The structure and function of the human FMR1 gene

Abstract The CGG repeat located in the 5' untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene has a risk of pathogenicity when it expands beyond the normal range (5-44 CGG repeats). The dynamic mutations that occur at this triplet repeat are classified as intermediate (45-54 CGG repeats), premutation (55-200 CGG repeats), and full mutation (greater than 200 CGG repeats). Three monogenic disorders are caused by the FMR1 CGG repeat mutation, fragile X syndrome (FXS), Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), and Fragile X-associated Primary Ovarian Insufficiency (FXPOI), but there are a number of other disorders and conditions that are associated with FMR1 including autism spectrum disorders (ASD), immune mediated disorders, anxiety, and depression.;While the underlying cause of fragile X associated disorders (FXADs) is predominantly the expansion of the CGG trinucleotide repeat (TNR) the mechanisms that become disrupted vary by syndrome. The focus of this dissertation is on the structure of the CGG trinucleotide repeat of the FMR1 gene and on the impact that it has both molecularly and phenotypically. The different CGG repeat classifications are considered in these findings, highlighting the importance of the CGG repeat structure in the function of the FMR1 gene.;The presence of AGG interruptions in the TNR of FMR1 was discovered shortly after identification of the gene, and it was hypothesized these interruptions had a protective function, which helped prevent instability of the TNR. In the first chapter the role of AGG interruptions in the stability and expression of FMR1 is investigated. The study shows that the risk of expansion from premutation to full mutation allele during maternal transmission decreases by increasing number of AGG interruptions. In addition, the risk of instability of an intermediate or premutation allele during maternal or paternal transmission is shown to be reduced with the presence of AGG interruptions, as is the magnitude of size change that occurs during transmission. The study also shows that in the premutation range AGG interruptions do not affect the expression of FMR1 mRNA, confirming previous studies. Additionally, variation in AGG interruption patterns were observed in normal alleles from different populations, which may explain the differences in prevalence for premutation and full mutation alleles in different ethnic groups.;The dynamic instability of the CGG repeat locus often leads to mosaicism in full mutations, which is the topic of Chapter 2. The results show that mosaicism can impact both the expression of the gene and the phenotypic manifestation of FXS due to the presence of partial FMRP expression, which can sometimes improve clinical outcomes.;The presence of elevated mRNA in premutation carriers is thought to result in RNA toxicity. Chapter 3 reports on the alternative splicing of FMR1 in premutation carriers showing an increase of approximately 2-fold in the expression levels of all isoforms detected. Interestingly, for two of them (Iso10 and Iso10b) the expression level was disproportionally increased (approximately 4-fold) compared to the other isoforms. The alternative splicing of these two isoforms suggest potential significant pathogenic consequence. Translation of these isoforms would lead to truncated proteins lacking two important FMRP functional domains, the phosphorylation sites and the RGG box, due to the frame shift created by the splicing of exon 14.;One of the core molecular perturbations that occurs when the CGG TNR expands to a full mutation is the increase of mGluR dependent long term depression (LTD), which leads to deficits in learning and memory formation in both Fmr1 knock-out (KO) mice and humans with FXS. Chapter 4 describes the characterization of two signaling pathways important for mGluR-LTD, the mammalian Target Of Rapamycin (mTOR) and the Extracellular signal-regulated kinases (ERK) in primary fibroblast cell lines derived from subjects with an FMR1 full mutation, and in one case with both an FMR1 full mutation and TSC1 loss of function mutation. The dysregulation that has been reported in brain tissue of Fmr1 KO mice and humans with FXS, and in peripheral tissue of humans with FXS was not observed in cultured fibroblasts. These findings also disagree with a recent report that translational dysregulation can be studied in vitro in primary fibroblast cell lines.;The whole work presented here reports on the impact of CGG repeat substructure by investigating the molecular consequences for each mutational category from normal to full mutation, including mosaicism and captures the many layers of complexities of the FMR1 gene related to the CGG repeat.;Although the FMR1 CGG TNR was identified over two decades ago, it continues to offer new insights into its role in the function of the gene and on pathogenesis related to it.