Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion

Abstract

Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations.

Figure 1

Age-related expansion of RF in mdx mouse muscles. (A) New born, (B) 4 wk, (C) 4 mo, and (D) 18 mo. The size of RF clusters increases from isolated single fibers in the new born mouse to a huge cluster containing >60 RFs in the 18-mo-old mouse. Variation of RFs in size and of staining intensity within a cluster is seen in mice aged 4 wk or older. Sections are stained for dystrophin with polyclonal antibody p6.

Figure 3

Expression of dystrophin and dystrophin-associated glycoprotein complex in RFs. A group of fibers are stained with p6 for dystrophin. The same group of fibers are clearly stained for β-dystroglycan, α- and β-sarcoglycan, and α-syntrophin and the staining intensity is correlated to that of dystrophin. The remaining fibers are only weakly or not stained for the Abs. All fibers are stained for spectrin (bottom left panel). Serial sections of TE muscle from 18-mo-old mdx mouse.

Figure 2

An overall age-related increase of RFs in TE muscle of mdx mouse. The number and length of RF clusters and maximum number of RFs within one cluster increase steadily, resulting in a huge increase in the number of RF sections within 1,000 μm of TE muscles. NB, new born mice; W, weeks; M, months; and Maxi No, maximum number. Vertical bars represent standard deviation. The total number of RFs counted in all 100 sections was referred to as RF sections.

Figure 5

Patterns of epitope loss in RF. Total of 279 RF clusters were examined with 14 Abs to dystrophin by three-step immunoperoxidase staining. Loss of 20 exons or more was found in >65% of RF clusters.

Figure 4

Exon mapping of RF. Staining of serial sections with a panel of 12 Abs (name above and exon specificity below the pictures). Sections are arranged in such an order from left to right with Ab recognizing exons from NH₂-terminal to COOH-terminal. The diagram below illustrates the position of the epitope(s) recognized by the Abs on the dystrophin molecule. Serial number of sections appears at the bottom right corners. Two isolated fiber clusters marked a and b with two and one fibers, respectively. Fibers a are recognized by Abs to NH₂-terminal exons up to 18, but negative with Abs to exons 20–26. These fibers are positive with Ab to exons 27–28, but negative again with Abs to exons from 26–29 to 38–39 followed by relatively weak staining with Ab to exons 40–41. Fiber b is recognized by Abs to NH₂-terminal exons up to 27–28, with relatively weak staining with Abs to exons 20–21. This fiber is not recognized with Ab against exons 26–29 to 38–39. Another revertant fiber of small caliber stained with Abs to exons 26–29 and 50 appears at the bottom of the sections, which are at the end of this serial sections. The primary Abs were detected with peroxidase method and the pictures are presented as negative image to enhance contrast. Nuclei appear positive due to the haematoxylin counterstaining.

Figure 7

Combined immunohistochemistry and in situ hybridization for detection of dystrophin protein and genomic sequence of intron 21 to exon 25. Dystrophin protein in RFs is stained with immunoperoxidase method and the positive staining is shown as false green color. Hybridization signals for the genomic region of dystrophin sequences are shown as false red color. Nuclei are counterstained with 4,6-diamidino-2-phenylindole (DAPI), showing as blue. Sections are first stained with antibody p6 for dystrophin on unfixed sections followed by in situ hybridization.