Damaggio, Gianluca (2023) Genomic Signatures and Cis-Modifications Underlying Neuronal Dysfunction in Huntington’s Disease: a comprehensive study using third-generation and single cell RNA sequencing. [Tesi di dottorato]

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Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder resulting from an expansion of CAG trinucleotide repeats in the HTT gene’s exon1. Recent evidence indicates that the CAG in HTT undergoes further expansion in somatic tissue, especially in the brain, and this may be a major cause of the selective neuronal vulnerability observed in the HD brain. Therefore precise sequencing of the HTT locus containing the repetitive CAG stretch, becomes essential. Yet this has so far been very challenging with traditional technologies. Oxford Nanopore Technology (ONT) offers a promising solution, allowing comprehensive coverage of this locus, including its sequence. In our research, we employed ONT to sequence the HTT locus in human stem cell lines carrying varying CAG repeat lengths, enabling us to monitor the number of repeats over time. We used the Straglr software to count repeats within reads and devised an adjusted Instability index for assessing repeat length changes in vitro. Our study comprises two key experiments. Firstly, we propagated stem cell lines with different CAG lengths in self-renewal for 120 days in vitro (DIV) monitoring repeat instability during this period of time. Secondly, we examined these cell lines during in vitro neuronal differentiation. In both experiments, our objective was to assess CAG instability over time and explore potential mechanisms contributing to in vitro instability. In a parallel work conducted in this thesis, we tested whether the CAG expansion in the HTT locus leads to abnormal development of the neurons in the striatum and cortex which is reflected in their vulnerability later in the adult life. To uncover potential neurodevelopmental insights related to specific cell types in the Lateral Ganglionic Eminence (LGE, the future striatum), we conducted single-cell RNA sequencing of the LGE from an HD-affected fetus and five control fetuses, analyzing the data at sixteen weeks postconception. Despite the limitations of a single HD sample, rare availability of HD fetal samples underscores the significance of this analysis. Our goals include identifying the cell types most affected by HD in comparison to control samples, potentially revealing a neurodevelopmental signature associated with HD. The results are expected to provide critical insights into the cell types primarily impacted by HD, paving the way for early diagnosis and the identification of potential targets for neurodegeneration prevention.

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