The identification of the FMR1 gene started the era of expanding triplet repeat diseases. In fragile X syndrome patients the repeat has more than 200 CGGs (full mutation) and due to this expansion of the repeat, present in the 5' untranslated region of the FMR1 gene, the promoter region is methylated resulting in silencing of the gene (Fig. 1). The expansion of the CGG repeat to a large repeat only occurs in oocytes and never in sperm, irrespectively of the repeat size in somatic cells. As a result of selection during forming and maturation of primordial germ cells only sperm producing cells with a premutation are present after puberty. The mechanism of the repeat instability occurring during gametogenesis and early embryogenesis is still unclear, mainly due to the lack of a human material. Therefore, we have developed an animal model with an expanded repeat. These mice will enable the study of timing and mechanism of amplification in the mouse germ line. At the same time these mice will be used to study the inclusion bodies seen in the nuclei of neurons in carriers of a premutation.

The identification of normal males with a long unmethylated repeat has learned that it is not the long repeat itself that is causing the disease but the methylation of the promoter of the FMR1 gene during embryogenesis. It has been shown that DNA methylation mediates the forming of a multiprotein repression complex by the interaction of MeCP2 with DNA at methylated cytosine residues and a number of factors including histone deacetylases. This complex formation results in modification of chromatin structure, which might explain the delay in replication of the mutated FMR1 gene. Treatment of cells with demethylating agents results in reactivation of the FMR1 gene. The repression could be alleviated by treatment with deacetylase inhibitors. Further studies will focus on the identification of the factors and enzymes that are involved in the (in)activation of the mutated FMR1 gene. This may eventually lead to strategies to prevent inactivation of the FMR1 gene.

The absence of the FMR1 protein is causing mental retardation, but what is the function of the FMR1 gene. Recent work has indicated that the FMR1 protein might regulate localized protein synthesis. The subcellular behavior of FMRP, including its selective mRNA binding, nucleocyplasmic shuttling, and predominant association with cytoplasmic polyribosomes, has led to the hypothesis that FMRP may play a role in nuclear export of its target mRNAs and further present these mRNAs to the translation machinery (Fig. 1 and 2). The discovery of FMRP-polyribosome association in the neuronal dendritic spine implies the possible involvement of FMRP in modulating localization and/or translation of its target mRNAs in the appropriate compartments. Regulation of localized protein synthesis has been demonstrated to be functionally important in cell growth and polarity development. There is still a lack of knowledge about the specific class of mRNAs whose transport/translation Is affected by the lack of FMRP. We are now examining in more detail the shuttling of FMRP and the identification of the specific mRNAs that can be bound to FMRP. Further studies will be directed towards the detection of differences in (cytoplasmic levels of) these mRNAs in patients and in a developed Fmr1 knockout animal model, using differential DNA chip expression screening. Identification of these mRNAs should help to define the influence of FMRP in mRNA metabolism, and finally to the elucidation of pathogenesis in mental retardation due to the lack of FMRP.

Fig1:
Schematic representation of the FMR1 gene with KH domain , RGG boxes and the nucleocyplasmic shuttling signals NLS and NES. In the 5’ part of the gene the CGG repeat is shown for control (N), premutation (P) and patients (F).

Fig 2: Model for the intracellular routing of FMRP