The Repeat Expansion Diseases are caused by the intergenerational expansion of a specific tandem repeat. The consequences of expansion depend on the gene involved, the location of the repeats within the gene, and the sequence of the repeat unit. Expansion of a CGG:CCG-repeat in the 5' UTR of the FMR1 gene is associated with 3 quite different clinical presentations: Individuals with >200 repeats have Fragile X mental retardation syndrome. Individuals with 60-200 repeats (the so-called premutation) are at risk for 2 quite different problems, namely Fragile X-associated tremor-ataxia syndrome and premature ovarian failure. GAA:TTC-repeat expansion in the first intron of the frataxin gene causes a deficit in frataxin mRNA. This results in Friedreich ataxia, a degenerative disease associated with cerebellar dysfunction, hypertrophic cardiomyopathy, and diabetes. We are interested in both the mechanism of expansion and the consequences of expansion in these disorders. The repeat is somatically stable in Fragile X premutation mice we generated. A small number of animals are mosaics having 2 alleles with different repeat numbers. Each allele is present in similar amounts suggesting that expansion occurs prezygotically or before the first cell division in the embryo. We also find that the repeat is significantly more prone to expansion when present on both alleles. This suggests a role for a homology-based process (recombination?) in repeat expansion. We have also shown that the flap endonuclease FEN-1 acts to prevent repeat expansion. This together with our previous demonstration that these repeats block DNA synthesis suggest a model in which expansions arise when enzymes like FEN-1 are unable to restart stalled replication forks. Expansion may then occur when attempts are made to repair the stalled fork by recombination. We have shown that binding of transcription factors to the FMR1 promoter leads to an altered promoter architecture that may be important for normal gene regulation. We have also shown that cells expressing RNA with large numbers of CGG-repeats have elevated levels of apoptosis. These cells are also prone to detachment from the growth surface in response to serum deprivation. Since some cells are sensitive to detachment-induced apoptosis (anoikis) this represents an additional way in which RNA with long CGG-tracts can be deleterious. In an effort to understand the molecular basis of the deficiency of the frataxin transcript in individuals with FRDA we have analyzed the frataxin gene in some detail. In doing so we have identified key regions downstream of the transcription start site that are important for normal transcription. Since transgenes containing long GAA?TTC-repeat tracts become heterochromatinized in mice this could in principle lead to frataxin gene silencing. We have thus also examined the methylation status of the frataxin gene in normal and affected individuals, and shown that it has a relatively subtle but potentially important effect on frataxin transcription.
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