Dataset Information

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.

SUBMITTER: Akimoto C

PROVIDER: S-EPMC4033024 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Publications

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 C Volk Alexander E AE van Blitterswijk Marka M Van den Broeck Marleen M Leblond Claire S CS Lumbroso Serge S Camu William W Neitzel Birgit B Onodera Osamu O van Rheenen Wouter W Pinto Susana S Weber Markus M Smith Bradley B Proven Melanie M Talbot Kevin K Keagle Pamela P Chesi Alessandra A Ratti Antonia A van der Zee Julie J Alstermark Helena H Birve Anna A Calini Daniela D Nordin Angelica A Tradowsky Daniela C DC Just Walter W Daoud Hussein H Angerbauer Sabrina S DeJesus-Hernandez Mariely M Konno Takuya T Lloyd-Jani Anjali A de Carvalho Mamede M Mouzat Kevin K Landers John E JE Veldink Jan H JH Silani Vincenzo V Gitler Aaron D AD Shaw Christopher E CE Rouleau Guy A GA van den Berg Leonard H LH Van Broeckhoven Christine C Rademakers Rosa R Andersen Peter M PM Kubisch Christian C

Journal of medical genetics 20140404 6

<h4>Background</h4>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.<h4>Methods</h4>The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or F ...[more]

PMID: 24706941

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Project description:The loss of chromosome 9 open reading frame 72 (C9ORF72) expression, associated with C9ORF72 repeat expansions, has not been examined systematically. Three C9ORF72 transcript variants have been described thus far; the GGGGCC repeat is located between two non-coding exons (exon 1a and exon 1b) in the promoter region of transcript variant 2 (NM_018325.4) or in the first intron of variant 1 (NM_145005.6) and variant 3 (NM_001256054.2). We studied C9ORF72 expression in expansion carriers (n = 56) for whom cerebellum and/or frontal cortex was available. Using quantitative real-time PCR and digital molecular barcoding techniques, we assessed total C9ORF72 transcripts, variant 1, variant 2, variant 3, and intron containing transcripts [upstream of the expansion (intron 1a) and downstream of the expansion (intron 1b)]; the latter were correlated with levels of poly(GP) and poly(GA) proteins aberrantly translated from the expansion as measured by immunoassay (n = 50). We detected a decrease in expansion carriers as compared to controls for total C9ORF72 transcripts, variant 1, and variant 2: the strongest association was observed for variant 2 (quantitative real-time PCR cerebellum: median 43 %, p = 1.26e-06, and frontal cortex: median 58 %, p = 1.11e-05; digital molecular barcoding cerebellum: median 31 %, p = 5.23e-10, and frontal cortex: median 53 %, p = 5.07e-10). Importantly, we revealed that variant 1 levels greater than the 25th percentile conferred a survival advantage [digital molecular barcoding cerebellum: hazard ratio (HR) 0.31, p = 0.003, and frontal cortex: HR 0.23, p = 0.0001]. When focusing on intron containing transcripts, analysis of the frontal cortex revealed an increase of potentially truncated transcripts in expansion carriers as compared to controls [digital molecular barcoding frontal cortex (intron 1a): median 272 %, p = 0.003], with the highest levels in patients pathologically diagnosed with frontotemporal lobar degeneration. In the cerebellum, our analysis suggested that transcripts were less likely to be truncated and, excitingly, we discovered that intron containing transcripts were associated with poly(GP) levels [digital molecular barcoding cerebellum (intron 1a): r = 0.33, p = 0.02, and (intron 1b): r = 0.49, p = 0.0004] and poly(GA) levels [digital molecular barcoding cerebellum (intron 1a): r = 0.34, p = 0.02, and (intron 1b): r = 0.38, p = 0.007]. In summary, we report decreased expression of specific C9ORF72 transcripts and provide support for the presence of truncated transcripts as well as pre-mRNAs that may serve as templates for RAN translation. We further show that higher C9ORF72 levels may have beneficial effects, which warrants caution in the development of new therapeutic approaches.

Dataset Information

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

Methods

Results

Conclusions

Publications

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

Similar Datasets

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