This story was published on June 28.
Exome sequencing in patients with heterogeneous genetic disorders currently yields a diagnosis between 20 percent and 50 percent of the time, researchers from the genetics department at Radboud University Nijmegen Medical Centre in the Netherlands reported last week.
At the European Society of Human Genetics annual meeting in Nuremberg, Germany, Marcel Nelen, head of the department's Core Genome Analysis Facility, presented results from the first 460 exomes analyzed through the center's diagnostic exome sequencing pipeline.
The project, which includes patients with movement disorders, hereditary blindness, hereditary deafness, oxidative phosphorylation disorders, and colorectal cancer, started last fall (CSN 10/19/2011). Each of these disorders was chosen because they are highly heterogeneous, meaning mutations in one of many different genes can cause the disease.
According to Nelen, one of the main questions the project was designed to answer is the diagnostic yield of an exome sequencing test. While single-gene tests based on Sanger sequencing are very sensitive and specific, for heterogeneous disorders, they are unlikely to lead to a diagnosis, so many separate tests are necessary. The promise of exome sequencing is that it "will give you a much higher diagnostic yield in a single test," Nelen said.
His team has set up a diagnostic exome sequencing pipeline that uses the Agilent SureSelect capture method, combined with sequencing on the Life Technologies 5500xl, of which the facility has three instruments. Last December, the diagnostic workflow was accredited by the Dutch Accreditation Council.
For all disorders except intellectual disability, the researchers analyze the patient's exome and restrict their analysis to so-called "gene packages" of known disease genes, ranging from about 100 genes to more than 200 genes per disorder. This focused analysis avoids incidental findings, Nelen said.
For intellectual disability, they employ a different strategy, sequencing the exomes of patient-parent trios and looking for de novo variants that have arisen in the patients.
If the analysis of the "gene packages" is able to establish or confirm a genetic diagnosis, a report is written that is discussed in a biweekly "patient meeting" that includes staff from the genetics department, clinicians, and members of the research center, before it is sent out to the treating physician.
If the researchers cannot find a diagnostic mutation, they report the result as negative. As a next step, they open up the entire exome for analysis, which may identify candidate genes that are also discussed in the biweekly meeting. Those candidates require further validation, though, to prove that they are causative.
Analyzing the entire exome may also lead to incidental findings, Nelen noted, which are discussed at the patient meeting as well. If the participants agree that an incidental finding needs further attention, a special committee convenes that includes clinicians, ethicists, a lawyer, and geneticists. If that committee concludes that the patient should be informed, it reports its recommendation to the patient meeting and a letter goes out to the treating clinician. Patients are told about the possibility of incidental findings as part of the informed consent process and agree to receive these results.
For disorders other than intellectual disability, the researchers have analyzed the exomes of about 160 patients so far. As a quality standard, they require a coverage of at least 30x, though the median coverage of 67 reads per target is much higher, Nelen said. They also perform a SNP test as well as a gender test to make sure the exome data from a sample belongs to a certain patient.
Not every exon is covered equally well by the test: for many disease genes, for example, exon 1 is not well covered, and some exons are missing entirely. Overall, though, the test is doing "a pretty good job," sequencing at least 90 percent of all targets with a coverage of 10x or higher, he said.
To analyze the exome data, the researchers have developed a graphical user interface that allows them to home in on the known disease genes, or the "gene package," for each disorder. They then apply a number of filter steps that are predefined but can also be applied manually. For example, these filters can select variants present with a frequency of less than 5 percent in dbSNP, non-synonymous variants, variants in canonical splice sites, and variants with a frequency of less than 1 percent in the center's in-house database.
In one patient, Nelen showed, exome sequencing delivered 41,000 variants, of which 504 were in the known disease gene, which filtering reduced to 10. Each potentially causative variant is then checked by Sanger sequencing, which reduces the number of variants further, to between zero and four per patient, Nelen said.
Overall, the diagnostic yield was highest for blindness, where 44 percent of patients could be diagnosed by the test. For deafness, movement disorders, and oxidative phosphorylation disorders, the yield was about 20 percent each.
No causal mutations were identified for any of the cancer patients, which could be explained by the fact that they had already undergone all standard testing prior to exome sequencing, and those with a positive test result had been removed.
The next step will be to "open up the exome" of those patients where the focused approach did not lead to a diagnosis, "which is expected to improve both yield and content of the gene packages," Nelen said.
The researchers also analyzed 100 patients with severe intellectual disability who did not have a disease syndrome or a family history of the disease, and who had a normal karyotype and genomic profile as assessed by SNP arrays.
The researchers generated at least 150 million reads per patient, equivalent to a median coverage of 30x. To rule out any sample mix-up, they also made sure the patient's array data matched the sequencing data, and they did a "trio check" on the parent and patient data to ensure they are members of the same family.
In 47 of the 100 patients, they did not find any de novo mutations. In each of the others, they found up to four de novo mutations, among them 10 novel variants in known intellectual disability genes, three variants in X-linked disability genes, three mutations in novel intellectual disability genes that they were able to confirm in other patients, and 19 variants in candidate genes.
"The striking thing is that we identified quite a number of known intellectual disability genes," Nelen said. As a result, they have now implemented a gene package for intellectual disability as well.
Nelen said that going forward, he and his colleagues are considering developing new gene packages for other diseases.
The Radboud University team's study is among the first to look at the diagnostic utility of exome sequencing in a cohort of patients, rather than individual cases.
Earlier this month, a group led by the University of California, San Diego, used exome sequencing in a group of patients with unexplained neurodevelopmental disorder. In almost 10 percent of patients, they found mutations in known disease genes that had been missed by prior tests, leading to a change in their diagnosis (CSN 6/20/2012).