Widespread severe myodegeneration in a compound heterozygote female dog with dystrophin deficiency

Abstract The University of Missouri (MU) has established a colony of dystrophin‐deficient dogs with a mixed breed background to mirror the variable pathologic effects of dystrophinopathies between persons of a given kindred to further the understanding of the genetic and molecular basis of the variable phenotype; thus to facilitate discovery of an effective therapeutic strategy. Herein we report the phenotype and genotype of a normal‐appearing 10‐month‐old colony female that died suddenly. At necropsy examination, there were reduced skeletal and laryngeal muscle volume and mild dilatation of the oesophagus. Microscopic findings consisted of extensive degeneration and regeneration of the axial skeletal, tongue, oesophageal, and laryngeal muscles that were characterized by considerable central nucleation, individual fibre mineralization and interstitial fibrosis. The myocardial findings were limited to infiltration of adipose cells in the interstitium. The female dog was a compound heterozygote with one X chromosome carrying a point mutation in intron 6 of the dystrophin gene and the other X chromosome carrying a repetitive element insertion in intron 13 of the dystrophin gene. Although the direct cause of death was uncertain, it might likely be due to sudden cardiac death as has been seen in Duchenne muscular dystrophy patients. This case demonstrated dystrophinopathy in female dogs that have no ameliorating normal X chromosome.


| INTRODUC TI ON
Muscular dystrophies are heritable degenerative diseases typically identified early in life (Guiraud et al., 2015). The most frequently reported muscle dystrophy occurs because of mutations in the dystrophin gene, a large gene on the X chromosome linked with dystrophin synthesis. Mutations resulting in dystrophin deficiency are denoted as Duchenne muscular dystrophy (DMD) (Guiraud et al., 2015). This muscular dystrophy typically affects males, and females serve as carriers when heterozygous for the mutation. Dystrophin normally localizes at the cytosolic side of the striated muscle sarcolemma protecting the sarcolemma from contraction-associated shear stress. An absence of dystrophin compromises cell integrity resulting in striated muscle cell injury. DMD occurs at an approximate world-wide incidence of one in 5,000 human male births (Hoffman et al., 1987;Stark, 2015). However, in DMD males, premature death ensues (Kornegay, 2017). Steroid drugs such as prednisolone and deflazacort, provide symptomatic relief but are not curative, thus DMD represents a disease with poorly met medical need.
Muscular dystrophies have been reported through case reports in numerous breeds of dogs including golden retrievers, Labrador retrievers, Rottweilers, Cavalier King Charles spaniels, French bulldogs, beagles, weimaraners, Samoyeds, miniature schnauzers, old English sheepdogs, Pembroke Welsh corgis and others (Kornegay, 2017;Smith et al., 2011). The most extensively studied muscular dystrophy in veterinary medicine involves dystrophin synthesis deficiency in the golden retriever, with several institutional colonies dedicated to basic research (McGreevy et al., 2015;Shin et al., 2013). In addition, important research colonies also involve Labrador retrievers, beagles and Pembroke Welsh corgis (McGreevy et al., 2015;Smith et al., 2011;Valentine et al., 1988). A driving factor for research funding reflects the surprisingly close homology of the golden retriever muscle dystrophy (GRMD) with Duchenne muscle dystrophy in humans (Smith et al., 2011). An in-depth comparison of the human, mouse and dog dystrophin-deficient phenotypes have been published by McGreevy et al., (2015).
Dystrophin gene replacement and repair therapies represent exciting avenues. These types of therapies will not replace the muscle that has already been degraded from significant bouts of degeneration, but will only lessen the severity and progression to a Becker-like phenotype. Requisite for such approaches will be the identification of the genetic and molecular basis for the DMD phenotype. The most widely used DMD research model is the mdx mouse, but this model does not exhibit good clinical homology to human patients.
To further the understanding of the genetic and molecular basis for the DMD phenotypes at the University of Missouri, one of the approaches involves outcrossing dogs with dystrophin deficiency in various breeds. The dystrophin mutation of golden retrievers was initially outcrossed onto a Labrador retriever background. Additional outcrossing to other breeds such as Pembroke Welsh corgi and beagle has produced a relatively outbred colony for investigation (Miyazato et al., 2011;Smith et al., 2011). In this quest, a sudden death occurred in a female compound heterozygote. This female was extensively characterized phenotypically and genotypically, as reported herein.

