Background: Boys with Duchenne muscular dystrophy (DMD) have DMD gene mutations, with associated loss of the dystrophin protein and progressive muscle degeneration and weakness. Corticosteroids and palliative support are currently the best treatment options. The long-term benefits of recently approved compounds such as eteplirsen and ataluren remain to be seen. Dogs with naturally occurring dystrophinopathies show progressive disease akin to that of DMD. Accordingly, canine DMD models are useful for studies of pathogenesis and preclinical therapy development. A dystrophin-deficient, male border collie dog was evaluated at the age of 5 months for progressive muscle weakness and dysphagia. Case presentation: Dramatically increased serum creatine kinase levels (41,520 U/L; normal range 59–895 U/L) were seen on a biochemistry panel. Histopathologic changes characteristic of dystrophinopathy were seen. Dystrophin was absent in the skeletal muscle on immunofluorescence microscopy and western blot. Whole genome sequencing, polymerase chain reaction, and Sanger sequencing revealed a frameshift, single nucleotide deletion in canine DMD exon 20, position 27,626,466 (c.2841delT mRNA), resulting in a stop codon six nucleotides downstream. Semen was archived for future line perpetuation. Conclusions: This spontaneous canine dystrophinopathy occurred due to a novel mutation in the minor DMD mutation hotspot (between exons 2 through 20). Perpetuating this line could allow for preclinical testing of genetic therapies targeted to this area of the DMD gene. Keywords: Whole genome sequencing, Next-generation sequencing, DMD, Duchenne muscular dystrophy, Dystrophin, CXMD, Animal model, Canine Background throughout the 79 exons of the DMD gene but concen- Duchenne muscular dystrophy (DMD) is an X-linked, trate in major (exons 45–53) and minor (exons 2–20) hot- degenerative muscle disease that affects ~ 1 in 5000 spot areas . According to Leiden’s database , ~ 40% males caused by DMD gene mutations and a resulting of DMD gene mutations are deletions of a mean size of lack of the protein dystrophin . Dystrophin anchors 6.5 exons, with exon 47 being most commonly affected the sarcolemmal membrane by connecting cytoskeletal . Duplications occur most frequently in exon 20. actin filaments to an associated glycoprotein complex There are several naturally occurring mammalian DMD . Untreated DMD boys typically lose ambulation by models, including the X-linked muscular dystrophy mouse 12 years of age and succumb to cardiopulmonary failure (mdx) , canine X-linked muscular dystrophy (CXMD) by their twenties or thirties . Mutations may occur dogs [7–9], pigs , and cats . Dystrophin-deficient dogs have progressive disease that largely parallels the * Correspondence: firstname.lastname@example.org course of DMD [8, 12]. The golden retriever (GRMD) ca- Department of Veterinary Integrative Biosciences, College of Veterinary nine model has been used most extensively for preclinical Medicine and Biomedical Sciences, Texas A&M University, College Station, TX testing . In GRMD, a splice site mutation in intron 6 77843-4458, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Mata López et al. Skeletal Muscle (2018) 8:16 Page 2 of 6 causes deletion (skipping) of exon 7 in the DMD tran- Masticatory, lingual, paraspinal, supraspinatus, and script, with a resulting frameshift and premature stop cranial tibial (CT) muscles were examined with bipolar codon in exon 8 . Several additional DMD mutations, needle electromyography while the dog was under gen- including variably sized deletions and insertions, have eral anesthesia (isoflurane and oxygen). Complex repeti- been characterized in other dogs [13, 15, 16]. tive discharges were detected, most pronounced in the Together, studies in mammalian models have provided a lingual and proximal thoracic limb muscles. better understanding of DMD pathogenesis and allowed At 6 months, blood was taken for biochemical and gen- for preclinical testing to determine both safety and poten- omic analysis and surgical biopsies were