Dystrophin (DMD) Missense Variant in Cats with Becker-Type Muscular Dystrophy.

Muscular dystrophy due to dystrophin deficiency in humans is phenotypically divided into a severe Duchenne and milder Becker type. Dystrophin deficiency has also been described in a few animal species, and few DMD gene variants have been identified in animals. Here, we characterize the clinical, histopathological, and molecular genetic aspects of a family of Maine Coon crossbred cats with clinically mild and slowly progressive muscular dystrophy. Two young adult male littermate cats exhibited abnormal gait and muscular hypertrophy with macroglossia. Serum creatine kinase activities were highly increased. Histopathologically, dystrophic skeletal muscle exhibited marked structural changes including atrophic, hypertrophic, and necrotic muscle fibers. Immunohistochemistry showed irregularly reduced expression of dystrophin but the staining of other muscle proteins such as ß- and γ-sarcoglycans as well as desmin was also diminished. Whole genome sequencing of one affected cat and genotyping of the littermate found both to be hemizygous mutant at a single DMD missense variant (c.4186C>T). No other protein-changing variants in candidate genes for muscular dystrophy were detected. In addition, one clinically healthy male littermate was hemizygous wildtype, while the queen and one female littermate were clinically healthy, but heterozygous. The predicted amino acid exchange (p.His1396Tyr) resides in a conserved central rod spectrin domain of dystrophin. Various protein modeling programs did not predict major disruption of the dystrophin protein by this substitution, but the altered charge of the region may still affect protein function. This study represents the first genotype-to-phenotype correlation of Becker-type dystrophin deficiency in companion animals.

COL6A1 related muscular dystrophy in Landseer dogs: A canine model for Ullrich congenital muscular dystrophy.

BACKGROUND: Collagen VI related myopathies are congenital diseases of variable phenotype. The severe phenotype is referred to as Ullrich congenital muscular dystrophy. In this study, we describe analoguos clinical signs and histopathological alterations in Landseer dogs. MATERIALS: We collected clinical data from two affected dogs and investigated the neuromuscular changes in five dogs from two different litters with immunohistochemistry and immunofluorescence. All affected dogs were homozygous for the p.Glu97* nonsense variant in the COL6A1 gene encoding the alpha-1 chain of collagen VI. RESULTS: Muscle biopsies revealed alterations similar to those in human patients with Ullrich congenital muscular dystrophy including the virtual absence of collagen VI in skeletal muscles. CONCLUSIONS: The clinical and pathological characterization of the affected Landseer dogs enhances the value of this animal model for human Ullrich congenital muscular dystrophy.

A SINE Insertion in ATP1B2 in Belgian Shepherd Dogs Affected by Spongy Degeneration with Cerebellar Ataxia (SDCA2).

Spongy degeneration with cerebellar ataxia (SDCA) is a genetically heterogeneous neurodegenerative disorder with autosomal recessive inheritance in Malinois dogs, one of the four varieties of the Belgian Shepherd breed. Using a combined linkage and homozygosity mapping approach we identified an ∼10.6 Mb critical interval on chromosome 5 in a Malinois family with four puppies affected by cerebellar dysfunction. Visual inspection of the 10.6 Mb interval in whole-genome sequencing data from one affected puppy revealed a 227 bp SINE insertion into the ATP1B2 gene encoding the ß2 subunit of the Na+/K+-ATPase holoenzyme (ATP1B2:c.130_131insLT796559.1:g.50_276). The SINE insertion caused aberrant RNA splicing. Immunohistochemistry suggested a reduction of ATP1B2 protein expression in the central nervous system of affected puppies. Atp1b2 knockout mice had previously been reported to show clinical and neurohistopathological findings similar to the affected Malinois puppies. Therefore, we consider ATP1B2:c.130_131ins227 the most likely candidate causative variant for a second subtype of SDCA in Malinois dogs, which we propose to term spongy degeneration with cerebellar ataxia subtype 2 (SDCA2). Our study further elucidates the genetic and phenotypic complexity underlying cerebellar dysfunction in Malinois dogs and provides the basis for a genetic test to eradicate one specific neurodegenerative disease from the breeding population in Malinois and the other varieties of the Belgian Shepherd breed. ATP1B2 thus represents another candidate gene for human inherited cerebellar ataxias, and SDCA2-affected Malinois puppies may serve as a naturally occurring animal model for this disorder.

