Long-read targeted sequencing uncovers clinicopathological associations for<i>C9orf72</i>-linked diseases
Mariely DeJesus‐Hernandez, Ross A. Aleff, Jazmyne L. Jackson, NiCole A. Finch, Matthew Baker, Tania F. Gendron, Melissa E. Murray, Ian McLaughlin, John Harting, Neill R. Graff‐Radford, Björn Oskarsson, David S. Knopman, Keith A. Josephs, Bradley F. Boeve, Ronald C. Petersen, John Denis Fryer, Leonard Petrucelli, Dennis W. Dickson, Rosa Rademakers, Mark Ebbert, Eric D. Wieben, Marka van Blitterswijk
Abstract
To examine the length of a hexanucleotide expansion in C9orf72, which represents the most frequent genetic cause of frontotemporal lobar degeneration and motor neuron disease, we employed a targeted amplification-free long-read sequencing technology: No-Amp sequencing. In our cross-sectional study, we assessed cerebellar tissue from 28 well-characterized C9orf72 expansion carriers. We obtained 3507 on-target circular consensus sequencing reads, of which 814 bridged the C9orf72 repeat expansion (23%). Importantly, we observed a significant correlation between expansion sizes obtained using No-Amp sequencing and Southern blotting (P = 5.0 × 10-4). Interestingly, we also detected a significant survival advantage for individuals with smaller expansions (P = 0.004). Additionally, we uncovered that smaller expansions were significantly associated with higher levels of C9orf72 transcripts containing intron 1b (P = 0.003), poly(GP) proteins (P = 1.3 × 10- 5), and poly(GA) proteins (P = 0.005). Thorough examination of the composition of the expansion revealed that its GC content was extremely high (median: 100%) and that it was mainly composed of GGGGCC repeats (median: 96%), suggesting that expanded C9orf72 repeats are quite pure. Taken together, our findings demonstrate that No-Amp sequencing is a powerful tool that enables the discovery of relevant clinicopathological associations, highlighting the important role played by the cerebellar size of the expanded repeat in C9orf72-linked diseases.