Long-read targeted sequencing uncovers clinicopathological associations forC9orf72-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.

Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by aC9orf72repeat expansion mutation in patient derived cells

“…Patients can have C9orf72 repeats into the thousands! We turned instead to single-molecule sequencing, which has been demonstrated to traverse the expanded repeats of C9orf72 in plasmid 58 and human tissue 59, 60 . We collected patient iPSC lines from previously published or publicly available sources 31, 42, 61, 62 and developed PacBio single-molecule sequencing of DNA from these lines to size their repeat expansions and identify surrounding SNPs ( Extended Data Fig.…”

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

“…For example, PacBio long-read sequencing was previously used to sequence repeat expansions in DM1, which is also characterized by long alleles of 4–6 kbp, but the microsatellites were first amplified by PCR(Mangin et al 2021; Cumming et al 2018). Even when coupled to PCR-free enrichment approach based on Cas9, the length of PacBio sequencing reads cannot exceed 20 kbp(Dejesus-Hernandez et al 2021; Ebbert et al 2018; Hafford-Tear et al 2019; Höijer et al 2018; Tsai et al 2017; Wieben et al 2019).…”

“…Targeted enrichment approaches coupled to long-read sequencing have already been used for the in-depth characterization of repeat expansions in fragile X syndrome ( FMR1 ), Huntington’s disease ( HTT ) and neuronal intranuclear inclusion disease ( NOTCH2NLC ), although these expansions are significantly shorter than the (CCTG) n repeats in DM2 patients(Dejesus-Hernandez et al 2021; Ebbert et al 2018; Giesselmann et al 2019; Grosso et al 2021; Hafford-Tear et al 2019; Höijer et al 2018; Mizuguchi et al 2021; Sone et al 2019; Tsai et al 2017; Wallace et al 2021; Wieben et al 2019; Mitsuhashi and Matsumoto 2020). The longest allele thus far characterized by third-generation sequencing is a 21-kbp allele of the C9orf72 gene that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (Dejesus-Hernandez et al 2021). In addition, many of the works cited above combined PacBio SMRT sequencing with LR-PCR(Mangin et al 2021; Ciosi et al 2021; Cumming et al 2018), which is unsuitable for the analysis of DM2 expansions due to their extreme length and high GC content.…”

“…In addition, many of the works cited above combined PacBio SMRT sequencing with LR-PCR(Mangin et al 2021; Ciosi et al 2021; Cumming et al 2018), which is unsuitable for the analysis of DM2 expansions due to their extreme length and high GC content. Importantly, PacBio reads do not exceed 20 kbp in a PCR-free enrichment(Dejesus-Hernandez et al 2021; Ebbert et al 2018; Hafford-Tear et al 2019; Höijer et al 2018; Tsai et al 2017; Wieben et al 2019), and would not completely span DM2 pathogenic expansions (20 kbp on average, but up to 50 kbp).…”

Profiling the Genome-Wide Landscape of Short Tandem Repeats by Long-Read Sequencing

“…Current LRS technologies, such as Pacific Biosciences sequencing and Oxford Nanopore Technologies (ONT) sequencing, have achieved reads longer than 10 kb on average, which have a high chance to cover whole tandem repeats, including flanking unique sequences ( Pollard et al, 2018 ; Midha et al, 2019 ; Amarasinghe et al, 2020 ; Logsdon et al, 2020 ). LRS has recently been applied to genotype long and complex repeats, such as the C9orf72 GGGGCC expansion implicated in frontotemporal lobar degeneration and a complex pentamer repeat in SAMD12 implicated in myoclonus epilepsy ( Zeng et al, 2019 ; Mitsuhashi and Matsumoto, 2020 ; DeJesus-Hernandez et al, 2021 ). More human diseases caused by STR expansions have also been reported in recently published studies with the utilization of LRS ( Sone et al, 2019 ; Tian et al, 2019 ; Zeng et al, 2019 ; Deng et al, 2020 ).…”