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Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing

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Title: Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing
Authors: Natsuga, Ken Browse this author →KAKEN DB
Furuta, Yoshikazu Browse this author
Takashima, Shota Browse this author
Nohara, Takuma Browse this author
Kosumi, Hideyuki Browse this author
Mai, Yosuke Browse this author
Higashi, Hideaki Browse this author
Ujiie, Hideyuki Browse this author →KAKEN DB
Keywords: epidermolysis bullosa
intragenic crossover
long-read sequencing
revertant mosaicism
Issue Date: 1-Apr-2022
Publisher: John Wiley & Sons
Journal Title: Human mutation
Volume: 43
Issue: 4
Start Page: 529
End Page: 536
Publisher DOI: 10.1002/humu.24331
Abstract: Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, but genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. Here, we introduce nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin. We took advantage of compound heterozygous COL7A1 mutations in a patient with recessive dystrophic epidermolysis bullosa who showed revertant skin spots. Cas9-mediated enrichment of genomic DNA (gDNA) covering the two mutation sites (>8 kb) in COL7A1 and subsequent MinION sequencing successfully detected intragenic crossover in the epidermis of the clinically revertant skin. This method enables the discernment of haplotypes of up to a few tens of kilobases of gDNA. Moreover, it is devoid of polymerase chain reaction amplification, which can technically induce recombination. We, therefore, propose that nCATS is a powerful tool for understanding complicated gene modifications, including RM.
Rights: This is the peer reviewed version of the following article: Natsuga, K., Furuta, Y., Takashima, S., Nohara, T., Kosumi, H., Mai, Y., Higashi, H., & Ujiie, H. (2022). Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing. Human Mutation, 43, 529– 536 which has been published in final form at doi:10.1002/humu.24331. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
Type: article (author version)
Appears in Collections:医学院・医学研究院 (Graduate School of Medicine / Faculty of Medicine) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 夏賀 健

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