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. 2023 Jun 15;13(1):9737.
doi: 10.1038/s41598-023-36654-8.

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

Céline Bonnet #  1   2 , David Pellerin #  3   4 , Virginie Roth  5 , Guillemette Clément  6   7 , Marion Wandzel  5 , Laëtitia Lambert  6   8 , Solène Frismand  7 , Marian Douarinou  7 , Anais Grosset  7 , Ines Bekkour  7 , Frédéric Weber  5 , Florent Girardier  5 , Clément Robin  5 , Stéphanie Cacciatore  5 , Myriam Bronner  5 , Carine Pourié  6 , Natacha Dreumont  6 , Salomé Puisieux  7 , Pablo Iruzubieta  3   9   10   11 , Marie-Josée Dicaire  4 , François Evoy  12 , Marie-France Rioux  12 , Armand Hocquel  7 , Roberta La Piana  4   13 , Matthis Synofzik  14   15 , Henry Houlden  3 , Matt C Danzi  16 , Stephan Zuchner  16 , Bernard Brais  4   17 , Mathilde Renaud  18   19   20
Affiliations

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

Céline Bonnet et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

Matthis Synofzik has received consultancy honoraria from Janssen, Ionis, Orphazyme, Servier, Reata, GenOrph, and AviadoBio, all unrelated to the present manuscript. Stephan Zuchner is consultant on drug targets for Aeglea BioTherapeutics and consultant on clinical trial design for Applied Therapeutics, all of them unrelated to the work in the present manuscript. Other authors have no relevant financial interests to disclose.

Figures

Figure 1
Figure 1
Testing strategy for the diagnosis of GAA-FGF14 ataxia. The number of samples in the French Cohort of 53 index patients processed at each step is indicated in the red circles. Normal alleles have (GAA)<250 repeats and expanded alleles have (GAA)≥250 repeats. *Two expanded alleles are possible. N allele, normal allele < 250 repeat units; LR-PCR, long-range polymerase chain reaction; RP-PCR, repeat-primed PCR.
Figure 2
Figure 2
Molecular analysis of the FGF14 repeat locus. (A) Fluorescent long-range PCR of the FGF14 repeat locus of four patients with late-onset cerebellar ataxia. The calculated number of repeat units of each allele (before correction) is indicated for each of the four patients. (B) 5′ RP-PCR and 3′ RP-PCR of the FGF14 repeat locus of two patients carrying a (GAA)≥250 repeat expansion in FGF14. (C) Sanger sequencing of a patient carrying a (GAA)≥250 repeat expansion showing GAA repeats: (GAA)n on forward strand and (TTC)n on reverse strand. (D) Agarose gel electrophoresis (1.5%), lanes 1 and 8: 2,000 bp molecular weight marker, 2: 17/132 repeat units, 3: 9/284 repeat units, 4: 9/510 repeat units, 5: 9/313 repeat units, 6: 9/467 repeat units, and 7: negative control. (E) Agarose gel electrophoresis (1.5%), lanes 1 and 9: 2,000 bp molecular weight marker, 2: 107/236 repeat units, 3: 40/132 repeat units, 4: 16/182 repeat units, 5: 8/9 repeat units, 6: 17/319 repeat units, 7: 43/370 repeat units, and 8: 28/94 repeat units. Agarose gels were imaged with the Uvidoc HD6 Gel Documentation System using the default UV-Gel settings in the Uvitec-1D software, which resulted in overexposure. The gels presented in D and E were cropped for clarity of presentation. The original gels are presented in Supplementary Fig. S10.
Figure 3
Figure 3
FGF14 allele size estimates by fluorescent LR-PCR, long-read nanopore sequencing, and agarose gel electrophoresis. Passing-Bablok regression (blue lines) with 95% confidence interval (shaded blue areas) for allele size measured by (A) fLR-PCR and nanopore sequencing, (B) fLR-PCR and gel electrophoresis, (E) fLR-PCR (with correction) and nanopore sequencing, and (F) fLR-PCR (with correction) and gel electrophoresis. The dashed black lines show the identity line and the dashed red lines show the pathogenic threshold of (GAA)≥250 repeats. Bland–Altman plots show the percentage difference between size estimates measured by fLR-PCR and (C) targeted nanopore sequencing or (D) agarose gel electrophoresis as a function of the average of the two measurements for each sample. Plots show the percentage difference between size estimates measured by corrected fLR-PCR and (G) targeted nanopore sequencing or (H) agarose gel electrophoresis as a function of the average of the two measurements for each sample. The dashed red lines show the mean bias between two techniques and the dashed gray lines show the limits of agreement, defined as the mean percentage difference ± 1.96 SD.
Figure 4
Figure 4
Pedigree and molecular analysis of the FGF14 repeat locus in a family with GAA-FGF14 ataxia and a patient compound heterozygous for two expansions. (A) Pedigree of an affected multigenerational French family showing autosomal dominant transmission of the disease. Patient II.1 carries a (GAA)467 allele and had a disease onset at age 76 years. Patients III.1 and III.2 carry a (GAA)492 allele and a (GAA)510 allele, respectively. They first manifested episodic ataxia at age 62 years and age 55 years, respectively. AAO indicates age at onset. (B) Agarose gel (1.5%), lanes 1 and 10: 2,000 bp molecular weight marker, 2: 58/300 repeat units, 3: 266/417 repeat units, 4: 9/9 repeat units, 5: 9/50 repeat units, 6: 9/492 repeat units, 7: 9/467 repeat units, 8: 9/510 repeat units, and 9: negative control. The agarose gel was imaged with the Uvidoc HD6 Gel Documentation System using the default UV-Gel settings in the Uvitec-1D software, which resulted in overexposure. The gel was cropped for clarity of presentation. The original gel is presented in Supplementary Fig. S11. (C) Fluorescent long-range PCR, (D) 5′ RP-PCR and 3′ RP-PCR, and (E) Sanger sequencing of a patient compound heterozygous for two expanded alleles (266/417 repeat units). (C) On fLR-PCR, the smallest expansion of 266 repeat units is detected whereas the larger expansion of 417 repeat units is not as it falls beyond the limit of detection of capillary electrophoresis. (E) Sanger sequencing shows polymorphism at the 5′ end of each expansion. Allele 1: (GAA)(GAAA)(GGA)(GAA)n and allele 2: (GAA)(GAAA)(GAAA)(GAA)n.
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