performed on the tial efficacy of a range of treatments. However, with the vastus lateralis (VL) and CT muscles. Muscle samples advent of gene replacement, exon skipping, and gene edit- were placed on a wooden tongue depressor, wrapped in a ing approaches that allow treatment of specific mutations, sterile saline-soaked gauze pad, and shipped overnight on additional large animal mammalian models with DMD a cold pack to Texas A&M University (laboratory of gene mutations paralleling those of DMD are needed. PPN). The samples were immediately frozen in liquid nitrogen-chilled isopentane and stored at − 80 °C. Case presentation As detailed above, no pedigree information was available A 5-month-old, male border collie dog was presented in on this dog. Hence, there were no carriers from which the September 2016 to a practicing veterinarian for clinical condition could be perpetuated through breeding. With signs consistent with neuromuscular disease. The owner this in mind, semen from the dog was collected at a re- had obtained the dog from a breeder and did not have check appointment and frozen in liquid nitrogen for fu- knowledge of his littermates or the sire and dam. He ture line perpetuation. By inseminating a normal dog, all was subsequently referred to a board-certified veterinary female progeny would be obligate carriers. neurologist (JJH) for further evaluation. Multiple at- Blood chemistry results showed elevated levels of AST, tempts by JJH to contact the breeder for more pedigree ALT, BUN/creatinine ratio, phosphorus, glucose, and information were unsuccessful. notably, creatine kinase (CK 41,520 U/L; Table 1). On examination, fatigue and a short-strided gait were Platelets were mildly elevated. Remaining blood count observed (Fig. 1). Postural reactions were normal when and chemistry values were within normal range. No anti- the dog’s body was supported. Muscle tone and spinal bodies were detected against Toxoplasma gondii or reflexes were normal, but generalized muscle atrophy Neospora caninum. was observed, most prominent in the distal limb mus- Tissue cryosections of the VL and CT (not shown) mus- culature. Muscles of the proximal thoracic limbs and at cles were stained with hematoxylin and eosin (H&E)  the base of the tongue were prominent. Cranial nerve and analyzed by light microscopy. There was myofiber size evaluation was normal. Drooling was reported by the variation, hyaline myofiber necrosis, increased primarily owners historically and was present during the exam. endomysial connective tissue, and increased mononuclear Neuroanatomical localization was consistent with a cells likely representing a mix of inflammatory cells and generalized neuromuscular disorder. activated satellite cells (Fig. 2b) (see further below). Immunofluorescence microscopy was performed on VL and CT samples. Cryosections co-stained using dystrophin rod (NCL-Dys 1 Leica) and C-terminus (NCL-Dys 2 Leica) domain antibodies at 1:100 dilution Table 1 Blood chemistry results for the affected border collie showed muscle-specific changes Lab finding Values Normal range AST (SGOT) (U/L) 671 15–66 ALT (SGPT) (U/L) 446 12–118 Creatinine (mg/dL) 0.4 0.5–1.6 BUN/creatinine ratio 40 4–27 Phosphorus (mg/dL) 7.6 2.5–6.0 Glucose (mg/dL) 149 70–138 Fig. 1 Postural changes. a At the age of 1.5 years, the dog had a Creatine kinase (U/L) 41,520 59–895 palmigrade and plantigrade stance in all limbs and the pelvis was shifted in a cranioventral direction Platelet count (10 /μL) 489 170–400 Mata López et al. Skeletal Muscle (2018) 8:16 Page 3 of 6 Fig. 2 Histopathological changes consistent with dystrophinopathy. a Normal dog at 6 months of age showing uniform fiber size and minimal endomysial connective tissue. b Affected border collie vastus lateralis muscle with dystrophic changes, including myofiber size variation owing partly to larger hyaline fibers (*), increased cellularity likely due to combined effects of inflammation and satellite cell activation (#), and increased connective tissue (+). Hematoxylin and eosin (H&E). Metric bar = 100 μm