In next-gen sequencing, panel versus exome

He showed additional data for the efficacy of panels in complex disorders. A publication from Ambry Genetics using WES in 500 unselected families with undiagnosed genetic conditions showed that a positive or likely positive result in a characterized gene was identified in 30 percent of patients (152/500) (Farwell KD, et al. Genet Med. 2015;17:578–586). Data from this study also underscored the importance of keeping up with newly identified pathogenic genes: Genes characterized within the past two years accounted for 23 percent of positive findings. “Rapid progress in human genetics means panels are often chasing a moving target,” Dr. Nussbaum said. Moreover, adding genes to a panel means revalidating it.

Turning to the issue of secondary findings, Dr. Nussbaum showed results from two surveys of patients undergoing diagnostic WES. In one study, 187 of 200 individuals (93.5 percent) chose to receive one or more categories of secondary findings. In a second study, parents of children undergoing WES were most receptive to learning about variants that predispose to disorders treatable or preventable in childhood.

Dr. Nussbaum noted that dilemmas regarding secondary findings are mostly avoided with panels, which return few, if any, such results. Of course, additional information that parents might want is also not available.

“I’ve been talking to a lot of payers lately,” Dr. Nussbaum said. “Payers are concerned about generating downstream costs from secondary findings.” Not surprisingly, payers are most comfortable with panels that stay within guidelines for specific indications. As Dr. Rehm had said, payers are less comfortable with exomes because of their higher expense.

Invitae’s method for genetic analysis is intermediate in complexity, between isolated panels and WES with reporting of only indicated genes. Exons for all genes for all panels that Invitae offers—about 600 genes—are captured in one step. However, in any given assay the company investigates and annotates only those genes relevant to the individual patient’s condition. “We sequence exons for all genes we capture,” Dr. Nussbaum explained, “but we only work up the relevant ones.” As for depth of coverage: “If we don’t have at least 50× coverage of all bases, we redo the sequencing.”

Dr. Nussbaum said in an interview that clinicians are frustrated over lack of consistency. “The genetic testing industry is terribly fragmented, with many different billing policies, as well as dozens and dozens of payers, each with different and inconsistent policies on coverage of genetic testing. When I see a patient and decide a genetic test is warranted, either I or one of my colleagues has to spend as much or more time than I spent with the patient figuring out what insurance the patient has, what their coverage policy is, what the out-of-pocket cost will be to the patient, and, often, ultimately coming to the conclusion the patient is either going to have to pay an exorbitant bill or forego what I think is a valid, warranted genetic test.” He ends up having to tell the patient, “I think this is medically necessary but you can’t have it unless you pay thousands of dollars.”

“There is no other area of clinical medicine that has to deal with this,” he says.

Mayo Clinic for now is mainly developing targeted panels for a variety of disorders, Dr. Dawson says. Mayo currently offers whole exome sequencing through the Center for Individualized Medicine but is preparing to offer whole exome sequencing in the Department of Laboratory Medicine and Pathology through the efforts of a team led by Matthew J. Ferber, PhD, and Eric W. Klee, PhD. “We will start with trio analysis, mainly for diagnostic odyssey cases,” Dr. Dawson says. “We have looked at trying to develop panels out of whole exome sequencing. We were just not real happy with depth of coverage.” He says there may be a way to do that down the line.

“We are discussing it.”

Where they are offering subpanels based on large gene panels, the Mayo laboratory is not including the genes for which the ACMG recommends reporting secondary findings unless those genes are already a part of the targeted panel due to the diseases of interest.

In Mayo’s molecular genetics laboratory, a minimum coverage of 100× is currently the goal for inherited disease target panels. “If we go lower than 100×,” Dr. Dawson says, “we need to be transparent about that to our clinicians.” Where mosaicism is known to be a possible cause, high depth of coverage is mandatory. “Some of the literature suggests that in some cases even small insertions and deletions need depth of coverage greater than 100× to detect them routinely.”

Commenting on cost, Dr. Dawson says, “Certainly with large panels we know exactly what genes we will be looking at, and we know the cost of the bioinformatic component up front.” With whole exome sequencing, that can change during the investigation. Then, too, “Some targeted panels are getting CPT codes. In that situation certain genes must be included, so hopefully you know about that ahead of time.”

