Disease onset in X-linked dystonia-parkinsonism correlates with expansion of a hexameric repeat within an SVA retrotransposon in TAF1

Abstract

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease associated with an antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1 This unique insertion coincides with six additional noncoding sequence changes in TAF1, the gene that encodes TATA-binding protein-associated factor-1, which appear to be inherited together as an identical haplotype in all reported cases. Here we examined the sequence of this SVA in XDP patients (n = 140) and detected polymorphic variation in the length of a hexanucleotide repeat domain, (CCCTCT)_n The number of repeats in these cases ranged from 35 to 52 and showed a highly significant inverse correlation with age at disease onset. Because other SVAs exhibit intrinsic promoter activity that depends in part on the hexameric domain, we assayed the transcriptional regulatory effects of varying hexameric lengths found in the unique XDP SVA retrotransposon using luciferase reporter constructs. When inserted sense or antisense to the luciferase reading frame, the XDP variants repressed or enhanced transcription, respectively, to an extent that appeared to vary with length of the hexamer. Further in silico analysis of this SVA sequence revealed multiple motifs predicted to form G-quadruplexes, with the greatest potential detected for the hexameric repeat domain. These data directly link sequence variation within the XDP-specific SVA sequence to phenotypic variability in clinical disease manifestation and provide insight into potential mechanisms by which this intronic retroelement may induce transcriptional interference in TAF1 expression.

Keywords: DYT3; Parkinson’s disease; TAF1; XDP; dystonia.

Fig. 1.

(Upper) The genomic segment previously associated with XDP on chromosome Xq13.1 with hg19 coordinates, seven known XDP-specific variants that comprise the disease haplotype (boxed region), and flanking markers used to narrow the region. Haplotype variants consist of five single-nucleotide substitutions annotated as DSC-1, 2, 3, 10, and 12; a 48-bp deletion (48 bp Del), and a SVA-type retrotransposon insertion. Eight genes are shown within the broader linkage region, including TAF1. (Lower) Canonical exons of TAF1, the relative position of the SVA inserted antisense to TAF1, and the domain structure of the SVA consisting of (5′–3′) a hexameric repeat (CCCTCT) of variable length, an Alu-like domain, a VNTR, a SINE domain, and a poly(A) tail.

Fig. 2.

Length of the hexameric repeat is polymorphic in affected XDP individuals and is inversely correlated with AO based on linear regression analysis. (A) Correlation between repeat length and AO in the entire cohort; n = 140, R² = 0.507, P = 3.54 × 10⁻²³. (B–D) Analysis of individual subgroups revealed similar correlations in probands seen in Philippines clinics (n = 67, R² = 0.5073, P = 1.39 × 10⁻¹¹) (B), probands seen in a US clinic (n = 14, R² = 0.519, P = 0.003658) (C), and archival DNA samples (20) (n = 59, R² = 0.505, P = 1.79 × 10⁻¹⁰) (D).

Fig. 3.

(A) A representative XDP pedigree with multiple haplotype-positive individuals, including an affected proband (black box) with 41 repeats of the SVA hexamer and four daughters (circles) with different tract lengths ranging from 37 to 44 hexameric repeats. (B) Contingency table of intergenerational pairs depicting the change in hexameric repeat tract length during transmission through the male (solid bars) vs. female (open bars) germ lines. Because the repeat is present on the X chromosome, mothers transmitted the allele to both sons and daughters, whereas fathers transmitted only to daughters. When inherited from females (n = 42), the hexameric repeat length primarily remained the same or increased, but when inherited from males (n = 11) there was an increased frequency of contractions. χ² analysis revealed a significant difference in these distributions (χ² = 23.35, df = 2; P < 0.0001), suggesting that parental sex may influence expansion vs. contraction of the hexamer.

Fig. 4.

Luciferase reporter assay to quantify intrinsic promoter activity of the XDP-specific SVA. (A) Schematic depicting the reporter constructs. Three versions of SVA forward and reverse orientations were generated, representing hexamers with 52, 41, or 35 repeats (Hex52, Hex41, and Hex35, respectively). A truncated SVA lacking the hexameric repeat domain (ΔHex) was also generated in both forward and reverse orientations. (B) Fold changes in luciferase activity in SH-SY5Y cells produced by SVA constructs relative to basal level induced by the pGL3b vector alone. Data represent fold-change values averaged across four replicate experiments, shown as SEM. Significance was assessed by one-way ANOVA followed by post hoc Student’s t tests with Bonferroni correction. Asterisks above bars denote the significance for the indicated SVA construct compared with vector alone. Asterisks above lines indicate the significance for additional comparisons. n.s., not significant. All four SVA forward constructs significantly repressed luciferase activity, although the ΔHex variant was less effective than the constructs bearing hexameric repeats. In the reverse orientation, the longer SVAs (Hex52 and Hex41) significantly increased luciferase activity, whereas the shorter variants (Hex35 and ΔHex) exhibited minimal effect. (C) In U2OS cells, the Hex52 and Hex35 SVAs in the forward orientation significantly repressed activity, whereas the truncated ΔHEX SVA significantly increased it. Increased activity was also produced by all SVA variants in the reverse orientation, with the greatest increase produced by the Hex35 variant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

(A and B) Predicted G4 formation by each position in the XDP-specific SVA on both forward (sense; 5′–3′) (A) and reverse (antisense; 3′–5′) (B) strands. Predicted boundaries are designated for the SVA functional domains: the hexameric repeat (Hex), Alu-like domain (Alu), VNTR, SINE-R domain, and a poly(A) or poly(T) sequence. The x axis depicts the nucleotide position within the SVA sequence of the individual bearing 35 hexameric repeats, as derived from the BAC clone. The y axis represents the G-score computed by QGRS Mapper software (60). The central VNTR region in both orientations includes positions predicted to form G4 structures, whereas the AGAGGG_n hexamer in the reverse orientation exhibits greater G4 potential than any other position in the SVA. (C) Increasing the number of hexameric repeats to 41 or 52 extends the length of the predicted quadruplex as shown.

Fig. 6.

Hypothesized sources of transcriptional interference associated with the XDP-specific SVA insertion in an intron of TAF1 between flanking exons 32 (Ex32) and 33 (Ex33). (A) In wild-type cells, RNAP II successfully traverses the intron, generating a transcript that splices sequences derived from exons 32 and 33. (B) In XDP cells, the intronic SVA insertion may create multiple barriers to RNAP II transcribing TAF1, including transcription factors and/or competing RNAP II complexes associated with the SVA on both strands as well as G4 formation by the VNTR and hexameric domains. Obstacles that slow or prevent progression of RNAP II transcribing TAF1 may potentially decrease transcription of downstream exons and/or alter splicing at these loci. (Inset) Organization of SVA inserted antisense to TAF1.