Stability of Mosaic Divergent Repeat Interruptions in X-Linked Dystonia-Parkinsonism.

BACKGROUND: X-Linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative disorder characterized by rapidly progressive dystonia and parkinsonism. Mosaic Divergent Repeat Interruptions affecting motif Length and Sequence (mDRILS) were recently found within the TAF1 SVA repeat tract and were shown to associate with repeat stability and age at onset in XDP, specifically the AGGG [5'-SINE-VNTR-Alu(AGAGGG)2AGGG(AGAGGG)n] mDRILS. OBJECTIVE: This study aimed to investigate the stability of mDRILS frequencies and stability of (AGAGGG)n repeat length during transmission in parent-offspring pairs. METHODS: Fifty-six families (n = 130) were investigated for generational transmission of repeat length and mDRILS. The mDRILS stability of 16 individuals was assessed at two sampling points 1 year apart. DNA was sequenced with long-read technologies after long-range polymerase chain reaction amplification of the TAF1 SVA. Repeat number and mDRILS were detected with Noise-Cancelling Repeat Finder (NCRF). RESULTS: When comparing the repeat domain, 51 of 65 children had either contractions or expansions of the repeat length. The AGGG frequency remained stable across generations at 0.074 (IQR: 0.069-0.078) (z = -0.526; P = 0.599). However, the median AGGG frequency in children with an expansion (0.072 [IQR: 0.066-0.076]) was lower compared with children with retention or contraction (0.080 [IQR: 0.073-0.083]) (z = -0.007; P = 0.003). In a logistic regression model, the AGGG frequency predicted the outcome of either expansion or retention/contraction when including repeat number and sex as covariates (ß = 80.7; z-score = 2.63; P = 0.0085). The AGGG frequency varied slightly over 1 year (0.070 [IQR: 0.063-0.080] to 0.073 [IQR: 0.069-0.078]). CONCLUSIONS: Our results show that a higher AGGG frequency may stabilize repeats across generations. This highlights the importance of further investigating mDRILS as a disease-modifying factor with generational differences. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

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

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.

Transcriptional Alterations in X-Linked Dystonia-Parkinsonism Caused by the SVA Retrotransposon.

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 TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i 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 TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons.

LINE1-mediated epigenetic repression of androgen receptor transcription causes androgen insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is a difference of sex development (DSD) characterized by different degrees of undervirilization in individuals with a 46,XY karyotype despite normal to high gonadal testosterone production. Classically, AIS is explained by hemizygous mutations in the X-chromosomal androgen receptor (AR) gene. Nevertheless, the majority of individuals with clinically diagnosed AIS do not carry an AR gene mutation. Here, we present a patient with a 46,XY karyotype, born with undervirilized genitalia, age-appropriate testosterone levels and no uterus, characteristic for AIS. Diagnostic whole exome sequencing (WES) showed a maternally inherited LINE1 (L1) retrotransposon insertion in the 5' untranslated region (5'UTR) of the AR gene. Long-read nanopore sequencing confirmed this as an insertion of a truncated L1 element of ≈ 2.7 kb and showed an increased DNA methylation at the L1 insertion site in patient-derived genital skin fibroblasts (GSFs) compared to healthy controls. The insertion coincided with reduced AR transcript and protein levels in patient-derived GSFs confirming the clinical diagnosis AIS. Our results underline the relevance of retrotransposons in human disease, and expand the growing list of human diseases associated with them.

Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in TAF1 with Nanopore Sequencing.

BACKGROUND: 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 TAF1 gene with a polymorphic (CCCTCT)n domain that acts as a genetic modifier of disease onset and expressivity. METHODS: 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. RESULTS: 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. CONCLUSIONS: Nanopore sequencing can reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly, variation in the repeat motif.

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept.

Objective: To establish a workflow for mitochondrial DNA (mtDNA) CpG methylation using Nanopore whole-genome sequencing and perform first pilot experiments on affected Parkin biallelic mutation carriers (Parkin-PD) and healthy controls. Background: Mitochondria, including mtDNA, are established key players in Parkinson's disease (PD) pathogenesis. Mutations in Parkin, essential for degradation of damaged mitochondria, cause early-onset PD. However, mtDNA methylation and its implication in PD is understudied. Herein, we establish a workflow using Nanopore sequencing to directly detect mtDNA CpG methylation and compare mtDNA methylation between Parkin-related PD and healthy individuals. Methods: To obtain mtDNA, whole-genome Nanopore sequencing was performed on blood-derived from five Parkin-PD and three control subjects. In addition, induced pluripotent stem cell (iPSC)-derived midbrain neurons from four of these patients with PD and the three control subjects were investigated. The workflow was validated, using methylated and unmethylated 897 bp synthetic DNA samples at different dilution ratios (0, 50, 100% methylation) and mtDNA without methylation. MtDNA CpG methylation frequency (MF) was detected using Nanopolish and Megalodon. Results: Across all blood-derived samples, we obtained a mean coverage of 250.3X (SD ± 80.5X) and across all neuron-derived samples 830X (SD ± 465X) of the mitochondrial genome. We detected overall low-level CpG methylation from the blood-derived DNA (mean MF ± SD = 0.029 ± 0.041) and neuron-derived DNA (mean MF ± SD = 0.019 ± 0.035). Validation of the workflow, using synthetic DNA samples showed that highly methylated DNA molecules were prone to lower Guppy Phred quality scores and thereby more likely to fail Guppy base-calling. CpG methylation in blood- and neuron-derived DNA was significantly lower in Parkin-PD compared to controls (Mann-Whitney U-test p < 0.05). Conclusion: Nanopore sequencing is a useful method to investigate mtDNA methylation architecture, including Guppy-failed reads is of importance when investigating highly methylated sites. We present a mtDNA methylation workflow and suggest methylation variability across different tissues and between Parkin-PD patients and controls as an initial model to investigate.