Lifestyle factors and clinical severity of Parkinson's disease.

peer reviewedGenetic factors, environmental factors, and gene-environment interactions have been found to modify PD risk, age at onset (AAO), and disease progression. The objective of this study was to explore the association of coffee drinking, aspirin intake, and smoking, with motor and non-motor symptoms in a cohort of 35,959 American patients with PD from the Fox Insight Study using generalized linear models. Coffee drinkers had fewer problems swallowing but dosage and duration of coffee intake were not associated with motor or non-motor symptoms. Aspirin intake correlated with more tremor (p = 0.0026), problems getting up (p = 0.0185), light-headedness (p = 0.0043), and problems remembering (p = 1 × 10-5). Smoking was directly associated with symptoms: smokers had more problems with drooling (p = 0.0106), swallowing (p = 0.0002), and freezing (p < 1 × 10-5). Additionally, smokers had more possibly mood-related symptoms: unexplained pains (p < 1 × 10-5), problems remembering (p = 0.0001), and feeling sad (p < 1 × 10-5). Confirmatory and longitudinal studies are warranted to investigate the clinical correlation over time

Coffee, smoking and aspirin are associated with age at onset in idiopathic Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder. Genetic modifiers, environmental factors and gene-environment interactions have been found to modify PD risk and disease progression. The objective of this study was to evaluate the association of smoking, caffeine and anti-inflammatory drugs with age at onset (AAO) in a large PD cohort. A total of 35,963 American patients with idiopathic PD (iPD) from the Fox Insight Study responded to health and lifestyle questionnaires. We compared the median AAO between different groups using the non-parametric Mann-Whitney U test. Non-parametric Spearman's correlation was used for correlation assessments and regression analysis was used to assess interaction between variables. We found that smoking (p < 0.0001), coffee drinking (p < 0.0001) and aspirin intake (p < 0.0001) show an exploratory association with AAO in PD, that was further supported by multivariate regression models. The association of aspirin with PD AAO was replicated in another cohort (EPIPARK) (n = 237 patients with PD)

Interaction of Mitochondrial Polygenic Score and Lifestyle Factors in LRRK2 p.Gly2019Ser Parkinsonism.

peer reviewed[en] BACKGROUND: A mitochondrial polygenic score (MGS) is composed of genes related to mitochondrial function and found to be associated with Parkinson's disease (PD) risk. OBJECTIVE: To investigate the impact of the MGS and lifestyle/environment on age at onset (AAO) in LRRK2 p.Gly2019Ser parkinsonism (LRRK2-PD) and idiopathic PD (iPD). METHODS: We included N = 486 patients with LRRK2-PD and N = 9259 with iPD from the Accelerating Medicines Partnership® Parkinson's Disease Knowledge Platform (AMP-PD), Fox Insight, and a Tunisian Arab-Berber founder population. Genotyping data were used to perform the MGS analysis. Additionally, lifestyle/environmental data were obtained from the PD Risk Factor Questionnaire (PD-RFQ). Linear regression models were used to assess the relationship between MGS, lifestyle/environment, and AAO. RESULTS: Our derived MGS was significantly higher in PD cases compared with controls (P = 1.1 × 10-8 ). We observed that higher MGS was significantly associated with earlier AAO in LRRK2-PD (P = 0.047, β = -1.40) and there was the same trend with a smaller effect size in iPD (P = 0.231, β = 0.22). There was a correlation between MGS and AAO in LRRK2-PD patients of European descent (P = 0.049, r = -0.12) that was visibly less pronounced in Tunisians (P = 0.449, r = -0.05). We found that the MGS interacted with caffeinated soda consumption (P = 0.003, β = -5.65) in LRRK2-PD and with tobacco use (P = 0.010, β = 1.32) in iPD. Thus, patients with a high MGS had an earlier AAO only if they consumed caffeinated soda or were non-smokers. CONCLUSIONS: The MGS was more strongly associated with earlier AAO in LRRK2-PD compared with iPD. Caffeinated soda consumption or tobacco use interacted with MGS to predict AAO. Our study suggests gene-environment interactions as modifiers of AAO in LRRK2-PD

Mitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1/PRKN-linked Parkinson’s disease

Biallelic mutations in PINK1/PRKN cause recessive Parkinson’s disease. Given the established role of PINK1/Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA (mtDNA) integrity and inflammation as disease modifiers in carriers of mutations in these genes. MtDNA integrity was investigated in a large collection of biallelic (n = 84) and monoallelic (n = 170) carriers of PINK1/PRKN mutations, idiopathic Parkinson’s disease patients (n = 67) and controls (n = 90). In addition, we studied global gene expression and serum cytokine levels in a subset. 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 (controls, n = 3; biallelic PRKN mutation carriers, n = 4) and postmortem (control, n = 1; biallelic PRKN mutation carrier, n = 1) midbrain neurons. Lastly, the heteroplasmic mtDNA variant load correlated 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

