Comprehensive genotyping of the <i>C9orf72</i> hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, long-read PCR assay

<i>Objective</i>: Expansion of the G₄C₂ repeat tract in the <i>C9orf72</i> gene is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we provide comprehensive genotyping of the <i>C9orf72</i> repeat region for the National Institute of Neurological Disorders and Stroke (NINDS) ALS collection (<i>n</i> = 2095), using a novel bimodal PCR assay capable of amplifying nearly 100% GC-rich sequences. <i>Methods</i>: 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 (&gt;145 repeats) up to ∼5.8 kb amplicons (∼950 G₄C₂ repeats). <i>Results</i>: 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. <i>Conclusion</i>: This study and PCR method may improve and standardize molecular characterization of the <i>C9orf72</i> locus, and have the potential to inform phenotype–genotype correlations and therapeutic development in ALS/FTD.