The Radboud University Medical Centre in the Netherlands is expanding its diagnostic exome sequencing program further, reflecting a more routine use of the technology for the diagnosis of genetically heterogeneous single-gene disorders.
So far, the Nijmegen-based center has completed approximately 1,000 diagnostic exomes in total – many of them patient-parent trios – and plans to sequence about 2,000 exomes in 2013 alone. The center is in a unique position because the Dutch government only allows academic hospitals to perform diagnostic genetic testing in the country, so there is no commercial competition.
To complement its in-house sequencing facility – consisting of three SOLiD 5500xl instruments, one of them owned by Life Technologies, and two Ion Torrent PGMs − the center is currently evaluating services from BGI-Europe in Copenhagen. Longer term, it plans to beef up its in-house capacity and is looking into several options for doing so.
Besides diagnosing inherited diseases, the center has been developing methods for non-invasive prenatal testing, which it plans to launch as soon as the Dutch government grants permission for NIPT in the Netherlands, expected later this year.
While the major patient group for the center's diagnostic exome test continues to be children with sporadic intellectual disability (CSN 10/3/2012), the test is now offered for a growing number of other disorders, including deafness, blindness, neurological disorders, movement disorders, mitochondrial diseases, renal disease, multiple congenital abnormalities without intellectual disability, hereditary cancer, and hereditary immune disorders.
In addition, the group recently started providing exome testing to patients with undiagnosed diseases.
"For the first 1,000 exomes, we often did patients that we had already seen before, but now it is getting more routine," with some new patients undergoing exome sequencing as a first-tier test instead of Sanger sequencing, said Joris Veltman, a professor of translational genomics in the department of human genetics at Radboud UMC.
Intellectual disability is an exception to that because patients currently still receive an array-based test for copy number variations first as the diagnostic yield of that test, 10 percent to 15 percent, is high. Patients that do not receive an array-based diagnosis go on to exome sequencing.
While the Radboud group recently showed that CNVs can also be determined from exome data, it has not replaced the diagnostic arrays yet, mainly because its exome sequencing capacity is too small at the moment.
The diagnostic yield from exome sequencing ranges between about 20 percent to 50 percent, depending on the type of disease and whether or not a patient has already undergone other genetic testing.
Veltman's team has found that for many diseases, the diagnostic yield is significantly higher than from routine Sanger testing, a study that is to be published soon. "The coverage is not perfect, you still miss mutations, but the overall diagnostic yield is much higher than what we see with traditional approaches," he said.
Diagnostic yield has been lower for certain types of inherited cancer, though, where the number of possible genes involved is apparently smaller than for other diseases. For those, the center is evaluating whether to switch from exome sequencing to a targeted gene panel, even though the exome could help discover novel cancer genes.
The center already offers gene panels on the Ion Torrent PGM for a number of diseases, including a breast cancer screening test for BRCA1 and BRCA2 (CSN 2/27/2013) and a 10-gene panel for Noonan disease.
As the number of patients eligible for exome sequencing has grown, the center's sequencing capacity has become "one of the major bottlenecks," Veltman said. "We're constantly looking for faster sequencing, better sequencing quality, and cheaper sequencing."
Because its three SOLiD machines no longer meet the demand, regarding both throughput and cost, the center recently started evaluating sequencing services from BGI-Europe in Copenhagen, which has done 100 exomes for it so far.
However, outsourcing will remain an interim solution because Dutch health insurance companies, which cover the exome test, require diagnostic tests to be performed in house. "Our goal is to move this back to Nijmegen as soon as possible," Veltman said. One possibility is to set up a joint laboratory with BGI in Nijmegen, he said, while another one is to purchase new sequencing equipment.
Despite successful diagnoses, Veltman's team, like other groups, has found that exome sequencing is incomplete, missing mutations even within the target regions of the exome.
The center currently uses Agilent's SureSelect to capture the exome and sequence the DNA with about 60x to 65x coverage on the SOLiD instrument. For a research project, they followed up 150 patients with negative exomes by whole-genome sequencing, which was performed by Complete Genomics, and found that the exome test had missed several variants within the exome.
Long term, the center would therefore like to switch to whole-genome sequencing, Veltman said, but this will take at least a couple of years because of the high cost and large amounts of data involved. "You would need to set up a very different infrastructure" he said. "With the exome, we can organize the data analysis within our hospital − it's not easy, but we can do this. If you go to whole-genome sequencing, there is another 100-fold increase in data, and that's challenging."