Limitations of the chemistry are preventing them from moving to whole exome or whole genome platforms. “What ends up happening,” he says, “is that the more sequences you do, the more the depth of coverage decreases. For a targeted panel, you can have much higher depth of coverage for specific genes of interest. And depth of coverage impacts our ability to detect genetic alterations.”

On the optimistic side, Dr. Dawson adds, “We are getting pretty close to reliably detecting copy number variants with some algorithms.” The methods that have been optimized for coverage, however, may not be the best for determining CNVs. “Better algorithms plus enhanced sequencing should allow us to start calling copy number variants soon.”

Leslie G. Biesecker, MD, chief and senior investigator in the Medical Genomics and Metabolic Genetics Branch of the National Human Genome Research Institute, National Institutes of Health, cautions about so-called virtual panels. In these panels, the entire exome is examined but only genes related to an individual patient’s condition are reported. “The clinician may say, ‘I ordered a virtual panel on intellectual disability, so there is no reason to look at colon cancer susceptibility genes,’ even though they are on ACMG’s list of variants to be considered as secondary findings,” says Dr. Biesecker, who attended the AMP workshop and spoke with CAP TODAY recently. “I think this will turn out badly. It won’t be taken kindly to by patients who suffer adverse outcomes in the future because of the absence of that information.” (Dr. Biesecker explicitly excludes from this caution the approaches of Dr. Rehm and Dr. Nussbaum.)

Ultimately, interpretation is the real challenge. Even with the best-designed methods it is still possible for laboratories to disagree on the interpretation of variants. “There can be legitimate reasons for labs to come to somewhat different conclusions about a variant,” Dr. Biesecker says. “It is overly simplistic to say that everyone’s categorization should be the same. There will be edge cases, and it is reasonable that labs might differ in interpretation of those cases.”

It is important for laboratories contemplating NGS for Mendelian disorders to understand that there is nothing automatic about this process. “There is an assumption that you can turn the NGS crank and variants will fall out and be apparent,” Dr. Biesecker says. “However, there is a lot of genetics and genomics behind that process. And that level of expertise is essential to the process. Many elements can be systematized and automated. On the other hand, the interpretive part will always be essential, and it is critical that labs have the expertise that equips them to make that determination.”

Dr. Rehm said it’s now known that variants were not always interpreted in the most accurate and consistent manner. She supported this with reference to data from ClinVar showing that of the nearly 13,000 variants in ClinVar with at least two submitters, 17 percent were interpreted differently (Rehm HL, et al. N Engl J Med. 2015;372:2235–2242). The problem is that it’s difficult to generate a standard for “truth.”

“If I want to compare how well my lab is doing in interpreting variants and making calls, there is no easy way for me to do that,” she says. “If I want to know whether my NGS calls are valid, I can compare them to Sanger data. But there is no gold standard for the interpretive part.”

What can help is for laboratories to share data and evidence. “Two groups can look at the same data and come to different conclusions, just as clinicians sometimes differ in a diagnosis,” Dr. Rehm says. “There is subjectivity to the process. It is an imperfect science. On the other hand, in some cases groups differ not because of expert opinion but because of access to data. If both groups had access to the same evidence, they might have come to the same conclusion.” She encourages laboratories to share data and communicate.

“This was a great session,” Dr. Biesecker says. “It brought up a number of challenges and showed how genomics differs from a lot of other kinds of testing. We really have to come to grips with the comprehensiveness of genomic interrogation, and there is resistance to that.”

Like change, comprehensiveness is also more of a challenge to clinicians, he says. “Most geneticists are generalists; we are disease agnostic.” As a result, comprehensiveness doesn’t perturb geneticists, he says. “It gets tougher with people who are disease or organ specialists—oncologists or cardiologists. They are focused on their field of interest. They may become perplexed or overwhelmed about other diseases that genomics can tell you about.” For instance, a cardiologist may not feel as comfortable about managing inherited cardiomyopathies as ischemic heart disease.

As a result, ordering clinicians may not want laboratories to include all data from a genetic test in a report. “They may want to shift responsibility to labs,” Dr. Biesecker cautions. “Labs need to be mindful that there is a tendency on the part of some people to make this somebody else’s problem and should be wary about accepting that kind of request.

“There is a balance to be struck and that’s a hard problem.”
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William Check is a writer in Ft. Lauderdale, Fla.