A Missense Variant in KCNJ10 in Belgian Shepherd Dogs Affected by Spongy Degeneration with Cerebellar Ataxia (SDCA1).

Spongy degeneration with cerebellar ataxia (SDCA) is a severe neurodegenerative disease with monogenic autosomal recessive inheritance in Malinois dogs, one of the four varieties of the Belgian Shepherd breed. We performed a genetic investigation in six families and seven isolated cases of Malinois dogs with signs of cerebellar dysfunction. Linkage analysis revealed an unexpected genetic heterogeneity within the studied cases. The affected dogs from four families and one isolated case shared a ∼1.4 Mb common homozygous haplotype segment on chromosome 38. Whole genome sequence analysis of three affected and 140 control dogs revealed a missense variant in the KCNJ10 gene encoding a potassium channel (c.986T>C; p.Leu329Pro). Pathogenic variants in KCNJ10 were reported previously in humans, mice, and dogs with neurological phenotypes. Therefore, we consider KCNJ10:c.986T>C the most likely candidate causative variant for one subtype of SDCA in Malinois dogs, which we propose to term spongy degeneration with cerebellar ataxia 1 (SDCA1). However, our study also comprised samples from 12 Malinois dogs with cerebellar dysfunction which were not homozygous for this variant, suggesting a different genetic basis in these dogs. A retrospective detailed clinical and histopathological analysis revealed subtle neuropathological differences with respect to SDCA1-affected dogs. Thus, our study highlights the genetic and phenotypic complexity underlying cerebellar dysfunction in Malinois dogs and provides the basis for a genetic test to eradicate one specific neurodegenerative disease from the breeding population. These dogs represent an animal model for the human EAST syndrome.

A Nonsense Variant in COL6A1 in Landseer Dogs with Muscular Dystrophy.

A novel canine muscular dystrophy in Landseer dogs was observed. We had access to five affected dogs from two litters. The clinical signs started at a few weeks of age, and the severe progressive muscle weakness led to euthanasia between 5 and 15 months of age. The pedigrees of the affected dogs suggested a monogenic autosomal-recessive inheritance of the trait. Linkage and homozygosity mapping indicated two potential genome segments for the causative variant on chromosomes 10 and 31 harboring a total of 4.8 Mb of DNA or 0.2% of the canine genome. Using the Illumina sequencing technology, we obtained a whole-genome sequence from one affected Landseer. Variants were called with respect to the dog reference genome and compared with the genetic variants of 170 control dogs from other breeds. The affected Landseer dog was homozygous for a single, private nonsynonymous variant in the critical intervals, a nonsense variant in the COL6A1 gene (Chr31:39,303,964G>T; COL6A1:c.289G>T; p.E97*). Genotypes at this variant showed perfect concordance with the muscular dystrophy phenotype in all five cases and more than 1000 control dogs. Variants in the human COL6A1 gene cause Bethlem myopathy or Ullrich congenital muscular dystrophy. We therefore conclude that the identified canine COL6A1 variant is most likely causative for the observed muscular dystrophy in Landseer dogs. On the basis of the nature of the genetic variant in Landseer dogs and their severe clinical phenotype these dogs represent a model for human Ullrich congenital muscular dystrophy.

Medial humeral epicondylitis in clinically affected cats.

OBJECTIVE: To describe the clinical signs and histologic changes in cats clinically affected with medial humeral epicondylitis (MHE) and evaluate long-term outcome after either conservative or surgical treatment. STUDY DESIGN: Prospective cohort study. ANIMALS: Client-owned cats (n = 17) with MHE. METHODS: Cats diagnosed with MHE, based on clinical signs, radiographs and computed tomography (CT), were prospectively recruited. Cats were treated conservatively for an initial 4 weeks, followed by either surgery or continued conservative treatment. Followup examinations were performed at 6 and 12 weeks and at 6-49 months. RESULTS: Cats had a mean age of 10.3 years and presented for chronic lameness. Examination revealed pain on palpation caudodistal to the medial epicondyle and by exerting antebrachial supination/pronation with elbow and carpal flexion. Lameness was restricted to 1 limb although CT revealed bilateral disease in 11/17 cats. Free mineralized joint bodies were identified in 9/17 cats. Nine cats were treated surgically and 8 cats were treated conservatively. Intraoperative findings included new bone formation at the origin of the humeral head of the flexor carpi ulnaris muscle with displacement and adhesions of the ulnar nerve. Microscopic examination revealed neurogenic myopathy in 4/9 cats treated surgically. Seven of 9 cats treated surgically were free from lameness by 12 weeks. Seven of 8 cats treated conservatively were chronically lame throughout the study. CONCLUSIONS: Cats with forelimb lameness should be evaluated for MHE. This condition is associated with free joint bodies and neurogenic myopathy. Surgical treatment is associated with excellent outcome in the majority of cats.

