Targeted long-read sequencing as a single assay improves diagnosis of spastic-ataxia disorders

Abstract

The hereditary spastic-ataxia spectrum disorders are a group of rare disabling neurological diseases. The genetic testing process is complex, and often requires multiple different assays to evaluate the many potential causative genes and variant types, including short tandem repeat expansions, single nucleotide variants, insertions/deletions, structural variants and copy number variants. This can be a protracted process and, even after all avenues are exhausted, many individuals do not receive a genetic diagnosis. Aiming to streamline and improve this process, we developed a targeted long-read sequencing strategy with capacity to characterise genetic variation of all types and sizes within 469 disease-associated genes, in a single assay. We applied this to a cohort of 34 individuals with genetically undiagnosed spastic-ataxia spectrum disorders. An additional five individuals with a known genetic diagnosis were included as positive controls. We identified causative pathogenic variants that would be sufficient for genetic diagnosis in 14/34 (41%) unsolved participants. The success rate was 5/11 (45%) in those who were naive to genetic testing and 9/23 (39%) in those who were undiagnosed after prior genetic testing, completed on a clinical basis. Short tandem repeat expansions in FGF14 were the most common cause, present in 7/34 (21%). Two individuals (2/34, 6%) had biallelic pathogenic short tandem repeat expansions in RFC1 and one individual had a monoallelic pathogenic short tandem repeat expansion in ATXN8OS/ATXN8. Causative pathogenic sequence variants other than short tandem repeat expansions were found in four individuals, including a heterozygous missense variant in VCP, a heterozygous in-frame deletion in STUB1, a homozygous splicing variant in ANO10, and compound heterozygous missense and nonsense variants in SPG7. In addition to these solved cases, a pathogenic or likely-pathogenic variant with uncertain clinical implications was identified in a further three individuals, including a single individual who was found to have a short tandem repeat expansion in BEAN1 in addition to biallelic expansions in FGF14 within the range of 200-249 repeats. Our results demonstrate the utility of targeted long-read sequencing in the genetic evaluation of patients with spastic-ataxia spectrum disorders, highlighting both the capacity to increase overall diagnostic yield and to streamline the testing pathway by capturing all known genetic causes in a single assay.