Objective: To uncover potentially causative Parkinson’s disease (PD) somatic mutations in post-mortem substantia nigra pars compacta (SNpc) tissues from sporadic PD patients using single cell DNA sequencing technologies.

Background: PD is a common neurodegenerative movement disorder resulting from dopaminergic neuronal loss in the SNpc. As known germline PD variants only account for <10% of PD cases and explain a small percentage of PD risk, sporadic PD cases may be attributed to somatic mutations within brains of affected individuals.

Method: We first evaluated single cell recovery rate of flow cytometry, microfluidics-based (Fluidigm C1) and droplet-based (10x Genomics Chromium) methods. We proceeded with single cell genome partitioning and library preparation of single nuclei isolated from SNpc sections of nine sporadic PD patients with the 10x Genomics CNV solution, followed by sequencing on HiSeq4000 and NovaSeq. Read alignment to GRCh38 and cell calling was conducted with cellranger-dna, followed by normalisation and copy number calling with SCOPE.

Results: We observed the highest single cell recovery with the 10x Genomics Chromium platform out of all the platforms tested, and applied it to nuclei isolated from SNpc sections of nine sporadic PD patients. We captured an average of 1469±710 single cells per patient and a total of 13,226 single cells across all nine patients. A total of 8,797 single cells (66.5%) passed read quality filtering (mapq≥40 and ≥20 reads per 500kb fixed bin) with 423,000 mean reads per cell. Initial cross-sample segmentation conducted showed potential shared breakpoints across some cells, however, the current dataset is limited by the low resolution of 2.42Mb. We are currently improving the resolution to 1.2Mb through additional sequencing to increase read depth per cell.

Conclusion: We eventually aim to identify recurrent CNVs among these 9 patients, and assay for their presence in up to 500 patients and age-matched controls. We will also evaluate all somatic CNVs which overlap known PD genes.  We will then correlate age of disease onset and disease severity with the degree of mosaicism observed in patients. Our efforts will ultimately allow us to gain insights into PD pathogenesis through the identification of disrupted genes and functional pathways in PD.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/interrogating-parkinsons-disease-associated-mutations-at-single-cell-resolution/