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1.  X-linked myotubular myopathy in Rottweiler dogs is caused by a missense mutation in Exon 11 of the MTM1 gene 
Skeletal Muscle  2015;5(1):1.
Congenital and inherited myopathies in dogs are faithful models of human muscle diseases and are being recognized with increasing frequency. In fact, canine models of dystrophin deficient muscular dystrophy and X-linked myotubular myopathy are of tremendous value in the translation of new and promising therapies for the treatment of these diseases. We have recently identified a family of Australian Rottweilers in which male puppies were clinically affected with severe muscle weakness and atrophy that resulted in early euthanasia or death. X-linked myotubular myopathy was suspected based on the early and severe clinical presentation and histopathological changes within muscle biopsies. The aim of this study was to determine the genetic basis for myopathy in these dogs and compare and contrast the clinical presentation, histopathology, ultrastructure, and mutation in this family of Rottweiler dogs with the previously described myotubular myopathy in Labrador retrievers.
Histopathology, histochemistry, and ultrastructural examination of muscle biopsies from affected Rottweiler puppies were consistent with an X-linked myotubular myopathy. An unusual finding that differed from the previously reported Labradors and similar human cases was the presence of excessive autophagy and prominent autophagic vacuoles. Molecular investigations confirmed a missense mutation in exon 11 of MTM1 that was predicted to result in a non-functional phosphatase activity. Although the clinical presentations and histopathology were similar, the MTM1 p.(Q384P) mutation is different from the p.(N155K) mutation in exon 7 affecting Labrador retrievers with X-linked myotubular myopathy.
Here we describe a second pathogenic mutation in MTM1 causing X-linked myotubular myopathy in dogs. Our findings suggest a variety of MTM1 mutations in dogs as seen in human patients. The number of MTM1 mutations resulting in similar severe and progressive clinical myopathy and histopathological changes are likely to increase as canine myopathies are further characterized.
PMCID: PMC4320619  PMID: 25664165
Congenital myopathy; Myotubularin; Canine myopathy; Animal model
2.  Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice 
Human Molecular Genetics  2013;23(1):209-225.
Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery–Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations.
PMCID: PMC3916749  PMID: 23975679
3.  A Novel Mutation in CLCN1 Associated with Feline Myotonia Congenita 
PLoS ONE  2014;9(10):e109926.
Myotonia congenita (MC) is a skeletal muscle channelopathy characterized by inability of the muscle to relax following voluntary contraction. Worldwide population prevalence in humans is 1∶100,000. Studies in mice, dogs, humans and goats confirmed myotonia associated with functional defects in chloride channels and mutations in a skeletal muscle chloride channel (CLCN1). CLCN1 encodes for the most abundant chloride channel in the skeletal muscle cell membrane. Five random bred cats from Winnipeg, Canada with MC were examined. All cats had a protruding tongue, limited range of jaw motion and drooling with prominent neck and proximal limb musculature. All cats had blepharospasm upon palpebral reflex testing and a short-strided gait. Electromyograms demonstrated myotonic discharges at a mean frequency of 300 Hz resembling the sound of a ‘swarm of bees’. Muscle histopathology showed hypertrophy of all fiber types. Direct sequencing of CLCN1 revealed a mutation disrupting a donor splice site downstream of exon 16 in only the affected cats. In vitro translation of the mutated protein predicted a premature truncation and partial lack of the highly conserved CBS1 (cystathionine β-synthase) domain critical for ion transport activity and one dimerization domain pivotal in channel formation. Genetic screening of the Winnipeg random bred population of the cats' origin identified carriers of the mutation. A genetic test for population screening is now available and carrier cats from the feral population can be identified.
PMCID: PMC4214686  PMID: 25356766
4.  An ARHGEF10 Deletion Is Highly Associated with a Juvenile-Onset Inherited Polyneuropathy in Leonberger and Saint Bernard Dogs 
PLoS Genetics  2014;10(10):e1004635.
