Objective: Nanopore whole-genome sequencing of native DNA to investigate mitochondrial DNA (mtDNA) CpG methylation from patients with Parkinson’s disease (PD) and healthy controls.

Background: PD is the fastest growing neurological disease currently affecting ~7 million patients worldwide. Mitochondria, including mtDNA, are an established key player in PD pathogenesis. However, mtDNA methylation and its implication in PD is understudied. Using Nanopore sequencing to directly detect mtDNA CpG methylation from the sequencing data, can overcome previous methodological limitations.

Method: Whole-genome Nanopore sequencing of native whole-cell DNA was performed. In this study, blood-derived DNA from five Parkin mutation carriers that are affected by PD and three control subjects were included. In addition, DNA of induced pluripotent stem cell (iPSC)-derived neurons from three patients with PD and the three control subjects were investigated. The workflow was validated, using methylated and unmethylated 897bp synthetic DNA samples at different dilution ratios (0%, 50%, 100% methylation). MtDNA CpG methylation frequency (MF) was detected using Nanopolish.

Results: The validation of the workflow, using synthetic DNA samples showed that highly methylated DNA molecules were prone to lower Guppy Phred quality scores. We obtained a mean coverage of 275X (SD±56) across blood-derived mtDNA and 785X (SD±458) across neuronal-derived mtDNA. We detected overall low-level CpG methylation. Nonetheless, the methylation frequency was significantly higher in the blood-derived mtDNA compared to the neuronal-derived mtDNA (Mann-Whitney U-test p<0.0001). We then investigated differences in methylation between PD and healthy control subjects. The detected CpG methylation in blood- and neuronal-derived DNA was significantly lower in PD patients (Mann-Whitney U-test p<0.05).

Conclusion: Nanopore sequencing is a useful method to investigate the mtDNA methylation architecture. For the reanalysis of highly methylated sites, inclusion of Guppy failed reads might be of importance. Although mtDNA methylation is rare, our preliminary results suggest that there is possible variability across different tissues and between patients with PD and controls. Thus, further investigation of the underlying molecular mechanism and its implication in PD pathogenesis is of importance.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/nanopore-single-molecule-sequencing-to-investigate-mitochondrial-dna-cpg-methylation-in-parkinsons-disease/