Figure 2. Allele Distribution and Long-Read Sequencing of the FGF14 GAA Repeat Locus.
Panels A and B show the allele distribution of the FGF14 repeat locus in 408 controls (816 chromosomes) (Panel A) and 128 patients with GAA-FGF14–related ataxia (122 normal and 134 expanded chromosomes) (Panel B). The repeat length was estimated by agarose-gel electrophoresis of PCR-amplification products. Among the patients with GAA-FGF14–related ataxia, 4 were homozygous or compound heterozygous for (GAA)≥250 expansions, and 2 were compound heterozygous for a (GAA)≥250 expansion and a (GAAGGA)≥125 expansion. The smallest number of GAA repeats among controls and patients was 8. The density plots show allele-size frequencies, with higher densities indicating greater frequencies. The box-and-whisker plots show the allelic distribution in controls and patients. In Panel B, the box-and-whisker plots above the graph show the distribution of the normal alleles (left-hand plot) and expanded alleles (right-hand plot) in patients. The box indicates the 25th percentile (first quartile), the median, and the 75th percentile (third quartile), and the whiskers indicate the 2.5th and 97.5th percentiles. Outliers are represented by black dots. In controls, expanded alleles consisting of non-GAA repeats are represented by red triangles. The dashed gray lines and the shaded gray areas indicate the incompletely penetrant range of (GAA)250–300, and the dashed red lines mark the threshold of (GAA)300 repeat units, above which the alleles are fully penetrant. The allelic distributions in the different control and patient cohorts are shown in Figure S4. Panel C shows swarm plots of 3000 randomly sampled individual nanopore reads containing at least 50 repeat units for 2 controls and 6 patients. Control G-C164 carries a subpathogenic (GAA)222 allele, and control FC-C57 carries a nonpathogenic (GAAGGA)152 allele. Patients I.22, II.6, and III.3 (French Canadian discovery cohort) carry a (GAA)388, (GAA)508, and (GAA)285 expansion, respectively. Patients G6 (German cohort) and A2 (Australian cohort) carry a (GAA)345 and (GAA)437 expansion, respectively. Patient FC2 (French Canadian cohort) carries a (GAA)223 allele and a (GAA)355 allele. Horizontal black bars indicate repeat size of the larger allele, as measured by nanopore sequencing. Expansion sizing by long-read nanopore sequencing and agarose-gel electrophoresis of PCR amplification products are highly similar (Pearson correlation coefficient, 0.96), as shown in Figure S8. The horizontal dashed gray line and the shaded gray area show the incompletely penetrant range of (GAA)250–300, and the dashed red line marks the threshold of (GAA)300 repeat units, above which the alleles are fully penetrant. The color of the data points is a function of the GAA repeat motif purity in each individual read, with dark blue indicating pure and lighter blue impure motif (a hue scale is shown on the right y axis). Panel D shows the percentages of patients with LOCA who carried an FGF14 (GAA)≥250 repeat expansion in the French Canadian (40 of 66 index patients), German (42 of 228), Australian (3 of 20), and Indian (3 of 31) cohorts. Panel E shows repeat-length variation across 15 maternal and 15 paternal meiotic events involving alleles of (GAA)≥250 repeats.