By Julia Karow
This article has been updated with additional information about funding for the Undiagnosed Diseases Program.
NIH researchers have found that exome sequencing can help provide diagnoses for patients suffering from diseases that doctors could not explain in any other way, and say that the approach may become a tool in standard clinical settings in the future.
As part of the National Institutes of Health's Undiagnosed Diseases Program, researchers had sequenced about 50 exomes from 11 patients and their family members as of last month, as well as full genomes of three individuals. They have so far found disease-causing variants in two patients, and variants likely to be causative in another two.
Given that many of these patients had previously undergone costly procedures and testing without success, "it's likely that it's going to be a cost-effective method, even if you only find the right answer in 10 or 20 percent of cases," said David Adams, a senior staff clinician at the National Human Genome Research Institute, who sees pediatric patients entering the program.
Launched in 2008, the Undiagnosed Diseases Program, which is organized by the NHGRI, the NIH Office of Rare Diseases, and the NIH Clinical Center and currently operates as a pilot project, aims to deliver a diagnosis to patients with "mysterious conditions" and to advance medical knowledge about rare as well as common diseases. The program started with $280,000 per year in funding from the ORD, though funding has since increased to $3.5 million per year, through 2012.
According to Adams, more than 300 patients have gone through the program so far, of about 4,000 who have inquired about it. Both children and adults are eligible, and after applying, staff members select them based on how likely the program will be able "to shed some light on their conditions," he said. This usually means that their disease has a strong genetic component, or a severe and measurable phenotype.
Patients selected to participate visit the NIH for a week or so to undergo a thorough evaluation, after which a diagnosis is made in about 10 to 15 percent of cases, for example because doctors had previously missed some evidence or because it turned out the condition was a rare presentation of a known disease.
But for the majority of cases, there is still no diagnosis, "and that's where the next-gen sequencing comes in" as one of several diagnostic tools, Adams said. Other tools include, for example, metabolic screens of spinal fluid, which have proven helpful for figuring out neurological syndromes. "The next-gen [sequencing] is one piece of the whole puzzle," he said.
As of last month, Adams and his colleagues had sequenced and analyzed approximately 50 exomes from 11 patients and their families, using the Agilent SureSelect capture method and the Illumina sequencing platform, and several dozen more were waiting to be sequenced. The NIH Intramural Sequencing Center performs sequencing for the program. NISC has developed a custom pipeline for variant calling and uses an in-house pathogenicity assessment software to prioritize variants. In addition, the researchers use data from the 1000 Genomes Project and from their own cohort to filter out common variants.
Sequencing the exomes of several family members — including, for example, the patient, his unaffected parents, and an unaffected sibling — has been the most powerful way of using next-gen sequencing, they found, because it allows them to zoom in on the most relevant parts of the genome, based on the genetic model of the disease and the inheritance pattern. Imposing constraints to determine which parts of the genome follow patterns of inheritance have been "extremely powerful," Adams said, allowing them to cut down the number of variants to study more closely by 60-fold in some cases.
Sequencing the exomes of families, rather than individuals, "makes a huge difference," he said. "Even from a whole exome, for a family, you get 100,000 variants or so. To be able to filter those down to a list of things that we can look at carefully … and evaluate for pathogenic potential is the challenge that everybody has with this technology."
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So far, the researchers have found disease-causing variants for two patients, and for another two, they are confident that they will be able to confirm the variants as causative with additional lab work. For the remaining seven patients, "we don't know one way or the other" and need to do more studies, he said.
In one of the confirmed cases, the researchers found variants in the beta-galactosidase gene, which prompted them to repeat a test of the enzyme that had previously shown normal activity. Retesting showed very low enzyme activity, allowing them to diagnose the disease. "Even though it's not a new disease, finding those variations was what caused us to make a diagnosis, and we would not have sequenced that gene otherwise because the enzymatic testing ….did not give the correct results the first time," Adams said.
For the second case they solved, a neurologic condition, they discovered a variation in a gene that was shortly afterwards described in a scientific publication by another group as the cause of a disease. Collaborating with that group allowed them to confirm that the variant also caused that disease in their patient, and to shed additional light on the gene and pathway involved.
In addition to sequencing the exomes of families, the scientists are also exploring exome sequencing for individuals in cases where they suspect a disease and want to sequence a large set of candidate genes that could be involved, which would be expensive to do by clinical-grade Sanger sequencing. "For instance, it would cost $40,000 to sequence all of the hereditary spastic paraplegia genes, and it costs $3,500 or $4,000 to do a whole-exome sequence," Adams said. "It's much more cost-effective to do the whole exome and see what you find, and then do clean-up with Sanger sequencing if you need to as a secondary step."
There is, of course, the danger of missing causative variants because they might not be in the coding regions, or not captured or sequenced effectively. "Absolutely, there is a chance that we're going to miss things," Adams said.
So far, all the exomes he and his colleagues have looked at "have generated pathogenic areas of interest, but we feel very strongly that it's very difficult to jump from a candidate variation to a strong assertion of disease pathogenicity without additional laboratory work." Also, for some families, "even though we've got a list of things that we have found, there is nothing obvious in that collection that attaches those variations to the disease," he said. "You always get variants. With the huge number of variations that come out of this type of analysis, they may require quite a bit of work to confirm after you found them."
The researchers have also explored whole-genome sequencing — so far on three individuals — but mostly to understand the characteristics of the data compared to exome sequencing, "rather than really using it as a diagnostic tool, like we are using whole exome for," he said.
Even though exome sequencing might only be able to render a diagnosis in a fraction of cases, at $4,000 per sample — not including the analysis — it is likely a cost-effective method, Adams said, since many of the patients had tests, hospital stays, and procedures totaling between $100,000 and a $1 million prior to coming to the NIH.
And even though obtaining a diagnosis does not always lead to a treatment, he said, many families are grateful just to learn what causes the disease. "It's hard to overstate how much families want and appreciate a diagnosis once it's found. It's actually still surprising to me, even after years of doing this work, that families just want to know what the diagnosis is," he said. Knowing the diagnosis reassures families, for example, that they have done everything they can for their relative. "But of course, we would love to find cases where we could use that [diagnostic] information to develop rational therapy."
Even though it has not been determined whether the Undiagnosed Diseases Program will be scaled up following the pilot phase — Adams said an external and internal review committee are currently drawing up guidelines for evaluating it — the organizers have learned about the importance of incorporating family data in clinical genetic testing.
"In most cases now, people do a lot of testing on the proband," he said. "It may be that from a very early step, we need to start thinking about gathering DNA from family members. Even in standard clinical settings, I think that it's extremely important to have that information. Once exome sequences are used in a standard clinical setting, I think that may be the way that DNA needs to be gathered, in contrast to the way it is now." This will have implications for health insurance, for example, which currently only pays for the patient to be tested.
And next-gen sequencing is likely to become a widely used diagnostic tool, he said. "I can imagine when people may have genomic sequencing done to screen for disease in some years. … If someone comes down with a medical condition, we'll be going back to that sequence and looking at it."
"It's a path to be gone down with some care, for sure," he said, due to ethical issues. "But I imagine that [next-gen sequencing] is going to be, eventually, at the very least a standard part of subspeciality medicine, if not a part of routine medical care."
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