NEW YORK (GenomeWeb) – Researchers at Harvard University Partners HealthCare's Laboratory of Molecular Medicine have demonstrated that they can improve the diagnostic rate of a 70-gene hearing loss panel by ensuring that they are not inadvertently analyzing the nearly identical pseudogene of a critical gene on the panel.
The strategy can also be applied to other medically relevant genes on next-gen sequencing panels and exomes, according to the researchers.
In a study published in the Journal of Molecular Diagnostics, the researchers were able to increase the clinical sensitivity of their non-syndromic hearing loss panel from 20.4 percent to 31.6 percent by using long-range PCR to capture the STRC gene and subsequently avoid capturing its pseudogene, pSTRC.
Birgit Funke, director of clinical R&D at the Laboratory of Molecular Medicine, told Clinical Sequencing News that the group has since examined the entire exome to see what medically relevant genes also have pseudogenes that could complicate identifying variants, and discovered around 300 genes affected by homology issues "to the extent that we believe it might inhibit proper analysis."
Even well-known genes can be affected by homology, Funke said; for instance, the Lynch syndrome gene, PMS2, which the American College of Medical Genetics recommends specifically testing for and reporting back to physicians that order diagnostic exomes or genomes. The VWF gene, which is associated with the blood clotting disorder von Willebrand's disease, also has a pseudogene.
When next-gen sequencing finds a variant in one of these genes, said Funke, it is currently very difficult to know whether the variant is in the actual disease-related gene or its pseudogene, because the genes are so similar. Conversely, even if sequencing does not find a variant, it could be just because the test sequenced the pseudogene.
"That just illustrates the importance of being able to deal with these properly," Funke said.
In the case of the STRC gene, it is 98.9 percent identical to its pseudogene, and more than 99 percent identical across the coding sequence. In addition, between 1 percent and 1.6 percent of the general population carry a heterozygous deletion in the gene, making it one of the largest contributors to autosomal recessive non-syndromic hearing loss, the authors wrote.
As an example of the problem, Funke said that the NGS assay had a false positive rate for SNVs in the STRC gene of around 18 percent. "That's way higher than what we typically find for SNVs," she said, which is usually around one in 1,300.
To counter this, the researchers designed a long-range PCR assay that could be combined with either traditional Sanger sequencing or next-gen sequencing. The long-range PCR assay amplifies the entire coding region of STRC using primers in the gene's flanking regions that had difference sequence from the pseudogene. The researchers confirmed that their assay targeted the STRC gene and not the pseudogene by evaluating allelic fraction. For instance, in a sample with a known heterozygous deletion to STRC, the NGS test without the long-range PCR assay found an allelic fraction of 0.69, while the long-range PCR assay yielded the expected allelic fraction of 1, showing that it successfully eliminated pseudogene contamination.
In addition, in order to confirm STRC copy number variants identified by next-gen sequencing, the team performs a droplet digital PCR reaction for the gene. Results are then combined for a final STRC genotype.
To evaluate the clinical effect of STRC analysis, the researchers analyzed 153 patients that came to the Laboratory of Molecular Medicine for hearing loss genetic testing. Of those, 98 had isolated non-syndromic hearing loss. The NGS panel, not including STRC analysis, identified the pathogenic variant in 20 of the 98 patients, or 20.4 percent. Then on the remaining negative or inconclusive cases, the researchers performed a copy number analysis of STRC, identifying biallelic alterations in 11 cases.
Funke said that the problem of pseudogenes will increase as laboratories move toward exome sequencing as a primary method for diagnosing genetic conditions. In addition, she said, in the past researchers often had expertise on the handful of genes that they tested, so they knew the various challenges each gene posed.
Now, however, "as we scale to exome sequencing, we're losing that knowledge," she said. "So we need something simple in place that we can utilize for all these genes" with homology issues, she said.
Currently, Funke said the lab is running the STRC assay as an add-on to the panel, but she envisions a more efficient way that would address the problem more comprehensively — running in parallel an assay that targets the top medically relevant genes with homology issues. Within the 70-gene hearing loss panel, another gene, OTOA, has homology issues, she said, although it is not quite as problematic as STRC. Homology issues tend to result in poor mapping for about 24 percent of the coding sequence of OTOA, while the mapping quality of STRC is "near zero," the authors wrote.
Nonetheless, Funke said that better analysis of OTOA might increase the assay's diagnostic rate even further, although not up to 100 percent. "The missing detection rate has multiple reasons," she said, including not knowing all of the disease-causing genes, not being able to detect the right variant type, not sequencing the regulatory regions, or the existence of variants in problematic genes.
Funke has also been involved in another effort to address the challenges associated with diagnostic sequencing. In a collaboration that includes Funke, Emory Genetics Laboratory's Madhuri Hegde, and Avni Santani of the Children's Hospital of Philadelphia, the team has been working on designing a "medical exome" to boost coverage of medically relevant genes. EGL has since been offering the medical exome test clinically, although at the time Hegde said that there were still problematic regions, particularly in pseudogenes.