NEW YORK (GenomeWeb) – A reference laboratory in Ontario, Canada has validated a next-generation sequencing gene panel to test individuals at high risk of hereditary breast, ovarian, colorectal, and gastric cancer and will use the panel to screen patient samples throughout Ontario.
In a study published last month in the Journal of Molecular Diagnostics, the researchers described the validation of a BRCA1 and BRCA2 test that uses NGS, showing that the technology can detect not only point mutations but also copy number alterations without requiring a secondary test. Since that initial publication, the lab has also validated a 25-gene hereditary cancer panel.
Bekim Sadikovic, head of molecular genetics at Western University in Ontario, told GenomeWeb that the laboratory is licensed by the Ontario Ministry of Health to provide hereditary cancer screening in the province and began offering the cancer panel earlier this year.
Previously, he said, the BRCA1 and BRCA2 genes were either analyzed via Sanger sequencing or would be sent to laboratories in the US for NGS-based testing. The Western University clinical lab began developing its own targeted NGS panel as part of an effort by the Canadian government to "repatriate genetic testing," as well as to make genetic testing "more cost-effective, sensitive, and higher throughput," Sadikovic said.
For its first genetic test offering, the lab focused on hereditary cancer risk. In the recent study, it validated mutation testing in the BRCA1 and BRCA2 genes, using targeted capture probes from Roche's NimbleGen and performing sequencing on Illumina's MiSeq instrument.
The panel covered 46 exons and some intronic regions in the BRCA genes. To validate the panel, the team analyzed 402 retrospective samples, comparing the NGS panel with previous Sanger sequencing and multiplex ligation-dependent probe amplification results. The researchers also ran the assay on 240 prospective patient samples.
The researchers multiplexed 24 patient samples in one NGS run and generated a mean depth of coverage of 6,500 reads per nucleotide per patient. In the retrospective cohort, the NGS panel identified 95 different variants, including intronic heterozygous SNPs, intronic homozygous SNPs, coding heterozygous SNPs, coding homozygous SNPs, deletions, insertions, and exon deletions/duplications. All variants that had previously been detected by Sanger sequencing and MLPA were also detected by NGS. In addition, the NGS panel detected five variants outside of the region analyzed by Sanger that were subsequently confirmed, demonstrating 100 percent sensitivity.
In the 240-patient prospective cohort, the NGS panel detected 101 variants that were classified as either pathogenic, likely pathogenic, or variants of unknown significance using the American College of Medical Genetics and Genomics guidelines. All variants were confirmed using Sanger or MLPA, demonstrating 100 percent specificity, the authors reported.
Since the researchers performed the validation study, they have expanded the panel to interrogate not only the BRCA1 and BRCA2 genes, but a total of 25 genes involved in risk of hereditary breast, ovarian, colorectal, and gastric cancer.
Sadikovic said that the NGS panel has helped reduce turnaround times for BRCA testing, which previously were either tested via Sanger or were sent to US labs for NGS-based testing, from several months to a few weeks. In addition, he said, the panel is a fraction of the cost of non-NGS testing. Previously, analyzing the BRCA1 and BRCA2 genes would have cost around C$1,500 ($1,152) using Sanger sequencing and MLPA, but that two-gene test has been replaced with a 25-gene panel, which costs C$890 ($684), Sadikovic said. The tests are all reimbursed by the provincial ministries of health and patients do not pay out of pocket.
One unique component of the lab's test is that it does not have to perform any parallel testing to detect copy number variants, Sadikovic said. Copy number variants are often difficult to detect with targeted sequencing protocols, and many labs run parallel tests in conjunction with NGS panels to screen for them, including MLPA, array CGH, or PCR. However, it is possible to detect CNVs from targeted sequencing panels as long as the depth of sequencing is high enough and coverage is uniform, Sadikovic said.
A key to detecting CNVs from targeted gene panels is the sample prep process, he said, adding that the lab spent considerable effort custom-designing its target capture using the NimbleGen probes, rather than relying on an off-the-shelf kit.
Aside from not having to run multiple tests, the NGS panel has an advantage over MLPA in CNV detection in that it is not susceptible to allele dropout, Sadikovic said. He added that his group is planning to submit a separate study to a journal that will focus specifically on the ability of its NGS assays to detect CNVs.
Aside from hereditary cancer, the lab also provides NGS-based testing for mitochondrial disorders and Charcot-Marie-Tooth disease for patients in the province of Ontario, and it is developing a clinical exome sequencing test that it plans to launch this fall.
Similar to the BRCA1 and BRCA2 test, Sadikovic said, these other panels are also able to detect CNVs without the use of an alternate test. In their recently published study, the researchers reported that its Charcot-Marie-Tooth disease panel was able to detect the deletion or duplication of the PMP2 gene, which is "the most common mutation" found in the disease and had previously required "stand-alone MLPA prescreening of all patients" suspected to have the disorder, the authors wrote. Similarly, the mitochondrial genome panel "demonstrated the ability to sensitively detect the common 5-kb deletion associated with the Kearns-Sayre syndrome," they noted.
Since reporting the results of its BRCA1 and BRCA2 validation, Sadikovic said, the group has developed nine different gene panel tests and has assessed around 3,000 patient samples.