An ARHGEF10 deletion is highly associated with a juvenile-onset inherited polyneuropathy in Leonberger and Saint Bernard dogs.

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.

Transient neuromyopathy after bromide intoxication in a dog with idiopathic epilepsy.

A seven-year old Australian Shepherd, suffering from idiopathic epilepsy under treatment with phenobarbitone and potassium bromide, was presented with generalised lower motor neuron signs. Electrophysiology and muscle-nerve biopsies revealed a neuromyopathy.The serum bromide concentration was increased more than two-fold above the upper reference value.Clinical signs disappeared after applying diuretics and reducing the potassium bromide dose rate. This is the first case report describing electrophysiological and histopathological findings associated with bromide induced lower motor neuron dysfunction in a dog.

Centronuclear myopathy in Labrador retrievers: a recent founder mutation in the PTPLA gene has rapidly disseminated worldwide.

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 PTPLA(cnm) mutation. Genotyping of an international panel of 7,426 Labradors led to the identification of PTPLA(cnm) carriers in 13 countries. Haplotype analysis demonstrated that the PTPLA(cnm) 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.

[Influence of physiological disturbances on treatment success of dietary therapy in dogs with chronic enteropathies]. / Einfluss körperlicher Mangelzustände auf den diätetischen Behandlungserfolg bei Hunden mit chronischen Enteropathien.

The aim of this retrospective study in 40 dogs with chronic inflammatory enteropathies was to investigate a possible influence of immunologic and metabolic deficiencies on the success of dietary treatments. At the time of initial presentation, routine clinical and laboratory methods were used to evaluate various metabolic (i.e., weight loss, measurement of serum total protein, albumin, and cobalamin concentrations) and immunological parameters (i.e., serum globulin concentration, immunoglobulin-fractionation by serum electrophoresis, and serum C-reactive protein concentration as measured by a species-specific ELISA). The dogs were classified as food-responsive (FR-group, n = 11), antibiotic-responsive (AR-group, n = 12), steroid-responsive (SR-group, n = 11), or dogs with various combinations of therapies due to protein-losing enteropathy (PLE-group, n = 6). Differences among the four treatment groups were evaluated by statistical analysis. Compared to dogs in the AR, SR, and PLE groups, dogs in the FR-group showed a significantly milder weight loss (p < 0.01 for each). Dogs in the FR-group also had significantly higher serum concentrations of total protein and albumin compared to the PLE-group (p < 0.001 for each). The FR-group had significantly higher median concentrations of total globulin and gamma-globulin fractions compared to the AR- and PLE-groups (p <0.005 and p < 0.01, respectively). Lower gamma-globulin concentrations correlated with increased weight loss (Spearman r = -0.53; p < 0.005), serum cobalamin (Spearman r = 0.38; p < 0.05), and albumin (Spearman r = 0.45; p < 0.01). Increased serum concentrations of C-reactive protein correlated inversely with serum concentrations of cobalamin (Spearman r = -0.58; p < 0.05) and gamma-globulins (Spearman r = -0.6; p < 0.005). This study provides evidence for a possible association between the severity of immunological disturbances and the development of deficiencies in dogs with chronic enteropathies. This study provides further evidence that less severe physiological disturbances, such as milder forms of weight loss and higher serum concentrations of albumin, globulin, and gamma-globulin are possibly associated with a more favorably response to dietary treatment alone.

Myopathy and alterations in serum 3-methylhistidine in dogs with liver disease.