An inherited polyneuropathy (PN) observed in Leonberger dogs has clinical similarities to a genetically heterogeneous group of peripheral neuropathies termed Charcot-Marie-Tooth (CMT) disease in humans. The Leonberger disorder is a severe, juvenile-onset, chronic, progressive, and mixed PN, characterized by exercise intolerance, gait abnormalities and muscle atrophy of the pelvic limbs, as well as inspiratory stridor and dyspnea. We mapped a PN locus in Leonbergers to a 250 kb region on canine chromosome 16 (Praw = 1.16×10−10, Pgenome, corrected = 0.006) utilizing a high-density SNP array. Within this interval is the ARHGEF10 gene, a member of the rho family of GTPases known to be involved in neuronal growth and axonal migration, and implicated in human hypomyelination. ARHGEF10 sequencing identified a 10 bp deletion in affected dogs that removes four nucleotides from the 3′-end of exon 17 and six nucleotides from the 5′-end of intron 17 (c.1955_1958+6delCACGGTGAGC). This eliminates the 3′-splice junction of exon 17, creates an alternate splice site immediately downstream in which the processed mRNA contains a frame shift, and generates a premature stop codon predicted to truncate approximately 50% of the protein. Homozygosity for the deletion was highly associated with the severe juvenile-onset PN phenotype in both Leonberger and Saint Bernard dogs. The overall clinical picture of PN in these breeds, and the effects of sex and heterozygosity of the ARHGEF10 deletion, are less clear due to the likely presence of other forms of PN with variable ages of onset and severity of clinical signs. This is the first documented severe polyneuropathy associated with a mutation in ARHGEF10 in any species.
Author Summary
Leonberger dogs are a breed originally produced by crossing large-bodied dogs, including Saint Bernards and Newfoundlands. A peripheral neuropathy has been described in Leonbergers that is similar to a group of inherited polyneuropathies known as Charcot-Marie-Tooth disease in humans. We collected a cohort of well-characterized Leonberger polyneuropathy cases and controls, conducted a genome-wide association study, and ultimately identified a highly associated and likely causative mutation in the AHGEF10 gene. This sequence variant is a 10-bp deletion encompassing a splice site, which forces use of a downstream splice site to create a processed mRNA with a premature stop codon, and represents a loss-of-function mutation. The identical mutation was also found in several polyneuropathy-affected Saint Bernards. When homozygous, this deletion results in the onset of clinical signs before four years of age. ARHGEF10 has not previously been associated with severe CMT, but comes from a family of genes shown to be involved in neuron morphology. This first-documented severe polyneuropathy associated with an ARHGEF10 mutation in any species provides an opportunity to gain further insights into the pathobiology of diseases associated with this gene. The ARHGEF10 mutation does not, however, by itself account for all cases of polyneuropathy in Leonbergers or Saint Bernards.
PMCID: PMC4183422  PMID: 25275565
5.  A COLQ Missense Mutation in Labrador Retrievers Having Congenital Myasthenic Syndrome 
PLoS ONE  2014;9(8):e106425.
Congenital myasthenic syndromes (CMSs) are heterogeneous neuromuscular disorders characterized by skeletal muscle weakness caused by disruption of signal transmission across the neuromuscular junction (NMJ). CMSs are rarely encountered in veterinary medicine, and causative mutations have only been identified in Old Danish Pointing Dogs and Brahman cattle to date. Herein, we characterize a novel CMS in 2 Labrador Retriever littermates with an early onset of marked generalized muscle weakness. Because the sire and dam share 2 recent common ancestors, CMS is likely the result of recessive alleles inherited identical by descent (IBD). Genome-wide SNP profiles generated from the Illumina HD array for 9 nuclear family members were used to determine genomic inheritance patterns in chromosomal regions encompassing 18 functional candidate genes. SNP haplotypes spanning 3 genes were consistent with autosomal recessive transmission, and microsatellite data showed that only the segment encompassing COLQ was inherited IBD. COLQ encodes the collagenous tail of acetylcholinesterase, the enzyme responsible for termination of signal transduction in the NMJ. Sequences from COLQ revealed a variant in exon 14 (c.1010T>C) that results in the substitution of a conserved amino acid (I337T) within the C-terminal domain. Both affected puppies were homozygous for this variant, and 16 relatives were heterozygous, while 288 unrelated Labrador Retrievers and 112 dogs of other breeds were wild-type. A recent study in which 2 human CMS patients were found to be homozygous for an identical COLQ mutation (c.1010T>C; I337T) provides further evidence that this mutation is pathogenic. This report describes the first COLQ mutation in canine CMS and demonstrates the utility of SNP profiles from nuclear family members for the identification of private mutations.
