English Springer Spaniel Dogs Research Articles

EHBP1L1 Frameshift Deletion in English Springer Spaniel Dogs with Dyserythropoietic Anemia and Myopathy Syndrome (DAMS) or Neonatal Losses.

Hereditary myopathies are well documented in dogs, whereas hereditary dyserythropoietic anemias are rarely seen. The aim of this study was to further characterize the clinical and clinicopathological features of and to identify the causative genetic variant for a dyserythropoietic anemia and myopathy syndrome (DAMS) in English springer spaniel dogs (ESSPs). Twenty-six ESSPs, including five dogs with DAMS and two puppies that died perinatally, were studied. Progressive weakness, muscle atrophy—particularly of the temporal and pelvic muscles—trismus, dysphagia, and regurgitation due to megaesophagus were observed at all ages. Affected dogs had a non-regenerative, microcytic hypochromic anemia with metarubricytosis, target cells, and acanthocytes. Marked erythroid hyperplasia and dyserythropoiesis with non-orderly maturation of erythrocytes and inappropriate microcytic metarubricytosis were present. Muscle biopsies showed centralized nuclei, central pallor, lipocyte infiltrates, and fibrosis, which was consistent with centronuclear myopathy. The genome sequencing of two affected dogs was compared to 782 genomes of different canine breeds. A homozygous frameshift single-base deletion in EHBP1L1 was identified; this gene was not previously associated with DAMS. Pedigree analysis confirmed that the affected ESSPs were related. Variant genotyping showed appropriate complete segregation in the family, which was consistent with an autosomal recessive mode of inheritance. This study expands the known genotype–phenotype correlation of EHBP1L1 and the list of potential causative genes in dyserythropoietic anemias and myopathies in humans. EHBP1L1 deficiency was previously reported as perinatally lethal in humans and knockout mice. Our findings enable the genetic testing of ESSP dogs for early diagnosis and disease prevention through targeted breeding strategies.

Exercise-induced hyperthermia syndrome (canine stress syndrome) in four related male English springer spaniels.

ObjectiveThis retrospective study describes the signalment, clinical presentation, diagnostic findings, and mode of inheritance in four young male English springer spaniel dogs with presumptive canine stress syndrome.Materials and methodsAppropriate cases were located through medical searches of medical records of two large private referral centers. Inclusion criteria comprised of English springer spaniel dogs with tachypnea and hyperthermia that subsequently developed weakness or collapse, with or without signs of hemorrhage, soon after a period of mild-to-moderate exercise. The pedigrees of the four affected dogs, as well as eleven related English springer spaniels, were then analyzed to determine a presumptive mode of genetic inheritance.ResultsFour dogs met the inclusion criteria. All four were male, suggesting the possibility of a recessive sex-linked heritable disorder. Pedigree analysis suggests that more dogs may be potentially affected, although these dogs may have never had the concurrent triggering drug/activity/event to precipitate the clinical syndrome. There was complete resolution of clinical signs in three of the four dogs with aggressive symptomatic and supportive therapy, with one dog dying during treatment.ConclusionDogs with canine stress syndrome have the potential for rapid recovery if treated aggressively and the complications of the disease (eg, coagulopathy) are anticipated. All four dogs were male, suggesting the possibility of a recessive sex-linked mode of inheritance. Further genetic analyses should be strongly considered by those involved with the English springer spaniel breed, either with a genome-wide association study using canine single-nucleotide polymorphism arrays or whole-genome sequencing of affected and closely related dogs.

Echocardiographic assessments of longitudinal left ventricular function in healthy English Springer spaniels

ObjectivesTo establish reference intervals for echocardiographic measures of longitudinal left ventricular function in adult English Springer spaniel (ESS) dogs. AnimalsThis study involved 42 healthy adult ESS. MethodsAnimals were prospectively recruited from a general practice population in the United Kingdom. Dogs were examined twice, at least 12 months apart, to exclude dogs with progressive cardiac disease. Mitral annular plane systolic excursion, tissue Doppler imaging mitral annular velocities and two-dimensional speckle-tracking echocardiographic left ventricular longitudinal strain and strain rate were measured. Intraoperator and intraobserver variability were examined and reference intervals were calculated. The potential effects of body weight, age and heart rate on these variables were examined. ResultsIntraoperator and intraobserver variability was <10% for all parameters except tissue Doppler imaging E′ (the peak velocity of early diastolic mitral annular motion as determined by pulsed wave Doppler) and two-dimensional speckle-tracking echocardiographic variables, which were all <20%. Thirty-nine dogs were used to create reference intervals. Significant (but mostly weak) effects of age, heart rate and body weight on were detected. Reference intervals were similar to previously published values in different breeds. Clinical significanceBreed specific reference intervals for measures of longitudinal left ventricular function in the ESS are presented.

