Background: The Repeat Expansion Diseases are caused by intergenerational expansions of a specific tandem repeat. More than 20 such diseases that belong to this group have been identified thus far. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. Carriers of alleles with 55-200 repeats, so-called premutation (PM) alleles, are at risk for a neurodegenerative disorder, Fragile X-associated tremor/ataxia syndrome (FXTAS) and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency (FXPOI). Furthermore, in females, the premutation allele can undergo expansion on intergenerational transfer that can result in their children having alleles with >200 repeats. This expanded allele is known as a full mutation (FM) and individuals who inherit such alleles almost always have Fragile X syndrome (FXS), which is the leading heritable cause of intellectual disability. FM alleles become silenced. This results in a deficiency of the protein product of this gene, FMRP, which is involved in the regulation of translation of a subset of mRNAs. The FMRP deficiency in brain results in aberrant dendritic spine morphology and a defective response to synaptic activation. The mechanism of gene silencing is unknown, but may show parallels to Friedreich ataxia, a related disorder that also shows repeat-mediated gene silencing. Progress report: In addition to our ongoing work on FXPOI using a mouse model we developed, we have used induced pluripotent stem cells (iPSCs) and a patient-derived embryonic stem cell (ESC) line to study the mechanism of gene silencing that is responsible for FXS. Our findings this year include the identification of small molecules that produce sustained FMR1 gene expression in FX cells treated with a single dose of 5-azadeoxycytidine. The importance of this finding is that it demonstrates that despite the expression of the FMR1 transcript that we previously showed was responsible for gene silencing, silencing can still be prevented by blocking the epigenetic events that are triggered by the FMR1 mRNA. One class of molecules that were effective at prolonging gene expression are inhibitors of EZH2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2) that we had previously hypothesized to be important for Fragile X gene silencing. The second class of molecules interact directly with the CGG-RNA and we hypothesize that they act by preventing binding to chromatin repressors.In the course of doing this work we also developed a set of tools for analyzing repeat length, AGG interruption patterns and the methylation status of the FMR1 gene. These tools, which are robust and inexpensive, should allow the routine monitoring of these parameters in studies of the effect of PM and FM alleles on early embryonic development. Without such tools it is impossible to distinguish between bona fide developmentally regulated changes and artifacts arising from the instability in repeat number and methylation commonly associated with these cells
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