Next-generation sequencing: Examining an often oversimplified & misunderstood technology–Part II

One of the biggest misunderstandings about genetic testing is a perception that once a variant is identified and analyzed thoroughly, using all the best tools available, it can be associated with a specific disease or condition. But many mutations are deemed “variants of unknown significance,” meaning there is no reported (or insufficient) evidence as to whether or not they cause disease. 

Benjamin Kipp, Ph.D.

“The fact of the matter is, if you're the only person in the world with a specific variant, no matter how hard we look, and no matter how deep we dive into the literature around this variant, at the end of the day, it will end up a variant of unknown significance in the patient’s report,” says Benjamin Kipp, Ph.D. “That's hard for people to understand. And people underestimate the amount of variants we may see that are of unknown significance. We're used to tasks where everything is black or white, or it's positive or negative, but genetic testing can identify variants where we just don’t know whether they cause disease or not, due to the lack of information about a specific alteration.”

The power of targeted versus exploratory testing

Genetic testing may be used for diagnostic or predictive (pre-symptomatic) indications, and the indication for testing informs the recommended testing strategy.

Jessica Balcom, CGC

“With any gene sequencing assay, we typically have the ability to do an analysis of the entire gene coding regions (full gene sequencing), to be used for diagnostic purposes when we're testing a patient with a particular clinical indication or phenotype,” says Jessica Balcom, a certified genetic counselor who manages the Genetic Counseling unit in Mayo Clinic’s Department of Laboratory Medicine and Pathology (DLMP). “Depending on how specifically a clinician can narrow the differential diagnosis, it may be appropriate to target a single gene or to order a multi-gene panel that’s geared toward a particular phenotype, like peripheral neuropathy, that may be caused by mutations in any one of several, dozens, or even hundreds of genes.”  

With diagnostic or exploratory genetic testing of this type, casting a broader net will increase the likelihood of identifying the genetic etiology. Conversely, a bigger gene panel also increases the likelihood of detecting variants of uncertain significance, which pose a significant interpretive and clinical management challenge for health care providers and their patients.

Once a diagnosis is genetically confirmed, the physician can use this information to manage the patient appropriately. Confirmation of a hereditary cause of disease in the patient also allows for targeted genetic testing in family members. This predictive testing is done to identify who in the family might be at risk for that disease before they're presenting clinically. With confirmation of the specific genetic marker in the family, pre-symptomatic testing can be performed in a “targeted” rather than exploratory manner, analyzing only the specific region of the gene in which the mutation occurs.                                  

“The power of this kind of targeted testing comes from the fact that once you've identified the exact genetic marker for risk in the family, it's no longer an exploratory diagnostic test for anyone else who gets tested,” says Balcom. “Now you know the exact marker you're looking for, and you're getting a yes or no answer for everyone else in the family. So to do that, it's important that we accurately identify what that genetic marker is.”

When testing is mis-ordered

Accurate and valuable genetic testing is contingent on appropriately targeting the gene(s) of interest.

“If we are not targeting the correct region, there's a significant risk for a false negative,” says Balcom. “So if the information provided with that test order is inaccurate in any way, and we run an analysis that essentially isn't covering the right region, then we're going to get a false-negative result, and that patient may be falsely reassured when they could still be at risk.”    

More and more, people are walking into their primary care physician’s office and asking for a genetic test, especially after finding out a cousin, parent, or other family member is at risk for a particular disease. But often, no other family member has had genetic testing, or those results aren’t available, and so there is no record of a targeted gene to look for. This becomes problematic if physicians choose to order a whole gene test anyway.

“And so that's a really common scenario that we see. Orders will come in as a full gene analysis or a multigene panel but from a utilization standpoint, this is really not the recommended approach for family testing for somebody who's asymptomatic.  The genetic counselor or lab specialist would flag that order based on the indication for testing.  We don't want to run a multi-thousand-dollar panel when we're not even sure it has the right gene”

Mitigating false assurances

One prevalent test women request is for BRCA genetic testing—referring to the BRCA1 and BRCA2 genes that are associated with a higher risk for breast and ovarian cancers. Because familiarity with this genetic testing has been mainstreamed in our culture, this is a commonly flagged familial risk concern for patients and their providers. But this testing is not as simple as it might seem. The BRCA1 and BRCA2 genes are not the only genes associated with familial breast and ovarian cancer risk.

“If somebody has a familial pattern of breast and ovarian cancer, we know that BRCA1 and BRCA2 mutations will account for about fifty percent of hereditary breast and ovarian cancer families, and mutations in other genes (such as TP53, CHEK2, or PALB2) account for the other half of families with a genetic etiology,” says Balcom. “And so that's a scenario we really worry about, where we have blood coming in from somebody who doesn't have cancer themselves. And the indication is, a sister, cousin, or whoever, has a ‘BRCA’ mutation.

“But, oftentimes, when we probe that further and talk with the clinician, they have not confirmed that the other family member has a mutation specifically in the BRCA1 or BRCA2 genes. Once we investigate and request that they actually track down that report for the relative, in a lot of cases, it turns out it was a CDH1 mutation, or it was some other gene. And so if we had tested only the BRCA1 and BRCA2 genes, we would have given them false reassurance, because a negative result would not mean they are risk free.”

Pushing boundaries beyond turnkey solutions

In order to stay on top of all the complexities, Mayo Clinic’s IT specialists and bioinformaticians work in constant collaboration with lab directors, consultants, and pathologists on next-generation sequencing (NGS) methodology.

Eric Klee, Ph.D.

“For any given test, there is a set of instructions, the particular genetic information we are asked to interrogate.  But in the process of running that test, we learn more and find additional information that we can detect with our tools,” says Eric Klee, Ph.D. “Over time we see things changing, both in the complexity of what is being asked for from a test and in the resolution of our tools and the clarity of information we can deliver”

Meanwhile, Mayo Clinic continues to invest significant resources in bioinformatics, to not only stay on the cutting edge of emerging complexities, but to also help create the edge itself. New discoveries, and the best of commercial applications, must be harnessed to benefit the patient.

Shawn McClelland, Ph.D.

“Mayo innovates on the most cutting-edge new tests, and when the tools need something more than the commoditized software, Mayo’s Bioinformatics continues to build the necessary pieces,” says Shawn McClelland, Ph.D. “In order to put more time into only building those pieces—and because the market solutions are now more mature and standardized—the hope is to be able to use market solutions on analyses that have become more standard and to continue to innovate on the newer pieces.”

Dr. Klee chimes in: “You only learn the challenges of ‘Are you calling the events right?’ by doing it, by investing in and exploring these complexities more than anyone else. If we continue this investment, we will always be a leader while sorting out what these commercial-driven applications can offer to the patient.”

More from the series

Chris Bahnsen

Chris J. Bahnsen covers emerging research and discovery for Mayo Clinic Laboratories. His writing has also appeared in The New York Times, Los Angeles Times, and Smithsonian Air & Space. He divides his time between Southern California and Northwest Ohio.