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New DNA Test Easily Diagnoses Difficult-To-Detect Mutations

By Deborah Borfitz

April 26, 2022 | Researchers in Australia played a leading role in development of a DNA-based test to catch what short-read Illumina sequencing often misses: long and repetitive genetic variants implicated in about 40 late-onset neurodegenerative and neuromuscular conditions. These highly complex short tandem repeat (STR) expansion disorders, which have proven difficult to profile with any technology, can now be handily diagnosed with using the Oxford Nanopore Technology (ONT) gene sequencing platform costing around $1,000, according to Ira Deveson, Ph.D., head of genomics technologies at the Garvan Institute of Medical Research in Sydney.   

In lieu of sequencing the whole genome, the targeted and programmable approach uses the platform’s ReadUntil function allowing for parallel genotyping of all known neuropathogenic STRs at the same time. Unlike previous targeted sequencing methods, Deveson points out, no laboratory process is needed to select for the 37 genes underpinning the STR expansion disorders, and any other genes of interest can be easily added in at no extra cost simply be amending a text file. “It’s a pretty crazy feat of engineering.”

Per a study newly published in Science Advances (DOI: 10.1126/sciadv.abm5386), the assay correctly found all pathogenic STRs when used on 37 patient-derived DNA samples, including those associated with Huntington’s disease, Fragile X syndrome, hereditary cerebellar ataxias, myotonic dystrophies, myoclonic epilepsies, and motor neuron disease. As a demonstration of the test’s flexibility, the researchers also added 28 pharmacogenomic genes to the panel as secondary targets providing additional clinical insights, notably an individual’s capacity to metabolize specific drugs and prevent adverse drug reactions.

All the necessary software, other than ONT’s based-calling software Guppy, is free and open source. “We hope labs around the world at least try it out,” says Deveson.

More than 50 diseases involve STR expansion mutations, he notes. The study focused only on those causing neurodegenerative and neuromuscular disorders.

An efficient and effective means of diagnosing STR expansion disorders is an unmet clinical need globally, so demand for the assay is presumably everywhere, he adds. Besides the nature of the repeat mutations, the challenges include the fact that patients present with progressive and often overlapping symptoms (e.g., tremors and muscle weakness) and existing genetic testing methods are all hit-and-miss.

To exemplify the shortcomings of the gold-standard diagnostic approach, the researchers pitted short read whole genome sequencing against ONT in identifying STR expansions in the RFC1 gene responsible for a neurodegenerative movement disorder called cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). “The short reads couldn’t properly resolve the repeat expansions,” Deveson reports.

Given the inadequacy of the mainstream method, the only option up to now has been one-at-a-time testing of each of the 37 genes known to underpin neurodegenerative and neuromuscular conditions caused by STR expansions, he says. “The real selling point of our assay is that you can test all these different repeat expansion genes, and other genes if you’re interested, in a single assay.”

The only STR expansion disorders that are likely to be missed are those caused by yet-to-be described genes, says Deveson. The number of these new-gene discoveries has been growing quickly over the last few years—and many more such breakthroughs are expected, given the volume of studies underway around the world. But some of the described disorders are so alike that they are sometimes considered one in the same.

Better Outcomes

The diagnostic odyssey for patients with STR expansion disorders can last months if not years or even a lifetime, says Deveson. While these conditions have no cure, an accurate diagnosis could help patients avoid unnecessary biopsies for diseases they don’t have or risky treatments that compromise their immune system, as well as enable any disease complications to be clinically managed.

It is anxiety-relieving for patients when they have answers and a diagnosis can lead to improved clinical outcomes for themselves and, potentially, family members who may have inherited the disorder but have yet to experience symptoms, he adds. For family planning purposes, patients may also want to know how likely it is that a particular genetic mutation could be passed on to their children.

The test could potentially be applied to people of any age, including children to identify their predisposition to developing one of the 40 or so STR expansion disorder later in life, says Deveson. But he believes it will primarily be used to test adults who present with symptoms and perhaps their family members.

In collaboration with NSW Health Pathology, which operates more than 60 clinical labs in Australia, researchers now plan to go through the multi-year process of validating the technology and bringing the test into diagnostic practice through accreditation by the National Association of Testing Authorities, the country’s equivalent to the Clinical Laboratory Improvement Amendments (CLIA) process in the U.S., he says.

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