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

Abstract

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.

Variation in TAF1 expression in female carrier induced pluripotent stem cells and human brain ontogeny has implications for adult neostriatum vulnerability in X-linked Dystonia Parkinsonism

“…The SVA region harbors extensive DNA methylation in XDP patient blood and the level of methylation is reduced in the basal ganglia of the single patient tissue examined thus far (Lüth et al, 2022). Our findings suggest dynamic regulation of SVA elements in the adult caudate nucleus.…”

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

“…Since the human genome contains >2700 SVA elements [27], we applied long-read nanopore sequencing to precisely target the XDP-specific SVA and bisulfite pyrosequencing to verify the results at selected CpGs in intron 32. We have previously shown that the SVA is heavily methylated in various XDP tissues and cell lines [28]. When comparing the methylation frequency of the regions proximal to the SVA insertion in patients and controls (which also includes the intronic region within the TAF1-32i transcript), a CpG site at genomic position chrX:70,659,134 (hg19) strikingly deviated from others in brain samples (Figure 3a).…”

Variation in TAF1 Expression in Female Carrier-Induced Pluripotent Stem Cells and Human Brain Ontogeny Has Implications for Adult Neostriatum Vulnerability in X-Linked Dystonia Parkinsonism

“…KZFP and KAP/TRIM28 are generally lowly expressed in postnatal striatum, for example the caudate, basal ganglia and putamen are among the tissue with the lowest expression of TRIM28, ZNF587, ZNF91/93 in GTEx ( GTEx Consortium, 2020 ). The SVA region harbors extensive DNA methylation in XDP patient blood and the level of methylation is reduced in the basal ganglia of the single patient tissue examined thus far ( Lüth et al, 2022 ). Our findings suggest dynamic regulation of SVA elements in the adult caudate nucleus.…”