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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

107 Citations (Scopus)


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
Issue number6
Publication statusPublished - Jun 2014


  • FTD
  • ALS

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