Comprehensive genotyping of the C9orf72 hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, longread PCR assay


Microsatellite repeat expansions are associated with neuromuscular and neurodegenerative disorders, including frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS).  Both FTD and ALS are considered part of the same clinical continuum and have been linked by a hexanucleotide repeat element (G4C2)n in intron 1 of the chromosome 9 open reading frame 72 (C9orf72) gene.  Expansions in the C9orf72 repeat region have been reported in approximately 0.14% (∼1:700) of the population, and are enriched in both familial FTD and ALS patients (∼25% and 20–67%, respectively). The expansion also appears in up to 7% of sporadic FTD and ALS patients, making it the most prevalent genetic mutation in both disorders.

Although a validated threshold for repeat expansion pathogenicity has yet to be established, normal alleles have fewer than 20 repeats whereas disease-associated C9orf72 expansions have more than 30, and more typically, hundreds to thousands of repeats. Expanded alleles also manifest extensive somatic size mosaicism.  The impact of repeat length and associated DNA methylation status on a range of clinical phenotypes requires further elucidation.  Moreover, C9orf72 expansions may influence other neurodegenerative disorders, including Alzheimer’s disease.

Molecular characterization of the hexanucleotide repeat can be challenging, as the large size and high GC content of pathogenic C9orf72 expansions impede polymerization during PCR and limit the utility of conventional PCR-based fragment sizing methods.

In this study, researchers demonstrate the analytical capabilities and performance of a novel two-mode, multiplexed PCR chemistry for the genotyping of the C9orf72 repeat tract. This assay balances co-amplification of GS and RP PCR products tailored for capillary electrophoresis (CE)-based, accurate sizing of C9orf72 repeats from genomic DNA (gDNA) samples. They show the utility of this GS/RP-PCR assay by genotyping the NINDSrepository’s ALS collection (n=2095), a valuable and readily accessible resource with annotated set of patient-derived cell lines and matched genetic materials for researchers studying ALS and other neurodegenerative disorders.  GS/RP-PCR/CE was used to quantify the number of hexanucleotide repeats in this collection at single-repeat resolution while also detecting 3′-SVs and low-level size-mosaicism.

This study and PCR method may improve and standardize molecular characterization of the C9orf72 locus, and have the potential to inform phenotype–genotype correlations and therapeutic development in ALS/FTD.

Click here to access the full publication. Additional information is available on the PubMed website.

Click here to access accompanying genotyping data for ALS samples.

PMID:  30430876

Amyotroph Lateral Scler Frontotemporal Degener. 2018 Nov 15:1-8. doi: 10.1080/21678421.2018.1522353. [Epub ahead of print]

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