Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in 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 gene with a polymorphic () 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.