Abstract-Text (inkl. Referenzen)

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 hereditary ataxias. The method combines target enrichment by CRISPR/Cas9, Oxford Nanopore long-read sequencing, and a bioinformatics pipeline for parallel detection of length, methylation, and sequence 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. We analyzed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients including very rare conditions. 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. 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.