Objective:

To assess the diagnostic yield of long-read genome sequencing (LR-GS) in ataxia patients with prior non-diagnostic genetic testing.

Background:

Hereditary cerebellar ataxias are genetically diverse disorders characterized by progressive cerebellar atrophy. Despite advances in conventional methods of genetic testing, such as exome sequencing, repeat-expansion testing, and targeted gene panels, many ataxia cases remain genetically undiagnosed. The diagnostic yield of next-generation sequencing and repeat-expansion panels remains limited, and multiple tests are often needed. LR-GS can detect structural variants, including tandem repeats, more robustly than short-read approaches and may consolidate testing and increase diagnostic yield.

Design/Methods:

We attempted to perform LR-GS on 100 previously extracted DNA samples from patients with negative prior clinical testing for cerebellar ataxia. Of these, 59 samples had adequate quality for LR-GS. Inclusion was not limited by prior test type. The standard PacBio pipeline was used for variant calling and annotation.

Results:

All 59 patients had negative ataxia repeat-expansion analysis, and a subset had prior exome testing. Six yielded a diagnostic or likely diagnostic finding, and one candidate variant was identified that requires further validation. In one case, LR-GS assisted in the interpretation of two previously identified variants by establishing their phase as in trans.

Conclusions:

LR-GS can accurately identify disease-causing variants in ataxia. While high-molecular-weight DNA extraction is recommended for LR-GS, over half of previously extracted DNA samples were of sufficient quality, suggesting the feasibility of reanalyzing previously stored specimens. In 8 cases (14%) a potentially diagnostic finding was identified; however, in seven of these eight cases, the finding would likely have been detected by short-read genome sequencing.