in both and goat anti-mouse Alexa Fluor 488 secondary anti- in some fibers with central nuclei in the border collie, but body (Life Technologies) at a 1:500 dilution were ana- almost undetectable in normal canine tissue (Fig. 3b, g, l). lyzed. Utrophin was stained with a primary antibody Cryosections from the affected dog stained for sarcospan (Developmental Studies Hybridoma Bank) at 3.5 μg/mL with a primary antibody (Origene) at 1:250 and Alexa 488 with the aforementioned secondary antibody. Dystrophin goat anti-rabbit (Life Technologies) at 1:500 (Fig. 3c, g) protein was absent on immunofluorescence microscopy showed increased expression, probably associated with (Fig. 3a, f, k) compared with a normal sample. Revertant utrophin upregulation . Spectrin (Abcam) at 1:100 as fibers were not observed. Utrophin staining was positive together with the same secondary mentioned above Fig. 3 Dystrophin deficiency in the affected border collie (BC) dog. Normal and dystrophic muscle were immunostained for DYS1 and 2 (a, f, k). Peri-membranous dystrophin expression was seen in each myofiber of normal muscle (a) but was absent in the affected dog (f, k). Utrophin (UTRN) was minimally expressed in normal muscle (b) but, by comparison, was increased in the affected dog (g, l). Similarly, sarcospan (SSPN) was minimally expressed in normal muscle (c) and comparably increased in the affected dog (h). Spectrin (SPTBN) was used as a cellular membrane marker (d, i). Myosin heavy chain developmental fibers (MHCd) positive myofibers were absent in normal muscle (e) but present in the affected dog (j). Nuclei were stained with DAPI. All images were taken with a × 20 objective. m Western blot showed absent dystrophin in the BC; GAPDH was used as a loading control. Metric bar = 100 μm Mata López et al. Skeletal Muscle (2018) 8:16 Page 4 of 6 (Fig. 3d, i) was used as a cellular membrane control. Mul- to a normal dog (Fig. 4c, d). Outside and inside forward tiple inflammatory cell markers were assessed with im- and reverse primers were designed to encompass the gen- munofluorescence, and no definite positive cells were omic DNA region containing the deletion identified by seen. Some myofibers in the dystrophic dog stained posi- WGS (Additional file 1 Table S1). Primary PCR was per- tive for myosin heavy chain developmental fibers (MHCd) formed using the outside primers and TaKaRa Ex Taq antibody (Leica) (Fig. 3e, j) at 1:100 and 1:500 Alexa 488 Polymerase Kit under the following conditions: 94 °C for goat anti-mouse (Life Technologies) antibody, consistent 1min;94°Cfor 30 s, 48.4 °C for 30 s,72 °Cfor with satellite cell activation. All slides were co-stained with 1 min (30 times); and 75 °C for 5 min. The product of this DAPI (Invitrogen) at 1:2000. reaction was used for secondary PCR with inside forward Western blotting methods have been described previ- and reverse primers designed (Additional file 1 Table S1). ously . NCL-Dys1NCL-Dys2antibodiesat1:200 dilu- The T7 sequence (TAATACGACTCACTATAG) was in- tion and goat anti-mouse IgG HRP (ABCam) were cluded on the 5′ end of the inside forward primer for incubated at a 1:5000 dilution. GAPDH was used as a load- Sanger sequencing. Secondary PCR was performed under ing control (Santa Cruz Technologies) after stripping the the following conditions: 94 °C for 1 min; 94 °C for 30 s; membrane (Thermo Fisher). Dystrophin protein was absent 45.3 °C for 30 s, 72 °C for 1 min (30 times); and 75 °C for on immunostaining analysis of muscle lysates (Fig. 3m). 