PMCID: PMC4148433  PMID: 25166616
7.  Altered Splicing of the BIN1 Muscle-Specific Exon in Humans and Dogs with Highly Progressive Centronuclear Myopathy 
PLoS Genetics  2013;9(6):e1003430.
Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.
Author Summary
The intracellular organization of muscle fibers relies on a complex membrane system important for muscle structural organization, maintenance, contraction, and resistance to stress. Amphiphysin 2, encoded by BIN1, plays a central role in membrane sensing and remodelling and is involved in intracellular membrane trafficking in different cell types. The ubiquitously expressed BIN1, altered in centronuclear myopathy (CNM) and myotonic dystrophy (DM), possesses a muscle-specific exon coding for a phosphoinositide binding domain. We identified splice mutations affecting the muscle-specific BIN1 isoform in humans and dogs presenting a clinically and histopathologically comparable highly progressive centronuclear myopathy. Our functional and ultrastructural data emphasize the importance of amphiphysin 2 in membrane remodeling and suggest that the defective maintenance of the triad structure is a primary cause for the muscle weakness. The canine Inherited Myopathy of Great Danes is the first faithful mammalian model for investigating other potential pathological mechanisms underlying centronuclear myopathy and for testing therapeutic approaches.
PMCID: PMC3675003  PMID: 23754947
8.  Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy 
PLoS ONE  2013;8(3):e57195.
Alaskan Husky Encephalopathy (AHE) has been previously proposed as a mitochondrial encephalopathy based on neuropathological similarities with human Leigh Syndrome (LS). We studied 11 Alaskan Husky dogs with AHE, but found no abnormalities in respiratory chain enzyme activities in muscle and liver, or mutations in mitochondrial or nuclear genes that cause LS in people. A genome wide association study was performed using eight of the affected dogs and 20 related but unaffected control AHs using the Illumina canine HD array. SLC19A3 was identified as a positional candidate gene. This gene controls the uptake of thiamine in the CNS via expression of the thiamine transporter protein THTR2. Dogs have two copies of this gene located within the candidate interval (SLC19A3.2 – 43.36–43.38 Mb and SLC19A3.1 – 43.411–43.419 Mb) on chromosome 25. Expression analysis in a normal dog revealed that one of the paralogs, SLC19A3.1, was expressed in the brain and spinal cord while the other was not. Subsequent exon sequencing of SLC19A3.1 revealed a 4bp insertion and SNP in the second exon that is predicted to result in a functional protein truncation of 279 amino acids (c.624 insTTGC, c.625 C>A). All dogs with AHE were homozygous for this mutation, 15/41 healthy AH control dogs were heterozygous carriers while 26/41 normal healthy AH dogs were wild type. Furthermore, this mutation was not detected in another 187 dogs of different breeds. These results suggest that this mutation in SLC19A3.1, encoding a thiamine transporter protein, plays a critical role in the pathogenesis of AHE.
PMCID: PMC3587633  PMID: 23469184
9.  A Gly98Val Mutation in the N-Myc Downstream Regulated Gene 1 (NDRG1) in Alaskan Malamutes with Polyneuropathy 
PLoS ONE  2013;8(2):e54547.
