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See detailMitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1- and PRKN-linked Parkinson's disease 2022.05.17.22275087
Trinh, Joanne; Hicks, Andrew A.; Koenig, Inke R. et al

E-print/Working paper (2022)

Biallelic mutations in PINK1 and PRKN cause recessively inherited Parkinson's disease (PD). Though some studies suggest that PINK1/PRKN monoallelic mutations may not contribute to risk, deep phenotyping ... [more ▼]

Biallelic mutations in PINK1 and PRKN cause recessively inherited Parkinson's disease (PD). Though some studies suggest that PINK1/PRKN monoallelic mutations may not contribute to risk, deep phenotyping assessment showed that PINK1 or PRKN monoallelic pathogenic variants were at a significantly higher rate in PD compared to controls. Given the established role of PINK1 and Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA (mtDNA) integrity and inflammation as potential disease modifiers in carriers of mutations in these genes. MtDNA integrity, global gene expression and serum cytokine levels were investigated in a large collection of biallelic (n=84) and monoallelic (n=170) carriers of PINK1/PRKN mutations, iPD patients (n=67) and controls (n=90). Affected and unaffected PINK1/PRKN monoallelic mutation carriers can be distinguished by heteroplasmic mtDNA variant load (AUC=0.83, CI:0.74-0.93). Biallelic PINK1/PRKN mutation carriers harbor more heteroplasmic mtDNA variants in blood (p=0.0006, Z=3.63) compared to monoallelic mutation carriers. This enrichment was confirmed in iPSC-derived and postmortem midbrain neurons from biallelic PRKN-PD patients. Lastly, the heteroplasmic mtDNA variant load was found to correlate with IL6 levels in PINK1/PRKN mutation carriers (r=0.57, p=0.0074). PINK1/PRKN mutations predispose individuals to mtDNA variant accumulation in a dose- and disease-dependent manner. MtDNA variant load over time is a potential marker of disease manifestation in PINK1/PRKN mutation carriers.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThe authors wish to thank the many patients and their families who volunteered, and the efforts of the many clinical teams involved. Funding has been obtained from the German Research Foundation (ProtectMove; FOR 2488, GR 3731/5-1; SE 2608/2-1; KO 2250/7-1), the Luxembourg National Research Fund in the ATTRACT (Model-IPD, FNR9631103), NCER-PD (FNR11264123) and INTER programmes (ProtectMove, FNR11250962; MiRisk-PD, C17/BM/11676395, NB 4328/2-1), the BMBF (MitoPD), the Hermann and Lilly Schilling Foundation, the European Community (SysMedPD), the Canadian Institutes of Health Research (CIHR), Peter and Traudl Engelhorn Foundation. Initial studies in Tunisia on familial parkinsonism were in collaboration with Lefkos Middleton, Rachel Gibson, and the GlaxoSmithKline PD Programme Team (2002-2005). We would like to thank Dr Helen Tuppen from the Welcome Trust Centre for Mitochondrial Research, Newcastle University, UK for providing us with the plasmid p7D1. Moreover, this project was supported by the high throughput/high content screening platform and HPC facility at the Luxembourg Centre for Systems Biomedicine, and the University of Luxembourg.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:University of Lubeck Ethics CommitteeI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors [less ▲]

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See detailBenchmarking Low-Frequency Variant Calling With Long-Read Data on Mitochondrial DNA
Lüth, Theresa; Schaake, Susen; Grünewald, Anne UL et al

in Frontiers in Genetics (2022), 13

Background: Sequencing quality has improved over the last decade for long-reads, allowing for more accurate detection of somatic low-frequency variants. In this study, we used mixtures of mitochondrial ... [more ▼]

Background: Sequencing quality has improved over the last decade for long-reads, allowing for more accurate detection of somatic low-frequency variants. In this study, we used mixtures of mitochondrial samples with different haplogroups (i.e., a specific set of mitochondrial variants) to investigate the applicability of nanopore sequencing for low-frequency single nucleotide variant detection.Methods: We investigated the impact of base-calling, alignment/mapping, quality control steps, and variant calling by comparing the results to a previously derived short-read gold standard generated on the Illumina NextSeq. For nanopore sequencing, six mixtures of four different haplotypes were prepared, allowing us to reliably check for expected variants at the predefined 5%, 2%, and 1% mixture levels. We used two different versions of Guppy for base-calling, two aligners (i.e., Minimap2 and Ngmlr), and three variant callers (i.e., Mutserve2, Freebayes, and Nanopanel2) to compare low-frequency variants. We used F<sub>1</sub> score measurements to assess the performance of variant calling.Results: We observed a mean read length of 11 kb and a mean overall read quality of 15. Ngmlr showed not only higher F<sub>1</sub> scores but also higher allele frequencies (AF) of false-positive calls across the mixtures (mean F<sub>1</sub> score = 0.83; false-positive allele frequencies < 0.17) compared to Minimap2 (mean F<sub>1</sub> score = 0.82; false-positive AF < 0.06). Mutserve2 had the highest F<sub>1</sub> scores (5% level: F<sub>1</sub> score >0.99, 2% level: F<sub>1</sub> score >0.54, and 1% level: F<sub>1</sub> score >0.70) across all callers and mixture levels.Conclusion: We here present the benchmarking for low-frequency variant calling with nanopore sequencing by identifying current limitations. [less ▲]