5 min. Gel electrophoresis (1.3% agarose) was used to de- Genomic DNA was extracted from the blood using a termine the quality of PCR products. The desired band Qiagen DNA extraction kit (QIAamp DNA Blood Mini (223 bp) was excised from the gel and the DNA purified Kit, QIAGEN) following methods provided by the (QIAEX II Gel Extraction Kit Qiagen). Purified secondary manufacturer. Subsequent molecular characterization of PCR product was submitted for Sanger sequencing (Eton the underlying DMD gene mutation was performed Bioscience; Texas A&M University). using whole genome sequencing (WGS) with methods In addition to this novel deletion in DMD exon 20, previously described . National Center for Biotech- two additional non-synonymous substitutions were iden- nology Information’s (NCBI) Genome Workbench soft- tified in DMD exons 15 (position 27,697,781; serine ware was used for data analysis. Single nucleotide AGC to asparagine AAC) and 34 (position 27,512,289; polymorphisms (SNPs), deletions, and insertions in the alanine GCG to serine TGC). Using the Ensembl data- DMD gene were compared to the CanFam3.1 whole base , there was also a T deletion at position genome shotgun sequence . Subsequent analysis of 26,290,826 in the untranslated region of exon 79. In con- this dog’s deleted base pair (bp) was performed with the trast, when the NCBI database was used, this deletion Leiden DMD database . fell outside the untranslated region of exon 79. Finally, For comparison purposes, the reference genome the previously published GRMD “escaper” single nucleo- length for the canine DMD gene is 2,392,715,236 (NCBI tide substitution in the gene Jag1  was not present. CanFam3.1)  with mapped reads for the affected border collie at 2,386,159,041 (99.73% of reference gen- ome). There was a mean depth read of 31X. The total Discussion and conclusions number of reads mapped to the reference genome This study describes a novel DMD gene mutation in a (608,164,144) was 573,083,874 (94.23%). There were border collie dog that could potentially be a valuable 6,072,297 (1% of reference genome) variants composed preclinical model. While this dog had several DMD gene of 612,599 deletions (10% of variants), 655,520 insertions mutations, we believe the T nucleotide deletion in exon (11% of variants), and 4,804,178 SNPs (79% of variants). 20 most likely led to the loss of dystrophin. Located in The overall genomic GC content was 41.75%. There the exon 2–20 minor hotspot for the DMD gene , this were 2531 variants within the DMD gene (X chromo- mutation would result in a stop codon 6 bp downstream some, NC_006621.3; 26,290,903…28,444,730 NCBI), . The other mutations in exons 15 and 34 were which was relatively higher than previously reported in a non-synonymous substitutions, expected to change the Cavalier King Charles spaniel dog with WGS . amino acid but not disrupt the reading frame. Exon 20 WGS revealed a 1-bp nucleotide (T) deletion in pos- is most frequently duplicated in both Becker’s muscular ition 27,626,466 (c.2841delT mRNA) in exon 20 of the dystrophy and DMD but can also be deleted with other canine DMD gene (Fig. 4a), corresponding to position exons. In Leiden’s database, exon 20 deletions have been 36,636,833 (c.2552delT mRNA) in exon 20 of the human reported in 27 cases of both DMD and Becker patients, DMD gene. According to the Leiden DMD database , having an incidence of 0.08% (total of 2432 BMD/DMD this nucleotide deletion would result in a stop codon six patients). Notably, even though this dog was alive at nucleotides downstream from the deletion site (Fig. 4b). 22 months and had a relatively mild phenotype, it did Polymerase chain reaction (PCR) and Sanger sequencing not have the “escaper mutation” in the Jag1 gene de- confirmed the single nucleotide deletion when compared scribed by Vieira et al. . Our laboratory has recently Mata López et al. Skeletal Muscle (2018) 8:16 Page 5 of 6 Fig. 4 Whole genome sequencing revealed a point mutation (1 base pair deletion) in exon 20 of the canine DMD gene. a Screen shot of NCBI Genome Workbench revealing 22 reads with the point mutation (nucleotide A; black rectangle). b Screen shot of Leiden DMD database with the deleted nucleotide highlighted in blue (red arrow). A stop codon (TGA) present six nucleotides downstream in exon 20 (red line). c Sanger sequencing screen shot of the mutated area (black arrow) with the reverse strain ACT stop codon six nucleotides downstream (black line). d Sanger sequencing screen shot of a normal dog in the same area. Black arrow points at the normal (non-mutated) sequence published on a large cohort of