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Sarcoglycan a Mutation in Miniature Dachshund Dogs Causes Limb-Girdle Muscular Dystrophy 2D

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Sarcoglycan a Mutation in Miniature Dachshund Dogs Causes Limb-Girdle Muscular Dystrophy 2D UC Davis UC Davis Previously Published Works Title Sarcoglycan A mutation in miniature dachshund dogs causes limb-girdle muscular dystrophy 2D. Permalink https://escholarship.org/uc/item/3r82r6wn Journal Skeletal muscle, 11(1) ISSN 2044-5040 Authors Mickelson, James R Minor, Katie M Guo, Ling T et al. Publication Date 2021-01-07 DOI 10.1186/s13395-020-00257-y Peer reviewed eScholarship.org Powered by the California Digital Library University of California Mickelson et al. Skeletal Muscle (2021) 11:2 https://doi.org/10.1186/s13395-020-00257-y RESEARCH Open Access Sarcoglycan A mutation in miniature dachshund dogs causes limb-girdle muscular dystrophy 2D James R. Mickelson1* , Katie M. Minor1, Ling T. Guo2, Steven G. Friedenberg3, Jonah N. Cullen3, Amanda Ciavarella4, Lydia E. Hambrook4, Karen M. Brenner5, Sarah E. Helmond6, Stanley L. Marks7 and G. Diane Shelton2 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, Genetics, Myopathy, Sarcoglycanopathy, Gene mutation Background discovery of their genetic loci. The sarcoglycan complex Muscular dystrophies are a heterogenous group of her- is part of the dystrophin-glycoprotein complex, com- editary degenerative myopathies with variable inherit- posed of four heavily glycosylated glycoproteins (α, β, γ, ance patterns. In people, the most common forms and δ-sarcoglycan) which help maintain sarcolemmal in- include the X-linked Duchenne and Becker muscular tegrity [3]. Disorders (also termed sarcoglycanopathies) dystrophies associated with mutations in the dystrophin resulting from mutated genes encoding the sarcoglycan- gene (DMD), and the limb-girdle muscular dystrophies sarcospan complex disrupt sarcolemmal integrity and (LGMD) associated with autosomal dominant (LGMD1) cause LGMD types 2C-2F, respectively. or autosomal recessive (LGMD2) mutations in several Mutations in the dystrophin gene (DMD) result in a genes [1, 2]. Further sub-classification of LGMDs is wide range of clinical severity in human patients, includ- based on an alphabetical system following the order of ing the milder Becker type MD (BMD) to the severe Du- chenne type MD (DMD). Similarly, patients with LGMD * Correspondence: [email protected] may have a wide range of clinical phenotypes including 1Department of Veterinary and Biomedical Sciences, College of Veterinary both the severe DMD and milder BMD types with onset Medicine, University of Minnesota, Saint Paul, MN 55113, USA from childhood to young adult. A phenotype with Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mickelson et al. Skeletal Muscle (2021) 11:2 Page 2 of 11 exercise intolerance, recurrent rhabdomyolysis, and MD for many years [20, 21]. We report here the discov- myoglobinuria has also been described [4, 5]. The serum ery of the first mutation in the canine sarcoglycan alpha creatine kinase (CK) activities are usually mildly to subunit (SGCA) gene in young adult miniature dachs- markedly elevated. Muscle biopsies from most sarcogly- hunds that leads to a form of LGMD with clinical signs canopathy patients show dystrophic features. Immunore- that include subclinical myopathy with hyperCKemia, activity for the mutated sarcoglycan subunit is often exercise intolerance, progressive weakness, variable dys- reduced or absent, but mutations in one sarcoglycan phagia and pneumonia, and myoglobinuria. often result in decreased or absence of the other sarco- glycans as well as dystrophin [6]. Thus, it is difficult to Methods reach a diagnosis of a specific sarcoglycanopathy based Animals on immunoreactivity alone. The initial diagnostic muscle biopsies or stained cryosec- Several well-described forms of muscular dystrophy tions from four young miniature dachshund dogs were are found in domestic dogs, with at least fifteen DMD submitted by veterinary specialists in Australia to the mutations found in over 10 breeds [7–18], as well as two Comparative Neuromuscular Laboratory, University of sarcoglycan delta subunit (SGCD) mutations [19], re- California San Diego. The veterinarians reported that all ported to cause dystrophic clinical phenotypes similar to four dogs had persistently elevated CK activities and a DMD, BMD, and LGMD, respectively, as described in myopathic clinical phenotype varying from generalized people. The DMD mutation responsible for X-linked weakness and exercise intolerance with myoglobinuria to muscular dystrophy in golden retriever dogs has been a pharyngeal dysphagia (Fig. 1). Additional familial infor- model for testing new therapeutics for Duchenne type mation revealed relationships in which one of the initial 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 Mickelson et al. Skeletal Muscle (2021) 11:2 Page 3 of 11 four affected dachshunds came from one small pedigree Western blotting was performed by standards methods (dog A1, Fig. 1), and three came from a large multi- using extracts from the triceps muscle of an affected dog generational pedigree with extensive interbreeding (dogs with a confirmed mutation in α-sarcoglycan (starred dog B1, B2, and B3, Fig. 1). in Fig. 1) or from archived cryopreserved canine control Further investigation found six additional affected dogs triceps muscle. Protein bands were separated using which could be placed in the pedigrees. One (dog A2) NuPage Bis-Tris 4–12% gradient gels (Invitrogen). Pri- was a sib to the original affected dog of pedigree A, mary antibodies included a rabbit polyclonal antibody while the five others (dogs B4, B5 B6, B7, and B8) could against α-sarcoglycan (1:2000; [25]) and monoclonal be placed in pedigree B, often as sibs to other affected antibodies against β-sarcoglycan (1:1000), γ-sarcoglycan dogs. Many parents and unaffected siblings were also (1:1000), and against β-actin (1:2000, Sigma A2066) as a identified. All parents were unaffected, and when tested, loading control. Secondary antibodies included CK activities from the parents and unaffected siblings peroxidase-conjugated goat anti-mouse IgG (1:20,000, were within the reference ranges. Jackson ImmunoResearch Lab, 115-035-062) and Muscle biopsies from two other dogs, originating in peroxidase-conjugated goat anti-rabbit IgG (1:20,000, California and South Africa, respectively, were submitted Thermo Scientific, 31460). Protein bands were detected to the Comparative Neuromuscular Laboratory. Both using Super Signal West Dura Extended Duration Sub- dogs also had markedly elevated CK activities, and clin- strate (Thermo Scientific). ical signs similar to the initial Australian dogs. Genomic data collection and analysis Eight cases and four unaffected dogs originating from Histopathology, immunofluorescence microscopy, and Australia, and the case
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