Optimized testing strategy for the diagnosis of GAA-FGF14ataxia

Abstract Background Dominantly inherited GAA repeat expansions in FGF14 are a common cause of spinocerebellar ataxia (GAA- FGF14 ataxia; SCA27B, late-onset). Molecular confirmation of FGF14 GAA repeat expansions has thus far mostly relied on long-read sequencing, a technology that is not yet widely available in clinical laboratories. Methods We developed and validated a strategy to detect FGF14 GAA repeat expansions using long-range PCR, bidirectional repeat-primed PCRs, and Sanger sequencing. We compared this strategy to targeted nanopore sequencing in a cohort of 22 French Canadian patients and next validated it in a cohort of 53 French index patients with unsolved ataxia. Results Diagnosis was accurately confirmed for all 22 French Canadian patients using this strategy. Method comparison showed that capillary electrophoresis of long-range PCR products significantly underestimated expansion sizes compared to nanopore sequencing (slope, 0.87 [95% CI, 0.81 to 0.93]; intercept, 14.58 [95% CI, -2.48 to 31.12]) and gel electrophoresis (slope, 0.84 [95% CI, 0.78 to 0.97]; intercept, 21.34 [95% CI, -27.66 to 40.22]). The latter techniques yielded similar size estimates. Following calibration with internal controls, expansion size estimates were similar between capillary electrophoresis and nanopore sequencing (slope: 0.98 [95% CI, 0.92 to 1.04]; intercept: 10.62 [95% CI, -7.49 to 27.71]), and gel electrophoresis (slope: 0.94 [95% CI, 0.88 to 1.09]; intercept: 18.81 [95% CI, -41.93 to 39.15]). We identified 9 French patients (9/53; 17%) and 2 of their relatives who carried an FGF14 (GAA) ≥250 expansion. Conclusion This novel strategy reliably detected and sized FGF14 GAA expansions. It compared favorably to long-read sequencing and can readily be implemented in clinical laboratories.