Jonas Möhner
Published: 2023
Total Pages: 0
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Cells of the soma, especially of the brain, generate genomic variations with region-specific differences in frequency, which leads to somatic mosaicism. This postzygotic phenomenon is, among others, a consequence of DNA damage or defective repair and may contribute to neurogenetic disorders. The present work provides two innovative approaches to investigate the role of retrotransposons and DNA double-strand breaks (DSBs) in the formation of somatic mosaicism in the human brain. Retrotransposons, including SVA and LINE-1, are mobile genetic elements that replicate in the genome by the "copy-and-paste" mechanism. Recent NGS-based studies demonstrated that the retrotransposon machinery is active in the human brain. This raises the question of whether SVA and LINE-1, respectively their presence at orthologous loci, can be used to track somatic differences in brain regions. For this purpose, a subtractive kinetic enrichment technique called Representational Difference Analysis (RDA) coupled with NGS is established. In addition, chromosomal DSB hotspots and their regional differences in the brain will be investigated. For one type of DSB repair, SINE/LINE information is known to be used in the context of non-homologous end-joining, i.e. typical signatures of SINE/LINE integrations at DSB sites are generated. To describe the 'breakome', a DSB labeling system based on Breaks Labeling In Situ and Sequencing (BLISS) is implemented. The RDA provides evidence for somatic mosaicism caused by differential retrotransposition of LINE-1 and SVAs in the human brain. In this context, SVAs as 'presence/absence' markers can reflect the development of telencephalon and metencephalon. De novo SVA and LINE-1 insertions have chromosome-wide rates and preferential integration in GC- and TE-rich regions and genes that tend to be involved in neural functions. The 'breakome' results show DSB hotspots occurring across the brain or in a brain region-specific manner. As a result, several known and novel recurrent DSB cluster (RDC) associated genes are detectable and can be linked to neurological diseases. Moreover, (epi-) genetic predictors of DSB formation can be identified, including DNA-binding proteins that play a role in DSB repair. Interestingly, retrotransposons and DSBs frequently occur in close proximity to each other, suggesting a possible involvement of mobile DNA in the induction or repair of DSBs. In summary, the methods presented in this work can be applied in various research areas, such as cell lineage tracing experiments or the analysis of potentially pathogenic DNA damage in the context of neurological or tumor diseases.