UC Davis

The human fragile X messenger ribonucleoprotein 1 (FMR1) gene contains an expansion-prone trinucleotide CGG repeat that alters gene expression, giving rise to various central nervous system (CNS)-centric disorders. Alleles with repeats between ~55-200 CGGs, termed premutation alleles, predispose an individual to the adult-onset neurodegenerative disorder, Fragile X-associated tremor/ataxia syndrome (FXTAS). Typically, premutation alleles result in increased transcription, a slight decrease in protein product (FMRP), and cellular toxicity. Unfortunately, the exact molecular mechanism underlying FXTAS pathogenesis is unknown and no targeted treatments exist. FMR1 alleles harboring expansions of more than 200 CGG repeats, termed full mutation alleles, trigger gene silencing. The resultant loss of FMRP expression causes Fragile X syndrome (FXS), the most common single gene form of autism and heritable intellectual disability. This dissertation describes investigations of aspects of FMR1 gene expression in FXTAS and FXS to assess models of pathogenesis and to further evaluate the interplay of various relevant molecular features, respectively.Chapter 1 of this dissertation addresses one aspect of the DNA damage repair (DDR) model of FXTAS pathogenesis, which proposes that unresolved DDR events triggered by R-loop formation result in cellular toxicity and death. R-loops are generated during transcription when nascent mRNA hybridizes with the template DNA strand, exposing the single stranded DNA to damage. DNA-RNA immunoprecipitation (DRIP)-qPCR was used to quantify FMR1 R-loop abundance in primary fibroblasts from control and premutation carriers with and without FXTAS. Importantly, this study is the first known effort to characterize R-loops in FXTAS patients. FMR1 was determined to be a low to moderate R-loop producing locus with an average steady-state frequency of ~2.5%. Furthermore, no associations between steady-state R-loop frequency and CGG repeat size, FMR1 mRNA level, or FXTAS were found. Therefore, R-loop frequency may not substantially trigger DDR in FXTAS although the DDR model more broadly should be further investigated. Chapter 2 of this dissertation studies the effect of CGG repeat size and methylation status on FMR1 protein (FMRP) production in FXS. FMRP expression in control and full mutation primary peripheral blood mononuclear cells (PBMCs) was quantified by time-resolved fluorescence resonance energy transfer (TR-FRET). FMR1 mRNA and methylation status were quantified by qRT-PCR and southern blot, respectively. Most full mutation PBMCs displayed a high degree of size and methylation mosaicism, which was necessary for significant FMRP production. Interestingly, mRNA and repeat size of unmethylated alleles successfully modeled non-significant levels of FMRP, suggesting that FMRP was present, but below the significance threshold for many samples. Thus, a more sensitive assay may provide deeper insights into the interaction between these molecular characteristics. The results presented here highlight the continued need to investigate the molecular mechanisms by which altered expression leads to FMR1-associated disorders.