UC Davis

The Fragile X-associated disorders are a group of genetic diseases resulting from the expansion of the CGG repeats in the 5’ untranslated region (5’ UTR) of the FMR1 gene, located on the X chromosome. Individuals with an expansion of greater than 200 CGG repeats have the full mutation, which leads to silencing of the gene and lack of the encoded protein, FMRP, causing Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Individual carriers of a premutation allele (55-200 CGG repeat) are at risk of developing Fragile X-associated tremor/ataxia syndrome (FXTAS), a late onset neurodegenerative condition, which affects both males and females. Currently, there is no approved therapy or reliable biomarkers to determine therapeutic efficacy in FXS and associated disorders, in particular of FXTAS.The first part of my dissertation (Chapters 2, 3 and 4) investigates the development of a potential biomarker for early diagnosis and progression of FXTAS. The second part of my dissertation (Chapters 5 and 6), reports on the development of a protocol to derive epithelial cells from urine samples of FXS patients and on the molecular methods to study and diagnose FXTAS. Chapter 1 is a detailed introduction to the genetics of FXS, its pathophysiology and on currently available molecular biomarkers. In Chapter 2, I report on an investigation of the alternative splicing landscape at the FMR1 locus in conjunction with brain measures. I show that increased levels of specific FMR1 mRNA isoforms, those that encode truncated proteins, are present in premutation carriers who developed symptoms of FXTAS, relative to non-carrier healthy controls, suggesting a potential role in the development of the disorder. Chapter 3 reports on the identification of biomarkers for early diagnosis and progression of FXTAS. Interestingly, lipid metabolism and, specifically, the sub-pathways involved in mitochondrial bioenergetics as observed in other neurodegenerative disorders; were found to be significantly altered in FXTAS. Further, in Chapter 4, I demonstrate that differential expression levels of some of the previously-identified metabolites are linked with areas within the pons. In addition, we observed a significant correlation of these metabolic signatures with the FXTAS stage, strengthening the likelihood that they contribute to the progression and pathogenesis of FXTAS. In the second part of my thesis, which begins with Chapter 5, I describe a cost-effective and straightforward method to derive epithelial cell lines from urine samples collected from participants with FXS and healthy controls (TD). These cell lines can be used as a new model to study the molecular mechanisms behind FXS, including the expression of surface markers, inter and the intra-tissue CGG mosaicism, FMR1 mRNA and FMRP expression levels. Finally, in Chapter 6, I provide the protocols for FXTAS diagnostic tools, in both humans and mouse.