Myotonic dystrophy (MtD) is a dominantly-inherited multisystem degenerative disease with features of premature aging. Although the genetic basis of MtD is now known to be a (CTG)n repeat expansion in non-coding sequence of the myotonin protein kinase (MtPK) gene, the disease mechanism remains unclear even in its general outline. There is little experimental support for the initial hypothesis that a change in the amount or function of MtPK peptide explains the phenotype. Alternative hypotheses that the disease involves an effect on the expression of flanking genes, or a gain-of-function by (CTG)n repeats in MtPK mRNA, are the focus of this proposal. The extent (8-12 kb in most tissues) and character (strongest known natural nucleosome positioning element) of the CTG repeat expansion suggest its potential for altering chromatin structure and expression of other genes in the region. The recently discovered homeodomain gene DMAHP is a candidate for cis-acting suppression by the CTG expansion and for involvement in MtD pathogenesis because of its proximity to the repeat expansion and its sequence homology to transcription factors involved in myogenesis and connective tissue patterning. Studies are proposed to compare the levels of DMAHP mRNA in myoblasts from MtD patients, disease controls, and healthy subjects using allele-specific RT/PCR and ribonuclease protection assays. MtPK expression patterns and tissue pathology in MtD are strongly concordant. The clearest immediate consequence of the CTG expansion is to increase the amount of (CUG)n repeat-containing RNA in cells that express MtPK. Preliminary data from RNA folding algorithms, thermal melting, gel retardation, and gel mobility shift experiments are consistent with the idea that long (CUG)n RNA tracts form stable folded structures that bind to PKR, the dsRNA dependent protein kinase which plays a central role in translational control. Activation of PKR by (CUG)n repeat RNA tracts could explain the reduced muscle protein synthetic rate and myofiber atrophy in MtD. Structural characteristics and PKR binding/activation of (CUG)n repeats will be studied by gel retardation, nuclease mapping, gel mobility shift, and in vitro phosphorylation experiments.