variably affected GRMD semen was collected to allow perpetuation of the line dogs without the Jag1 mutation . myoblasts which were extracted for future Blood values and histopathological changes in this immortalization. border collie were consistent with those of other Over and above the potential preclinical value of this new dystrophin-deficient dogs [7, 25]. He was seen in the clinic model, our work further demonstrates the value of WGS as at 5 months with signs of muscle atrophy, macroglossia, a tool to characterize canine DMD gene mutations . fatigue during ambulation, drooling, and “bunny hopping” Whole genome and exome sequencing provide valuable gait. At the time of this study, the dog continued to live techniques to detect mutations ranging from a single bp to with his owners. His clinical signs had largely stabilized, in multi-exon deletions. We have previously utilized WGS to keeping with mildly affected dystrophic dogs seen in our identify a7-basepairmutationin DMD exon 42 of a own lab [7, 13, 24] and by others [8, 9, 19, 25]. Cavalier King Charles spaniel (CKCS) dog , distinct Duetothe rangeof DMD gene mutations observed from the splice site mutation reported earlier by Walmsley in patients, this new potential canine model could be et al. . A recent study utilized whole exome sequencing useful in testing genetic therapies. Novel exon skipping to identify two distinct mutations in the sarcoglycan-δ compounds, whereby exons 19 and 20 or 20 and 21 are (SGCD) gene of Boston terrier dogs with a condition akin skipped to restore the reading frame, could potentially to limb girdle muscular dystrophy of humans . be tested. In addition, techniques such as TALEN or In conclusion, WGS was used to characterize a novel CRISPR/Cas9 could be utilized for single bp restoration single bp deletion in exon 20 of the canine DMD gene. (i.e., homology directed repair) or exon "snipping" (i.e. These studies provide another example of the power of non-homologous end joining). With this in mind, the next-generation sequencing technology in the diagnosis Mata López et al. Skeletal Muscle (2018) 8:16 Page 6 of 6 of genetic animal diseases. If perpetuated, this 3. Yiu EM, Kornberg AJ. Duchenne muscular dystrophy. J Paediatr Child Health. 2015;51(8):759–64. condition could serve as a valuable model for testing 4. Flanigan KM, et al. Mutational spectrum of DMD mutations in genetic therapies. dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009;30(12):1657–66. 5. White SJ, den Dunnen JT. Copy number variation in the genome; the human Additional file DMD gene as an example. Cytogenet Genome Res. 2006;115(3–4):240–6. 6. Bulfield G, et al. X chromosome-linked muscular dystrophy (mdx) in the Additional file 1: Table S1. Nested PCR primers. (DOCX 14 kb) mouse. Proc Natl Acad Sci U S A. 1984;81(4):1189–92. 7. Kornegay JN, et al. Muscular dystrophy in a litter of golden retriever dogs. Muscle Nerve. 1988;11(10):1056–64. Abbreviations 8. Walmsley GL, et al. A duchenne muscular dystrophy gene hot spot Bp: Base pair; CKCS: Cavalier King Charles Spaniel; CT: Cranial tibialis; mutation in dystrophin-deficient cavalier king charles spaniels is amenable CXMD: Canine X-linked muscular dystrophy; DMD: Duchenne muscular to exon 51 skipping. PLoS One. 2010;5(1):e8647. dystrophy; GDNA: Genomic DNA; GRMD: Golden retriever muscular 9. Smith BF, K.J. Independent canine models of Duchenne muscular dystrophy dystrophy; H&E: Hematoxylin and eosin; Mdx: Murine X-linked muscular dys- due to intronic insertions of repetitive DNA. Mol Ther. 2007;15:S51. trophy; MHCd: Myosin heavy chain developmental; NCBI: National Center for 10. Selsby JT, et al. Porcine models of muscular dystrophy. ILAR J. 2015;56(1): Biotechnology Information; PCR: Polymerase chain reaction; 116–26. SGCD: Sarcoglycan-δ; SNPs: Single nucleotide polymorphism; VL: Vastus 11. Carpenter JL, et al. Feline muscular dystrophy with dystrophin deficiency. lateralis; WES: Whole exome sequencing; WGS: Whole genome sequencing Am J Pathol. 1989;135(5):909–19. 