| C A S E HIS TORY
A 10-month-old mixed breed female dog in a muscular dystrophy research colony at the University of Missouri died suddenly without premonitory symptoms. This female was derived through the artificial insemination mating of a carrier female and an affected male.
The genotype of the sire is WY. W refers to frame-shifting mutation in the dystrophin gene due to insertion of a long interspersed repetitive element-1 (LINE-1) in intron 13, as reported previously (Smith et al., 2011). The genotype of the dam is XG. G refers to out-of-frame mutation in the dystrophin gene due to a point mutation in intron 6 as defined by Cooper et al . The creatine kinase levels of the female dog were 322,635 U/L and 84,456 U/L at the age of 1.9 and 6.1 months, respectively.

| RE SULTS
At necropsy, axial skeletal muscles were slightly pale and reduced in volume. All skeletal muscles were pale and reduced in volume. Pharyngeal and laryngeal muscles were atrophic. The oesophagus was dilated and flaccid. The lungs were mottled, viscera were congested and the spleen was contracted. Tissue samples were collected into 10% neutral buffered formalin and submitted for processing into paraffin blocks.
Tissues in blocks were cut in 5-micron sections and stained with haematoxylin and eosin (HE). Special stains and immunohistochemistry for dystrophin demonstration were applied on selected tissues. Genotype examination methodology was performed as previously published (Smith et al., 2011) and the PCR/gel is included in Pembroke Welsh Corgis with DMD, respectively (Kornegay, 2017;Smith et al., 2011). In this dog, both alleles are mutated at different gene loci, producing a compound heterozygote.