Liver disease can influence the metabolism of various other organs. Regarding the influence of liver diseases on muscles, only a few studies done on people exist. The goal of our study was to investigate the influence of liver diseases on muscles in dogs. Twenty-eight dogs with different liver diseases were investigated in this study. The diagnosis of muscle alteration was based on electromyography (EMG), creatine kinase serum activity, 3-methylhistidine serum concentration and a muscle biopsy in some cases. Our results suggest that liver diseases in dogs can be accompanied with muscle alteration. 3-Methylhistidine serum concentration as a new parameter for muscle destruction in dogs was significantly increased compared to clinical healthy dogs and was comparable to those concentrations in dogs with histologically confirmed myopathy of different types. The differentiation of the liver diseases into severe hepatitis, moderate hepatitis and liver tumours showed a significant elevation of 3-methylhistidine serum concentration in cases of liver tumours (P=0.03) and a tendency in cases of severe hepatitis (P=0.07). Based on our study we can conclude that liver diseases have an influence on muscles in dogs and 3-methylhistidine could be a useful parameter for muscle destruction.

[Assessment of disease severity and outcome of dietary, antibiotic, and immunosuppressive interventions by use of the canine IBD activity index in 21 dogs with chronic inflammatory bowel disease]. / Bestimmung des Krankheitsschweregrades und des Ergebnisses diätetischer, antibiotischer und immunsuppressiver Interventionen durch Anwendung des caninen IBD Aktivitätsindex bei 21 Hunden mit chronisch entzündlicher Darmkrankheit.

Recently, the canine IBD activity index (CIBDAI) was developed for evaluation of the severity of illness, therapeutic strategies, and efficacy of therapy. The aim of the present study was to assess the severity of illness and the therapeutic strategy in dogs with IBD by the use of CIBDAI, serum albumin concentration, and histologic score (HPEG). Furthermore the use of CIBDAI and the efficacy of therapy in a prospective study during a 3 month treatment period were evaluated. Twentyone dogs with inflammatory bowel disease (lymphocytic-plasmacytic enteritis and enterocolitis) were examined in this study. In 11 dogs with IBD the severity of illness was assessed as low, according to CIBDAI and HPEG (CIBDAI score 4 or between 5 and 10 with HPEG score between 1 and 1.5). Six dogs were treated with hypoallergenic diet (Group D), five dogs were treated with hypoallergenic diet and metronidazole (15.6-22,3 mg/kg/day) (Group M). In 10 dogs with IBD the severity of illness was assessed as high (CIBDAI <10, or CIBDAI between 5 and 10 with HPEG score between 2 and 3 or hypoalbuminemia (< or = 2.5 g/dl)). This group (Group I) was treated with immunosuppressive therapy. Treatment consisted of prednisolone (n=10; 0.9-2 mg/kg/day), azathioprine (n=5; 0.9-2.3 mg/kg/day), sulfasalazine (n=4; 18.2-25 mg/kg/day) and hypoallergenic diet (n=10). Efficacy of therapy was evaluated prospectively 3 times in a 12 weeks treatment period. Remission (CIBDAI score < 4) indicated good therapeutic response, chronic or recurrent disease (CIBDAI score persistent or recurrent > or =4) indicated poor therapeutic response. Age, CIBDAI score and HPEG score were significantly different in IBD dogs with low severity of illness (age: median 60 months; CIBDAI score: median 5; HPEG score: median (1) and IBD dogs with high severity of illness (age: median 90 months; CIBDAI score: median 9.5; HPEG score: median 2.25) (p = 0.0101 and p = 0.0099, respectively). The presence of hypoalbuminemia was not significantly different between these two groups (p = 0.3108). There was no significant correlation between CIBDAI score and serum albumin concentration (r = 0.0394; p = 0.0802) or between CIBDAI score and HPEG score (r = 0.2587; p = 0.2574). In the treatment groups, HPEG score was only significantly different between D-group and group I (p < 0.01). The CIBDAI score decreased significantly in group I after 4 weeks of treatment (median 4th week: 3; p < 0.05), and in the D-group after 8 weeks of treatment (median 8" week 1; p < 0.05). No significant decrease of CIBDAI score was seen in the M-group (median 12th week: 1.75; p > 0.05). All dogs in group D, four of five dogs in group M, and six from ten dogs in group I went into remission. Poor therapeutic response (1 dog in group M and 5 dogs in group I; one dog died) was seen in 6 dogs, where as 15 dogs showed good therapeutic response. There was no significant association between efficacy of therapy and age (p = 0.8455), CIBDAI score (p = 0.3293), or serum albumin concentraton (p = 0.8455). Poor therapeutic response was weekly associated with HPEG score > or =2 (p = 0.0635). Using CIBDAI in dogs with IBD as a single parameter to assess the severity of illness and the therapeutic response, misinterpretations are possible. The assessment of the severity of illness by the combination of CIBAI, HPEG, and serum albumin concentration is leading to adaequate therapeutic results. Dogs with low grade IBD benefit from hypoallergenic diet, whereas dogs with high grade IBD benefit from immunosuppressive therapy. The effect of antibiotic treatment is questionable.