12. Kornegay JN, et al. Canine models of Duchenne muscular dystrophy and Acknowledgements their use in therapeutic strategies. Mamm Genome. 2012;23(1–2):85–108. We would like to thank Dr. Ann Huntington and Kathy Loughman for collecting 13. Kornegay JN. The golden retriever model of Duchenne muscular dystrophy. the samples at Suffield Veterinary Hospital in Suffield, CT and Dr. Jean Kucia Skelet Muscle. 2017;7(1):9. from the Fox Memorial Clinic in Newington, CT for primary care of the dog. 14. Sharp NJ, et al. An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular Funding dystrophy. Genomics. 1992;13(1):115–21. All studies performed at Texas A&M were fully funded by a start-up package 15. Jeandel A, et al. Late-onset Becker-type muscular dystrophy in a Border to PPN from the Department of Veterinary Integrative Biosciences at Texas terrier dog. J Small Anim Pract. 2018. https://doi.org/10.1111/jsap.12824. A&M University, College of Veterinary Medicine and Biomedical Sciences. 16. McAtee BB, et al. Dysphagia and esophageal dysfunction due to dystrophin deficient muscular dystrophy in a male Spanish water spaniel. Vet Q. 2018; Availability of data and materials 38(1):28–32. The data generated from the WGS is available from the corresponding 17. Nghiem PP, et al. Sparing of the dystrophin-deficient cranial sartorius author on reasonable request. muscle is associated with classical and novel hypertrophy pathways in GRMD dogs. Am J Pathol. 2013;183(5):1411–24. Authors’ contributions 18. Marshall JL, Crosbie-Watson RH. Sarcospan: a small protein with large SML generated, analyzed, and interpreted most of the data and drafted the potential for Duchenne muscular dystrophy. Skelet Muscle. 2013;3(1):1. manuscript. JJH assessed the dog clinically and provided the muscle samples 19. Nghiem PP, et al. Whole genome sequencing reveals a 7 base-pair deletion for characterization. MBR completed the nested PCR reactions and sequencing in DMD exon 42 in a dog with muscular dystrophy. Mamm Genome. 2017; data. CBA and JNK provided guidance on the project and experiments. PPN 28(3–4):106–13. provided funding and overall direction for the project. All authors read and 20. Hoeppner MP, et al. An improved canine genome and a comprehensive catalogue approved the final manuscript. of coding genes and non-coding transcripts. PLoS One. 2014;9(3):e91172. 21. Aken BL, et al. The Ensembl gene annotation system. Database (Oxford). Ethics approval and consent to participate 2016;2016:baw093. The owners of the animal consented to provide tissue for the analysis at the 22. Vieira NM, et al. Jagged 1 rescues the Duchenne muscular dystrophy Texas A&M muscular dystrophy laboratory. phenotype. Cell. 2015;163(5):1204–13. 23. Spitali P, et al. DMD transcript imbalance determines dystrophin levels. Competing interests FASEB J. 2013;27(12):4909–16. PPN is a scientific consultant for Agada Biosciences. SML is in a PhD graduate 24. Nghiem PP, et al. Changes in muscle metabolism are associated with program at Texas A&M University under the mentorship of Dr. Joe Kornegay. Her phenotypic variability in golden retriever muscular dystrophy. Yale J Biol position is funded by SOLID Biosciences. The other authors declare that they have Med. 2017;90(3):351–60. no competing interests. 25. Schatzberg SJ, et al. Molecular analysis of a spontaneous dystrophin ‘knockout’ dog. Neuromuscul Disord. 1999;9(5):289–95. 26. Cox ML, et al. Exome sequencing reveals independent SGCD deletions causing Publisher’sNote limb girdle muscular dystrophy in Boston terriers. Skelet Muscle. 2017;7:15. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4458, USA. Department of Neurology and Neurosurgery, Pieper Memorial Veterinary Center, Middletown, CT 06457, USA. Received: 15 March 2018 Accepted: 15 May 2018 References 1. Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987;51(6):919–28. 2. Ehmsen J, Poon E, Davies K. The dystrophin-associated protein complex. J Cell Sci. 2002;115(Pt 14):2801–3.
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Published: May 29, 2018
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