To characterize variants within and downstream of the C9orf72 hexanucleotide repeat and to investigate their correlation with repeat length of C9orf72 expansion.

A hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of both familial amyotrophic lateral sclerosis and frontotemporal dementia. Small insertion/deletions (indels) and sequence variants frequently occur in flanking sequences downstream of the repeat region and strongly correlate with the C9orf72 expansion. However, the causal effects of the variants have not been well elucidated.

Penn-C9 assay, including sizing and repeat-primed PCR (RP-PCR) followed by capillary electrophoresis, was performed to detect hexanucleotide repeat size and small indels of C9orf72. Variants within the repeat sequence appeared as low amplitude RP-PCR peaks due to primer mismatch. The PCR products of all cases with atypical patterns underwent Sanger sequencing. Southern blot analysis, restriction enzyme digestion, and variant-specific RP-PCR were performed to validate variants. Statistical analyses determined the association of variants with clinical data and repeat size.

In a cohort of 2077 neurodegenerative disease patients, family members, and controls, 0.89% of non-expanded alleles (n=3952), 22% of expanded alleles (n=192), and 10% of intermediate alleles (n=10) had variants in the region. Of those, 5 large expansion cases and 1 intermediate case had variants within the hexanucleotide repeat. Expanded alleles exhibited variant mosaicism and intrafamilial heterogeneity, and had more diverse variants overall than non-expanded with greater GC-content and contiguity flanking the expansion. Furthermore, expansions with variants had significantly smaller repeat length in the brain than those without, but there was no significant difference in peripheral.

Variants flanking the C9orf72 repeat expansion are more frequent and heterogeneous than those without expansion, and are only seen in the repeat sequence itself in alleles with at least 25 rpts. And while expansion variants increase downstream GC-content, they associate with smaller expansion lengths in the brain.