Myoclonus dystonia (MD) is a rare autosomal-dominant combined dystonia movement disorder characterised by quick, involuntary muscle jerks (myoclonus) paired with sustained muscular contraction (dystonia). Although known to be genetically heterogeneous, the most common genetic factor is mutations within SGCE, the gene encoding ε-sarcoglycan, accounting for approximately 45% of cases. Previous linkage analyses conducted on a family displaying inherited MD without SGCE mutations lead to the identification of another critical region, DYT15. Preliminary data suggested that mutations within the long non-coding RNA (lncRNA) CD108131, found within the DYT15 locus, resulted in decreased expression of both the SGCE transcript, as well as the SGCE protein. Validation of the remaining variants of interest yielded no new candidate genes. A low coverage area coinciding with the entire sequence of TMEM200C was discovered, however subsequent sequencing data revealed no potential disease-causing variants. Therefore, to further characterise the relationship between CD108131 and SGCE suggested by the preliminary data, a CRISPR-Cas9 knockout was developed in HEK293 cells using a double-cut strategy that allowed for complete excision of the CD108131 gene. Stable CD108131 knockout mutant cell lines were examined for differences in gene expression. QRT-PCR analysis was conducted and revealed a significant decrease in SGCE expression in the absence of CD108131. Additionally, expression also trended towards a decrease for ZBTB14, however ARHGAP28 and RPPH1 were not significantly altered. This data demonstrates that the lncRNA CD108131 is likely to have a regulatory effect on SGCE, and perhaps ZBTB14, transcription.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/39418 |
| Date | 15 July 2019 |
| Creators | Jamieson-Williams, Rhiannon |
| Contributors | Bulman, Dennis |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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