Mosaic divergent repeat interruptions in XDP influence repeat stability and disease onset

Abstract

While many genetic causes of movement disorders have been identified, modifiers of disease expression are largely unknown. X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease caused by a SINE-VNTR-Alu(AGAGGG)n retrotransposon insertion in TAF1, with a polymorphic (AGAGGG)n repeat. Repeat length and variants in MSH3 and PMS2 explain ∼65% of the variance in age at onset (AAO) in XDP. However, additional genetic modifiers are conceivably at play in XDP, such as repeat interruptions. Long-read nanopore sequencing of PCR amplicons from XDP patients (n = 202) was performed to assess potential repeat interruption and instability. Repeat-primed PCR and Cas9-mediated targeted enrichment confirmed the presence of identified divergent repeat motifs. In addition to the canonical pure SINE-VNTR-Alu-5'-(AGAGGG)n, we observed a mosaic of divergent repeat motifs that polarized at the beginning of the tract, where the divergent repeat interruptions varied in motif length by having one, two, or three nucleotides fewer than the hexameric motif, distinct from interruptions in other disease-associated repeats, which match the lengths of the canonical motifs. All divergent configurations occurred mosaically and in two investigated brain regions (basal ganglia, cerebellum) and in blood-derived DNA from the same patient. The most common divergent interruption was AGG [5'-SINE-VNTR-Alu(AGAGGG)2AGG(AGAGGG)n], similar to the pure tract, followed by AGGG [5'-SINE-VNTR-Alu(AGAGGG)2AGGG(AGAGGG)n], at median frequencies of 0.425 (IQR: 0.42-0.43) and 0.128 (IQR: 0.12-0.13), respectively. The mosaic AGG motif was not associated with repeat number (estimate = -3.8342, P = 0.869). The mosaic pure tract frequency was associated with repeat number (estimate = 45.32, P = 0.0441) but not AAO (estimate = -41.486, P = 0.378). Importantly, the mosaic frequency of the AGGG negatively correlated with repeat number after adjusting for age at sampling (estimate = -161.09, P = 3.44 × 10-5). When including the XDP-relevant MSH3/PMS2 modifier single nucleotide polymorphisms into the model, the mosaic AGGG frequency was associated with AAO (estimate = 155.1063, P = 0.047); however, the association dissipated after including the repeat number (estimate = -92.46430, P = 0.079). We reveal novel mosaic divergent repeat interruptions affecting both motif length and sequence (DRILS) of the canonical motif polarized within the SINE-VNTR-Alu(AGAGGG)n repeat. Our study illustrates: (i) the importance of somatic mosaic genotypes; (ii) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability; and (iii) that these variations may remain undetected without assessment of single molecules.

Keywords: X-linked dystonia parkinsonism; genetic modifiers‌; mosaic; repeat interruptions; single-molecule sequencing.

Figure 1

Detection of divergent repeat interruptions within the TAF1 SVA hexanucleotide repeat domain. (A) Plus-strand reads show deletions or insertions within the SVA hexanucleotide repeat domain. (B) Minus-strand reads show deletions or insertion within the SVA hexanucleotide repeat domain. (C) Single-nucleotide resolution of the deletions detected at the 5′ end of the SVA hexanucleotide repeat domain. COV = coverage (number of reads covering the TAF1 SVA hexanucleotide repeat domain); DEL = deletion; INS = insertion.

Figure 2

Overview of mDRILS combinations within the TAF1 SVA hexanucleotide repeat motif and the corresponding detected frequencies. (A) Deleted nucleotides change the size and sequence of a single repeat unit of the hexameric tract. Boxed outline: change in repeat unit. Shaded highlight: common haplotypes. Black dots: indicating shifts in the repeat tract frame. The mosaic frequency of each DRIL is displayed alongside the interrupted repeat units. (B) The box plot shows frequencies of the combinations of deletions. The line and box represent median and interquartile range, respectively, and the whiskers represent the range.

Figure 3

Comparison of mDRILS frequencies detected in blood- and brain-derived DNA, enriched for the SVA insertion by PCR or amplification-free Cas9-enrichment. The bars represent a single frequency value for each mDRILS detected from (A) blood-derived DNA, (B) basal ganglia (BG)-derived DNA and (C) cerebellum (CRB)-derived DNA. (D) Repeat number distribution of each mDRILS, and (E) quartile coefficient of dispersion of each mDRILS. The bar charts show the detected frequencies of the different combinations of mDRILS. Box plot centre line represents median and box limits are upper and lower quartiles.

Figure 4

Relationship between mDRILs, AAO, and repeat number in patients with XDP. (A) The correlation between hexanucleotide repeat number and the mosaic frequency for divergent motif AGGG [5′-SINE-VNTR-Alu(AGAGGG)₂AGGG(AGAGGG)_n]. (B) The correlation between hexanucleotide repeat number and mosaic frequency for divergent motif AGG [5′-SINE-VNTR-Alu(AGAGGG)₂AGG(AGAGGG)_n]. (C) The correlation between AAO and mosaic frequency for divergent motif AGGG [5′-SINE-VNTR-Alu(AGAGGG)₂AGGG(AGAGGG)_n]. (D) The correlation between age at onset (AAO) mosaic frequency for divergent motif AGG [5′-SINE-VNTR-Alu(AGAGGG)₂AGG(AGAGGG)_n]. r = Spearman’s rank correlation coefficient; P = Spearman’s exploratory P-value.