Exome sequencing reveals independent SGCD deletions causing limb girdle muscular dystrophy in Boston terriers

Abstract

Background: Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited autosomal myopathies that preferentially affect voluntary muscles of the shoulders and hips. LGMD has been clinically described in several breeds of dogs, but the responsible mutations are unknown. The clinical presentation in dogs is characterized by marked muscle weakness and atrophy in the shoulder and hips during puppyhood.

Methods: Following clinical evaluation, the identification of the dystrophic histological phenotype on muscle histology, and demonstration of the absence of sarcoglycan-sarcospan complex by immunostaining, whole exome sequencing was performed on five Boston terriers: one affected dog and its three family members and one unrelated affected dog.

Results: Within sarcoglycan-δ (SGCD), a two base pair deletion segregating with LGMD in the family was discovered, and a deletion encompassing exons 7 and 8 was found in the unrelated dog. Both mutations are predicted to cause an absence of SGCD protein, confirmed by immunohistochemistry. The mutations are private to each family.

Conclusions: Here, we describe the first cases of canine LGMD characterized at the molecular level with the classification of LGMD2F.

Keywords: Dog; LGMD; Muscle; Myopathy; Sarcoglycanopathy.

Fig. 1

Histopathology of muscle biopsies from a female Boston terrier affected with sarcoglycanopathy (case 3). A hunch back stance was evident in the dog (a). H&E stained cryosections from a representative limb muscle (b) showed degenerative changes and calcific deposits (black arrow). Similar degenerative changes and calcific deposits were observed in the tongue (c). The calcific deposits in the tongue were highlighted bright orange using the alizarin stain for calcium (d)

Fig. 2

Loss of SGC staining in cases 3 and 4. Representative H&E and immunofluorescence of cryosections from the muscle of cases 3 and 4, as well as of a control dog muscle. In the control muscle, antibodies to the SGC (α-, β-, δ-, γ-sarcoglycans: αSG, βSG, δSG, γSG), as well as sarcospan (SSPN), localize to the sarcolemma of the muscle fibers. Staining from each of these antibodies is reduced in muscle from cases 3 and 4

Fig. 3

Representative immunofluorescence of cryosections from muscle of cases 3 and 4 and control dog muscle. Staining of α-dystroglycan (αDG), β-dystroglycan (βDG), dystrophin (DMD), caveolin 3 (CAV3), collagen VI (COL6), and perlecan (PCAN) in cases 3 and 4. Antibodies to α- and β-dystroglycans, dystrophin, caveolin 3, and perlecan demonstrate sarcolemmal localization and intensity that is comparable to control tissue. An antibody to collagen VI shows increased localization to the endomysium compared to the control tissue, consistent with endomysial fibrosis

Fig. 4

Electropherogram showing the 2-bp SGCD deletion in case 3. The top panel shows the sequence from case 3, while the lower panel shows the sequence from a healthy non-related Boston terrier. The SGCD c.534_535delGA mutation leads to a frameshift and a premature stop codon two amino acids later

Fig. 5

Schematic and sequence showing the breakpoints of the 19,403-bp SGCD deletion in case 1. Note that SGCD is annotated on the minus strand. Whole exome sequence from a healthy dog and case 1 are aligned to the reference genome, visualized in Golden Helix GenomeBrowse ® [21, 22]. Case 1 has no coverage of exons 7 and 8 and flanking regions. Sequence of the wild-type and case 1 alleles show the precise breakpoints. Nucleotides 5′ and 3′ of the breakpoint are in bold blue and orange typeface, respectively. A substitution (chr4:53262020-53262018, CAT > GG) is found 9 bp downstream of the microdeletion and is shown in bold red typeface