Deciphering X-chromosome inactivation and the role of MECP2e1 in Rett Syndrome patient induced pluripotent stem cells
Year: 2013
Type: dissertation
Abstract: Rett Syndrome (RTT) is a neurodevelopmental disorder that affects girls due primarily to heterozygous mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). MECP2 encodes four exons that are alternatively spliced into two isoforms, MECP2e1 and MECP2e2. MECP2 isoform-specific functions are unknown, but due to the higher abundance in the brain and MECP2e1-specific mutations associated with RTT, MECP2e1 is hypothesized to be the functional isoform in the brain. MECP2 is an X-linked gene subject to random X-chromosome inactivation (XCI) resulting in mosaic expression of mutant MECP2. The lack of readily accessible human brain tissue motivates the need for alternative human cellular models to study RTT. In this thesis, I established a novel human in vitro model of RTT by isolating human induced Pluripotent Stem cells (hiPSCs) from RTT patient fibroblasts. RTT-hiPSCs retained the MECP2 mutation, are pluripotent and fully reprogrammed, and retained an inactive X-chromosome in a nonrandom pattern. Taking advantage of the latter characteristic, I obtained a pair of isogenic wild-type and mutant MECP2 expressing RTT-hiPSC lines that retained this MECP2 expression pattern upon differentiation into neurons. Phenotypic analysis of mutant RTT-hiPSC-derived neurons demonstrated a reduction in soma size compared with the isogenic control RTT-hiPSC-derived neurons from the same RTT patient validating RTT-hiPSC-derived neurons for disease phenotyping. To further understand the role of MECP2e1 in RTT, I took advantage of RTT-hiPSCs carrying a MECP2 mutation that specifically disrupts MECP2e1 while MECP2e2 remains intact (RTTe1). RTTe1-hiPSC-derived neurons exhibited a soma size defect compared to wild-type neurons. Furthermore, this phenotype was rescued by MECP2e1 vectors in a cell autonomous manner suggesting that disruption of MECP2e1 alone is sufficient to cause a cellular RTT phenotype. Altogether, in this thesis, I isolated mutant and isogenic control RTT-hiPSCs by taking advantage of their unique XCI pattern. Upon differentiation, mutant RTT-hiPSC-derived neurons exhibited a RTT phenotype compared to isogenic control hiPSC-derived neurons from the same patient. Analysis of mutant and isogenic control hiPSC-derived neurons represents a novel human in vitro model for understanding the pathogenesis of RTT and the role of MECP2 and its isoforms in human neurons.
Source: TSpace (University of Toronto)
Author: Aaron Cheung
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2754410072