Uncovering new genetic modifiers of monogenic Parkinson’s disease and parkinsonism-related disorders

Nanopore technology allows single-molecule detection, which engages a whole new level of hypotheses on somatic variation.

Oxford Nanopore technology has opened up our doors to explore single-molecule, long-read genetic data to identify new modifiers of disease onset in the nuclear and mitochondrial genome. We have been very fortunate to unravel specific somatic and epigenetic variations in mitochondrial DNA to distinguish affected and unaffected PRKN/PINK1-linked PD, somatic repeat interruptions within TAF1 in X-linked dystonia parkinsonism (XDP), and now even expanding our efforts genome-wide.

The start of our journey to success

The journey to these successes has not been straightforward. In 2019, the group consisted of one very experienced research technician, Susen Schaake, and me, and we had one MinION in the lab (amongst other shared equipment with other groups at the Institute of Neurogenetics). Susen and I went to Oxford together to receive some training, then we worked day and night to get the MinION running well. I would check the flow cell capacity and Susen made libraries in the lab, then walked the MinION device down the hall to my office to plug into my laptop for analyses. Our initial genome run had a grand total of only 1 Gb of data and a whole lot of clogged pores. With persistence, whether it was a different enzyme that we needed, more or less DNA, different Cas9 designs, bioinformatic issues...we figured it out! And, we even managed to publish some small collaborative papers in dystonia (PMIDs: 34250228, 32018151)1,2. However, these were targeted approaches and, at the time, not easy to obtain sequences for.

In 2020, after securing more funding with our preliminary data, Theresa Lüth (now a senior PhD student) joined us to look at mitochondrial DNA and environmental effects on Parkinson’s disease. We were fortunate to buy a GridION, and at that point we were getting around 10 Gb of data per flow cell! Things were starting to work more efficiently. We established a workflow to assess the epigenetic architecture of the mitochondrial genome (PMID: 34650424)3. Utilising the direct read-out of DNA methylation from the nanopore data, we could show overall low-level mitochondrial DNA (mtDNA) methylation. However, there was evidence for subtle but significant differences between blood- and neuron-derived mtDNA methylation levels as well as between Parkin-linked PD patients and healthy controls. We also set up and carefully benchmarked a pipeline to detect mtDNA low-frequency variants (i.e., heteroplasmy) at an allele frequency down to 1%, using deep and targeted mtDNA nanopore sequencing (PMID: 35664331)4. In fact, our work on mitochondrial heteroplasmic variant load is now published in Brain (PMID: 36478228)5, where we demonstrated higher variant load in heterozygous PINK1/Parkin mutation carriers affected with PD compared to unaffected and even higher mtDNA variant load in biallelic PINK1/Parkin mutation carriers.

New members and new ideas

This technological and scientific expansion also allowed the growth of project ideas and new blood: Carolin Gabbert (PhD student) and Joshua Laß (PhD student) joined us. We expanded our analysis on carriers of the strongest genetic risk factors for PD, variants in GBA1. A challenge that arises when sequencing the GBA1 gene is the nearby pseudogene GBAP1, which can be overcome by using long-read nanopore sequencing. We additionally compared and evaluated different analysis pipelines and determined the frequency of GBA1 variants in the Norwegian population (PMID: 37312046)6.

Another new area of interest is our work on structural variants in neurodegeneration using nanopore technology. We are currently working on identifying pathogenic structural variants and repeat expansions in PD using whole-genome sequencing data to find novel genetic bases in well-characterised and genetically pre-screened PD patients. We have established workflows in our lab to investigate repeat expansions with nanopore sequencing with a PCR amplification or Cas9 enrichment (PMID: 35052466)7. Nanopore sequencing opens new possibilities for the investigation of repeat expansions. Recently, we have identified somatic repeat interruptions in XDP, which are correlating with the disease onset (PMID: 35481544)8. We are currently expanding our investigations on other repeat expansion disorders, like Huntington's disease or ataxias.

Lastly, Christoph Much (research technician) joined in 2022 with single-cell sequencing experience, aiming to look into brain-derived DNA from mice and humans. The group now consists of seven members; we have grown as the adoption of nanopore technologies has also expanded, and we have been fortunate to uncover some key mechanisms of genetic modifiers that correlate with disease onset, such as those published in Brain (PMID: 35481544)8.

Our seminal work with nanopore technology illustrates 1) the importance of somatic mosaic genotypes, 2) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability, and 3) that these variations may remain undetected without assessment of single molecules. Nanopore technology allows single-molecule detection, which engages a whole new level of hypotheses on somatic variation.

The PhD students, Theresa, Carolin, and Joshua passionately drive their independent research projects in our team and are now bioinformatic experts on long reads, Susen is our lab mother and Christoph brings on functional hands in brain work. Our hopes are to continue to expand the technology in Northern Germany, being a large academic hub for Parkinson’s disease. We have broad interests using single-molecule sequencing. We would like to understand the microbiome in Parkinson’s and how it is interconnected with mitochondrial DNA, and we would like to know how the environment plays a role in somatic variation in parkinsonism-related disorders. These are all new avenues unexplored using our approach in a somatic mosaic manner.

Lastly, we are grateful for all the support we receive from the Institute of Neurogenetics at the University of Lübeck. We appreciate our key collaborators: Drs. Anne Grünewald, Inke König, Hauke Busch, Hansi Weißensteiner, Patrick May, Ana Westenberger, and Christine Klein on these nanopore projects. The German Foundation, Michael J. Fox Foundation, Else Kroener Fresenius Foundation were key funders. We are extremely grateful for the patients and families who continue to support our research goals.

1. Jourdan Reyes C, Laabs B-H, Schaake S, et al. Brain Regional Differences in Hexanucleotide Repeat Length in X-Linked Dystonia-Parkinsonism Using Nanopore Sequencing. Neurol Genet. 7(4):e608 (2021).

2. Bally JF, Breen DP, Schaake S, et al. Mild dopa-responsive dystonia in heterozygous tyrosine hydroxylase mutation carrier: Evidence of symptomatic enzyme deficiency? Parkinsonism Relat Disord. 71:44-45 (2020).

3. Lüth T, Wasner K, Klein C, et al. Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept. Front Aging Neurosci. 13:713084 (2021).

4. Lüth T, Schaake S, Grünewald A, et al. Benchmarking Low-Frequency Variant Calling With Long-Read Data on Mitochondrial DNA. Front Genet. 13:887644 (2022).

5. Trinh J, Hicks AA, König IR, et al. Mitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1/PRKN-linked Parkinson's disease. Brain. 146(7):2753-2765 (2023).

6. Gabbert C, Schaake S, Lüth T, et al. GBA1 in Parkinson's disease: variant detection and pathogenicity scoring matters. BMC Genomics. 24(1):322 (2023).

7. Lüth T, Laß J, Schaake S, et al. Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in TAF1 with Nanopore Sequencing. Genes. 13(1):126 (2022).

8. Trinh J, Lüth T, Schaake S, et al. Mosaic divergent repeat interruptions in XDP influence repeat stability and disease onset. Brain. 146(3):1075-1082 (2023).