Benchmarking Low-Frequency Variant Calling With Long-Read Data on Mitochondrial DNA

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 F1 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 F1 scores but also higher allele frequencies (AF) of false-positive calls across the mixtures (mean F1 score = 0.83; false-positive allele frequencies 1 score = 0.82; false-positive AF 1 scores (5% level: F1 score >0.99, 2% level: F1 score >0.54, and 1% level: F1 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

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept

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

Interaction of mitochondrial polygenic score and environmental factors in LRRK2 p.Gly2019Ser parkinsonism 2023.01.02.23284113

The objective of our study was to investigate the impact of the mitochondrial polygenic score (MGS) and lifestyle/environmental data on age at onset in LRRK2 p.Gly2019Ser parkinsonism (LRRK2-PD) and idiopathic Parkinson\textquoterights disease (iPD).In this study, we included N=486 patients with LRRK2-PD and N=9259 patients with iPD from AMP-PD, Fox Insight, and a Tunisian Arab-Berber founder population. Genotyping data was utilized to perform the MGS analysis, using 14 Single Nucleotide Polymorphisms (SNPs) from genes causally associated with mitochondrial function and PD risk. Additionally, lifestyle and environmental data were obtained from the PD risk factor questionnaire (PD-RFQ). Correlation analyses and linear regression models were used to assess the relationship between MGS, lifestyle/environment, and AAO.We observed that higher MGS was associated with earlier AAO in patients with LRRK2-PD (p=4.0\texttimes10-4, β=-0.18) but not in patients with iPD. A correlation between MGS and AAO was visibly stronger in European ancestry LRRK2-PD patients (p=0.01, r=-0.16) than in Tunisian Arab-Berber patients (p=0.44, r=-0.05). We found that the MGS interacted with coffee (p=0.03, β=-0.38) and caffeinated soda consumption (p=0.03, β=-0.37) in LRRK2-PD and with caffeine soda consumption (p=0.047, β=-0.22) and pesticide exposure (p=0.02, β=-0.37) in iPD. Thus, patients with a high MGS had an earlier AAO only if they consumed caffeine or were exposed to pesticides.The MGS related to mitochondrial function was associated with AAO in LRRK2-PD but not iPD with an ethnic-specific effect. Caffeine consumption or pesticide exposure interacted with MGS to predict PD AAO. Our study suggests gene-environment interactions as modifiers of AAO in LRRK2-PD.Competing Interest StatementCK serves as a medical advisor to Centogene and Retromer Therapeutics and received speaking honoraria from Desitin. The remaining authors declare no conflict of interest.Funding StatementThis project was supported by the DFG RU ProtectMove (DFG FOR2488), the Michael J. Fox Foundation (MJFF-021227 \& MJFF-019271), and the Else Kroener-Fresenius-Stiftung.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:Ethical permission was given by the Ethical Committee of the Institut National de Neurologie and certified by the Ministry of Health, Tunisia.I 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).YesI 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.YesData sharing is not applicable to this article as no new data were created or analysed in this study. Data used in the preparation of this manuscript were obtained from the Fox Insight database (https://foxinsight-info.michaeljfox.org/insight/explore/insight.jsp) on 18/10/2020. For up-to-date information on the study, visit https://foxinsight-info.michaeljfox.org/insight/explore/insight.jsp. Data used in the preparation of this article were obtained from the Accelerating Medicine Partnership (AMP) Parkinson\textquoterights Disease (AMP PD) Knowledge Platform. For up-to-date information on the study, visit https://www.amp-pd.org

Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in <i>TAF1</i> with Nanopore Sequencing

Background: X-linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative disorder characterized by progressive dystonia and parkinsonism. It is caused by a SINE-VNTR-Alu (SVA) retrotransposon insertion in the TAF1 gene with a polymorphic (CCCTCT)n domain that acts as a genetic modifier of disease onset and expressivity. Methods: Herein, we used Nanopore sequencing to investigate SVA genetic variability and methylation. We used blood-derived DNA from 96 XDP patients for amplicon-based deep Nanopore sequencing and validated it with fragment analysis which was performed using fluorescence-based PCR. To detect methylation from blood- and brain-derived DNA, we used a Cas9-targeted approach. Results: High concordance was observed for hexanucleotide repeat numbers detected with Nanopore sequencing and fragment analysis. Within the SVA locus, there was no difference in genetic variability other than variations of the repeat motif between patients. We detected high CpG methylation frequency (MF) of the SVA and flanking regions (mean MF = 0.94, SD = ±0.12). Our preliminary results suggest only subtle differences between the XDP patient and the control in predicted enhancer sites directly flanking the SVA locus. Conclusions: Nanopore sequencing can reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly, variation in the repeat motif