Abstract
Background The gene C9orf72 harbors a non-coding hexanucleotide repeat expansion known to cause amyotrophic lateral sclerosis and frontotemporal dementia. While previous studies have estimated the length of this repeat expansion in multiple tissues, technological limitations have impeded researchers from exploring additional features, such as methylation levels.
Methods We aimed to characterize C9orf72 repeat expansions using a targeted, amplification-free long-read sequencing method. Our primary goal was to determine the presence and subsequent quantification of observed methylation in the C9orf72 repeat expansion. In addition, we measured the repeat length and purity of the expansion. To do this, we sequenced DNA extracted from blood for 27 individuals with an expanded C9orf72 repeat.
Results For these individuals, we obtained a total of 7,765 on-target reads, including 1,612 fully covering the expanded allele. Our in-depth analysis revealed that the expansion itself is methylated, with great variability in total methylation levels observed, as represented by the proportion of methylated CpGs (13 to 66%). Interestingly, we demonstrated that the expanded allele is more highly methylated than the wild-type allele (P-Value = 2.76E-05) and that increased methylation levels are observed in longer repeat expansions (P-Value = 1.18E-04). Furthermore, methylation levels correlate with age at collection (P-Value = 3.25E-04) as well as age at disease onset (P-Value = 0.020). Additionally, we detected repeat lengths up to 4,088 repeats (~ 25 kb) and found that the expansion contains few interruptions in the blood.
Conclusions Taken together, our study demonstrates robust ability to quantify methylation of the expanded C9orf72 repeat, capturing differences between individuals harboring this expansion and revealing clinical associations.
Document Type
Article
Publication Date
12-2024
Digital Object Identifier (DOI)
https://doi.org/10.1186/s13024-024-00790-0
Funding Information
The results and data included in this manuscript are funded by RF1 NS123052, R21 NS099631, P01 NS084974, as well as the Muscular Dystrophy Association, ALS Association, and Robert Packard Center for ALS Research.
Repository Citation
Udine, Evan; Finch, NiCole; DeJesus-Hernandez, Mariely; Jackson, Jazmyne L.; Baker, Matthew C.; Saravanaperumal, Siva Arumugam; Wieben, Eric; Ebbert, Mark T. W.; Shah, Jaimin; Petrucelli, Leonard; Rademakers, Rosa; Oskarsson, Björn; and van Blitterswijk, Marka, "Targeted long-read sequencing to quantify methylation of the C9orf72 repeat expansion" (2024). Neurology Faculty Publications. 103.
https://uknowledge.uky.edu/neurology_facpub/103
Included in
Molecular and Cellular Neuroscience Commons, Molecular Biology Commons, Neurology Commons
Notes/Citation Information
© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.