The first cases of early-onset progressive polyneuropathy appeared in the Alaskan Malamute population in Norway in the late 1970s. Affected dogs were of both sexes and were ambulatory paraparetic, progressing to non-ambulatory tetraparesis. On neurologic examination, affected dogs displayed predominantly laryngeal paresis, decreased postural reactions, decreased spinal reflexes and muscle atrophy. The disease was considered eradicated through breeding programmes but recently new cases have occurred in the Nordic countries and the USA. The N-myc downstream-regulated gene (NDRG1) is implicated in neuropathies with comparable symptoms or clinical signs both in humans and in Greyhound dogs. This gene was therefore considered a candidate gene for the polyneuropathy in Alaskan Malamutes. The coding sequence of the NDRG1 gene derived from one healthy and one affected Alaskan Malamute revealed a non-synonymous G>T mutation in exon 4 in the affected dog that causes a Gly98Val amino acid substitution. This substitution was categorized to be “probably damaging” to the protein function by PolyPhen2 (score: 1.000). Subsequently, 102 Alaskan Malamutes from the Nordic countries and the USA known to be either affected (n = 22), obligate carriers (n = 7) or healthy (n = 73) were genotyped for the SNP using TaqMan. All affected dogs had the T/T genotype, the obligate carriers had the G/T genotype and the healthy dogs had the G/G genotype except for 13 who had the G/T genotype. A protein alignment showed that residue 98 is conserved in mammals and also that the entire NDRG1 protein is highly conserved (94.7%) in mammals. We conclude that the G>T substitution is most likely the mutation that causes polyneuropathy in Alaskan Malamutes. Our characterization of a novel candidate causative mutation for polyneuropathy offers a new canine model that can provide further insight into pathobiology and therapy of human polyneuropathy. Furthermore, selection against this mutation can now be used to eliminate the disease in Alaskan Malamutes.
PMCID: PMC3564917  PMID: 23393557
10.  Centronuclear Myopathy in Labrador Retrievers: A Recent Founder Mutation in the PTPLA Gene Has Rapidly Disseminated Worldwide 
PLoS ONE  2012;7(10):e46408.
Centronuclear myopathies (CNM) are inherited congenital disorders characterized by an excessive number of internalized nuclei. In humans, CNM results from ∼70 mutations in three major genes from the myotubularin, dynamin and amphiphysin families. Analysis of animal models with altered expression of these genes revealed common defects in all forms of CNM, paving the way for unified pathogenic and therapeutic mechanisms. Despite these efforts, some CNM cases remain genetically unresolved. We previously identified an autosomal recessive form of CNM in French Labrador retrievers from an experimental pedigree, and showed that a loss-of-function mutation in the protein tyrosine phosphatase-like A (PTPLA) gene segregated with CNM. Around the world, client-owned Labrador retrievers with a similar clinical presentation and histopathological changes in muscle biopsies have been described. We hypothesized that these Labradors share the same PTPLAcnm mutation. Genotyping of an international panel of 7,426 Labradors led to the identification of PTPLAcnm carriers in 13 countries. Haplotype analysis demonstrated that the PTPLAcnm allele resulted from a single and recent mutational event that may have rapidly disseminated through the extensive use of popular sires. PTPLA-deficient Labradors will help define the integrated role of PTPLA in the existing CNM gene network. They will be valuable complementary large animal models to test innovative therapies in CNM.
PMCID: PMC3465307  PMID: 23071563
11.  A canine BCAN microdeletion associated with episodic falling syndrome 
Neurobiology of Disease  2012;45(1):130-136.
Episodic falling syndrome (EFS) is a canine paroxysmal hypertonicity disorder found in Cavalier King Charles spaniels. Episodes are triggered by exercise, stress or excitement and characterized by progressive hypertonicity throughout the thoracic and pelvic limbs, resulting in a characteristic 'deer-stalking' position and/or collapse. We used a genome-wide association strategy to map the EFS locus to a 3.48 Mb critical interval on canine chromosome 7. By prioritizing candidate genes on the basis of biological plausibility, we found that a 15.7 kb deletion in BCAN, encoding the brain-specific extracellular matrix proteoglycan brevican, is associated with EFS. This represents a compelling causal mutation for EFS, since brevican has an essential role in the formation of perineuronal nets governing synapse stability and nerve conduction velocity. Mapping of the deletion breakpoint enabled the development of Multiplex PCR and Multiplex Ligation-dependent Probe Amplification (MLPA) genotyping tests that can accurately distinguish normal, carrier and affected animals. Wider testing of a larger population of CKCS dogs without a history of EFS from the USA revealed that carriers are extremely common (12.9%). The development of molecular genetic tests for the EFS microdeletion will allow the implementation of directed breeding programs aimed at minimizing the number of animals with EFS and enable confirmatory diagnosis and pharmacotherapy of affected dogs.
