In next-gen sequencing, panel versus exome

William Check, PhD

January 2016—As next-generation sequencing takes its place in clinical laboratory medicine, a difference is developing between its use when there is a defined phenotype, as with hereditary oncology syndromes or hereditary cardiovascular disorders, and its use in diagnosing hereditary developmental disorders. In oncology, targeted panels remain the optimal mode of application. In medical genetics, NGS is moving beyond panels to whole exome sequencing and perhaps soon even to whole genome sequencing. A recent Association for Molecular Pathology workshop on the clinical utility of genomes versus exomes versus targeted panels spotlighted how decisions are being made for one or the other in diagnosing inherited disorders.

Heidi L. Rehm, PhD, an associate professor of pathology at Brigham and Women’s Hospital and Harvard Medical School, addressed the laboratory component of the topic. Her main message: In genetic testing for Mendelian disorders, it is not a question of panels or exomes. Rather, the challenge is how to combine the two to support the most useful services. To do that, Dr. Rehm told CAP TODAY in an interview, “we may actually decide to offer defined panels which we interpret off of an exome platform where the rest of the data are available for reflex testing or research.”

Dr. Rehm says it is much less costly to offer many rare disease tests by doing them on the same technical platform. “We are moving in that direction. To do that,” she says, “we have to ensure a very high-quality exome backbone and ensure that clinically relevant genetic regions are covered fully for each indication.” Much of her presentation was about how to achieve that.

Dr. Rehm, who is also director of the Partners HealthCare laboratory for molecular medicine and clinical director of the Broad Institute Clinical Research Sequencing Platform, says she and colleagues are likely to move to whole genome sequencing in the future. “It is inevitable. The quality of data coming from PCR-free whole genome sequencing is impressive. We have data at the Broad on this.” She doesn’t know yet when the transition will take place: “It is a cost question. My guess is within the next two years.” Balancing genome versus panel testing will entail the same strategy as exome versus panel testing.

Robert Nussbaum, MD, chief medical officer at Invitae, speaking at the same workshop, addressed the question, How is a clinician to decide? Dr. Nussbaum was a clinician for many years and is now Holly Smith professor of medicine emeritus in the Department of Medicine and Institute for Human Genetics, University of California, San Francisco. He sounded a Heraclitean note. “Technologies are in flux. I can stand up here and say anything I want,” he said, “but it will all change by next year. This is more of a problem for clinicians because they don’t fundamentally understand the technical aspects of sequencing.”

Dr. Nussbaum focused on patient choice and cost as driving forces for choosing between exomes and panels.

Workshop organizer D. Brian Dawson, PhD, of Mayo Clinic, says the views the speakers presented were “more because of the structure of what I had asked them to present. I asked them to speak on areas that we are all dealing with right now: Is it better to do targeted panels or to use a whole exome sequencing backbone?”

“From that standpoint, they presented strengths and weaknesses of both approaches”—which is helpful to hear for those who are just getting into whole exome sequencing or targeted panels using next-generation sequencing, says Dr. Dawson, co-director of Mayo’s molecular genetics laboratory and an associate professor of laboratory medicine and pathology and medical genetics.

In choosing between panels and whole exome sequencing (WES) for rare inherited disease testing, several factors are important, Dr. Rehm told attendees. First is certainty of diagnosis. Exome analysis is best suited to conditions with multiple clinical features that have no clear diagnosis, such as neurodevelopmental and other neurological presentations. Gene panel testing is best suited to patients with a clear diagnosis and for which panel testing yields a reasonable detection rate, such as cardiomyopathy, retinal disease, and hearing loss.

She showed data from two 2014 publications, one from Baylor and one from UCLA, on clinical findings with WES among patients referred for genetic identification of rare Mendelian disorders (Lee H, et al. JAMA. 2014;312:1880–1887; Yang Y, et al. JAMA. 2014;312:1870–1879). Both groups showed that “Exome testing is a good primary test for cases with unusual presentations for which no panel is available or for which currently available tests are very low yield,” Dr. Rehm tells CAP TODAY. For pediatric neurologic presentations, for example, “There is no really good targeted panel that gives you a high-yield answer.”

A second factor in choosing a testing modality is the analytical performance of exomes versus panels. Completeness and depth of coverage are critical for good analytical performance. “Whole exomes are not whole,” Dr. Rehm says. Using 51 genes for inherited cardiomyopathy as an example, analysis in the Partners laboratory for molecular medicine showed that with a panel, less than one percent of exons were not fully covered (0.7 percent of base pairs had less than 20× coverage), in contrast to 15 percent of exons not being fully covered with standard exome capture (3.7 percent base pairs had less than 20× coverage). However, with probe supplementation of its hybrid capture, Dr. Rehm’s laboratory was able to improve its exome platform coverage to less than one percent of exons requiring Sanger fill-in, enabling panel-based analysis of an exome backbone to be a high-quality, cost-effective first approach for many indications.