Consent required for incidental findings
To avoid incidental findings, the center focuses its initial data analysis on known disease genes and only considers the entire exome if no variants of interest are found. This is not true for intellectual disability, however, where they look for de novo mutations anywhere in the exome right from the start.
Patients, after receiving pre-test counseling by a clinical geneticist, need to sign a consent form in which they acknowledge the possibility of incidental findings and agree to receive them.
If patients do not want to receive any incidental findings, they currently cannot receive the exome test. But the center is in the process of changing its informed consent procedure, which will allow patients to opt for receiving mutations in disease genes related to their phenotype only. The remainder of the exome will not be analyzed in those patients. "It is clear that exome sequencing has a lot of advantages for people, so we do want to give them the opportunity to do exome sequencing if they do not want to have incidental findings," Veltman said.
According to Ilse Feenstra, a clinical geneticist at Radboud UMC, whether or not patients or their parents agree to receive incidental findings often depends on the type of disease. "If it's a severe disease, like severe intellectual disability, parents really want to know, it doesn't matter what it is," she said.
"It really differs between patients. Some say, 'I don't want to know anything,' and I see other patients who say 'I want to know everything.'"
While the American College of Medical Genetics and Genomics issued guidelines on incidental findings earlier this year in which they recommended the return of mutations found in specific genes (CSN 5/18/2013), European labs tend to value patient autonomy more highly, Veltman said. "If the patient only wants to know about his or her disease cause, then he has a right not to know their future or risk of other diseases."
A requirement to check for mutations in genes unrelated to the patient's phenotype would also be a task for which the center is currently unprepared. "Our system is not ready for that," Veltman said. "First of all, our clinical geneticists have no expertise in adult-onset diseases, and the risk for those. So, we would really need to change that." In addition, the extra time required for the analysis would currently not be reimbursed by insurance.
Nevertheless, the center might decide in the future to start scanning the exome data for mutations in genes that are linked to treatable diseases, he said.
So far, the number of incidental findings at the UMC has been small — only two in 1,000 exomes. One was a mutation in a cancer gene, the other the presence of an additional Y chromosome, which was discovered during a quality control test.
In cases of incidental findings – mutations that are likely pathogenic – an independent committee of experts convenes, which includes a clinical geneticist, a lawyer, an ethics expert, and a social worker, to discuss the result before it is reported back to the clinical geneticist who ordered the test.
Widening the reach of diagnostic NGS
The center has also developed methods for non-invasive prenatal testing for trisomies and X-chromosomal disorders on the SOLiD system and is "ready to go" with NIPT testing, in collaboration with its gynecology department, though not for large numbers of patients due to its limited sequencing capacity.
The Dutch government has not yet granted permission for NIPT, which it regards as a screening test, to any laboratory in the Netherlands. In addition, Dutch laboratories are not allowed to refer pregnant women to international providers of the test.
According to Veltman, permission from the government is expected later this year, at which point the center plans to start offering NIPT for high-risk pregnancies where the conventional combination test suggests a high risk of disease.
Reducing the cost and increasing the throughput of sequencing will be required to make diagnostic exome testing more pervasive. "That is a major bottleneck at this moment to get this really established as a standard test," Veltman said. "We now talk about 2,000 exomes per year — in a few years' time, we will laugh about that."
The test, which has a turnaround time of about three months to four months, is currently reimbursed by Dutch health insurance companies at a standard rate of about €700 ($940), the same reimbursement centers receive for other molecular diagnostic tests, be it a single-gene test or a diagnostic array.
This is one of the reasons why the Radboud center remains the only one in the Netherlands offering diagnostic exome sequencing routinely, Veltman said. So far, the group has been subsidizing the test, which has an all-in cost of close to €1,500 when performed on the SOLiD platform, with internal funding. "That's something you can do for a year or so, but you can't do that for much longer," he said.
Most other Dutch centers also do not have the bioinformatics capacity and expertise to analyze exome data at present, focusing on targeted next-gen sequencing panels instead, though that might change in a few years. "Our group is the largest department of human genetics in the Netherlands and we have a lot of research involved," Veltman said. "This requires a lot of bioinformatics, which most groups simply don't have."
To take advantage of this, the group has also started to offer its data analysis expertise to others. As part of a pilot study, it is interpreting exome data provided by outside groups. "That's something we see as a goal, that we are the experts in the interpretation of all genetic disease, and people can come to us and we can help them with the interpretation," Veltman said.