Genetic 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
). Smoking was directly associated with symptoms: smokers had more problems with drooling (p = 0.0106), swallowing (p = 0.0002), and freezing (p < 1 × 10
). Additionally, smokers had more possibly mood-related symptoms: unexplained pains (p < 1 × 10
), problems remembering (p = 0.0001), and feeling sad (p < 1 × 10
). Confirmatory and longitudinal studies are warranted to investigate the clinical correlation over time.
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.
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
score measurements to assess the performance of variant calling.
We observed a mean read length of 11 kb and a mean overall read quality of 15. Ngmlr showed not only higher F
scores but also higher allele frequencies (AF) of false-positive calls across the mixtures (mean F
score = 0.83; false-positive allele frequencies < 0.17) compared to Minimap2 (mean F
score = 0.82; false-positive AF < 0.06). Mutserve2 had the highest F
scores (5% level: F
score >0.99, 2% level: F
score >0.54, and 1% level: F
score >0.70) across all callers and mixture levels.
We here present the benchmarking for low-frequency variant calling with nanopore sequencing by identifying current limitations.
Abstract The objective of this study was to investigate the association between a Parkinson’s disease (PD)-specific polygenic score (PGS) and protective lifestyle factors on age at onset (AAO) in PD. ...We included data from 4367 patients with idiopathic PD, 159 patients with GBA1 -PD, and 3090 healthy controls of European ancestry from AMP-PD, PPMI, and Fox Insight cohorts. The association between PGS and lifestyle factors on AAO was assessed with linear and Cox proportional hazards models. The PGS showed a negative association with AAO ( β = − 1.07, p = 6 × 10 –7 ) in patients with idiopathic PD. The use of one, two, or three of the protective lifestyle factors showed a reduction in the hazard ratio by 21% ( p = 0.0001), 44% ( p < 2 × 10 –16 ), and 55% ( p < 2 × 10 –16 ), compared to no use. An additive effect of aspirin ( β = 7.62, p = 9 × 10 –7 ) and PGS ( β = − 1.58, p = 0.0149) was found for AAO without an interaction ( p = 0.9993) in the linear regressions, and similar effects were seen for tobacco. In contrast, no association between aspirin intake and AAO was found in GBA1 -PD ( p > 0.05). In our cohort, coffee, tobacco, aspirin, and PGS are independent predictors of PD AAO. Additionally, lifestyle factors seem to have a greater influence on AAO than common genetic risk variants with aspirin presenting the largest effect.
GBA1 variants are the strongest genetic risk factor for Parkinson's disease (PD). However, the pathogenicity of GBA1 variants concerning PD is still not fully understood. Additionally, the frequency ...of GBA1 variants varies widely across populations.
To evaluate Oxford Nanopore sequencing as a strategy, to determine the frequency of GBA1 variants in Norwegian PD patients and controls, and to review the current literature on newly identified variants that add to pathogenicity determination.
We included 462 Norwegian PD patients and 367 healthy controls. We sequenced the full-length GBA1 gene on the Oxford Nanopore GridION as an 8.9 kb amplicon. Six analysis pipelines were compared using two aligners (NGMLR, Minimap2) and three variant callers (BCFtools, Clair3, Pepper-Margin-Deepvariant). Confirmation of GBA1 variants was performed by Sanger sequencing and the pathogenicity of variants was evaluated.
We found 95.8% (115/120) true-positive GBA1 variant calls, while 4.2% (5/120) variant calls were false-positive, with the NGMLR/Minimap2-BCFtools pipeline performing best. In total, 13 rare GBA1 variants were detected: two were predicted to be (likely) pathogenic and eleven were of uncertain significance. The odds of carrying one of the two common GBA1 variants, p.L483P or p.N409S, in PD patients were estimated to be 4.11 times the odds of carrying one of these variants in controls (OR = 4.11 1.39, 12.12).
In conclusion, we have demonstrated that Oxford long-read Nanopore sequencing, along with the NGMLR/Minimap2-BCFtools pipeline is an effective tool to investigate GBA1 variants. Further studies on the pathogenicity of GBA1 variants are needed to assess their effect on PD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
While many genetic causes of movement disorders have been identified, modifiers of disease expression are largely unknown. X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease ...caused by a SINE-VNTR-Alu(AGAGGG)n retrotransposon insertion in TAF1, with a polymorphic (AGAGGG)n repeat. Repeat length and variants in MSH3 and PMS2 explain ∼65% of the variance in age at onset (AAO) in XDP. However, additional genetic modifiers are conceivably at play in XDP, such as repeat interruptions.
Long-read nanopore sequencing of PCR amplicons from XDP patients (n = 202) was performed to assess potential repeat interruption and instability. Repeat-primed PCR and Cas9-mediated targeted enrichment confirmed the presence of identified divergent repeat motifs.
