NEW YORK (GenomeWeb) – Researchers at Harvard University's Ocular Genomics Institute have found that for diagnosing inherited eye disorders, a gene panel may be better than an exome test, even though exome sequencing is more comprehensive and is more likely to identify novel mutations and disease genes.
In a clinical validation study of the panel published recently in Genetics in Medicine, the group found that it is more sensitive than doing a whole exome.
Eric Pierce, director of the Ocular Genomics Institute, told GenomeWeb that the group began to look into genetic testing for inherited retinal disorders several years ago because there were promising gene therapies for the disease in clinical trials.
"We wanted to develop a cost effective and efficient method of genetic diagnostic testing for these patients," he said. At the time, exome sequencing was too expensive, so they decided to try a targeted panel and "quickly realized that we liked it better than exome [sequencing] for diagnostic testing and we went on to try to perfect it."
The panel, dubbed GEDi, contains 214 known genes that cause inherited retinal disease, eight early-onset glaucoma and optic-atrophy genes, 24 candidate IRD genes, nine age-related macular degeneration genes, and one nonsyndromic hearing loss gene.
Overall, Pierce said the lab has tested around 250 clinical samples and is now receiving several dozen per month. The overall diagnostic rate is just over 50 percent. In a subset of 50 families for which GEDi did not find an answer, the lab has conducted exome sequencing. In addition, Pierce said that the group recently received funding to do whole-genome sequencing and transcriptome analysis in a handful of families to try and identify new causes of the disease.
In its validation study, the group first analyzed four samples: three randomly chosen patient samples and one HapMap sample. The samples were sequenced in triplicate on three separate days. The investigators also genotyped the four samples using the Omni 2.5 array, which served as their gold standard for assessing accuracy, and did exome sequencing on the samples with an Agilent kit.
To assess sensitivity of GEDi, they compared the 2,443 SNPs that overlapped between the panel and the SNP array and found sensitivity was between 96.4 percent and 97.9 percent while specificity was between 99.9 percent and 100 percent. Overall, accuracy for calling both SNPs and indels was 99.9 percent.
In addition, GEDi was more sensitive than exome sequencing, which had a sensitivity of 88.3 percent, primarily because it failed to call a number of variants due to low or no coverage at those positions.
"There were not probes for about 10 percent of the variants we used for our quantitative analyses," Pierce said.
The researchers next went on to evaluate GEDi in 192 clinical patient samples and found genetic diagnoses for 98 of them, for a diagnostic rate of 51 percent.
While the majority of the diagnoses were consistent with the patient's clinical presentation and family history, two without a family history had mutations in known dominant genes, suggesting that they were de novo. Dominant mutations in those genes, PRPH2 and IMPDH1, had not previously been reported.
The test also missed diagnoses in five individuals who were later diagnosed. In two cases, the panel identified a single potentially pathogenic variant, but a second variant was initially not detected. Subsequent Sanger sequencing in each case found "deep intronic mutations known to alter splicing."
Pierce said that the lab is now updating its test to include that region as well as about a dozen other new disease genes that his team and other investigators have identified over the last year and he thinks those additions will help bump up the diagnostic rate to between 55 percent and 60 percent.
He added that since conducting the study, his team has done copy number analysis and exome sequencing in about 50 families that did not originally receive a diagnosis from the GEDi test, and exome sequencing did uncover several novel mutations; however, half of those patients still did not receive a diagnosis.
Pierce said that the lab recently secured funding to do whole-genome sequencing and transcriptome analysis in three patients for whom the panel, exome sequencing, and copy number analysis all failed to identify the molecular cause of the disease. Pierce said the team is calling it the "Elusive Genes Project," and that it will do whole-genome sequencing and transcriptome analysis of induced pluripotent stem cells from those patients to both look for noncoding variants and gene expression alterations.
Improving the diagnostic rate
Other groups have similarly found that for certain conditions where many disease genes are already known, panels are more successful than exomes. For instance, Madhuri Hegde, executive director of Emory Genetics Laboratory, has previously told GenomeWeb that especially for very specific phenotypes, gene panels often have a higher diagnostic rate. EGL's neuromuscular panel has a diagnostic rate of 63 percent and its eye disorders panel has a diagnostic rate of 71 percent, she said at the time.
To bridge the discrepancy between panels and exomes while also taking advantage of exome sequencing's ability to uncover novel mutations, Hegde, along with collaborators from Harvard University and Children's Hospital Philadelphia, designed a so-called medical exome, which provides enhanced coverage in about 4,600 medically relevant genes.
The company Personalis has also taken a similar approach, enhancing coverage of over 8,000 genes in its ACE Clinical Exome. Both designs were done as means to improve on the diagnostic rate of standard clinical exome sequencing, which has been hovering in the 25 percent to 30 percent range.
Pierce said that his group has not tested any of the enhanced exome sequencing approaches. He noted, however, another advantage of panel testing over exome sequencing is cost. In the study, the researchers reported that the cost of reagents for the panel test was around $430, while for exome sequencing it was $1,325. The actual clinical test is substantially more expensive, Pierce said, currently costing around $2,500 for GEDi, which also includes Sanger validation, confirmation in family members, and testing for segregation.