Researchers at the Garvan Institute of Medical Research in Sydney and collaborators in Australia, the U.K., and Israel have developed a DNA test that studies suggest can identify a range of hard-to-diagnose neurological and neuromuscular genetic diseases faster and more accurately than existing methods. The diseases covered by the new test belong to a class of over 50 disorders caused by unusually long repetitive DNA sequences in a person’s genes—known as short tandem repeat (STR) expansion disorders.

Using a single DNA sample, usually extracted from blood, the test works by scanning a patient’s genome using Nanopore sequencing technology, from Oxford Nanopore Technologies (ONT). “We correctly diagnosed all patients with conditions that were already known, including Huntington’s disease, fragile X syndrome, hereditary cerebellar ataxias, myotonic dystrophies, myoclonic epilepsies, motor neuron disease, and more,” said Ira Deveson, PhD, head of genomics technologies at the Garvan Institute. “They are often difficult to diagnose due to the complex symptoms that patients present with, the challenging nature of these repetitive sequences, and limitations of existing genetic testing methods.” The team aims to start validations so that the test can ultimately be made available in pathology services around the world.

Deveson is senior author of the researchers’ reported study, which is published in Science Advances, and titled, “Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing.”

An STR is a short DNA sequence motif, typically 2 to 6 base pairs, repeated consecutively at a given position in the genome, the authors explained. STRs make up ~7% of the human genome sequence and are highly polymorphic, commonly varying in length between unrelated individuals. “Unusually long or ‘expanded’ STR alleles are an important class of pathogenic variants in human populations,” the team continued. “To date, STR expansions in more than 40 genes have been shown to cause heritable disorders, with the majority of these exhibiting primary neurological or neuromuscular presentations … With each of the >40 STR-associated neurogenetic diseases estimated to affect ~1 to 10 individuals per 100,000, their collective prevalence is high.” In fact, the list of disorders in which STR expansions have been implicated is growing, the team noted.

Kishore Kumar, MD [Garvan Institute of Medical Research]

Repeat expansion disorders can be passed on through families, and can be life threatening. However, current genetic testing for expansion disorders can be “hit and miss,” said Kishore Kumar, MD, a co-author of the study and clinical neurologist at Concord Hospital. “When patients present with symptoms, it can be difficult to tell which of these 50-plus genetic expansions they might have, so their doctor must decide which genes to test for based on the person’s symptoms and family history. If that test comes back negative, the patient is left without answers. This testing can go on for years without finding the genes implicated in their disease. We call this the ‘diagnostic odyssey,’ and it can be quite stressful for patients and their families.”

Ira Deveson, PhD, of the Garvan Institute of Medical Research [Garvan Institute of Medical Research]

Although repeat expansion disorders cannot be cured, a quicker diagnosis can help doctors identify and treat disease complications earlier, such as heart issues associated with Friedreich’s ataxia.

For their newly developed test, based on targeted long-read sequencing, the investigators harnessed ONT’s “ReadUntil” functionality, whereby, they explained, the ONT sequencing device can be programmed to recognize and accept/reject specific DNA sequence fragments during the sequencing experiment. “Target selection is fully flexible and requires no additional laboratory processes beyond standard library preparation. Here, we demonstrate that ONT ReadUntil can be used to achieve accurate molecular characterization of all known neuropathogenic STRs in a single assay.”

The Nanopore technology used in this newly developed test approach is also smaller and cheaper than standard tests. The team hopes that this will aid uptake into pathology labs. “With Nanopore, the gene sequencing device has been reduced from the size of a fridge to the size of a stapler, and costs around $1000, compared with hundreds of thousands needed for mainstream DNA sequencing technologies, commented Deveson.

Kumar continued, “We’ve programmed the Nanopore device to hone in on the roughly 40 genes known to be involved in these disorders and to read through the long, repeated DNA sequences that cause disease. By unraveling the two strands of DNA and reading the repeated letter sequences (combinations of A, T, G, or C), we can scan for abnormally long repeats within the patient’s genes, which are the hallmarks of disease.”

Deveson added, “In the one test, we can search for every known disease-causing repeat expansion sequence, and potentially discover novel sequences likely to be involved in diseases that have not yet been described.”

The team expects to see the new technology used in diagnostic practice within the next two to five years. One of the key steps towards that goal is to gain appropriate clinical accreditation for the method. Once accredited, the test will also transform research into genetic diseases, the authors suggested. “While the potential benefits for genetic diagnosis of patients with STR expansion disorders are clear, targeted STR sequencing will be similarly useful as a research tool. STRs are highly polymorphic and exhibit pathogenicity through an array of different mechanisms.”

Gina Ravenscroft, PhD, a co-author of the study and a researcher working on rare disease genetics at the Harry Perkins Institute of Medical Research, noted, “Adult-onset genetic disorders haven’t received as much research attention as those that appear in early life. By finding more people with these rare adult-onset diseases, and those who may be pre-symptomatic, we’ll be able to learn more about a whole range of rare diseases through cohort studies, which would otherwise be hard to do.”

The scientists commented, “ … by resolving STR expansions that are not amenable to existing techniques, long-read sequencing promises to accelerate the discovery of repeat expansion genes and disorders … Moreover, repeat expansions need not be the only pathogenic variants found by this approach, with targeted long-read sequencing also suitable for the detection of other types of structural variation. We anticipate that this will be a powerful approach to STR gene discovery and provide molecular diagnoses for many previously unsolved cases in the future.”

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