Objective
X‐linked dystonia parkinsonism (XDP) is a neurodegenerative movement disorder caused by a single mutation: SINE‐VNTR‐Alu (SVA) retrotransposon insertion in TAF1. Recently, a (CCCTCT)n ...repeat within the SVA insertion has been reported as an age‐at‐onset (AAO) modifier in XDP. Here we investigate the role of this hexanucleotide repeat in modifying expressivity of XDP.
Methods
We genotyped the hexanucleotide repeat in 355 XDP patients and correlated the repeat number (RN) with AAO (n = 295), initial clinical manifestation (n = 294), site of dystonia onset (n = 238), disease severity (n = 28), and cognitive function (n = 15). Furthermore, we investigated i) repeat instability by segregation analysis and Southern blotting using postmortem brain samples from two affected individuals and ii) relative TAF1 expression in blood RNA from 31 XDP patients.
Results
RN showed significant inverse correlations with AAO and with TAF1 expression and a positive correlation with disease severity and cognitive dysfunction. Importantly, AAO (and not RN) was directly associated with whether dystonia or parkinsonism will manifest at onset. RN was lower in patients affected by mouth/tongue dystonia compared with blepharospasm. RN was unstable across germline transmissions with an overall tendency to increase in length and exhibited somatic mosaicism in brain.
Interpretation
The hexanucleotide repeat within the SVA insertion acts as a genetic modifier of disease expressivity in XDP. RN‐dependent TAF1 repression and subsequent differences in TAF1 mRNA levels in patients may be potentiated in the brain through somatic variability leading to the neurological phenotype. ANN NEUROL 2019;85:812–822.
Abstract
X-linked dystonia-parkinsonism is a neurodegenerative disorder caused by a founder retrotransposon insertion, in which a polymorphic hexanucleotide repeat accounts for ~50% of age at onset ...variability. Employing a genome-wide association study to identify additional factors modifying age at onset, we establish that three independent loci are significantly associated with age at onset (
p
< 5 × 10
−8
). The lead single nucleotide polymorphisms collectively account for 25.6% of the remaining variance not explained by the hexanucleotide repeat and 13.0% of the overall variance in age at onset in X-linked dystonia-parkinsonism with the protective alleles delaying disease onset by seven years. These regions harbor or lie adjacent to
MSH3
and
PMS2
, the genes that were recently implicated in modifying age at onset in Huntington’s disease, likely through a common pathway influencing repeat instability. Our work indicates the existence of three modifiers of age at onset in X-linked dystonia-parkinsonism that likely affect the DNA mismatch repair pathway.
X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an ...intron of the
gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the
intron retention transcript
can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the
promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons.
Neuroinflammation plays a pathogenic role in neurodegenerative diseases and recent findings suggest that it may also be involved in X-linked Dystonia-Parkinsonism (XDP) pathogenesis. Previously, ...fibroblasts and neuronal stem cells derived from XDP patients demonstrated hypersensitivity to TNF-α, dysregulation in NFκB signaling, and an increase in several pro-inflammatory markers. However, the role of inflammatory processes in XDP patient brain remains unknown. Here we demonstrate that there is a significant increase in astrogliosis and microgliosis in human post-mortem XDP prefrontal cortex (PFC) compared to control. Furthermore, there is a significant increase in histone H3 citrullination (H3R2R8R17cit3) with a concomitant increase in peptidylarginine deaminase 2 (PAD2) and 4 (PAD4), the enzymes catalyzing citrullination, in XDP post-mortem PFC. While there is a significant increase in myeloperoxidase (MPO) levels in XDP PFC, neutrophil elastase (NE) levels are not altered, suggesting that MPO may be released by activated microglia or reactive astrocytes in the brain. Similarly, there was an increase in H3R2R8R17cit3, PAD2 and PAD4 levels in XDP-derived fibroblasts. Importantly, treatment of fibroblasts with Cl-amidine, a pan inhibitor of PAD enzymes, reduced histone H3 citrullination and pro-inflammatory chemokine expression, without affecting cell survival. Taken together, our results demonstrate that inflammation is increased in XDP post-mortem brain and fibroblasts and unveil a new epigenetic potential therapeutic target.
•Astrogliosis and microgliosis are increased in post-mortem XDP prefrontal cortex.•Histone H3 citrullination and PADs are increased in XDP prefrontal cortex.•Myeloperoxidase levels are increased in post-mortem XDP prefrontal cortex.•Increases in histone H3 citrullination contribute to neuroinflammation in XDP.
X-linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative disorder characterized by progressive dystonia and parkinsonism. It is caused by a SINE-VNTR-Alu (SVA) retrotransposon ...insertion in the
gene with a polymorphic (
)
domain that acts as a genetic modifier of disease onset and expressivity.
Herein, we used Nanopore sequencing to investigate SVA genetic variability and methylation. We used blood-derived DNA from 96 XDP patients for amplicon-based deep Nanopore sequencing and validated it with fragment analysis which was performed using fluorescence-based PCR. To detect methylation from blood- and brain-derived DNA, we used a Cas9-targeted approach.
High concordance was observed for hexanucleotide repeat numbers detected with Nanopore sequencing and fragment analysis. Within the SVA locus, there was no difference in genetic variability other than variations of the repeat motif between patients. We detected high CpG methylation frequency (MF) of the SVA and flanking regions (mean MF = 0.94, SD = ±0.12). Our preliminary results suggest only subtle differences between the XDP patient and the control in predicted enhancer sites directly flanking the SVA locus.
Nanopore sequencing can reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly, variation in the repeat motif.
X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant ...TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.
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.
While many genetic causes of movement disorders have been identified, modifiers of disease expression are largely unknown. Trinh et al. identify a novel somatic divergent repeat interruption within the TAF1 repeat expansion that influences repeat stability, and indirectly the age at onset of X-linked dystonia parkinsonism.
Deep brain stimulation (DBS) is indisputable in improving motor symptoms of Parkinson's Disease (PD) and X-Linked Dystonia Parkinsonism (XDP)(4,9,22,23,26). However, a discrepancy between this ...improvement and the perceived quality of life (QoL) has been observed. This study aims to investigate changes and correlation between quality of life, motor symptoms and medication dosing.
This prospective observational study enrolled 13 patients (6 PD, 7 XDP) who underwent DBS from 2017 to 2018. Quality of life changes were determined by Parkinson's Disease - 39 (PDQ-39 English and Filipino versions) at baseline, 6 months and 12 month after DBS. Motor symptoms and medication dosing were also evaluated within the same period and correlated with QoL changes.
There is a significant reduction of PDQ-39 mean scoresF(1.06,11.64) = 18.235; p = 0.001; ηp2 = 0.624 between baseline and 6 months among XDP patients (p = 0.018) and baseline and 12 months among PD patients (p = 0.027) and XDP patients (p < 0.001). Specific domains with significant improvement were stigma, cognition, mobility, ADLs, communication and bodily discomfort. Correlating these with changes in motor symptoms, only mobility for PD and ADLs for XDP were positively related.
This study has shown the positive impact of DBS in improving QoL among PD and XDP patients over a 12-month period.
•Deep brain stimulation positively impacts QoL of PD and XDP patients.•Improvement of parkinsonian and dystonic symptoms among PD and XDP patients are comparable after undergoing DBS.•Medication doses and medication-induced complications were reduced after deep brain stimulation.•DBS effects regress with respect to time.
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