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
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
Institute of Human Genetics, Ulm University, Ulm, Germany.
J Med Genet. 2014 Jun;51(6):419-24. doi: 10.1136/jmedgenet-2014-102360. Epub 2014 Apr 4.
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.
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.
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.
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.
C9orf72基因中的GGGGCC重复序列扩增是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）患者中最常见的突变。大多数关于C9orf72的研究都依赖重复引物PCR（RP-PCR）方法来检测这种扩增。为了研究该技术的内在局限性，我们比较了14个实验室的方法和结果。
14个实验室以盲法对78例（诊断为ALS或FTD）个体的DNA进行基因分型。11个实验室使用扩增子长度分析和RP-PCR相结合的方法，而3个实验室仅使用RP-PCR；Southern印迹技术用作参考。
使用基于PCR的技术，14个实验室中有5个的结果与Southern印迹结果完全一致。78个DNA样本中只有50个在所有14个实验室中得到相同的基因型结果。存在高度的假阳性和假阴性结果，14个实验室中有9个至少有一个样本根本无法进行基因分型。扩增子长度分析和RP-PCR相结合的平均灵敏度为95.0%（73.9 - 100%），平均特异性为98.0%（87.5 - 100%）。总体而言，只有7个实验室观察到灵敏度和特异性超过95%。
由于基因分型结果差异很大，我们建议在研究环境中至少使用扩增子长度分析和RP-PCR相结合的方法。我们建议Southern印迹技术应作为金标准，并在临床诊断环境中强制使用。
