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.