| D ISCUSS I ON
In humans, DMD is an X-linked recessive mutation in the gene coding for the protein dystrophin. Symptoms develop in boys around the age of two to five years. Most are unable to walk by age 12. The main symptom is voluntary muscle weakness and wasting. Other symptoms include awkward walking, frequent falls, fatigue, poor motor skills, lumbar hyperlordosis and muscle contractures. The average life expectancy is 26 years, with death resulting from cardiorespiratory failure. Well-managed patients may live into the third or fourth decade of life. Steroid therapy provides symptomatic benefits but is not curative. The mother of the patient may be normal or serve as a carrier. Mothers may show mild symptoms but are often clinically normal. Although 70% of female carriers [people] have histologic abnormalities in skeletal muscle, only 5%-10% manifest mild/moderate correlative symptoms (Dubowitz, 1982). However, sudden death in female carriers has been reported as a consequence of myocardial dysfunctions (Grain et al., 2001;Hoogerwaard et al., 1999;Politano et al., 1996;Schade van Westrum et al., 2011) and histopathological lesions (Kane et al., 2013;Moise et al., 1991;Takano et al., 2011;Yugeta et al., 2006) are present.
The pathologic effects of dystrophinopathies vary between persons of a given kindred; thus the purpose of using cross-bred animals is to achieve comparable variability in clinical presentations. The University of Missouri has established a colony of dystrophin-deficient dogs in a mixed breed background to further the understanding of the genetic and molecular basis for the DMD phenotypes, as an approach leading to the development of a gene replacement therapy or CRISPR gene editing therapy. Here we reported the sudden death in a relatively normal-appearing 10-month-old female mixed breed dog from the colony, that had severe and widespread striated muscle myodegeneration characteristic of DMD. Immunohistochemically, we confirmed the lack of dystrophin in muscle. In this case, the female dog had different mutations at different gene loci on each X chromosome. Double mutation indeed happens in humans. Several different cases have been reported. There is one patient with compound mutation (one X chromosome has a deletion of exons 8-13 and the other X chromosome has a splice site mutation (c.10086 + 2T>C) (Soltanzadeh et al., 2010). Another interesting case was reported from a patient who carries the same mutation in both X chromosomes (homozygous for exon 44-45 deletion) (Fujii et al., 2009). Dystrophin labelling of muscle biopsies from heterozygous female carriers of DMD mutations who develop symptoms (manifesting carriers) shows a mosaic pattern, i.e. a nonuniform staining of fibres which represent normal and abnormal fibres (Soltanzadeh et al., 2010).
A mosaic pattern was not observed in the study case presented herein. It is possible that the antibody used to detect dystrophin in the muscle histological section may not recognize the truncated dystrophin protein.
Clinical laboratory findings in cDMD male dogs are limb weakness and exercise intolerance that appear around 2-3 months of age . At approximately 6 months of age, muscle atrophy, joint contracture, hypersalivation, dysphagia, abnormal gait and signs of impaired heart function appear (Bergman et al., 2002;Kornegay, 2017). Death occurs most frequently around 1-3 years of age (McGreevy et al., 2015;Vieira et al., 2015). In contrast to DMD in humans, 20%-30% of cDMD dogs die within 2 weeks of birth. The cDMD dogs also show variation in symptoms. For example, cDMD dogs may be asymptomatic despite absence of dystrophin in striated muscle (McGreevy et al., 2015).
Heterozygous cDMD female dog carriers have been reported to have electrocardiographic abnormalities (Moise et al., 1991;Takano et al., 2011;Yugeta et al., 2006). Sudden deaths have also been reported Sharp et al., 1992;Valentine et al., 1992). With Holter monitoring, 10 of 11 carriers were reported to exhibit ventricular ectopy, even though baseline electrocardiograms (EKGs) were normal, and each had myocardial lesions identified post mortem (Kane et al., 2013). In controlled experiments effects on growth occur as early as 5 days of age (Smith et al., 2011). In this case, electrocardiography was not assessed.
In clinical laboratory assessments of cDMD dogs, creatine kinase levels are consistently elevated whether symptomatic or not (Bergman et al., 2002;Cooper et al., 1988). The creatine kinase levels were elevated in the mixed breed female dog described in this report. The reduction in the CK level at later age (6.1 months versus 1.9 months herein) is due to the loss of muscle. Due to the disease, significant amount of muscle tissue has been replaced by fat or fibrotic tissues. Hence the total CK level was reduced (Hathout et al., 2015).
The consistent alterations found with routine light microscopy preparations of cDMD dog striated muscle include myofibre F I G U R E 4 PCR/gel electrophoresis of this study case genotype which exhibits the golden retriever muscular dystrophy (GRMD) and Pembroke Welsh Corgis (WCMD) mutations (GW)  (Shiga et al., 2017). These muscle histologic changes are not specific for, but are typical of cDMD dogs. Lesions are severe in males but mild in carrier females, except in the compound heterozygotes, as presented here. Immunohistochemical staining for dystrophin is negative in males and negative in a mosaic pattern in carrier females. IHC for utrophin in sarcolemma is increased in affected dogs (Smith et al., 2011). Utrophin is a membrane protein that compensates for lack of dystrophin (Miyazato et al., 2011). In the heart, fatty infiltration, myofibre hypereosinophilia/fragmentation/vacuolation/shrinkage, myocardial fibrosis, myofiber mineralization, fatty infiltration, and multifocal inflammatory infiltrates are reported to occur. Dystrophin IHC on heart tissues reflects absence in affected males and mosaic areas of positivity in carrier females (Kane et al., 2013;Smith et al., 2011).
Although the cause of death in this animal was not found, we suspect it may very likely be due to ventricular arrhythmia. The female dog that was a compound heterozygote (of the X-linked dystrophin gene) with widespread myodegeneration. This case demonstrated that more severe pathology can occur in female dogs that have no ameliorating normal X chromosome.

ACK N OWLED G EM ENT
The authors wish to acknowledge Susan Helming and Candace Kassel (VDL) for histologic technical support, Keqing Zhang for excellent technical assistance and Dr. Christina J. Ramirez (Paw Print Genetics) for confirmation of mutations identified previously by Dr. Duan.

CO N FLI C T O F I NTE R E S T
DD is a member of the scientific advisory board for Solid Biosciences LLC and an equity holder of Solid Biosciences LLC. The other authors declare no conflict of interest with respect to the publication of this manuscript.

E TH I C A L S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethics approval was required as this is an investigation of an animal at post-mortem examination.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.433.