| Myotonic dystrophy type 2 (DM2) is a multisystemic adult onset disease caused by a CCTG expansion mutation in intron 1 of the zinc finger protein 9 (ZNF9) gene. While evidence for a RNA gain-of-function mechanism involving DM1 CUG or DM2 CCUG expansion transcripts grows, still widely debated is the potential role that flanking sequences within the DMPK 3' UTR may have on disease pathogenesis and whether or not decreased expression of DMPK, ZNF9 or neighboring genes at these loci contribute to disease. Using DM2 patient derived cell lines either haploid or homozygous for the expansion, as well as skeletal muscle biopsy tissue, I demonstrate that pre-mRNAs containing even very large CCUG expansions are normally spliced and exported from the nucleus, that the expansions do not decrease ZNF9 expression at the mRNA or protein level, and that the ribonuclear inclusions are enriched for the CCUG expansion, but not intronic flanking sequences.; To test the hypotheses that CCUG expansion transcripts alone, in the absence of any gene context, are sufficient to replicate the skeletal muscle features of the disease and that many of these features will be reversible after transgene expression is turned off, I developed an inducible murine model of DM2. Analysis of skeletal muscle from transgenic animals expressing 300 CCUGs shows a number of phenotypic changes characteristic of myotonic dystrophy including: (1) variation in fiber size and centrally located nuclei indicative of myopathy; (2) electrical myotonia; (3) ribonuclear inclusions; and (4) aberrant splicing of the insulin receptor. Additionally, I show several of these phenotypes are reversible after expression of the CCUG transgene has been turned off. Moreover, expression analysis of CUGBP1, one of the key RNA binding proteins implicated in DM pathogenesis, revealed dramatic Cugbp1 overexpression in a chloride channel knock-out mouse, but not in the CCUG expansion mice. This suggests that overexpression of CUGBP1 is not only caused by expression of repeat containing transcripts, and therefore is not unique to DM.; Taken together, these data support a RNA gain-of-function mechanism for DM1 and DM2 in which the repeats themselves are sufficient to cause the multisystemic features of these diseases. |
