A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories.
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.
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
Chizuru
C
Akimoto
Institute of Human Genetics, Ulm University, Ulm, Germany.
Alexander E
AE
Volk
Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA.
Marka
M
van Blitterswijk
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.
Marleen
M
Van den Broeck
Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada.
Claire S
CS
Leblond
Department of Biochemistry, Nimes University Hospital, Nimes Cedex 9, France.
Serge
S
Lumbroso
Center SLA, Montpellier University Hospital, Hôpital Gui-de-Chauliac, Montpellier Cedex 5, France.
William
W
Camu
Medizinisch Genetisches Zentrum, München, Germany.
Birgit
B
Neitzel
Department of Neurology, Brain Research Institute, Niigata University,  Niigata, Japan.
Osamu
O
Onodera
Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
Wouter
W
van Rheenen
Faculty of Medicine-University of Lisbon, Instituto de Medicina Molecular, Hospital de Santa Maria, University of Lisbon, Alameda Universidade, Lisbon, Portugal.
Susana
S
Pinto
Department of neurology, Kantonsspital St. Gallen and University Hospital, St. Gallen, Switzerland.
Markus
M
Weber
Institute of Psychiatry, King's College London and King's Health Partners, London, UK.
Bradley
B
Smith
Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, England.
Melanie
M
Proven
Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe hospital, Oxford, UK.
Kevin
K
Talbot
Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Pamela
P
Keagle
Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
Alessandra
A
Chesi
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.
Antonia
A
Ratti
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.
Julie
J
van der Zee
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
Helena
H
Alstermark
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
Anna
A
Birve
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.
Daniela
D
Calini
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
Angelica
A
Nordin
Institute of Human Genetics, Ulm University, Ulm, Germany.
Daniela C
DC
Tradowsky
Institute of Human Genetics, Ulm University, Ulm, Germany.
Walter
W
Just
Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada.
Hussein
H
Daoud
Medizinisch Genetisches Zentrum, München, Germany.
Sabrina
S
Angerbauer
Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA.
Mariely
M
DeJesus-Hernandez
Department of Neurology, Brain Research Institute, Niigata University,  Niigata, Japan.
Takuya
T
Konno
Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, England.
Anjali
A
Lloyd-Jani
Faculty of Medicine-University of Lisbon, Instituto de Medicina Molecular, Hospital de Santa Maria, University of Lisbon, Alameda Universidade, Lisbon, Portugal.
Mamede
M
de Carvalho
Department of Biochemistry, Nimes University Hospital, Nimes Cedex 9, France.
Kevin
K
Mouzat
Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
John E
JE
Landers
Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
Jan H
JH
Veldink
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.
Vincenzo
V
Silani
Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
Aaron D
AD
Gitler
Institute of Psychiatry, King's College London and King's Health Partners, London, UK.
Christopher E
CE
Shaw
Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada.
Guy A
GA
Rouleau
Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
Leonard H
LH
van den Berg
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.
Christine
C
Van Broeckhoven
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,
Rosa
R
Rademakers
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden Department of Neurology, University of Ulm, Ulm, Germany.
Peter M
PM
Andersen
Institute of Human Genetics, Ulm University, Ulm, Germany.
Christian
C
Kubisch
D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics; D000073885:C9orf72 Protein; D064790:Clinical Laboratory Services / Q000592:standards*; D005260:Female; D057180:Frontotemporal Dementia / Q000235:genetics; D005820:Genetic Testing / Q000379:methods* / Q000592:standards*; D006801:Humans; D008297:Male; D011506:Proteins / Q000235:genetics*; D015203:Reproducibility of Results
D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D011506:Proteins
Molecular genetics; Motor neurone disease; Neurology