Current Classification of Canine Muscular Dystrophies and Identification of New Variants

Abstract

The spectrum of canine muscular dystrophies has rapidly grown with the recent identification of several more affected breeds and associated mutations. Defects include those in genes and protein products associated with the sarcolemma (dystrophin deficient X-linked muscular dystrophy and sarcoglycan-deficient limb-girdle muscular dystrophy) and with the extracellular matrix (collagen 6, laminin α2, and α-dystroglycan-deficient congenital muscular dystrophies). With the increasing application of whole genome sequencing and whole exome sequencing, the clinical and pathological spectra associated with specific neuromuscular genetic defects are constantly evolving. In this report, we provide a brief overview of the current status of gene defects reported in canine muscular dystrophies. We also report the causative mutations for novel forms of X-linked muscular dystrophy in Brittany spaniels and in a French bulldog.

Keywords: animal model; dog; muscle; myopathy; whole genome sequencing.

Figure 1

Histopathology and immunofluorescent staining of muscle cryosections from two Brittany spaniels with dystrophin-deficient muscular dystrophy. (A) is Case 1, a 6-month-old intact male Brittany from the USA, and (B) is Case 2, a 7-month-old intact male Brittany from Japan. In (A), H&E stained cryosections (left) show variability in myofiber size, myonecrosis (arrowhead) and calcific deposits (arrows) consistent with a degenerative myopathy. Immunofluorescence staining for dystrophy-associated proteins (right) shows absent or markedly decreased staining for the rod and carboxy terminus of dystrophin, upregulation of utrophin, and clusters of regenerating fibers using the dMHC antibody for regenerating fibers. In (B), H&E stained cryosections (left) show variability in myofiber size and calcific deposits (arrows). Immunofluorescent staining for dystrophy-associated proteins showed a staining pattern similar to that of Case 1, with undetectable staining for the rod and carboxy terminus of dystrophin, upregulation of utrophin, and clusters of regenerating fibers (dMHC antibody). Staining for other dystrophy-associated proteins, including those for laminin α2, collagen 6, and caveolin 3, was similar to control in both cases.

Figure 2

Histology and Immunofluorescent staining of muscle cryosections from a French Bulldog. (A) Cryosections from the dystrophic French bulldog biceps femoris muscle and diaphragm showing degenerative and regenerative changes consistent with a form of muscular dystrophy. Variability in myofiber size was present in both muscles, with scattered and pale staining necrotic fibers (arrows) present in the diaphragm. H&E stain, bar = 100 µm for both images. (B) Immunofluorescent staining of muscle cryosections from the dystrophic French bulldog and an archived non-age matched control muscle using antibodies against dystrophy-associated proteins. Staining was absent for the rod (DYS1) and carboxy-terminus (DYS2) of dystrophin, with upregulation of utrophin (DRP2) consistent with a diagnosis of a dystrophin-deficient muscular dystrophy. Regenerating muscle fibers are highlighted with the antibody against developmental myosin heavy chain (dMCH). Results of staining for caveolin 3, laminin α2 and collagen VI are similar to those for control muscle. Bar in the lower right corner = 50 µm for all images.

Figure 3

IGV view of a DMD point mutation in exon 55 of Brittany spaniel Case 1. The green base identified in all reads covering this base in the case is the replacement of a G at genomic position 26,939,052 with an A, resulting in a p.Q2687* mutation in the DMD protein.

Figure 4

WGS reads from Brittany spaniel Case 2 aligned to dog chromosome 2 (left and center) and chromosome X (right) in the vicinities of the Ring Finger and SPRY Domain Containing 1 (RSPRY1) and DMD genes, respectively. The case is the top dog in all three panels. The colored bars circled in the case dog on the right represent reads that lie in intron 20 of DMD in which their mates map to the 3′ UTR or 5′ UTR (clusters of colored reads) of RSPRY1. WGS read mate pairs aligning to two different chromosomes can be indicative of a structural variant.

Figure 5

Evidence for the presence of a RSPYR1 retrogene in Brittany spaniel Case 2. The red mate pairs aligned over consecutive exons of RSPYR1 (skipping over intronic sequence but connecting red lines indicating their mates) are expected from reads derived from a retrogene-mediated RSPYR1 cDNA insertion elsewhere in the genome. A similar result is not observed in the control dog.

Figure 6

IGV view of a DMD 1 bp insertion mutation in exon 25 of a French bulldog with dystrophin-deficient MD. The purple “I” identified in all but one aligned read in the case is the insertion of a T at genomic DNA position 27,774,668, resulting in a p.F1125* mutation in the DMD protein.