Long-read targeted sequencing uncovers clinicopathological associations for C9orf72-linked diseases

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.

Keywords: C9orf72; amyotrophic lateral sclerosis; frontotemporal lobar degeneration; long-read sequencing; motor neuron disease.

Figure 1

C9orf72 expansion size as determined by long-read sequencing. (A) A histogram is displayed showing the expansion size of all 814 expanded reads obtained using No-Amp sequencing for C9orf72 expansion carriers included in this study (n = 28). The number of repeats ranges from approximately 500 to 3500. The dashed grey line denotes the median (1261 repeats) of the expansion size estimates for each individual. (B) A significant correlation exists between the number of repeats as measured by No-Amp sequencing and Southern blotting. Each C9orf72 expansion carrier is symbolized by a filled circle; the colour is an indication of the number of reads for a given individual [from low (dark blue) to high (dark red)]. The solid grey line denotes the linear regression line. (C) The expansion size determined by No-Amp sequencing (light blue) is compared to that determined by Southern blotting (dark blue). (D) When using the median expansion size as a cut-off point, smaller expansions (light blue) appear to be associated with a higher number of reads as compared to longer expansions (dark blue). For each box plot, the median is represented by a solid black line, each box spans the interquartile range (IQR; 25th percentile to 75th percentile), and every individual is visualized as a filled circle.

Figure 2

Clinicopathological associations with C9orf72 expansion size. (A) A Kaplan-Meier curve is shown when comparing the bottom 50% (light blue) to the top 50% (dark blue), depending on the size of the C9orf72 repeat expansion. Individuals with a smaller expansion seem to demonstrate an increased survival after onset (median: 4.9 years) as compared to individuals with a longer expansion (median: 1.8 years). The black dashed line denotes the median for each group. Of note, since only one individual received a primary diagnosis of Alzheimer’s disease, this individual was excluded from our survival analysis where we adjusted for disease subgroup as well as age at onset. (B–D) An inverse correlation is detected between the number of repeats as measured by No-Amp sequencing and C9orf72 transcripts containing intron 1b (downstream of the expansion), total poly(GP) protein levels, and total poly(GA) protein levels, respectively. Each C9orf72 expansion carrier is symbolized by a filled circle. The solid dark blue line denotes the linear regression line. Notably, a non-parametric Spearman’s test of correlation was used to assess these correlations, which is based on rank and less sensitive to outliers than parametric equivalents of this test.