► Episodic falling is a canine autosomal recessive paroxysmal hypertonicity disorder. ► We mapped the episodic falling locus to a 3.48 Mb region on canine chromosome 7. ► We found a novel microdeletion in BCAN encoding the proteoglycan brevican. ► Multiplex PCR and MLPA tests can distinguish normal, carrier and affected animals. ► BCAN mutations may underlie human exercise-induced hypertonicity disorders.
PMCID: PMC3898273  PMID: 21821125
Brevican; BCAN; Episodic falling syndrome; Cavalier King Charles spaniels; Microdeletion
12.  Startle disease in Irish wolfhounds associated with a microdeletion in the glycine transporter GlyT2 gene 
Neurobiology of Disease  2011;43(1):184-189.
Defects in glycinergic synaptic transmission in humans, cattle, and rodents result in an exaggerated startle reflex and hypertonia in response to either acoustic or tactile stimuli. Molecular genetic studies have determined that mutations in the genes encoding the postsynaptic glycine receptor (GlyR) α1 and β subunits (GLRA1 and GLRB) and the presynaptic glycine transporter GlyT2 (SLC6A5) are the major cause of these disorders. Here, we report the first genetically confirmed canine cases of startle disease. A litter of seven Irish wolfhounds was identified in which two puppies developed muscle stiffness and tremor in response to handling. Although sequencing of GLRA1 and GLRB did not reveal any pathogenic mutations, analysis of SLC6A5 revealed a homozygous 4.2 kb microdeletion encompassing exons 2 and 3 in both affected animals. This results in the loss of part of the large cytoplasmic N-terminus and all subsequent transmembrane domains due to a frameshift. This genetic lesion was confirmed by defining the deletion breakpoint, Southern blotting, and multiplex ligation-dependent probe amplification (MLPA). This analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, confirming recessive inheritance. Wider testing of related animals has identified a total of 13 carriers of the SLC6A5 deletion as well as non-carrier animals. These findings will inform future breeding strategies and enable a rational pharmacotherapy of this new canine disorder.
Research Highlights
► Startle disease is caused by mutations in glycine receptor and transporter genes. ► We report the first genetically confirmed cases of canine startle disease. ► We found a novel microdeletion in SLC6A5 encoding the glycine transporter GlyT2. ► We can now offer diagnostic testing of carriers and potentially treat this disorder. ► Deletion detection techniques should be applied in human startle disease genetics.
PMCID: PMC4068303  PMID: 21420493
Glycine transporter; GlyT2; Hyperekplexia; Irish wolfhound; SLC6A5; Startle disease
13.  Nesprin 1 is critical for nuclear positioning and anchorage 
Human Molecular Genetics  2009;19(2):329-341.
Nesprin 1 is an outer nuclear membrane protein that is thought to link the nucleus to the actin cytoskeleton. Recent data suggest that mutations in Nesprin 1 may also be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy. To investigate the function of Nesprin 1 in vivo, we generated a mouse model in which all isoforms of Nesprin 1 containing the C-terminal spectrin-repeat region with or without KASH domain were ablated. Nesprin 1 knockout mice are marked by decreased survival rates, growth retardation and increased variability in body weight. Additionally, nuclear positioning and anchorage are dysfunctional in skeletal muscle from knockout mice. Physiological testing demonstrated no significant reduction in stress production in Nesprin 1-deficient skeletal muscle in either neonatal or adult mice, but a significantly lower exercise capacity in knockout mice. Nuclear deformation testing revealed ineffective strain transmission to nuclei in muscle fibers lacking Nesprin 1. Overall, our data show that Nesprin 1 is essential for normal positioning and anchorage of nuclei in skeletal muscle.
