Stevanovski I, Chintalaphani SR, Gamaarachchi H, Ferguson JM, Pineda SS, Scriba CK, Tchan M, Fung V, Ng K, Cortese A, Houlden H, Dobson-Stone C, Fitzpatrick L, Halliday G, Ravenscroft G, Davis MR, Laing NG, Fellner A, Kennerson M, Kumar KR, Deveson IW. Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing. Sci Adv. 2022 Mar 4;8(9):eabm5386. doi: 10.1126/sciadv.abm5386. Epub 2022 Mar 4. PMID: 35245110.
More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care.
Depending on where they occur, unusually long forms of these repeating sequences can drive neurological or neuromuscular degeneration. Yet because there are 37 known genes that are linked to short tandem repeat disorders, it can take multiple tests before identifying the ones responsible for an individual's symptoms.
For patients like John – one of the participants involved in a new study – it can take over a decade to whittle down the options.
John was ultimately diagnosed with cerebellar ataxia, neuropathy, and vestibular areflexia syndrome, or CANVAS for short. This is a neurodegenerative movement disorder, which is linked to an expansion of repeat DNA sequences in the gene RFC1.
Just in this one gene, however, there are a diversity of ways short DNA sequences can be repeated, which makes a blanket diagnostic test difficult.
"I had test after test for over 10 years and absolutely no answers as to what was wrong,' says John.
Neurologist Kishore Kumar says he and his colleagues at the Garvan Institute for Medical Research in Australia refer to this stressful process as the 'diagnostic odyssey'. As patients like John wait years and years for answers, their symptoms grow gradually worse.
While there's currently no cure for tandem repeat disorders, early diagnosis can help patients manage their symptoms, and hopefully stall some of the disease progression, so the newly developed test should make a big difference to patients.
"This new test will completely revolutionize how we diagnose these diseases, since we can now test for all the disorders at once with a single DNA test and give a clear genetic diagnosis," says Kumar, "helping patients avoid years of unnecessary muscle or nerve biopsies for diseases they don't have, or risky treatments that suppress their immune system."
The newly developed assessment is based on nanopore technology, which can analyze long DNA or RNA fragments in known repetitive regions of the human genome.
Taking a single DNA sample from an individual's blood, researchers can pass the nucleic acids through a protein nanopore, using changes in electrical current arising from the molecular interactions to decode in real time the sequences from 40 genes known to be connected to 25 tandem repeat diseases.
Among 37 patients who were tested using this method, including John, all were correctly matched to their neurogenetic disease.
"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," says genomicist Ira Deveson, also from the Garvan Institute.
Current gene sequencing tests require machines as large as fridges, whereas the nanopore technology is no bigger than a stapler. It also costs hundreds of thousands of dollars less, meaning it could be easily scaled up and distributed.
Researchers are now trying to get the method clinically approved. They hope in two to five years the diagnostic test will be used regularly.