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

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, an amplification-free method for simultaneous analysis of 10 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 using the tools STRique and Megalodon for parallel detection of length, sequence, methylation and composition of the repeat loci. Clinical nanopore Cas9-targeted sequencing 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 clinical nanopore Cas9-targeted sequencing we analysed 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 on the basis of 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 clinical nanopore Cas9-targeted sequencing as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.

• Authors: Hannes Erdmann, Florian Schöberl, Mădălina Giurgiu, Rafaela Magalhaes Leal Silva, Veronika Scholz, Florentine Scharf, Martin Wendlandt, Stephanie Kleinle, Marcus Deschauer, Georg Nübling, Wolfgang Heide, Sait Seymen Babacan, Christine Schneider, Teresa Neuhann, Katrin Hahn, Benedikt Schoser, Elke Holinski-Feder, Dieter A Wolf, Angela Abicht
• Journal: Brain : a journal of neurology (Brain) Vol. 146 Issue 5 Pg. 1831-1843 (05 02 2023) ISSN: 1460-2156 [Electronic] England
• PMID: 36227727 (Publication Type: Journal Article)
• Copyright: © The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: [email protected].

Chemical References

• FMR1 protein, human
• Fragile X Mental Retardation Protein

Topics

• Adult
• Humans
• Ataxia (genetics)
• Cerebellar Ataxia (genetics)
• Computational Biology
• High-Throughput Nucleotide Sequencing
• Spinocerebellar Degenerations
• Fragile X Mental Retardation Protein