Objective: To detect relevant somatic “point mutations” (single nucleotide variants- SNVs) in brain-derived DNA in synucleinopathies.

Background: There is increasing recognition that neurons may harbour a wide range of somatic (post-zygotic) mutations, with a possible role in neurodegeneration [1]. We recently reported somatic DNA copy number gains of alpha-synuclein (SNCA) in substantia nigra [2]. Our previous work on somatic SNVs was limited by the modest resolution of the method, and targeting of SNCA coding exons only [3,4].

Method: We analysed DNA from up to three brain regions, including substantia nigra, from 27 Parkinson’s, 12 control, 1 incidental lewy body and 3 multiple system atrophy cases. We captured the SNCA locus, and coding exons of other PD genes, APP and MAPT, using a “molecular barcoding” approach to minimise false positives, and sequenced these at coverage >2,000x. For validation, amplicons targeting the candidate variants were re-sequenced at higher coverage. For further validation, droplet digital PCR assays targeting specific mutations were designed.

Results: We used artificial mosaics to validate the ability to detect variants at allelic fractions <0.5%. After excluding common SNPs, we selected twenty-three candidate variants with allele frequencies 0.33-5% for further study. Two coding variants were validated by amplicon sequencing. Further analysis by targeted droplet-digital PCR did not detect them in other regions from the same brains. Further review suggested that these were likely contaminants from other samples carrying them in the heterozygous state.

Conclusion: We have not detected any evidence of low-level somatic SNVs in SNCA and other relevant genes, despite using an assay capable of detecting alleles at fractions <0.5%. We cannot exclude negative results due to loss of neurons carrying somatic mutations, or due to not using purely neuronal-derived DNA. Unlike somatic CNVs, there is so far no evidence for a role of somatic point mutations in PD.

References: [1] Leija-Salazar M, Piette C, Proukakis C. Somatic mutations in neurodegeneration. Neuropathol Appl Neurobiol. 2018 Apr;44(3):267-285. [2] Mokretar K, Pease D, Taanman J-W, Soenmez A, Ejaz A, Lashley T, Ling H, Gentleman S, Houlden H, Holton JL, Schapira AH, Nacheva E, Proukakis C. Somatic copy number gains of α-synuclein (SNCA) in Parkinson’s disease and multiple system atrophy brains. Brain 2018; 141(8): 2419–2431. [3] Proukakis C, Houlden H., Schapira AH. Somatic SNCA mutations in Parkinson’s disease: hypothesis and preliminary data. Mov Disord 2013; 28:705-12. [4] Proukakis C , Shoaee M, Morris J, Brier T, Kara E, Sheerin UM, Charlesworth G, Tolosa E, Houlden H, Wood NW, Schapira AH. Analysis of Parkinson’s disease brain-derived DNA for alpha-synuclein coding somatic mutations. Mov Disord 2014;29:1060-1064.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/targeted-deep-sequencing-of-brain-dna-for-detection-of-somatic-point-mutations-in-synucleinopathies/