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, Alexander E. Volk, Marka van Blitterswijk, Marleen Van den Broeck, Claire S. Leblond, Serge Lumbroso, William Camu, Birgit Neitzel, Osamu Onodera, Wouter van Rheenen, Susana Pinto, Markus Weber, Bradley Smith, Melanie Proven, Kevin Talbot, Pamela Keagle, Alessandra Chesi, Antonia Ratti, Julie van der Zee, Helena AlstermarkAnna Birve, Daniela Calini, Angelica Nordin, Daniela C. Tradowsky, Walter Just, Hussein Daoud, Sabrina Angerbauer, Mariely DeJesus-Hernandez, Takuya Konno, Anjali Lloyd-Jani, Mamede de Carvalho, Kevin Mouzat, John E. Landers, Jan H. Veldink, Vincenzo Silani, Aaron D. Gitler, Christopher E. Shaw, Guy A. Rouleau, Leonard H. van den Berg, Christine Van Broeckhoven, Rosa Rademakers, Peter M. Andersen, Christian Kubisch

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)419-424
Number of pages6
JournalJournal of Medical Genetics
Volume51
Issue number6
DOIs
Publication statusPublished - 2014

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Genetic Testing
Polymerase Chain Reaction
Southern Blotting
Amyotrophic Lateral Sclerosis
DNA
Genotype
Sensitivity and Specificity
Mutation
Research

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)
  • Medicine(all)

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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. / Akimoto, Chizuru; Volk, Alexander E.; van Blitterswijk, Marka; Van den Broeck, Marleen; Leblond, Claire S.; Lumbroso, Serge; Camu, William; Neitzel, Birgit; Onodera, Osamu; van Rheenen, Wouter; Pinto, Susana; Weber, Markus; Smith, Bradley; Proven, Melanie; Talbot, Kevin; Keagle, Pamela; Chesi, Alessandra; Ratti, Antonia; van der Zee, Julie; Alstermark, Helena; Birve, Anna; Calini, Daniela; Nordin, Angelica; Tradowsky, Daniela C.; Just, Walter; Daoud, Hussein; Angerbauer, Sabrina; DeJesus-Hernandez, Mariely; Konno, Takuya; Lloyd-Jani, Anjali; de Carvalho, Mamede; Mouzat, Kevin; Landers, John E.; Veldink, Jan H.; Silani, Vincenzo; Gitler, Aaron D.; Shaw, Christopher E.; Rouleau, Guy A.; van den Berg, Leonard H.; Van Broeckhoven, Christine; Rademakers, Rosa; Andersen, Peter M.; Kubisch, Christian.

In: Journal of Medical Genetics, Vol. 51, No. 6, 2014, p. 419-424.

