A prospective trial comparing programmable targeted long-read sequencing and short-read genome sequencing for genetic diagnosis of cerebellar ataxia

  1. Paul J. Lockhart3,5,44
  1. 1Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia;
  2. 2Department of Medical Biology, University of Melbourne, Parkville, Victoria 3052, Australia;
  3. 3Bruce Lefroy Centre, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia;
  4. 4Department of Neuroscience, Central Clinical School, Monash University, The Alfred Centre, Melbourne, Victoria 3004, Australia;
  5. 5Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia;
  6. 6Austin Health, Heidelberg, Victoria 3084, Australia;
  7. 7Monash Medical Centre, Clayton, Victoria 3168, Australia;
  8. 8Department of Neurology, Alfred Hospital, Melbourne, Victoria 3004, Australia;
  9. 9Department of Medicine, St Vincent's Hospital, University of Melbourne, Fitzroy, Victoria 3065, Australia;
  10. 10Florey Institute of Neuroscience and Mental Health, Parkville, Victoria 3052, Australia;
  11. 11Hunter Genetics, Hunter New England Health Service, Waratah, New South Wales 2298, Australia;
  12. 12University of Newcastle, Callaghan, New South Wales 2308, Australia;
  13. 13Neurology Department, Royal Prince Alfred Hospital, Camperdown, New South Wales 2050, Australia;
  14. 14Central Clinical School, University of Sydney, Camperdown, New South Wales 2050, Australia;
  15. 15Department of Neurology, Westmead Hospital, Hawkesbury Westmead, New South Wales 2145, Australia;
  16. 16Brain and Nerve Research Centre, Concord Clinical School, University of Sydney, Camperdown, New South Wales 2050, Australia;
  17. 17Department of Neurology, Concord Repatriation General Hospital, Concord, New South Wales 2139, Australia;
  18. 18Department of Clinical Neurosciences, St Vincent's Hospital, University of Melbourne, Fitzroy, Victoria 3065, Australia;
  19. 19Molecular Medicine Laboratory and Neurology Department, Concord Repatriation General Hospital, Concord, New South Wales 2139, Australia;
  20. 20Faculty of Medicine and Health, The University of Sydney, Camperdown, New South Wales 2050, Australia;
  21. 21Genomics and Inherited Disease Program, The Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia;
  22. 22School of Medicine, University of New South Wales, Sydney, New South Wales 2052, Australia;
  23. 23Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia;
  24. 24Department of Clinical Genetics, Austin Health, Viewbank, Victoria 3084, Australia;
  25. 25Department of Neurology, Launceston General Hospital, Launceston, Tasmania 7250, Australia;
  26. 26Department of Neuroscience, University Hospital Geelong, Geelong, Victoria 3220, Australia;
  27. 27Department of Neurology, Royal Brisbane and Women's Hospital, Herston, Queensland 4006, Australia;
  28. 28Neurology Footscray, Footscray, Victoria 3011, Australia;
  29. 29Department of Neurology, St Vincent's Hospital, University of Melbourne, Fitzroy, Victoria 3065, Australia;
  30. 30Albury Wodonga Health, West Albury, New South Wales 2640, Australia;
  31. 31Newcastle Medical Genetics, Lambton, New South Wales 2299, Australia;
  32. 32St Vincent's Clinical Genomics, St Vincent's Hospital, Darlinghurst, New South Wales 2010, Australia;
  33. 33Tasmanian Clinical Genetics Service, Tasmanian Health Service, Royal Hobart Hospital, Hobart, Tasmania 7001, Australia;
  34. 34School of Medicine and Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania 7000, Australia;
  35. 35Department of Neurology, John Hunter Hospital, New Lambton Heights, New South Wales 2305, Australia;
  36. 36Genomic Medicine, The Royal Melbourne Hospital, Parkville, Victoria 3052, Australia;
  37. 37Department of Neurology, Calvary Health Care Bethlehem, Caulfield South Victoria 3162, Australia;
  38. 38Department of Neurology, The Royal Melbourne Hospital, Parkville, Victoria 3052, Australia;
  39. 39School of Medicine, University of Notre Dame, Darlinghurst, New South Wales 2010, Australia;
  40. 40Discipline of Genomic Medicine, Faculty of Medicine and Health, University of Sydney, Camperdown, New South Wales 2050, Australia;
  41. 41Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria 3002, Australia;
  42. 42Bionics Institute, East Melbourne, Victoria 3002, Australia

  1. ↵43 These authors are joint first authors and contributed equally to this work.

  2. ↵44 These authors are joint senior authors and contributed equally to this work.

  • Corresponding author: paul.lockhart{at}mcri.edu.au

    • Abstract

    The cerebellar ataxias (CAs) are a heterogeneous group of disorders characterized by progressive incoordination. Seventeen repeat expansion (RE) loci have been identified as the primary genetic cause and account for >80% of genetic diagnoses. Despite this, diagnostic testing is limited and inefficient, often utilizing single gene assays. This study evaluates the effectiveness of long- and short-read sequencing as diagnostic tools for CA. We recruited 110 individuals (48 females, 62 males) with a clinical diagnosis of CA. Short-read genome sequencing (SR-GS) was performed to identify pathogenic RE and also non-RE variants in 356 genes associated with CA. Independently, long-read sequencing with adaptive sampling (LR-AS) was performed to identify pathogenic RE. SR-GS provided a genetic diagnosis for 38% of the cohort (40/110) including seven non-RE pathogenic variants. RE causes disease in 33 individuals, with the most common condition being SCA27B (n = 24). In comparison, LR-AS identified pathogenic RE in 29 individuals. RE identification for the two methods was concordant apart from four SCA27B cases not detected by LR-AS due to low read depth. For both technologies manual review of the RE alignment enhances diagnostic outcomes. Orthogonal testing for SCA27B revealed a 15% and 0% false positive rate for SR-GS and LR-AS, respectively. In conclusion, both technologies are powerful screening tools for CA. SR-GS is a mature technology currently used by diagnostic providers, requiring only minor changes in bioinformatic workflows to enable CA diagnostics. LR-AS offers considerable advantages in the context of RE detection and characterization but requires optimization before clinical implementation.

    Footnotes

    • Received June 11, 2024.
    • Accepted November 21, 2024.

    This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see https://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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    1. Published in Advance February 27, 2025, doi: 10.1101/gr.279634.124 Genome Res. © 2025 Rafehi et al.; Published by Cold Spring Harbor Laboratory Press

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    1. Profile for Bahlo, M.http://orcid.org/0000-0001-5132-0774
    2. Profile for Lockhart, P. J.http://orcid.org/0000-0003-2531-8413

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