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Parallel in-depth analysis of repeat expansions in ataxia patients by long-read sequencing

Item Type:Article
Title:Parallel in-depth analysis of repeat expansions in ataxia patients by long-read sequencing
Creators Name:Erdmann, H. and Schöberl, F. and Giurgiu, M. and Leal Silva, R.M. and Scholz, V. and Scharf, F. and Wendlandt, M. and Kleinle, S. and Deschauer, M. and Nübling, G. and Heide, W. and Babacan, S.S. and Schneider, C. and Neuhann, T. and Hahn, K. and Schoser, B. and Holinski-Feder, E. and Wolf, D.A. and Abicht, A.
Abstract:Instability of simple DNA repeats has been known as a common cause of hereditary ataxias for over 20 years. Routine genetic diagnostics of these phenotypically similar diseases still rely on an iterative workflow for quantification of repeat units by PCR-based methods of limited precision. We established and validated clinical nanopore Cas9-targeted sequencing (Clin-CATS), an amplification-free method for simultaneous analysis of ten repeat loci associated with clinically overlapping hereditary ataxias. The method combines target enrichment by CRISPR/Cas9, Oxford Nanopore long-read sequencing, and a bioinformatics pipeline utilizing the tools STRIque and Megalodon for parallel detection of length, sequence, methylation, and composition of the repeat loci. Clin-CATS allowed for the precise and parallel analysis of 10 repeat loci associated with adult-onset ataxia and revealed additional parameter such as FMR1 promotor methylation and repeat sequence required for diagnosis at the same time. Using Clin-CATS we analyzed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients. Parallel repeat analysis enabled a molecular diagnosis of ataxias independent of preconceptions based on clinical presentation. Biallelic expansions within RFC1 were identified as the most frequent cause of ataxia. We characterized the RFC1 repeat composition of all patients and identified a novel repeat motif, AGGGG. Our results highlight the power of Clin-CATS as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.
Keywords:Repeat Analysis, Oxford Nanopore Sequencing, Hereditary Ataxia, Hereditary Ataxia, RFC1, Spinocerebellar Ataxia
Publisher:Oxford University Press
Page Range:1831-1843
Date:May 2023
Official Publication:https://doi.org/10.1093/brain/awac377
PubMed:View item in PubMed

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