NEW YORK (GenomeWeb) – A clinical lab at Newcastle University Medical School has converted its Sanger-based tests for a mitochondrial disorder called complex 1 deficiency disease to next-generation sequencing, a move that has enabled the lab to screen many more genes and improve on its diagnostic rate.
Following several research studies, including a publication in the Journal of the American Medical Association that found exome sequencing could help diagnose mitochondrial disease, and a more recent publication in the American Journal of Human Genetics that validated the TMEM126B gene as playing a role specifically in complex 1 deficiency, the laboratory is now offering a clinical NGS test that includes around 50 genes to patients suspected of having complex 1 disorder.
Charlotte Alston, lead author of the recent AJHG study and an associate researcher at Newcastle University, told GenomeWeb that the lab has been offering its clinical NGS gene panel for the last 10 to 12 months. For patients who do not receive a diagnosis via the gene panel, she said, researchers can use exome sequencing to analyze samples from consenting families. Those exome sequencing results can be used both for research and diagnostic purposes, she said. If a causative mutation is found and can be validated and tested for and identified by a clinical lab, it can be reported back to the patient's physician.
Eventually, Alston said, the goal is to move from the gene panel to an exome-based test, but that would not happen in the near term due to cost and turnaround time. The lab runs its current gene panel test on Thermo Fisher Scientific's Ion Torrent PGM, which has a turnaround time of 10 to 12 weeks, although actual turnaround times are often much quicker, Alston said. For urgent cases it can be as short as a few days, she added.
Complex 1 deficiency is the most common mitochondrial disorder, but can be hard to diagnose due to the variability in how it presents in patients. This variability can be explained by the various structural units that make up complex 1 — currently there are 44 known structural subunits and 10 known assembly factors. However, researchers believe there are still additional secondary proteins that play a role in assembling complex 1.
In the recent AJHG study, the researchers reported on using both the gene panel as well as exome sequencing to identify variants in the TMEM126B gene, and then a variety of functional and proteomic methods to validate the gene's role in complex 1 deficiency.
The researchers evaluated six individuals from four unrelated families with suspected mitochondrial disorder but without a molecular diagnosis. The individuals had various clinical presentations including exercise intolerance, muscle weakness, kidney failure, and retinitis pigmentosis. Five individuals were adults at the time of the study and one was 6 years old. Two were wheelchair bound.
Researchers and clinicians performed biochemical, histochemical, and molecular analyses for each individual, establishing first that they all likely had a mitochondrial disorder, which they narrowed down to a suspected complex 1 deficiency. The final molecular diagnosis was reached through a targeted AmpliSeq panel for two individuals and through exome sequencing for three individuals. All had mutations in the TMEM126B gene. In addition, all six individuals had one of two genotypes: a specific homozygous missense variant or the same compound heterozygous mutations.
The researchers confirmed that those mutations were indeed pathogenic through functional studies in cell lines as well as by doing complexome profiling — a tool that analyzes assembly of mitochondrial proteins.
Prior to offering the NGS panel, Alston said that these six individuals may not have ever received a molecular diagnosis. And, although there is no treatment for complex 1 deficiency, a molecular diagnosis can enable the parents to seek testing, especially helpful if they want to have additional children. "That's the power of having a genetic diagnosis," Alston said. "You know what to test for."
The lab can offer such testing both prior to conception or as a prenatal test, Alston said, and patients are then offered genetic counseling to discuss their options.
Alston said that the Newcastle diagnostic lab runs around 20 of its complex 1 deficiency panel tests per month, and that the diagnostic rates have improved dramatically, compared to when the lab only offered Sanger-based testing. "It's more than tripled our detection rate," she said.
The lab is able to diagnose about 20 percent of cases with the panel test. For samples that are then consented for exome sequencing in a research setting, they find the molecular cause about half of the time. For cases that receive research-based exome sequencing, results must be confirmed in a clinical setting before being returned to the physician, Alston said.
Although exome sequencing tends to have a higher diagnostic success rate, Alston said that it would still be some time before the lab transitioned to offering exome sequencing as a clinical test.
Cost is still a big factor, she said. The UK's National Health Service funds the lab's diagnostic testing service and the lab must meet certain requirements set by the NHS for its tests. For instance, Alston said, when it switched from Sanger sequencing to NGS, it had to ensure that the test would have an equivalent cost and turnaround time. Aside from the cost of running the test, the lab would need to invest in a higher throughput sequencer. Currently, it operates on PGM, which does not have enough throughput to run exomes.
Nonetheless, Alston said that in the future, she envisions a clinical exome test that would use bioinformatics to analyze only a subset of genes. For instance, she said, the set of 50 genes that make up the current diagnostic panel could be analyzed first, with additional suspected genes analyzed if a diagnosis was not found. "That would allow us to incorporate additional genes [into the initial analysis] as they were validated, but still on a targeted basis," she said.