Locoregional opening of the rodent blood-brain barrier for paclitaxel using Nd:YAG laser-induced thermo therapy: a new concept of adjuvant glioma therapy?

BACKGROUND AND OBJECTIVES: Nd:YAG laser-induced thermo therapy (LITT) of rat brains is associated with blood-brain barrier (BBB) permeability changes. We address the question of whether LITT-induced locoregional disruption of the BBB could possibly allow a locoregional passage of chemotherapeutic agents into brain tissue to treat malignant glioma. STUDY DESIGN/MATERIALS AND METHODS: CD Fischer rats were subject to LITT of the left forebrain. Disruption of the BBB was analyzed using Evans blue and immunohistochemistry (IH). Animals were perfused with paclitaxel, and high-pressure liquid chromatography (HPLC) was employed to analyze the content of paclitaxel in brain and plasma samples. RESULTS: LITT induces an opening of the BBB as demonstrated by locoregional extravasation of Evans blue, C3C, fibrinogen, and IgM. HPLC proved the passage of paclitaxel across the disrupted BBB. CONCLUSIONS: LITT induces a locoregional passage of chemotherapeutic agents into the brain tissue. This is of potential interest for the treatment of brain tumors.

Precursors of Borna disease virus-specific T cells in secondary lymphatic tissue of experimentally infected rats.

Borna disease in rats represents an experimental model to study the immunopathological role of T cells in central nervous system disease. Adoptive transfer experiments were performed to investigate homing properties of T cells that infiltrate the brains of infected animals. Lymphocytes isolated from the brains of diseased rats were labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and transferred into immunosuppressed infected recipients. In recipient rats displaying neurological disease, labeled lymphocytes were demonstrated in the vicinity of brain cell lesions, suggesting that the neuronal destruction was dependent on the presence of transferred lymphocytes. Furthermore, the presence of virus-specific cytotoxic T cells was scrutinized in secondary lymphatic tissue and the functional activity of lymphocytes isolated from spleens, cervical lymph nodes, and mesenteric lymph nodes of infected animals was tested immediately after isolation and after in vitro restimulation. The data presented here indicate that precursors of Borna disease virus (BDV)-specific CD8(+) T cells are present and cytotoxic activity was demonstrated after in vitro cocultivation with infected cells in cervical lymph nodes and spleens but not in mesenteric lymphoid tissue. Adoptive transfer of in vitro restimulated T cells induced alterations in BDV-infected, immunosuppressed rats that resemble the well-defined clinical symptoms and neuropathology of Borna disease. This report provides for the first time formal evidence that virus-specific cytotoxic T cells are primed in the periphery after BDV infection, a disease that exclusively manifests itself in the central nervous system.

The immunopathogenesis of Borna disease virus infection.

Borna disease virus (BDV) infection represents an excellent model system to study immunopathological mechanisms based on a T cell-mediated immune reaction in the central nervous system. The single-stranded RNA Borna disease virus, a member of Bornaviridae in the order of Mononegavirale, lacks cytopathogenicity both in vitro and in vivo. After experimental infection BDV causes a persistent infection of the central nervous system and induces Borna disease, an immune-mediated encephalomyelitis. The infiltrating immune cells have been characterized as CD4-positive, CD8-positive T-cells, macrophages and B cells. CD8-positive T cells represent the effector cell population exhibiting antigen specificity for the nucleoprotein.