PMCID: PMC2796894  PMID: 19864491
14.  An ADAMTSL2 Founder Mutation Causes Musladin-Lueke Syndrome, a Heritable Disorder of Beagle Dogs, Featuring Stiff Skin and Joint Contractures 
PLoS ONE  2010;5(9):e12817.
Musladin-Lueke Syndrome (MLS) is a hereditary disorder affecting Beagle dogs that manifests with extensive fibrosis of the skin and joints. In this respect, it resembles human stiff skin syndrome and the Tight skin mouse, each of which is caused by gene defects affecting fibrillin-1, a major component of tissue microfibrils. The objective of this work was to determine the genetic basis of MLS and the molecular consequence of the identified mutation.
Methodology and Principal Findings
We mapped the locus for MLS by genome-wide association to a 3.05 Mb haplotype on canine chromosome 9 (CFA9 (50.11–54.26; praw <10−7)), which was homozygous and identical-by-descent among all affected dogs, consistent with recessive inheritance of a founder mutation. Sequence analysis of a candidate gene at this locus, ADAMTSL2, which is responsible for the human TGFβ dysregulation syndrome, Geleophysic Dysplasia (GD), uncovered a mutation in exon 7 (c.660C>T; p.R221C) perfectly associated with MLS (p-value = 10−12). Murine ADAMTSL2 containing the p.R221C mutation formed anomalous disulfide-bonded dimers when transiently expressed in COS-1, HEK293F and CHO cells, and was present in the medium of these cells at lower levels than wild-type ADAMTSL2 expressed in parallel.
The genetic basis of MLS is a founder mutation in ADAMTSL2, previously shown to interact with latent TGF-β binding protein, which binds fibrillin-1. The molecular effect of the founder mutation on ADAMTSL2 is formation of disulfide-bonded dimers. Although caused by a distinct mutation, and having a milder phenotype than human GD, MLS nevertheless offers a new animal model for study of GD, and for prospective insights on mechanisms and pathways of skin fibrosis and joint contractures.
PMCID: PMC2941456  PMID: 20862248
15.  Main immunogenic region structure promotes binding of conformation-dependent myasthenia gravis autoantibodies, nicotinic acetylcholine receptor conformation maturation, and agonist sensitivity 
The main immunogenic region (MIR) is a conformation-dependent region at the extracellular apex of α1 subunits of muscle nicotinic acetylcholine receptor (AChR) that is the target of half or more of the autoantibodies to muscle AChRs in human myasthenia gravis and rat experimental autoimmune myasthenia gravis. By making chimeras of human α1 subunits with α7 subunits, both MIR epitopes recognized by rat mAbs and by the patient-derived human mAb 637 to the MIR were determined to consist of two discontiguous sequences, which are adjacent only in the native conformation. The MIR, including loop α1 67–76 in combination with the N-terminal α helix α1 1–14, conferred high-affinity binding for most rat mAbs to the MIR. However, an additional sequence corresponding to α1 15–32 was required for high-affinity binding of human mAb 637. A water soluble chimera of Aplysia acetylcholine binding protein with the same α1 MIR sequences substituted was recognized by a majority of human, feline, and canine MG sera. The presence of the α1 MIR sequences in α1/α7 chimeras greatly promoted AChR expression and significantly altered the sensitivity to activation. This reveals a structural and functional, as well as antigenic, significance of the MIR.
PMCID: PMC2787250  PMID: 19890000
myasthenia gravis; autoimmunity; AChR; antigenic structure
16.  A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping 
PLoS ONE  2010;5(1):e8647.
Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion “hot spot” is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD “hot spot”.
Methodology/Principal Findings
Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5′ donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression.
Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD.