In addition to the canonical pure SINE-VNTR-Alu-5′-(AGAGGG)n, we observed a mosaic of divergent repeat motifs that polarized at the beginning of the tract, where the divergent repeat interruptions varied in motif length by having one, two, or three nucleotides fewer than the hexameric motif, distinct from interruptions in other disease-associated repeats, which match the lengths of the canonical motifs. All divergent configurations occurred mosaically and in two investigated brain regions (basal ganglia, cerebellum) and in blood-derived DNA from the same patient. The most common divergent interruption was AGG 5′-SINE-VNTR-Alu(AGAGGG)2AGG(AGAGGG)n, similar to the pure tract, followed by AGGG 5′-SINE-VNTR-Alu(AGAGGG)2AGGG(AGAGGG)n, at median frequencies of 0.425 (IQR: 0.42–0.43) and 0.128 (IQR: 0.12–0.13), respectively. The mosaic AGG motif was not associated with repeat number (estimate = −3.8342, P = 0.869). The mosaic pure tract frequency was associated with repeat number (estimate = 45.32, P = 0.0441) but not AAO (estimate = −41.486, P = 0.378). Importantly, the mosaic frequency of the AGGG negatively correlated with repeat number after adjusting for age at sampling (estimate = −161.09, P = 3.44 × 10−5). When including the XDP-relevant MSH3/PMS2 modifier single nucleotide polymorphisms into the model, the mosaic AGGG frequency was associated with AAO (estimate = 155.1063, P = 0.047); however, the association dissipated after including the repeat number (estimate = −92.46430, P = 0.079).
We reveal novel mosaic divergent repeat interruptions affecting both motif length and sequence (DRILS) of the canonical motif polarized within the SINE-VNTR-Alu(AGAGGG)n repeat. Our study illustrates: (i) the importance of somatic mosaic genotypes; (ii) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability; and (iii) that these variations may remain undetected without assessment of single molecules.
While many genetic causes of movement disorders have been identified, modifiers of disease expression are largely unknown. Trinh et al. identify a novel somatic divergent repeat interruption within the TAF1 repeat expansion that influences repeat stability, and indirectly the age at onset of X-linked dystonia parkinsonism.
Purpose
Subclinical alterations of the vaginal microbiome have been described to be associated with female infertility and may serve as predictors for failure of in vitro fertilization treatment. ...While large prospective studies to delineate the role of microbial composition are warranted, integrating microbiome information into clinical management depends on economical and practical feasibility, specifically on a short duration from sampling to final results. The currently most used method for microbiota analysis is either metagenomics sequencing or amplicon-based microbiota analysis using second-generation methods such as sequencing-by-synthesis approaches (Illumina), which is both expensive and time-consuming. Thus, additional approaches are warranted to accelerate the usability of the microbiome as a marker in clinical praxis.
Methods
Herein, we used a set of ten selected vaginal swabs from women undergoing assisted reproduction, comparing and performing critical optimization of nanopore-based microbiota analysis with the results from MiSeq-based data as a quality reference.
Results
The analyzed samples carried varying community compositions, as shown by amplicon-based analysis of the V3V4 region of the bacterial
16S rRNA gene
by MiSeq sequencing. Using a stepwise procedure to optimize adaptation, we show that a close approximation of the microbial composition can be achieved within a reduced time frame and at a minimum of costs using nanopore sequencing.
Conclusions
Our work highlights the potential of a nanopore-based methodical setup to support the feasibility of interventional studies and contribute to the development of microbiome-based clinical decision-making in assisted reproduction.
To establish a workflow for mitochondrial DNA (mtDNA) CpG methylation using Nanopore whole-genome sequencing and perform first pilot experiments on affected
biallelic mutation carriers (Parkin-PD) ...and healthy controls.
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.
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.
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
< 0.05).
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.
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
gene with a polymorphic (
)
domain that acts as a genetic modifier of disease onset and expressivity.
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.
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.
Nanopore sequencing can reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly, variation in the repeat motif.
Our study investigated the presence of regional differences in hexanucleotide repeat number in postmortem brain tissues of 2 patients with X-linked dystonia-parkinsonism (XDP), a combined ...dystonia-parkinsonism syndrome modified by a (CCCTCT)
repeat within the causal SINE-VNTR-
retrotransposon insertion in the
gene.
Genomic DNA was extracted from blood and postmortem brain samples, including the basal ganglia and cortex from both patients and from the cerebellum, midbrain, and pituitary gland from 1 patient. Repeat sizing was performed using fragment analysis, small-pool PCR-based Southern blotting, and Oxford nanopore sequencing.
The basal ganglia (
< 0.001) and cerebellum (
< 0.001) showed higher median repeat numbers and higher degrees of repeat instability compared with blood.
Somatic repeat instability may predominate in brain regions selectively affected in XDP, thereby hinting at its potential role in disease manifestation and modification.
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