Abstract
Objective: Expansion of the G4C2 repeat tract in the C9orf72 gene is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we provide comprehensive genotyping of the C9orf72 repeat region for the National Institute of Neurological Disorders and Stroke (NINDS) ALS collection (n = 2095), using a novel bimodal PCR assay capable of amplifying nearly 100% GC-rich sequences. Methods: A single-tube 3-primer PCR assay mode, resolved using capillary electrophoresis, was used for sizing up to 145 repeats with single-repeat accuracy, for detecting expansions irrespective of their overall size, and for flagging confounding 3′ sequence variations (SVs). A modified two-primer PCR mode, resolved via agarose gel electrophoresis, provided further size information for hyper-expanded samples (>145 repeats) up to ∼5.8 kb amplicons (∼950 G4C2 repeats). Results: Within the evaluated cohort, 177 (8.4%) samples were expanded, with 175 (99%) samples being hyper-expanded. 3′-SVs were identified in 64 (3.1%) samples, and were most common in expanded alleles. Genotypes of all 606 (29%) homozygous samples were confirmed using an orthogonal PCR assay. Conclusion: 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.
Highlights
Microsatellite repeat expansions are associated with neuromuscular and neurodegenerative disorders, including frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) [1]
We show the utility of this GS/RP-PCR assay by genotyping the National Institute of Neurological Disorders and Stroke (NINDS) repository’s ALS collection
A PCR/capillary electrophoresis (CE) control admixture sample comprised of Chromosome 9 open reading frame 72 (C9orf72) alleles with 2, 5, 8, and 10 repeats and a no template control were used in all experiments
Summary
Microsatellite repeat expansions are associated with neuromuscular and neurodegenerative disorders, including frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) [1]. 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 [1]. Expansions in the C9orf repeat region have been reported in approximately 0.14% (~1:700) of the population [2], and are enriched in both familial FTD and ALS patients (~25% and 20–67%, respectively). The expansion appears in up to 7% of sporadic FTD and ALS patients, making it the most prevalent genetic mutation in both disorders [3–5]. C9orf expansions may influence other neurodegenerative disorders, including Alzheimer’s disease [2]
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