Functional characterization of R294X and C-terminal MECP2 mutations in Rett syndrome

Abstract

This dissertation work comprises a functional characterization of R294X and representative C-terminal stop loss MECP2 mutations that cause Rett syndrome (RTT) in people. In Chapter 2, we show that RTT-like phenotypes induced by the partial loss-of-function R294X mutation can be rescued by genetic MECP2 supplementation in mice. These results dissuade concerns about potential dominant negative interactions between full-length MeCP2 and an R294X truncated protein product with altered chromatin binding properties. Additionally, we present evidence of subtle motor-specific MECP2 duplication syndrome (MDS)-like behavioral effects of genetic MECP2 supplementation in female mice, suggesting that the motor circuitry is particularly susceptible to elevated gene dosage. Thus, we find that MeCP2 supplementation is safe and effective in males and largely so in females; however, careful consideration for potential motor-specific effects may be warranted for girls and women with RTT. With the knowledge that the R294X mutation can be genetically rescued, we wondered whether mutations in the C-terminal region of the protein, which retain intact methyl binding and NCOR/SMRT interaction functional domains, would also be amenable to genetic rescue. In Chapter 3, we develop an in vitro LUHMES-based cellular modelling system and demonstrate its utility in evaluating a unique stop loss X487R C-terminal MECP2mutation. We present evidence that the X487R mutation yields reduced protein levels in an endogenous context and provide preliminary data showing differences in dopaminergic phenotype during differentiation. While there remains much to be explored, this dissertation work advances an understanding of mutation-specific susceptibility to putative genetic supplementation therapy and develops a foundation for mechanistic study of the heterogeneous and understudied group of C-terminal MECP2 mutations.