Investigation of Somatic Mutations in Human Brains Targeting Genes Associated With Parkinson's Disease.

Background: Somatic single nucleotide variant (SNV) mutations occur in neurons but their role in synucleinopathies is unknown. Aim: We aimed to identify disease-relevant low-level somatic SNVs in brains from sporadic patients with synucleinopathies and a monozygotic twin carrying LRRK2 G2019S, whose penetrance could be explained by somatic variation. Methods and Results: We included different brain regions from 26 Parkinson's disease (PD), one Incidental Lewy body, three multiple system atrophy cases, and 12 controls. The whole SNCA locus and exons of other genes associated with PD and neurodegeneration were deeply sequenced using molecular barcodes to improve accuracy. We selected 21 variants at 0.33-5% allele frequencies for validation using accurate methods for somatic variant detection. Conclusions: We could not detect disease-relevant somatic SNVs, however we cannot exclude their presence at earlier stages of degeneration. Our results support that coding somatic SNVs in neurodegeneration are rare, but other types of somatic variants may hold pathological consequences in synucleinopathies.

Arachidonyl-2'-chloroethylamide (ACEA), a synthetic agonist of cannabinoid receptor, increases CB1R gene expression and reduces dyskinesias in a rat model of Parkinson's disease.

l-Dopa is the most effective drug used for Parkinson's disease (PD), but after long-term treatment, the vast majority of PD patients develop abnormal involuntary movements (AIMs) termed l-Dopa-induced dyskinesia (LID). Cannabinoid receptors in the basal ganglia can modulate motor functions, but their role in the treatment of LID is controversial. Therefore, the aim of this study is to evaluate the motor behavior and mRNA expression of the cannabinoid receptor-1 (CB1R), encoded by the Cnr1 gene, in the striatum and globus pallidus of a 6-hydroxydopamine rat model of PD. The evaluated rats had 6-hydroxydopamine-induced injury, LID, and LID treated with arachidonyl-2'-chloroethylamide (ACEA), a cannabinoid receptor agonist. Contralateral turns and AIMs were recorded to assess motor behavior. Gene expression was quantified by reverse transcription coupled with quantitative polymerase chain reaction using TaqMan probes. Behavioral evaluations demonstrated that dyskinetic rats treated with ACEA had a significant reduction in AIMs compared to the dyskinetic group. The expression of CB1R mRNA was significantly decreased in the 6-hydroxydopamine-injured and dyskinetic rats, compared to intact rats. The striata of dyskinetic rats treated with ACEA exhibited highly significant increases in CB1R mRNA expression. Contrary to results in the striatum, a lower CB1R expression was observed in globus pallidus from dyskinetic ACEA-treated group. In summary, significant differences in mRNA expression of CB1R were found between the evaluated groups of rats, suggesting the occurrence of compensatory mechanisms that may result in the ACEA-mediated reduction of dyskinesias in a rat model of PD.

Investigation of somatic CNVs in brains of synucleinopathy cases using targeted SNCA analysis and single cell sequencing.

Synucleinopathies are mostly sporadic neurodegenerative disorders of partly unexplained aetiology, and include Parkinson's disease (PD) and multiple system atrophy (MSA). We have further investigated our recent finding of somatic SNCA (α-synuclein) copy number variants (CNVs, specifically gains) in synucleinopathies, using Fluorescent in-situ Hybridisation for SNCA, and single-cell whole genome sequencing for the first time in a synucleinopathy. In the cingulate cortex, mosaicism levels for SNCA gains were higher in MSA and PD than controls in neurons (> 2% in both diseases), and for MSA also in non-neurons. In MSA substantia nigra (SN), we noted SNCA gains in > 3% of dopaminergic (DA) neurons (identified by neuromelanin) and neuromelanin-negative cells, including olig2-positive oligodendroglia. Cells with CNVs were more likely to have α-synuclein inclusions, in a pattern corresponding to cell categories mostly relevant to the disease: DA neurons in Lewy-body cases, and other cells in the striatonigral degeneration-dominant MSA variant (MSA-SND). Higher mosaicism levels in SN neuromelanin-negative cells may correlate with younger onset in typical MSA-SND, and in cingulate neurons with younger death in PD. Larger sample sizes will, however, be required to confirm these putative findings. We obtained genome-wide somatic CNV profiles from 169 cells from the substantia nigra of two MSA cases, and pons and putamen of one. These showed somatic CNVs in ~ 30% of cells, with clonality and origins in segmental duplications for some. CNVs had distinct profiles based on cell type, with neurons having a mix of gains and losses, and other cells having almost exclusively gains, although control data sets will be required to determine possible disease relevance. We propose that somatic SNCA CNVs may contribute to the aetiology and pathogenesis of synucleinopathies, and that genome-wide somatic CNVs in MSA brain merit further study.

Evaluation of the detection of GBA missense mutations and other variants using the Oxford Nanopore MinION.

BACKGROUND: Mutations in GBA cause Gaucher disease when biallelic and are strong risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA using long reads. METHODS: We sequenced GBA on the Oxford Nanopore MinION as an 8.9 kb amplicon from 102 individuals, including patients with Parkinson's and Gaucher diseases. We used NanoOK for quality metrics, NGMLR to align data (after comparing with GraphMap), Nanopolish and Sniffles to call variants, and WhatsHap for phasing. RESULTS: We detected all known missense mutations in these samples, including the common p.N409S (N370S) and p.L483P (L444P) in multiple samples, and nine rarer ones, as well as a splicing and a truncating mutation, and intronic SNPs. We demonstrated the ability to phase mutations, confirm compound heterozygosity, and assign haplotypes. We also detected two known risk variants in some Parkinson's patients. Rare false positives were easily identified and filtered, with the Nanopolish quality score adjusted for the number of reads a very robust discriminator. In two individuals carrying a recombinant allele, we were able to detect and fully define it in one carrier, where it included a 55-base pair deletion, but not in another one, suggesting a limitation of the PCR enrichment method. Missense mutations were detected at the correct zygosity, except for the case where the RecNciI one was missed. CONCLUSION: The Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantages of phasing and intronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants.