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A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories
  1. Chizuru Akimoto1,
  2. Alexander E Volk2,
  3. Marka van Blitterswijk3,
  4. Marleen Van den Broeck4,5,
  5. Claire S Leblond6,
  6. Serge Lumbroso7,
  7. William Camu8,
  8. Birgit Neitzel9,
  9. Osamu Onodera10,
  10. Wouter van Rheenen11,
  11. Susana Pinto12,
  12. Markus Weber13,
  13. Bradley Smith14,
  14. Melanie Proven15,
  15. Kevin Talbot16,
  16. Pamela Keagle17,
  17. Alessandra Chesi18,
  18. Antonia Ratti19,20,
  19. Julie van der Zee4,5,
  20. Helena Alstermark1,
  21. Anna Birve1,
  22. Daniela Calini19,20,
  23. Angelica Nordin1,
  24. Daniela C Tradowsky2,
  25. Walter Just2,
  26. Hussein Daoud6,
  27. Sabrina Angerbauer9,
  28. Mariely DeJesus-Hernandez3,
  29. Takuya Konno10,
  30. Anjali Lloyd-Jani15,
  31. Mamede de Carvalho12,
  32. Kevin Mouzat7,
  33. John E Landers17,
  34. Jan H Veldink11,
  35. Vincenzo Silani19,20,
  36. Aaron D Gitler18,
  37. Christopher E Shaw14,
  38. Guy A Rouleau6,
  39. Leonard H van den Berg11,
  40. Christine Van Broeckhoven4,5,
  41. Rosa Rademakers,
  42. Peter M Andersen1,21,
  43. Christian Kubisch2
  1. 1Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden
  2. 2Institute of Human Genetics, Ulm University, Ulm, Germany
  3. 3Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA
  4. 4Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, University of Antwerp—CDE, Antwerp, Belgium
  5. 5Diagnostic Service Facility, Laboratory of neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium
  6. 6Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada
  7. 7Department of Biochemistry, Nimes University Hospital, Nimes Cedex 9, France
  8. 8Center SLA, Montpellier University Hospital, Hôpital Gui-de-Chauliac, Montpellier Cedex 5, France
  9. 9Medizinisch Genetisches Zentrum, München, Germany
  10. 10Department of Neurology, Brain Research Institute, Niigata University,  Niigata, Japan
  11. 11Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands
  12. 12Faculty of Medicine-University of Lisbon, Instituto de Medicina Molecular, Hospital de Santa Maria, University of Lisbon, Alameda Universidade, Lisbon, Portugal
  13. 13Department of neurology, Kantonsspital St. Gallen and University Hospital, St. Gallen, Switzerland
  14. 14Institute of Psychiatry, King's College London and King's Health Partners, London, UK
  15. 15Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, England
  16. 16Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe hospital, Oxford, UK
  17. 17Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
  18. 18Department of Genetics, Stanford University School of Medicine, Stanford, California, USA
  19. 19Department of Pathophysiology and Transplantation, “Dino Ferrari” Center, Universtà degli Studi di Milano, Milan, Italy
  20. 20Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, , Milan, Italy
  21. 21Department of Neurology, University of Ulm, Ulm, Germany
  1. Correspondence to Dr Peter M Andersen, Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå SE-90185, Sweden; peter.andersen{at}


Background The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories.

Methods The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference.

Results Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9–100%), and the mean specificity was 98.0% (87.5–100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories.

Conclusions Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.

  • Motor neurone disease
  • Molecular genetics
  • Neurology

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