other parameters, she said, and the lab is still working on optimizing those. For one gene in particular that is known to harbor many indels, the researchers are debating whether to continue with Sanger sequencing in order to make sure they can detect indels with confidence.
Substitution discovery is also better for the new test than for the chip-based tests, though that is "not a big differentiator," she said.
The turnaround time, however, might increase from about six weeks for the chip-based tests to about eight weeks, though it will likely improve over time.
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Variant interpretation is not very different from that for the chip-based tests, but more upfront curation of the data is required for the new test in order to identify variants that are benign, for example by comparing the data to dbSNP and whole-exome data sets from collaborators.
Probably the biggest challenge for launching the new test was developing the IT pipeline to support the data processing and variant calling, Rehm said.
Another challenge is to manage the capacity of the HiSeq platform, which is larger than the LMM's requirements. In order to make the test cost-effective, the scientists need to barcode and pool several samples per lane. Since they only have enough clinical samples for about one lane per week, they also need to fill up the other seven lanes with research samples from other groups.
"The run time for the instrument is almost a week, so just the logistics of dealing with that dependency on non-clinical volume, the cost of the instrument, the turnaround time — those have all been major challenges," Rehm said.
Also, developing and validating the different parts of the test — starting from library preparation to data analysis — has been "enormous work on many different levels, but we are getting close," she said.
After launching the test — currently planned for June — the researchers will start to look into other, smaller sequencing platforms, such as the Illumina MiSeq or the Life Technologies Ion Torrent. "We definitely are interested in those instruments" and are starting to model their costs, Rehm said, though "we have not come to a conclusion yet as to whether they will solve the need."
At the moment, "we've committed to the HiSeq, and all of our effort is focused on getting that platform launched right now. As soon as it's launched, we will turn our efforts to evaluating the next iteration of it," she said.
Whether or not a next-gen sequencer is approved by the US Food and Drug Administration-is of little importance to the lab, since it has to validate all aspects of its laboratory-developed tests anyway, she said. In fact, the LMM runs its chip-based tests on an Affymetrix GeneChip platform that is for research use only, even though an FDA-cleared instrument exists, because the research system is less expensive to run.
How often the new test will be updated will depend on the significance of the additional content. "Certainly, if there is important content that is discovered, we will make a pretty significant effort to include it," Rehm said, whereas genes with weaker associations may not be included immediately.
The lab anticipates that insurance companies will reimburse for the new test at a similar level as they do for the current tests. "It remains a major challenge, although we have successfully had many people get coverage, really on the basis of the clinical need, not necessarily what codes are included and what the technology is," she said.
In addition to the cardiomyopathy test, the LMM is already working on another next-gen sequencing test, the Otogenome test, which will replace its OtoChip test. That test will include all genes for non-syndromic and some forms of syndromic hearing loss.
The lab is also thinking about developing next-gen sequencing-based cancer tests, both for inherited cancers and for analyzing tumors. Sequencing tumors, however, presents additional challenges, Rehm said, such as preparing libraries from formalin-fixed samples and lowering the turnaround time.
Certain tests, however, will probably not migrate to next-gen sequencing, at least in the near term. These include tests where the number of genes is too small — fewer than about 10 — or the testing volume too low to warrant the cost of developing, validating, and running a next-gen sequencing test. The lab will continue to run such tests on Sanger "until the costs really go down and the turnaround time goes down, and all the other complexities of the platform [are reduced]," she said.
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