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

Chizuru Akimoto¹, Alexander E Volk², Marka van Blitterswijk³, Marleen Van den Broeck⁴, Claire S Leblond⁵, Serge Lumbroso⁶, William Camu⁷, Birgit Neitzel⁸, Osamu Onodera⁹, Wouter van Rheenen¹⁰, Susana Pinto¹¹, Markus Weber¹², Bradley Smith¹³, Melanie Proven¹⁴, Kevin Talbot¹⁵, Pamela Keagle¹⁶, Alessandra Chesi¹⁷, Antonia Ratti¹⁸, Julie van der Zee⁴, Helena Alstermark¹, Anna Birve¹, Daniela Calini¹⁸, Angelica Nordin¹, Daniela C Tradowsky², Walter Just², Hussein Daoud⁵, Sabrina Angerbauer⁸, Mariely DeJesus-Hernandez³, Takuya Konno⁹, Anjali Lloyd-Jani¹⁴, Mamede de Carvalho¹¹, Kevin Mouzat⁶, John E Landers¹⁶, Jan H Veldink¹⁰, Vincenzo Silani¹⁸, Aaron D Gitler¹⁷, Christopher E Shaw¹³, Guy A Rouleau⁵, Leonard H van den Berg¹⁰, Christine Van Broeckhoven⁴, Rosa Rademakers¹⁹, Peter M Andersen²⁰, Christian Kubisch²

Affiliations

¹ Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
² Institute of Human Genetics, Ulm University, Ulm, Germany.
³ Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA.
⁴ 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.
⁵ Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada.
⁶ Department of Biochemistry, Nimes University Hospital, Nimes Cedex 9, France.
⁷ Center SLA, Montpellier University Hospital, Hôpital Gui-de-Chauliac, Montpellier Cedex 5, France.
⁸ Medizinisch Genetisches Zentrum, München, Germany.
⁹ Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan.
¹⁰ Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
¹¹ Faculty of Medicine-University of Lisbon, Instituto de Medicina Molecular, Hospital de Santa Maria, University of Lisbon, Alameda Universidade, Lisbon, Portugal.
¹² Department of neurology, Kantonsspital St. Gallen and University Hospital, St. Gallen, Switzerland.
¹³ Institute of Psychiatry, King's College London and King's Health Partners, London, UK.
¹⁴ Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, England.
¹⁵ Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe hospital, Oxford, UK.
¹⁶ Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
¹⁷ Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
¹⁸ 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.
¹⁹ Department 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,
²⁰ Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden Department of Neurology, University of Ulm, Ulm, Germany.

PMID: 24706941
PMCID: PMC4033024
DOI: 10.1136/jmedgenet-2014-102360

Free PMC article

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

Chizuru Akimoto et al. J Med Genet. 2014 Jun.

Free PMC article

. 2014 Jun;51(6):419-24.

doi: 10.1136/jmedgenet-2014-102360. Epub 2014 Apr 4.

Authors

Affiliations

¹ Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
² Institute of Human Genetics, Ulm University, Ulm, Germany.
³ Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA.
⁴ 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.
⁵ Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada.
⁶ Department of Biochemistry, Nimes University Hospital, Nimes Cedex 9, France.
⁷ Center SLA, Montpellier University Hospital, Hôpital Gui-de-Chauliac, Montpellier Cedex 5, France.
⁸ Medizinisch Genetisches Zentrum, München, Germany.
⁹ Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan.
¹⁰ Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
¹¹ Faculty of Medicine-University of Lisbon, Instituto de Medicina Molecular, Hospital de Santa Maria, University of Lisbon, Alameda Universidade, Lisbon, Portugal.
¹² Department of neurology, Kantonsspital St. Gallen and University Hospital, St. Gallen, Switzerland.
¹³ Institute of Psychiatry, King's College London and King's Health Partners, London, UK.
¹⁴ Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, England.
¹⁵ Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe hospital, Oxford, UK.
¹⁶ Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
¹⁷ Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
¹⁸ 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.
¹⁹ Department 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,
²⁰ Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden Department of Neurology, University of Ulm, Ulm, Germany.

PMID: 24706941
PMCID: PMC4033024
DOI: 10.1136/jmedgenet-2014-102360

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.

Keywords: Molecular genetics; Motor neurone disease; Neurology.

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.

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Figures

**Figure 1**
Results from RP-PCR and amplicon-length analysis of sample 6.5. (A) A-1 is the full shape of RP-PCR and A-2 is a 16-times scale-up (Y-axis) shape of A-1. It is possible to see the clear saw-tooth pattern after scale-up. A-3 is the result of amplicon-length analysis and there is a number of small peaks before the highest last peak. It may be hard to identify the amplicon numbers because of these small peaks. (B) The RP-PCR figures and classifications of the same sample in different laboratories. The scale of the Y-axis is the same as in A-2.

**Figure 2**
Flow chart for *C9orf72* genotyping in a scientific setting.

See this image and copyright information in PMC

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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

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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

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