Research output: Contribution to journalArticle

Akimoto, C, Volk, AE, van Blitterswijk, M, Van den Broeck, M, Leblond, CS, Lumbroso, S, Camu, W, Neitzel, B, Onodera, O, van Rheenen, W, Pinto, S, Weber, M, Smith, B, Proven, M, Talbot, K, Keagle, P, Chesi, A, Ratti, A, van der Zee, J, Alstermark, H, Birve, A, Calini, D, Nordin, A, Tradowsky, DC, Just, W, Daoud, H, Angerbauer, S, DeJesus-Hernandez, M, Konno, T, Lloyd-Jani, A, de Carvalho, M, Mouzat, K, Landers, JE, Veldink, JH, Silani, V, Gitler, AD, Shaw, CE, Rouleau, GA, van den Berg, LH, Van Broeckhoven, C, Rademakers, R, Andersen, PM & Kubisch, C 2014, 'A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories', Journal of Medical Genetics, vol. 51, no. 6, pp. 419-424. https://doi.org/10.1136/jmedgenet-2014-102360
Akimoto, Chizuru ; Volk, Alexander E. ; van Blitterswijk, Marka ; Van den Broeck, Marleen ; Leblond, Claire S. ; Lumbroso, Serge ; Camu, William ; Neitzel, Birgit ; Onodera, Osamu ; van Rheenen, Wouter ; Pinto, Susana ; Weber, Markus ; Smith, Bradley ; Proven, Melanie ; Talbot, Kevin ; Keagle, Pamela ; Chesi, Alessandra ; Ratti, Antonia ; van der Zee, Julie ; Alstermark, Helena ; Birve, Anna ; Calini, Daniela ; Nordin, Angelica ; Tradowsky, Daniela C. ; Just, Walter ; Daoud, Hussein ; Angerbauer, Sabrina ; DeJesus-Hernandez, Mariely ; Konno, Takuya ; Lloyd-Jani, Anjali ; de Carvalho, Mamede ; Mouzat, Kevin ; Landers, John E. ; Veldink, Jan H. ; Silani, Vincenzo ; Gitler, Aaron D. ; Shaw, Christopher E. ; Rouleau, Guy A. ; van den Berg, Leonard H. ; Van Broeckhoven, Christine ; Rademakers, Rosa ; Andersen, Peter M. ; Kubisch, Christian. / A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories. In: Journal of Medical Genetics. 2014 ; Vol. 51, No. 6. pp. 419-424.
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title = "A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories",
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.",
author = "Chizuru Akimoto and Volk, {Alexander E.} and {van Blitterswijk}, Marka and {Van den Broeck}, Marleen and Leblond, {Claire S.} and Serge Lumbroso and William Camu and Birgit Neitzel and Osamu Onodera and {van Rheenen}, Wouter and Susana Pinto and Markus Weber and Bradley Smith and Melanie Proven and Kevin Talbot and Pamela Keagle and Alessandra Chesi and Antonia Ratti and {van der Zee}, Julie and Helena Alstermark and Anna Birve and Daniela Calini and Angelica Nordin and Tradowsky, {Daniela C.} and Walter Just and Hussein Daoud and Sabrina Angerbauer and Mariely DeJesus-Hernandez and Takuya Konno and Anjali Lloyd-Jani and {de Carvalho}, Mamede and Kevin Mouzat and Landers, {John E.} and Veldink, {Jan H.} and Vincenzo Silani and Gitler, {Aaron D.} and Shaw, {Christopher E.} and Rouleau, {Guy A.} and {van den Berg}, {Leonard H.} and {Van Broeckhoven}, Christine and Rosa Rademakers and Andersen, {Peter M.} and Christian Kubisch",
year = "2014",
doi = "10.1136/jmedgenet-2014-102360",
language = "English",
volume = "51",
pages = "419--424",
journal = "Journal of Medical Genetics",
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TY - JOUR

T1 - A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories

AU - Akimoto, Chizuru

AU - Volk, Alexander E.

AU - van Blitterswijk, Marka

AU - Van den Broeck, Marleen

AU - Leblond, Claire S.

AU - Lumbroso, Serge

AU - Camu, William

AU - Neitzel, Birgit

AU - Onodera, Osamu

AU - van Rheenen, Wouter

AU - Pinto, Susana

AU - Weber, Markus

AU - Smith, Bradley

AU - Proven, Melanie

AU - Talbot, Kevin

AU - Keagle, Pamela

AU - Chesi, Alessandra

AU - Ratti, Antonia

AU - van der Zee, Julie

AU - Alstermark, Helena

AU - Birve, Anna

AU - Calini, Daniela

AU - Nordin, Angelica

AU - Tradowsky, Daniela C.

AU - Just, Walter

AU - Daoud, Hussein

AU - Angerbauer, Sabrina

AU - DeJesus-Hernandez, Mariely

AU - Konno, Takuya

AU - Lloyd-Jani, Anjali

AU - de Carvalho, Mamede

AU - Mouzat, Kevin

AU - Landers, John E.

AU - Veldink, Jan H.

AU - Silani, Vincenzo

AU - Gitler, Aaron D.

AU - Shaw, Christopher E.

AU - Rouleau, Guy A.

AU - van den Berg, Leonard H.

AU - Van Broeckhoven, Christine

AU - Rademakers, Rosa

AU - Andersen, Peter M.

AU - Kubisch, Christian

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

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U2 - 10.1136/jmedgenet-2014-102360

DO - 10.1136/jmedgenet-2014-102360

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