Sarcoglycan A mutation in miniature dachshund dogs causes limb-girdle muscular dystrophy 2D

Abstract

Background: A cohort of related miniature dachshund dogs with exercise intolerance, stiff gait, dysphagia, myoglobinuria, and markedly elevated serum creatine kinase activities were identified.

Methods: Muscle biopsy histopathology, immunofluorescence microscopy, and western blotting were combined to identify the specific pathologic phenotype of the myopathy, and whole genome SNP array genotype data and whole genome sequencing were combined to determine its genetic basis.

Results: Muscle biopsies were dystrophic. Sarcoglycanopathy, a form of limb-girdle muscular dystrophy, was suspected based on immunostaining and western blotting, where α, β, and γ-sarcoglycan were all absent or reduced. Genetic mapping and whole genome sequencing identified a premature stop codon mutation in the sarcoglycan A subunit gene (SGCA). Affected dachshunds were confirmed on several continents.

Conclusions: This first SGCA mutation found in dogs adds to the literature of genetic bases of canine muscular dystrophies and their usefulness as comparative models of human disease.

Keywords: Canine; Gene mutation; Genetics; Myopathy; Sarcoglycanopathy.

Fig. 1

Miniature dachshund dystrophic phenotype and pedigrees. A A miniature dachshund from Australia that was evaluated for persistently elevated creatine kinase activity and exercise intolerance. B Representative H&E stained cryosection from an affected dachshund showed degenerative (asterisk) and regenerative (arrow) changes consistent with a dystrophic phenotype. C One small and one large family that formed the primary basis for this report are presented. Males are designated as squares and females as circles. Cases are solid symbols, controls are open symbols labeled with an “N,” and dogs with unknown phenotypes are open symbols. The case (dog A1) with whole genome sequence data is indicated with an asterisk. Genotypes for the functional SGCA variant are provided for all dogs with available samples

Fig. 2

Immunofluorescent staining and western blotting from a dystrophic miniature dachshund. A Immunofluorescent staining of muscle cryosections from a representative dystrophic miniature dachshund. Staining for the rod-domain of dystrophin and laminin α-2 was similar to control muscle while staining for the dystrophin C-terminus was patchy. Utrophin was not increased. Numerous regenerating myofibers were highlighted with the antibody against developmental myosin heavy chain (dMHC). Staining for α- and γ-sarcoglycans was absent and decreased for β-sarcoglycan. B Western blotting confirmed all three sarcoglycans were absent. β-actin was used as loading control

Fig. 3

Haplotypes in the region of interest on CFA9. SNP coordinates are provided along the top row. A pink cell indicates the minor allele and a white cell indicates the major allele. Identification numbers on the far left refer to the dogs within the pedigrees of Fig. 1. The CFA9 haplotype in which all 9 cases were homozygous spanned from 24,792,165–26,644,060 Mb. The affected haplotype is denoted with a red highlight (left side), unaffected haplotypes have a blue highlight, and gray haplotypes are identical to the affected haplotype but do not have the SGCA c.G224A mutation. The SGCA gene, located from positions (26,164,863–26,174,864 Mb), is indicated by an orange bar along the top