FGF14 repeat length and mosaic interruptions: modifiers of spinocerebellar ataxia 27B?

Deep intronic FGF14 repeat expansions have been identified as a frequent genetic cause of late-onset cerebellar ataxias, explaining ≤30% of patients. Interruptions between repeats have previously been identified to impact the penetrance in other repeat expansion disorders. Repeat interruptions within FGF14 have yet to be characterized in detail. We used long-range PCR, Sanger sequencing, repeat-primed PCR, Nanopore and PacBio sequencing to distinguish the repeat motifs, mosaicism and number of repeat interruptions present in FGF14-related ataxia patients and unaffected individuals. A total of 304 patients with late-onset ataxia and 190 unaffected individuals were previously screened for repeat expansions in FGF14 by long-range PCR, identifying 37 individuals with expanded repeat lengths (≥250 repeats). These, along with three newly identified expansion carriers were included in the present study, and advanced genetic methods were applied to investigate the repeat composition in 27 patients and 13 unaffected individuals. The expansions, based on Nanopore data, ranged from 236 to 486 repeats (standard deviation = 60), with 20 individuals showing repeat interruptions, including complex motifs such as <ani:underline>GAG</ani:underline>, GAA<ani:underline>GGA</ani:underline>, GAAGAA<ani:underline>A</ani:underline>GAA, GAAA<ani:underline>A</ani:underline>GAAGAA<ani:underline>G</ani:underline>GAAGAA<ani:underline>G</ani:underline>GAA, GAAA<ani:underline>A</ani:underline>GAAGAA<ani:underline>G</ani:underline>GAA and <ani:underline>GCA</ani:underline>GAAGAAGAAGAA. We calculated the longest pure GAA length from the long-read data for all 40 individuals. When comparing the pure GAA tract between patients and unaffected individuals, clusters were apparent based on >200 or <200 repeats. Five ataxia patients with interruptions still had a remaining pure GAA expansion <200. We observed an association of the pure GAA length with age at onset (P = 0.016, R² = 0.256). Somatically incurred mosaic divergent repeat interruptions were discovered that affect motif length and sequence (mDRILS), which varied in number and mosaicism (frequency: 0.37–0.93). The mDRILS were correlated with pure GAA length (P = 0.022, R² = 0.334), with a higher mosaic frequency of interruptions in unaffected individuals compared with patients (unaffected: 0.90; patients: 0.67; P = 0.009). We demonstrate that: (i) long-read sequencing is required to detect complex repeat interruptions accurately; (ii) repeat interruptions in FGF14 are mosaic, have various lengths and start positions in the repeat tract, and can thereby be annotated as mDRILS; which (iii) enabled us to establish a categorization based on remaining pure GAA repeats quantifying the impact of mDRILS on pathogenicity or age at onset, dependent on the interruption length and position, with high accuracy; and (iv) we provide evidence that mosaicism stabilizes pure GAA repeats in interrupted FGF14 repeat expansions.