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See detailNanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept
Lüth, Theresa; Wasner, Kobi UL; Klein, Christine et al

in Frontiers in Aging Neuroscience (2021)

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 ... [more ▼]

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. [less ▲]

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See detailLIPAD (LRRK2/Luebeck International Parkinson's Disease) Study Protocol: Deep Phenotyping of an International Genetic Cohort
Usnich, Tatiana; Vollstedt, Eva-Juliane; Schell, Nathalie et al

in Frontiers in Neurology (2021), 12

Background: Pathogenic variants in the Leucine-rich repeat kinase 2 ( LRRK2) gene are the most common known monogenic cause of Parkinson's disease (PD). LRRK2 -linked PD is clinically indistinguishable ... [more ▼]

Background: Pathogenic variants in the Leucine-rich repeat kinase 2 ( LRRK2) gene are the most common known monogenic cause of Parkinson's disease (PD). LRRK2 -linked PD is clinically indistinguishable from idiopathic PD and inherited in an autosomal dominant fashion with reduced penetrance and variable expressivity that differ across ethnicities and geographic regions. Objective: To systematically assess clinical signs and symptoms including non-motor features, comorbidities, medication and environmental factors in PD patients, unaffected LRRK2 pathogenic variant carriers, and controls. A further focus is to enable the investigation of modifiers of penetrance and expressivity of LRRK2 pathogenic variants using genetic and environmental data. Methods: Eligible participants are invited for a personal or online examination which comprises completion of a detailed eCRF and collection of blood samples (to obtain DNA, RNA, serum/plasma, immune cells), urine as well as household dust. We plan to enroll 1,000 participants internationally: 300 with LRRK2 -linked PD, 200 with LRRK2 pathogenic variants but without PD, 100 PD patients with pathogenic variants in the GBA or PRKN genes, 200 patients with idiopathic PD, and 200 healthy persons without pathogenic variants. Results: The eCRF consists of an investigator-rated (1 h) and a self-rated (1.5 h) part. The first part includes the Movement Disorder Society Unified Parkinson's Disease Rating, Hoehn \&Yahr, and Schwab \& England Scales, the Brief Smell Identification Test, and Montreal Cognitive Assessment. The self-rating part consists of a PD risk factor, food frequency, autonomic dysfunction, and quality of life questionnaires, the Pittsburgh Sleep Quality Inventory, and the Epworth Sleepiness as well as the Hospital Anxiety and Depression Scales. The first 15 centers have been initiated and the first 150 participants enrolled (as of March 25th, 2021). Conclusions: LIPAD is a large-scale international scientific effort focusing on deep phenotyping of LRRK2 -linked PD and healthy pathogenic variant carriers, including the comparison with additional relatively frequent genetic forms of PD, with a future perspective to identify genetic and environmental modifiers of penetrance and expressivity Clinical Trial Registration: ClinicalTrials.gov , NCT04214509. [less ▲]

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See detailGenotype-phenotype relations for the Parkinson’s disease genes SNCA, LRRK2, VPS35: MDSGene Review.
Trinh, Joanne; Zeldenrust, Florentine M. J.; Huang, Jana et al

in Movement Disorders (2018), 33(12), 1857-1870

This comprehensive MDSGene review is devoted to the three autosomal-dominant PD forms: PARK-SNCA, PARK-LRRK2, and PARK-VPS35. It follows MDSGene's standardized data extraction protocol, screened a total ... [more ▼]

This comprehensive MDSGene review is devoted to the three autosomal-dominant PD forms: PARK-SNCA, PARK-LRRK2, and PARK-VPS35. It follows MDSGene's standardized data extraction protocol, screened a total of 2,972 citations, and is based on fully curated phenotypic and genotypic data on 937 patients with dominantly inherited PD attributed to 44 different mutations in SNCA, LRRK2, or VPS35. All of these data are also available in an easily searchable online database (www.mdsgene.org), which additionally provides descriptive summary statistics on phenotypic and genetic data. Despite the high degree of missingness of phenotypic features and unsystematic reporting of genotype data in the original literature, the present review recapitulates many of the previously described findings including later onset of disease (median age at onset: ∼49 years) compared to recessive forms of PD of an overall excellent treatment response. Our systematic review validates previous reports showing that SNCA mutation carriers have a younger age at onset compared to LRRK2 and VPS35 (P < 0.001). SNCA mutation carriers often have additional psychiatric symptoms, and although not exclusive to only LRRK2 or VPS35 mutation carriers, LRRK2 mutation carriers have a typical form of PD, and, lastly, VPS35 mutation carriers have good response to l-dopa. [less ▲]

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