Despite the discovery in 1992 that myotonic dystrophy (DM), the most common adult muscular dystrophy, results from an unstable expansion of CTG repeats in the 3' untranslated region of a gene encoding a serine/threonine kinase (DMPK), we still do not have a clear understanding of the pathomechanism responsible for this dominantly inherited, multisystem disorder. As yet there are no truly representative animal models of DM. Studies of heterozygous and homozygous knock-out mice demonstrate that a deficiency or lack of DMPK does not lead to the DM phenotype. In DM there is transcription of the abnormally expanded repeat; however, the mutant DMPK transcripts are retained in the myonuclei. We are proposing that muscle degeneration in DNA is RNA-mediated, i.e., that expanded CUG repeats in mutant transcripts are toxic for muscle fibers. To test this hypothesis, we have used the human skeletal actin (HSA) gene to overexpress an untranslated CTG repeat in the muscle of transgenic mice. The long-repeat HSA constructs have 250 CTG repeats inserted in the HSA 3' untranslated region. Short-repeat constructs have 5 CTG repeats. We have found that five independent founder lines of mice with long repeats develop myotonia and myopathy. No phenotypic abnormality has been observed in mice having short repeats, or nontransgenic mice. The disease manifestations cannot be ascribed to overexpression of actin protein. Although long-repeat transcripts are spliced normally, and polyadenylated, they remain in the myonuclei. To explore the disease mechanism. we have carried out in vitro studies which show that CUG repeat RNAs form highly stable hairpins that bind to double stranded RNA (dsRNA) binding proteins. Furthermore, pathologically expanded CUG repeats activate PKR, the dsRNA-activated protein kinase. A possible scenario for the pathogenesis of DM, therefore, is that expanded CUG repeats activate mediators of the dsRNA induced stress/antiviral response, such as P&R or RNAse L. The goals of this proposal are to (l) determine if the pathogenic effect of an expanded CTG repeat is mediated by mutant mRNA, (2) characterize the natural history of myotonic myopathy in long-repeat mice, (3) assess the effects of genetic background on the disease manifestations, (4) develop homozygous lines that have a consistent. severe, phenotype; (5) evaluate the effects of expanded CUG repeats on myonuclear morphology and survival, and (6) determine whether the effects of long -repeat transgenes are ameliorated on a PkR or RNAse L null genetic background. This work mad establish RNA gain-of-function as a new genetic mechanism, and provide the first animal model for DM.
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