Optimized testing strategy for the diagnosis of GAA-FGF14ataxia

Abstract

Background Dominantly inherited GAA repeat expansions inFGF14are a common cause of spinocerebellar ataxia (GAA-FGF14ataxia; SCA27B, late-onset). Molecular confirmation ofFGF14GAA 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 detectFGF14GAA 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 anFGF14(GAA)≥250 expansion. Conclusion This novel strategy reliably detected and sizedFGF14GAA expansions. It compared favorably to long-read sequencing and can readily be implemented in clinical laboratories.

Frequency and phenotypic spectrum of spinocerebellar ataxia 27B and other genetic ataxias in a Spanish cohort of late‐onset cerebellar ataxia

“…Specifically, we showed that SCA27B was particularly frequent in patients with pure cerebella ataxia. SCA27B accounted for 28% of cases in our cohort of patients with unsolved LOCA, a frequency similar to that reported in other European series [5,6,8,11,12]. Patients with SCA27B consistently presented with a slowly progressive pan-cerebellar syndrome predominantly affecting the gait, with frequent downbeat nystagmus and episodic symptoms at disease onset.…”

“…Thus, the phenotype of our patients did not differ from previous descriptions [5,6,[11][12][13][14]. Although neuropathy does not appear to be a core feature of SCA27B [5,6,8], an observation further supported by our study, hypopallaesthesia (without corresponding abnormalities on nerve conduction studies) was present in 50% of our patients. Similar findings were recently identified in a German cohort, suggesting impairment of the afferent sensory tracts [13].…”

“…In all, 40% of patients had a previously known molecular diagnosis (43/107). The remaining 64 patients with genetically undefined LOCA were screened for FGF14 GAA expansions, as previously described [8]. In patients without a molecular diagnosis, 83% (53/64) were screened for intronic FXN GAA repeat expansions, 92% (59/64) for SCA 1, 2, 3, 6, 7, 12, 17, and DRPLA, 100% (64/64) for RFC1 expansions, and 61% (39/64) for FMR1 premutation.…”

Natural History and Phenotypic Spectrum of GAA‐FGF14 Sporadic Late‐Onset Cerebellar Ataxia (SCA27B)

“…FGF14 GAA expansions were screened as reported elsewhere 13 using a three‐step procedure including fluorescent long‐range polymerase chain reaction (PCR), bidirectional repeated‐primed PCRs (RP‐PCRs), and agarose gel electrophoresis of long‐range PCR products and Sanger sequencing. The detailed procedures are described in Data S1.…”

The FGF14GAA repeat expansion in Greek patients with late‐onset cerebellar ataxia and an overview of the SCA27B phenotype across populations

“…The experimental methodology was performed as described previously, 10 with a modification of the 5 0 repeat-primed PCR (RP-PCR) for cases genotyped in Athens (Suppl. methods).…”