Investigation of somatic mutations in synucleinopathies

Neurons are susceptible to harbour somatic mutations, which may underlie vulnerability to degeneration or initiate a ‘prion-like’ spread of mutated proteins causing aggregation and disease. The present study aims to identify low-level somatic single nucleotide variants (SNVs) potentially involved in sporadic α- synucleinopathies. Different brain regions from twenty-seven Parkinson’s, twelve control, one Incidental Lewy body and three Multiple system atrophy cases were analysed using Haloplex HS and Illumina targeted sequencing for twelve genes associated mainly with Parkinson’s. Data analysis used the manufacturer’s software and other widely-validated bioinformatic tools. Twentytwo potentially pathological variants at 0.33-5% levels were selected for validation, using deeper sequencing at the variant sites and droplet digital PCR. Novel mosaic SNVs were not validated in our cohort, but results cannot exclude somatic variation occurring in earlier disease stages or in specific neuronal populations masked by this analysis. Therefore, part of the project focused on developing a methodology to study somatic variation in sorted dopaminergic nuclei, due to their relevance for Parkinson’s disease. Additionally, a Nanopore-sequencing protocol was designed to sequence the GBA gene, not fully covered by Illumina-sequencing, and to test the Nanopore potential use for diagnostics. Nine Parkinson's, four Gaucher’s, one multiple system atrophy and three controls or healthy carriers were analysed, from which all their previously recorded mutations were confirmed. The protocol enabled the detection of common, intronic and recombinant variants. An additional 55-base pair deletion and a coding variant were found in a carrier previously described as RecNciI, helping to better characterise the patient’s recombinant genotype. The optimised methodology for GBA Nanopore- 4 sequencing allows for haplotype and intronic analyses, not previously done by common short-read protocols. Useful advice is given to discriminate true from false positives and with further improvements in the technology and enrichment methods, this work can become a powerful tool for research and diagnostics

Somatic mutations in neurodegeneration

Somatic mutations are post-zygotic mutations which may lead to mosaicism, the presence of cells with genetic differences in an organism. Their role in cancer is well established, but detailed investigation in health and other diseases has only been recently possible. This has been empowered by the improvements of sequencing techniques, including single cell sequencing, which can still be error-prone but is rapidly improving. Mosaicism appears relatively common in the human body, including the normal brain, probably arising in early development, but also potentially during ageing. In this review, we first discuss theoretical considerations and current evidence relevant to somatic mutations in the brain. We present a framework to explain how they may be integrated with current views on neurodegeneration, focusing mainly on sporadic late onset neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis). We review the relevant studies so far, with the first evidence emerging in Alzheimer's in particular. We also discuss the role of mosaicism in inherited neurodegenerative disorders, particularly somatic instability of tandem repeats. We summarise existing views and data to present a model whereby the time of origin and spatial distribution of relevant somatic mutations, combined with any additional risk factors, may partly determine the development and onset age of sporadic neurodegenerative diseases

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

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

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

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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