Leija Salazar, Melissa Ivonne; (2020) Investigation of somatic mutations in synucleinopathies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

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.

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