There is increasing evidence that the mitochondrial genome may be regulated by DNA modifications, but their extent, identity and functional importance remain unclear. This uncertainty partly reflects technical limitations, as conventional approaches for measuring DNA methylation can generate artefacts when applied to mtDNA. Long-read sequencing technologies now provide an opportunity to directly investigate multiple DNA modifications in native mtDNA. This is particularly relevant to Parkinson’s disease (PD), where mitochondrial dysfunction is strongly implicated in disease pathogenesis. We hypothesise that mtDNA contains reproducible patterns of DNA modification that influence mitochondrial genome regulation and are altered in PD.
We will address this through 3 objectives.
- To develop and validate Oxford Nanopore Technologies (ONT) approaches to accurately characterise mtDNA modifications. ONT sequencing will be performed on native enriched mtDNA, enabling genetic variation and DNA modifications to be measured on individual molecules. We will generate artificial mtDNA molecules containing known levels of specific modifications and use these to establish the sensitivity, specificity and reproducibility of current modification-calling approaches. This will allow us to look at a range of DNA modifications (e.g. 5mC, 5hmC, 6mA).
- To define the landscape of mtDNA modifications in PD brain. We will apply the validated approach to an existing, well-characterised PD case-control cohort to determine the distribution and abundance of modifications across the mitochondrial genome and identify differences associated with disease. The single-molecule nature of ONT sequencing will also enable DNA modifications to be considered alongside mtDNA sequence variation and heteroplasmy, providing an integrated view of mitochondrial genetic and epigenetic variation. Analyses will encompass the entire mitochondrial genome while specifically testing the hypothesis that modification of regulatory regions, including the D-loop, is important in PD.
- To determine the functional relevance of mtDNA modifications by integrating them with molecular phenotypes in the same brains. We will initially test whether variation in mtDNA modifications, particularly within regulatory regions, is associated with mitochondrial transcription and mtDNA abundance. We will then integrate modification profiles with existing bulk and snRNA seq, small RNA-seq, nuclear DNA methylation and genetic data to determine how mtDNA modification states relate to cellular pathways and PD-associated molecular changes. This will prioritise candidate regulatory modifications for future causal investigation using emerging mitochondrial epigenome-editing approaches.
The project will establish a validated experimental and computational framework for studying mtDNA modifications, and determine their relationship with mitochondrial genome activity and wider cellular phenotypes in PD brain. It is highly interdisciplinary, combining long-read sequencing, mitochondrial genetics, epigenetics, bioinformatics and multiomic data integration. The project will exploit existing PD brain samples and extensive matched genomic and transcriptomic datasets, substantially reducing the requirement for costly new data generation.
The supervisory team brings complementary skills that directly reflects the project: Prof Lunnon has extensive expertise in neurodegenerative disease epigenetics and has led research investigating the mitochondrial epigenome in human brain; Prof Chinnery is an international leader in mitochondrial genetics, including the application of long-read and multiomic technologies; Dr Kouhsar brings expertise in bioinformatics, long-read sequencing analysis and multiomic data integration, whilst Dr Smith has experience in developing novel ONT approaches. Together, these existing samples, datasets, expertise and sequencing and computational infrastructure provide a strong PhD environment.
Funded by: MRC Medical Research Council
