Open Access

Hannes Erdmann, Annalisa Schaub, Morghan C. Lucas, Veronika Scholz, Anna Benet-Pages, Kerstin Becker, Christine Dineiger, Veronika Mayer, Inga van Buren, Eva Breithausen, Karl Akbari, Isabell Cordts, Mayra Sauer, Christine Schneider, Rosanna Krakowsky, Franziska Schnabel, Konstanze Dunker, Lena Fabritius, Johannes Gerb, Denis Grabova, Ken Möhwald, Marius Näher, Karoline Steinmetz, Franziska Thiessen, Alexander Jäck, Christiane Schneider-Gold, Simone Zittel, Christina Petersen, Isolde Schreyer, Larissa Mämecke, Sibylle Wilfling, Gilbert Wunderlich, David Brenner, Yorck Hellenbroich, Kirsten Muhle, Tessa Huchtemann, Inga Claus, Thomas Klopstock, Michael Strupp, Johannes Levin, Günter Höglinger, Doreen Huppert, Sandra Becker-Bense, Filipp Filippopulos, Fabian Kilpert, Elsa Leitão, Sabine Kaya, Christel Depienne, Florian Schöberl, Teresa Neuhann, Elke Holinski-Feder, Andreas Zwergal, Angela Abicht

• Hereditary adult-onset ataxias are a heterogeneous group of phenotypically overlapping conditions, often caused by pathogenic expansions of short tandem repeats. Currently, 18 repeat disorders with a core phenotype of adult-onset ataxia are known. Diagnosis typically relies on sequential PCR-based methods, which are labour-intensive and lack precision. Long-read sequencing (LRS) has the potential to overcome these limitations and is currently implemented and validated in clinical genetics. Using clinical nanopore Cas9-targeted sequencing (Clin-CATS) for parallel in-depth repeat analysis, we evaluated a diagnostic cohort of 513 adult-onset ataxia patients, determining frequencies of all known repeat-associated ataxias except Spinocerebellar ataxia 4 (SCA4), as well as the carrier frequencies for autosomal-recessive disorders, RFC1 spectrum disorder and Friedreich’s ataxia (FRDA). Additionally, phenotypes of patients with established genetic diagnoses were characterized, especially thoseHereditary adult-onset ataxias are a heterogeneous group of phenotypically overlapping conditions, often caused by pathogenic expansions of short tandem repeats. Currently, 18 repeat disorders with a core phenotype of adult-onset ataxia are known. Diagnosis typically relies on sequential PCR-based methods, which are labour-intensive and lack precision. Long-read sequencing (LRS) has the potential to overcome these limitations and is currently implemented and validated in clinical genetics. Using clinical nanopore Cas9-targeted sequencing (Clin-CATS) for parallel in-depth repeat analysis, we evaluated a diagnostic cohort of 513 adult-onset ataxia patients, determining frequencies of all known repeat-associated ataxias except Spinocerebellar ataxia 4 (SCA4), as well as the carrier frequencies for autosomal-recessive disorders, RFC1 spectrum disorder and Friedreich’s ataxia (FRDA). Additionally, phenotypes of patients with established genetic diagnoses were characterized, especially those of patients living with RFC1 spectrum disorder and SCA27B. Repeat-associated ataxias were confirmed in 33.3% of cases, including rare ataxias, such as SCA10, SCA36 and SCA37, alongside as the most prevalent conditions SCA27B and RFC1 spectrum disorder. Potentially pathogenic expansions in FGF14 were identified in an additional 4.7% of patients. Testing of another 347 patients for ZFHX3 expansions linked to SCA4 did not identify any cases. Dual diagnoses were frequent, occuring in 6.4% of patients with repeat-associated ataxia. We confirmed a high RFC1 spectrum disorder carrier frequency (7.2%) and reclassified certain FXN expansions as likely non-pathogenic, resulting in a lower than estimated carrier frequency for FRDA of 0.8%. We also identified novel repeat configurations in several loci and illustrated the high heterogeneity of repeat expansions in RFC1, highlighting it as a potential source of false results when using PCR-based methods. This study underscores the diagnostic advantages of LRS for comprehensive repeat analysis and recommends its adoption as a standard in clinical genetics, replacing Southern blot and PCR-based approaches. Furthermore, based on our findings in a large patient cohort a re-evaluation of existing phenotype-genotype correlations is recommended as well as evaluating additional parameters besides repeat length to improve diagnostic precision of repeat analysis.…