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

Abstract

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.

Keywords: Alpha-synuclein; Mosaicism; Multiple system atrophy; Parkinson’s disease; SNCA; Single cell sequencing; Somatic mutation.

Fig. 1

Mosaicism for SNCA gains. a, b. Combined FISH and NeuN IHC images of a neuron (a) and a non-neuronal cell (b) from cingulate cortex showing 3 copies of SNCA. Scale bar 5 μm. c, d. The % of mosaicism in cingulate cortex, in neurons (c) and non-neurons (d). p values were corrected for 2 comparisons. e, f. The % of mosaicism in the SN in NM+ cells (e) and NM- cells (f). LB cases included four ILBD and one DLB. The medians and interquartile ranges are shown in (c-f)

Fig. 2

Further investigation of SNCA gains. a, b. Investigation of possible correlations of the level of mosaicism. Mosaicism relation to age of death in PD cingulate cortex (a), and to age of onset in MSA-SND (b). Best-fit line is shown for each cell type. Further details in text and Additional file 3: Table S3. c, d Combinations of FISH and α-synuclein IHC in DA neurons (identified by neuromelanin in brightfield) in ILBD (c), and in oligodendrocytes (identified by olig2) in MSA-SND (d). In both cases, cells with inclusions are shown, without SNCA gains at the top, and with gains at the bottom. Scale bar (c) 10 μm, (d) 5 μm. Note that the reference FISH probe was not used where olig2 was used

Fig. 3

Single cell WGS profiles of cells with known germline SNCA CNVs (arrow). a Fibroblast with triplication. Note additional calls (gain in chr2, telomeric losses in chr4 and 8). b Lower quality fibroblast with triplication, which narrowly fails confidence score filter, and would thus not be analysed. Note increased “waviness”, and likely false positive losses in regions of a negative wave, or near centromeres. c Cortical neuron with duplication. Note the clear differentiation of the XY chromosome copy number in this male

Fig. 4

Examples of single cell WGS profiles showing CNVs. The WGS profile is shown for each, with a picture of the nucleus on the right. Scale bar 20 μm. Gains are losses are marked by dots at the respective copy numbers. The cell number is in brackets. a Pontine neuron with gains including SNCA (blue arrow), and adjacent to GBA (red arrow) (K3). b Pontine neuron with a nuclear inclusion and a gain over GRID2 (arrowed) (X21). c Nigral neuron with a cytosolic inclusion and two gains (H11). The dots representing losses are CNVs that were filtered based on the copy number criterion, and therefore have not been included in the analysis. d Putaminal neuron with a nuclear inclusion and multiple losses, including the SNCA region (L33). See also Additional file 2: Figure S6d. e Pontine non-neuronal cell with a cytoplasmic inclusion and likely tetraploidy with superimposed losses (D8)

Fig. 5

CNV size and pathway enrichment. a Comparison of size in Mb of sub-chromosomal CNVs in neurons and non-neuronal cells. b-d Gene Ontology maps showing biological processes enriched for CNVs in MSA in: (b) all neurons, (c) SN neurons, (d) SN non-neurons