Another consideration is whether the type of variant known to cause the disorder is reliably detected by next-generation sequencing, which is the underlying technique for both panels and exomes. “Most labs supplement” NGS assays, she says. Typical supplements are Sanger sequencing as fill-in for incomplete coverage, add-on triplet expansion assays for such conditions as Fragile X and spinocerebellar ataxias, inversion/breakpoint assays for factor VIII, and add-on copy number variant assays for genes known or likely to be subject to deletions and duplications. Copy number variants still present a challenge to NGS, she notes.

Analysis of results with the hearing loss panel illustrates some of these problems. “The two most common genes require supplemental testing,” one for a noncoding deletion and one to differentiate pseudogene variants, Dr. Rehm says. In addition, 30 percent of pathogenic variants are copy number variants.

Detecting all clinically relevant variants can require a complex algorithm, as illustrated in an article on detection of germline mutations in the DNA mismatch repair gene PMS2 that underlie Lynch syndrome (Fig. 1 in: Li J, et al. J Mol Diagn. 2015;17:545–553). Fortunately, Dr. Rehm says, “We usually don’t need such a complex algorithm for most diseases.”

Gene homology can also confound NGS-based methods. A pathogenic gene can be homologous with a pseudogene, which is the case with PMS2. In other conditions there may be more than one functional gene, such as hereditary cardiomyopathy and spinal muscular atrophy (SMN1 and SMN2). Dr. Rehm’s colleague, Diana Mandelker, MD, PhD, has identified 286 homologous genes of medical relevance.

A fourth consideration in test choice is clinical experience. A hearing loss panel produces an inconclusive result in almost 60 percent of cases. In one patient Dr. Rehm described, a known pathogenic variant was found but it is not associated with profound hearing loss, which the patient had. Dr. Rehm recommended reflex to broader panel testing, which identified two novel pathogenic variants in MYO7A, a gene for Usher syndrome (deafness and retinitis pigmentosa). Diagnosis of Usher syndrome was confirmed through electroretinography testing. A laboratory director must be not only experienced enough to recognize this type of situation but also willing and able to pursue novel genes or variants.

Secondary findings can also pre­sent problems. “In our experience a lot of physicians don’t want to deal with secondary findings. It’s information irrelevant to the care of their patient for the indication they showed up with. On the other hand,” she says, “that clinician perspective is not necessarily in the best interest of the patient, who may very well want or need to know secondary findings. It’s a bit of a conundrum sometimes to know how much information to return.” In practice, she adds, “In individual cases we rely on the physician to order the best test for each patient.”

Cost is an important issue. Dr. Rehm noted that insurers may cover panel tests but not exomes.

A further complication is that new disease genes are being discovered at a steady rate. Over the past four years, 915 new disease genes have been reported (Chong JX, et al. Am J Hum Genet. 2015;97:199–215). “Updating and revalidating panel tests is costly and time-consuming.”

“Can we make it simpler as a lab industry?” she asks. This is possible, in her view, using panels on an exome backbone. In this approach, the technical platform is the same for all tests but the genes analyzed and reported are distinct. The entire exome is sequenced, but the analysis pipeline only returns variants in the genes relevant to the condition for which the clinician ordered testing. Tests are offered to physicians as classic disease panels. Such an approach is less costly to validate and quicker to update through analysis pipeline modification. On the negative side, coverage may be lower per gene and variable costs (per test) are higher, though some of this may be offset by lower fixed costs.

Dr. Rehm showed that incomplete coverage with WES can be largely overcome by adding more capture probes. Coverage rose from less than 95 percent to more than 99.5 percent on several of her laboratory’s exome-based panels.

Over several months in 2014, Dr. Rehm and her colleagues compared WES to panels for 160 patients with genetic sendouts. They assessed clinical sensitivity and cost of the physician-ordered test versus using a panel-based analysis of their exome assay.

Results were mixed. There were a number of cases where exome analysis could improve clinical sensitivity and costs would be lower. However, copy number detection was critical for a number of tests ordered, and the added clinical sensitivity provided by exome assay did not save costs for small panels covering the most common causes.

Dr. Rehm says they are moving some panel tests to exome backbone near term (for example, cardiomyopathy, pulmonary) but leaving others on panels (such as hearing loss due to CNVs and RASopathies due to rapid turnaround time requirements). Meanwhile, they are validating their exome algorithm for detecting CNVs.

For panels performed on an exome backbone, Dr. Rehm does not think the recommendation of the American College of Medical Genetics and Genomics to report secondary findings on 56 disease-related genes applies. “When a clinician orders a panel test, such as an 80-gene panel for hearing loss, in my opinion you are only ordering analysis of genes on that panel. So there is no issue of secondary findings.” Where it is relevant, she says, is if a physician orders a full exome test and the whole exome is sequenced and analyzed.

Dr. Nussbaum agrees that a defined phenotype is needed to consider using a panel. He agrees, too, that exome or genome sequencing is preferred for undiagnosed disorders and for resolving a diagnostic odyssey.