Dystrophin‐Deficient Muscular Dystrophy in a Jack Russell Terrier With a Large Deletion in the Canine DMD Gene

Figures

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  FIGURE 1. Histological staining of skeletal muscles. H&E‐stained cryosections of the M. biceps femoris and M. triceps brachii from the Jack Russell Terrier with dystrophin‐deficient muscular dystrophy. Variability in myofiber size was present with numerous degenerating and regenerating fibers, scattered myofibers with internal nuclei and calcific deposits (arrows). These changes are consistent with a dystrophic phenotype. Bar in triceps muscle image = 100 µm for both muscles.

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  FIGURE 2. Immunofluorescent staining of skeletal muscle cryosections. Immunofluorescence staining of cryosections from the M. biceps femoris of the dystrophic dog and archived control canine limb muscle using monoclonal antibodies against dystrophin (DYS1 and DYS2), against spectrin for membrane integrity (SPEC2), against utrophin (DRP2), against dMHC for regenerating fibers, against α, β, and γ‐sarcoglycans (αSG, βSG, and γSG), against laminin α2, and collagen VI. Staining with both DYS1 and DYS2 was absent or markedly reduced with upregulation of utrophin on the muscle sarcolemma. Staining for the three sarcoglycans was moderately reduced. Staining for laminin α2 was normal in appearance, and mild endomysial fibrosis was shown in the staining for collagen VI. Bar in lower right image = 50 µm for all images.

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  FIGURE 3. Multi‐exon deletion within the DMD gene in the affected Jack Russell Terrier. (A) Overview (IGV screenshot) across the entire canine DMD gene from the whole genome sequence data in comparison to a control dog. The 25.5 kb deletion is indicated. It is located immediately adjacent to a region with high repeat content that cannot be reliably sequenced with short‐read data. (B) Details of the 25.5 kb‐sized deletion with breakpoints. (C) The deletion spans DMD exons 8 and 9 (highlighted in red) and part of the flanking introns. (D) Details of the breakpoints. The proximal breakpoint is in a repetitive region with low mapping quality of sequencing reads. The distal breakpoint is located at the position of an independent polymorphic ∼6.4 kb LINE‐1 insertion in dogs. This insertion is present in the affected dog and the first control dog shown, but absent from the second control dog shown and the UU_Cfam_GSD_1.0 reference genome. The repetitive nature of the flanking sequences prevented successful amplification of the mutant allele.

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  FIGURE 4. Pedigree of the family and PCR‐based determination of the presence or absence of the DMD wild‐type allele. Genotypes are indicated for all four dogs for which samples were available. The genotypes of the four other littermates remain unknown. The early deceased male puppy is indicated by a strikethrough symbol. We assume that this puppy was also affected, but the puppy was not examined in detail. In the genotype designation of hemizygous male dogs, only one allele is indicated. In the female dogs, the presence of the wild‐type allele was confirmed, but zygosity and carrier status are unknown. The unknown second allele is indicated by a hyphen. Note that the Fdel/R amplicon is only absent in the affected dog (location of primers is shown in Figure 3C).