Fragile X syndrome is a frequent cause of mental retardation that is inherited as an X-linked dominant with reduced penetrance. The mutational change in nearly all affected patients is the unstable expansion of a CGC trinucleotide repeat in the 5' untranslated region of the FMRI gene. This repeat is normally polymorphic in length and content, exhibiting a mode of 30 cryptic repeats in the normal population, but the triplet is found in excess of approximately 230 repeats in affected patients, often approaching 1,000 copies. Male, and most female, carriers have a FMRI premutation with an intermediate number of repeats, between about 60 and 200 triplets. In most penetrant males with full mutation alleles containing >230 repeats, the FMRI gene is abnormally methylated and late replicating. In such a state, the lengthy CGG repeat is believed to constitute the chromosomal fragile site. As a consequence, the FMRI gene is transcriptionally repressed in full mutation patients and the absence of the encoded protein, FMRP, a selective RNA-binding protein, is responsible for the clinical phenotype. Much of the understanding of this previously quite puzzling disorder emerged in the past 4 years, in part due to research supported under this award. Proposed below is a research program to continue our studies, initiated over a decade ago, to more fully understand this syndrome. We plan three broad specific aims. We will characterize the FMRI repeat in order to more fully understand molecular influences over its stability and evolution. We will continue our characterization of the FMR protein, absent in fragile X syndrome, with the hope that understanding its normal function will give insight into the pathophysiology of fragile X syndrome and human cognition. Finally, we will continue development of clinically useful diagnostic tools for this syndrome, in particular development of ELISA methods to rapidly and accurately quantitate FMRP. We will measure FMRP in women with the full fragile X mutation, who exhibit incomplete penetrance and extremely broad expressivity. Thus, the goal of this proposal is to capitalize upon the successful positional cloning of the FMRI gene and now use it as a molecular reagent to explore the mechanisms and consequences of the remarkable trinucleotide repeat expansion at the fragile X site.
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