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J Med Genet. 2014 Jun;51(6):419-24. doi: 10.1136/jmedgenet-2014-102360. Epub 2014 Apr 4.

A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories.

Author information

  • 1Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
  • 2Institute of Human Genetics, Ulm University, Ulm, Germany.
  • 3Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA.
  • 4Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, University of Antwerp-CDE, Antwerp, Belgium Diagnostic Service Facility, Laboratory of neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium.
  • 5Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada.
  • 6Department of Biochemistry, Nimes University Hospital, Nimes Cedex 9, France.
  • 7Center SLA, Montpellier University Hospital, Hôpital Gui-de-Chauliac, Montpellier Cedex 5, France.
  • 8Medizinisch Genetisches Zentrum, München, Germany.
  • 9Department of Neurology, Brain Research Institute, Niigata University,  Niigata, Japan.
  • 10Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
  • 11Faculty of Medicine-University of Lisbon, Instituto de Medicina Molecular, Hospital de Santa Maria, University of Lisbon, Alameda Universidade, Lisbon, Portugal.
  • 12Department of neurology, Kantonsspital St. Gallen and University Hospital, St. Gallen, Switzerland.
  • 13Institute of Psychiatry, King's College London and King's Health Partners, London, UK.
  • 14Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, England.
  • 15Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe hospital, Oxford, UK.
  • 16Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
  • 17Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
  • 18Department of Pathophysiology and Transplantation, "Dino Ferrari" Center, Universtà degli Studi di Milano, Milan, Italy Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, , Milan, Italy.
  • 19Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden Institute of Human Genetics, Ulm University, Ulm, Germany Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, University of Antwerp-CDE, Antwerp, Belgium Diagnostic Service Facility, Laboratory of neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada Department of Biochemistry, Nimes University Hospital, Nimes Cedex 9, France Center SLA, Montpellier University Hospital, Hôpital Gui-de-Chauliac, Montpellier Cedex 5, France Medizinisch Genetisches Zentrum, München, Germany Department of Neurology, Brain Research Institute, Niigata University,  Niigata, Japan Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands Faculty of Medicine-University of Lisbon, Instituto de Medicina Molecular, Hospital de Santa Maria, University of Lisbon, Alameda Universidade, Lisbon, Portugal Department of neurology, Kantonsspital St. Gallen and University Hospital, St. Gallen, Switzerland Institute of Psychiatry, King's College London and King's Health Partners, London, UK Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, England Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe hospital, Oxford, UK Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA Department of Genetics, Stanford University School of Medicine, Stanford, California, USA Department of Pathophysiology and Transplantation, "Dino Ferrari" Center, Universtà degli Studi di Milano, Milan, Italy Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, , Milan, Italy Department of Neurology, University of Ulm,
  • 20Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden Department of Neurology, University of Ulm, Ulm, Germany.

Abstract

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.

Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

KEYWORDS:

Molecular genetics; Motor neurone disease; Neurology

PMID:
24706941
[PubMed - in process]
PMCID:
PMC4033024
Free PMC Article

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