Optimized testing strategy for the diagnosis of GAA-FGF14 ataxia/spinocerebellar ataxia 27B

Dominantly inherited GAA repeat expansions in FGF14 are a common cause of spinocerebellar ataxia (GAA-FGF14 ataxia; spinocerebellar ataxia 27B). 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. 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. Method comparison showed that capillary electrophoresis of long-range PCR amplification 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]). Diagnosis was accurately confirmed for all 22 French Canadian patients using this strategy. We also identified 9 French patients (9/53; 17%) and 2 of their relatives who carried an FGF14 (GAA)≥250 expansion. This novel strategy reliably detected and sized FGF14 GAA expansions, and compared favorably to long-read sequencing.


Optimization of polymerase chain reaction protocols
The performance of the fluorescent long-range polymerase chain reaction (fLR-PCR) and repeat-primed PCR (RP-PCR) protocols was assessed using different formulations of Taq Supplementary Table S4.
DNA concentrations were measured with the NanoDrop Spectrophotometer.

Performance of the PCR protocols with different formulations of Taq DNA polymerase
We compared the performance of the fLR-PCR and RP-PCR protocols on specimens from persons carrying an FGF14 GAA expansion using 5 different formulations of Taq DNA polymerase. Our findings showed that the Phusion Flash High-Fidelity PCR Master Mix and the GoTaq G2 Hot Start Polymerase performed best on fLR-PCR of expanded FGF14 alleles, as assessed by relative fluorescence units (RFU) peak intensity (Supplementary Figure S2). However, compared to the Phusion Flash High-Fidelity PCR Master Mix, the GoTaq G2 Hot Start Polymerase provided less consistent amplification of expanded alleles. The Phusion High-Fidelity DNA Polymerase and the ThermoPrime Taq DNA Polymerase yielded low to undetectable RFU peak intensity of expanded alleles. Furthermore, we also found that the Phusion Flash High-Fidelity PCR Master Mix performed best on RP-PCRs of expanded alleles compared to other Taq DNA polymerase formulations, which yielded less well-defined and resolute electrophoretic profiles of lower intensity (Supplementary Figure S2). Overall, our results showed that the Phusion Flash High-Fidelity PCR Master Mix was the only Taq DNA polymerase studied that performed well and consistently in both fLR-PCR and RP-PCR protocols.

Effect of different input amounts of genomic DNA on the performance of the PCR protocols
We assessed the performance of the fLR-PCR and RP-PCR protocols along a range of input amounts of genomic DNA, ranging from 200ng to 5ng, using specimens from 3 persons respectively carrying an  Figure S3). In comparison, we found the 3' RP-PCR to perform best and yield consistent sawtoothed profiles with DNA input amounts of at least 20ng (Supplementary Figure S3). Lower input amounts in 3' RP-PCR yielded low-intensity to undetectable saw-toothed profiles.

Performance of the PCR protocols on genomic DNA extracted from different tissues
The genomic DNA isolated from peripheral blood of patients included in this study was extracted using one of four different extraction kits, depending on the local clinical laboratory performing the extraction as listed in the Supplementary Methods. None of the extraction kits affected the performance of the PCR protocols negatively.
We also assessed the performance of the LR-PCR and RP-PCR protocols using DNA extracted from saliva, FFPE cerebellar cortex, and fresh frozen cerebellar cortex. We used DNA extracted from the saliva of a person with FGF14 (GAA)11/354 alleles, DNA extracted from FFPE cerebellar cortex of a person with FGF14 (GAA)9/474 alleles, and DNA extracted from fresh frozen cerebellar cortex of a person with FGF14 (GAA)9/331 alleles. We found that the expanded alleles of DNA extracted from saliva and fresh frozen cerebellar tissue were adequately resolved by LR-PCR and RP-PCRs, while the expanded allele of DNA extracted from FFPE cerebellar cortex could not be resolved by any PCR technique (Supplementary Figure S4).

Assessment of the effect of DNA shearing on the performance of the PCR protocols
Our objective was to determine whether shearing DNA from patients with an FGF14 GAA expansion could cause false negative results on LR-PCR and RP-PCRs. We sheared genomic DNA from 3 patients carrying an FGF14 (GAA)371, (GAA)383, and (GAA)508 expansion, respectively, and assessed DNA integrity using the Agilent 4200 TapeStation instrument (Supplementary Figure S5). We found that shearing did not result in allele dropout and false negative results on LR-PCR, fLR-PCR, and RP-PCR, although RFU peak intensity of sheared samples was lower than that of non-sheared samples on capillary electrophoresis (Supplementary Figure S6). However, these results do not rule out the possibility that DNA degradation in patients with FGF14 expansions could lead to allele dropout and false negative results on LR-PCR and RP-PCRs.

Comparison of GeneMapper and Peak Scanner software
We compared the performance of GeneMapper (version 6.0, Applied Biosystems) and Peak Scanner (version 3.0, Applied Biosystems) to analyze the results of capillary electrophoresis. Results were analyzed using the built-in microsatellite default settings in GeneMapper and the default settings in Peak Scanner. We found both software to be suitable for the analysis of capillary electrophoresis results of LR-PCR and RP-PCRs (Supplementary Figure   S7). In both software, high molecular weight peaks associated with alleles (GAA)≥250 repeat units had a characteristic bell-shaped appearance that was readily distinguishable from the background on capillary electrophoresis of fluorescent LR-PCR amplification products (see