PMCID: PMC2800183  PMID: 20072625
17.  Vacuolar myopathy in a dog resembling human sporadic inclusion body myositis 
Acta Neuropathologica  2009;118(5):711-717.
Sporadic inclusion body myositis (sIBM) is the most common myopathy in people over the age of 50 years. While immune-mediated inflammatory myopathies are well documented in dogs, sIBM has not been described. An 11-year-old dog with chronic and progressive neuromuscular dysfunction was evaluated for evidence of sIBM using current pathologic, immunohistochemical and electron microscopic diagnostic criteria. Vacuoles and congophilic intracellular inclusions were identified in cryostat sections of multiple muscle biopsies and immunostained with antibodies against amyloid-β peptide, amyloid-β precursor protein, and proteosome 20S of the ubiquitin–proteosome system. Cellular infiltration and increased expression of MHC Class I antigen were observed. Cytoplasmic filamentous inclusions, membranous structures, and myeloid bodies were identified ultrastructurally. These observations constitute the first evidence that both the inflammatory and degenerative features of human sIBM can occur in a non-human species.
PMCID: PMC2773121  PMID: 19718499
Canine; Muscle; Myopathy; Inflammatory; Aging
18.  Loss of Modifier of Cell Adhesion Reveals a Pathway Leading to Axonal Degeneration 
Axonal dysfunction is the major phenotypic change in many neurodegenerative diseases, but the processes underlying this impairment are not clear. Modifier of cell adhesion (MOCA) is a presenilin binding protein that functions as a guanine nucleotide exchange factor for Rac1. The loss of MOCA in mice leads to axonal degeneration and causes sensorimotor impairments by decreasing cofilin phosphorylation and altering its upstream signaling partners LIM kinase and p21-activated kinase, an enzyme directly downstream of Rac1. The dystrophic axons found in MOCA-deficient mice are associated with abnormal aggregates of neurofilament protein, the disorganization of the axonal cytoskeleton, and the accumulation of autophagic vacuoles and polyubiquitinated proteins. Furthermore, MOCA deficiency causes an alteration in the actin cytoskeleton and the formation of cofilin-containing rod-like structures. The dystrophic axons show functional abnormalities, including impaired axonal transport. These findings demonstrate that MOCA is required for maintaining the functional integrity of axons and define a model for the steps leading to axonal degeneration.
PMCID: PMC2669744  PMID: 19129390
MOCA; DOCK180; guanine nucleotide exchange factor; axonal degeneration; neurodegeneration; sensorimotor defects
19.  Endoneurial Microvascular Pathology in Feline Diabetic Neuropathy 
Microvascular research  2007;75(3):403-410.
Endoneurial capillaries in nerve biopsies from 12 adult diabetic cats with varying degrees of neurological dysfunction were examined for evidence of microvascular pathology and compared to nerves obtained at autopsy from 7 adult non-diabetic cats without clinical evidence of neurological dysfunction. As reported previously (Mizisin et al., 2007), the diabetic cats had elevated glycosylated hemoglobin and serum fructosamine levels, decreased motor nerve conduction velocity and compound muscle action potentials (CMAP), and markedly decreased myelinated nerve fiber densities. Compared to non-diabetic cats, there was a non-significant 26% increase in capillary density and a significant (P<0.009) 45% increase in capillary size in diabetic cats. Capillary luminal size was also significantly (P<0.001) increased, while an index of vasoconstriction was significantly decreased (P<0.001) in diabetic cats compared to non-diabetic controls. No differences in endothelial cell size, endothelial cell number or pericyte size were detected between non-diabetic and diabetic cats. In diabetic cats, basement membrane thickening, seen as a reduplication of the basal lamina, was significantly (P<0.0002) increased by 73% compared to non-diabetic controls. Regression analysis of either myelinated nerve fiber density or CMAP amplitude against basement membrane size demonstrated a negative correlation with significant slopes (P<0.03 and P<0.04, respectively). These data demonstrate that myelinated nerve fiber injury in feline diabetic neuropathy is associated with microvascular pathology and that some of these changes parallel those documented in experimental rodent and human diabetic neuropathy.
