Targeted Long-Read Sequencing as a Single Assay Improves the Diagnosis of Spastic-Ataxia Disorders

Abstract

Objective: The hereditary spastic-ataxia spectrum disorders are a group of disabling neurological diseases. The traditional genetic testing pathway is complex, multistep and leaves many cases unsolved. We aim to streamline and improve this process using long-read sequencing.

Methods: We developed a targeted long-read sequencing strategy with the capacity to characterise the 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 unsolved spastic-ataxia. An additional five individuals with a known genetic diagnosis were included as positive controls.

Results: 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 naïve 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 (7/34, 21%). Two individuals (2/34, 6%) had biallelic pathogenic expansions in RFC1 and one individual had a monoallelic pathogenic expansion in ATXN8OS/ATXN8. Causative pathogenic sequence variants other than short tandem repeat expansions were found in four individuals, including in VCP, STUB1, ANO10 and SPG7. Furthermore, all five positive controls were identified.

Interpretation: 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.

Keywords: hereditary cerebellar ataxia; hereditary spastic paraplegia; nanopore sequencing; spinocerebellar ataxia.

FIGURE 1

Improved diagnosis of spastic‐ataxia spectrum disorders with targeted long‐read sequencing. (A) Summary of genetic diagnoses in our cohort of 34 genetically undiagnosed patients with spastic‐ataxia spectrum disorders. (B) Sequence bar charts show STR alleles identified in 13 patients with expanded STRs in FGF14. Seven individuals, denoted with asterisks, had pure (GAA) expansions longer than 250 copies, sufficient for SCA27B diagnosis. The remaining six patients had (GAA) expansions that did not reach 250 copies or expanded (GAA) repeats interrupted by other motifs (GCA or GGA), not considered pathogenic. (C) Genome browser view shows detection of two pathogenic heterozygous variants within SPG7 in a single patient. Alignments are phased into separate haplotypes (pink = haplotype 1; blue = haplotype 2), confirming the two pathogenic variants are on alternative haplotypes (i.e., in trans).

FIGURE 2

Clinical features of spastic‐ataxia patients receiving a genetic diagnosis via long‐read sequencing. (A) Chart provides an overview of clinical characteristics for all patients who received a genetic diagnosis (plus patient 15, with biallelic FGF14 GAA expansions < 250 repeats, of uncertain clinical significance). The upper bar chart shows patient ages at time of analysis (yellow) and their age at symptom onset (green). The heatmap below indicates symptoms identified in each patient (blue = positive; white = negative; grey = not assessed). Patient sex is encoded with blue (male) or pink (female) tiles and genetic diagnoses are coded as follows: FGF14 = red; RFC1 = yellow; ATXN8 = orange; STUB1 = navy; ANO10 = purple; VCP = blue; SPG7 = green; biallelic FGF14 (uncertain clinical significance) = grey. Patients with SCA27B are sorted by FGF14 expansion size, in matched order to Figure 1B. (B) Bar chart summarises the frequency of clinical phenotypes observed among patients diagnosed with SCA27B, based on STR expansions in FGF14 (n = 7 patients).

FIGURE 3

Overview of outcomes. Flow chart provides a schematic overview of diagnostic outcomes across the study cohort, which included 34 genetically undiagnosed patients with spastic‐ataxia spectrum disorders and five patients with known genetic diagnoses included as positive controls.