PMCID: PMC2413429  PMID: 18207200
Basement membrane; Endoneurial capillaries; Endothelial cells; Feline diabetes mellitus; Microangiopathy; Pericytes
20.  A possible new inherited myopathy in a young Labrador retriever 
The Canadian Veterinary Journal  2008;49(4):393-397.
A 5-month-old, male, Labrador retriever was evaluated for progressive weakness and muscle atrophy. Histologic evaluation of fresh frozen muscle revealed distinct cytoarchitectural changes and central mitochondrial accumulations indistinguishable from those found in the inherited myopathy described in Great Danes. Multiple male littermates and half-siblings were similarly affected.
PMCID: PMC2275345  PMID: 18481550
21.  The ER-Bound RING Finger Protein 5 (RNF5/RMA1) Causes Degenerative Myopathy in Transgenic Mice and Is Deregulated in Inclusion Body Myositis 
PLoS ONE  2008;3(2):e1609.
Growing evidence supports the importance of ubiquitin ligases in the pathogenesis of muscular disorders, although underlying mechanisms remain largely elusive. Here we show that the expression of RNF5 (aka RMA1), an ER-anchored RING finger E3 ligase implicated in muscle organization and in recognition and processing of malfolded proteins, is elevated and mislocalized to cytoplasmic aggregates in biopsies from patients suffering from sporadic-Inclusion Body Myositis (sIBM). Consistent with these findings, an animal model for hereditary IBM (hIBM), but not their control littermates, revealed deregulated expression of RNF5. Further studies for the role of RNF5 in the pathogenesis of s-IBM and more generally in muscle physiology were performed using RNF5 transgenic and KO animals. Transgenic mice carrying inducible expression of RNF5, under control of β-actin or muscle specific promoter, exhibit an early onset of muscle wasting, muscle degeneration and extensive fiber regeneration. Prolonged expression of RNF5 in the muscle also results in the formation of fibers containing congophilic material, blue-rimmed vacuoles and inclusion bodies. These phenotypes were associated with altered expression and activity of ER chaperones, characteristic of myodegenerative diseases such as s-IBM. Conversely, muscle regeneration and induction of ER stress markers were delayed in RNF5 KO mice subjected to cardiotoxin treatment. While supporting a role for RNF5 Tg mice as model for s-IBM, our study also establishes the importance of RNF5 in muscle physiology and its deregulation in ER stress associated muscular disorders.
PMCID: PMC2229664  PMID: 18270596
22.  Treatment of canine pediatric Neospora caninum myositis following immunohistochemical identification of tachyzoites in muscle biopsies 
The Canadian Veterinary Journal  2007;48(5):506-508.
A 7-week-old Irish wolfhound was evaluated for an abnormal hind limb gait. Quadriceps muscle atrophy was pronounced and patellar reflexes were absent bilaterally. Neospora caninum myositis was diagnosed by histopathologic and serologic examination and immunohistochemical staining of muscle. Substantial clinical improvement was noted after 18 weeks of treatment with clindamycin.
PMCID: PMC1852590  PMID: 17542369
23.  Inherited myopathy in a young Great Dane 
The Canadian Veterinary Journal  2006;47(9):899-901.
A 3-year-old, male Great Dane was evaluated for an 18-month history of progressive weakness. Histologic evaluation of muscle biopsies revealed distinct cytoarchitectural changes that were indistinguishable from the central “core-like” structures previously described as central core myopathy in this breed. Clinical features of this inherited myopathy are described.
PMCID: PMC1555684  PMID: 17017657
24.  Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy 
The Journal of Cell Biology  2001;155(4):605-612.
Cypher is a member of a recently emerging family of proteins containing a PDZ domain at their NH2 terminus and one or three LIM domains at their COOH terminus. Cypher knockout mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles. Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function. In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with α-actinin or other Z-line components. These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.
PMCID: PMC2198871  PMID: 11696561
congenital myopathy; cypher; LIM; PDZ; Z-line

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