Genome-Wide Analysis Identifies Germ-Line Risk Factors Associated with Canine Mammary Tumours.

Canine mammary tumours (CMT) are the most common neoplasia in unspayed female dogs. CMTs are suitable naturally occurring models for human breast cancer and share many characteristics, indicating that the genetic causes could also be shared. We have performed a genome-wide association study (GWAS) in English Springer Spaniel dogs and identified a genome-wide significant locus on chromosome 11 (praw = 5.6x10-7, pperm = 0.019). The most associated haplotype spans a 446 kb region overlapping the CDK5RAP2 gene. The CDK5RAP2 protein has a function in cell cycle regulation and could potentially have an impact on response to chemotherapy treatment. Two additional loci, both on chromosome 27, were nominally associated (praw = 1.97x10-5 and praw = 8.30x10-6). The three loci explain 28.1±10.0% of the phenotypic variation seen in the cohort, whereas the top ten associated regions account for 38.2±10.8% of the risk. Furthermore, the ten GWAS loci and regions with reduced genetic variability are significantly enriched for snoRNAs and tumour-associated antigen genes, suggesting a role for these genes in CMT development. We have identified several candidate genes associated with canine mammary tumours, including CDK5RAP2. Our findings enable further comparative studies to investigate the genes and pathways in human breast cancer patients.

Hemolysis, myopathy, and cardiac disease associated with hereditary phosphofructokinase deficiency in two Whippets

Two male castrated Whippet littermates were presented at 1 year of age for pallor, tachycardia, systolic heart murmur, dark yellow to orange feces, intermittent lethargy, pigmenturia, and muscle shivering or cramping after exercise. Persistent macrocytic hypochromic anemia with marked reticulocytosis and metarubricytosis was found when CBC results were compared with reference values for Whippets. Increased serum creatine kinase activity and hyperkalemia also were sometimes present over the 4-year period of evaluation. Progressively increasing serum concentrations of N-terminal prohormone brain natriuretic peptide suggested cardiac disease. Erythrocytes from the whippets were less osmotically fragile but more alkaline fragile than those from control dogs. Erythrocyte phosphofructokinase (PFK) activities and 2,3-diphosphoglycerate concentrations were decreased. Restriction enzyme-based DNA test screening and DNA sequencing revealed the same mutation in the muscle-PFK gene of the Whippets as seen in English Springer Spaniel dogs with PFK deficiency. This is the first report of PFK deficiency in Whippet dogs. In addition to causing hemolysis and exertional myopathy, heart disease may be a prominent clinical component of PFK deficiency in this breed and has not been previously recognized in PFK-deficient English Springer Spaniels.

The molecular defect underlying canine fucosidosis.

Fucosidosis is a lysosomal storage disease which affects humans and English springer spaniel dogs. The disease is recessively inherited in both species and results from a deficiency of the enzyme alpha-L-fucosidase. We have recently cloned and sequenced the canine fucosidase gene (EMBL sequence admission number X92448 (cDNA) and X92671-X92678 (individual exonic data)). The gene spans 12 kb and consists of eight exons. SSCP based mutation analysis of affected animals was carried out on the coding region of this gene both with exonic primers, and intronic primer pairs for each exon. A 14 base pair deletion of the cDNA was identified at the 3' end of exon 1 in fucosidosis affected animals. Surprisingly, PCR based genomic cloning of DNA from these animals showed an identical deletion in this DNA, ending at the start of intron 1. This change causes a frameshift and, in consequence, 25 novel codons are transcribed in exon 2 before the first of two adjacent premature stop codons is encountered.

Haematology and clinical chemistry of english springer spaniel dogs with phosphofructokinase deficiency

Haematologic and serum clinical chemical values were compared from five to nine phosphofructokinase (PFK)-deficient (when not in haemolytic crisis) and five normal adult English springer spaniel dogs. Affected dogs had macrocytic hypochromic compensated haemolytic anaemias (haematocrit 0.35 ± 0.021/l, mean ± standard deviation), with markedly increased absolute reticulocyte counts of 859 ± 320 × 109/l (normal control dogs, 29 ± 10 × 109/l). Bone marrow evaluation of affected dogs revealed erythroid hyperplasia with normal or increased amounts of stainable iron, as would be expected in response to a haemolytic anaemia. Although the platelet count was not different from that of normal control dogs, the mean platelet volume was nearly 50% higher in affected dogs. Absolute numbers of neutrophils, lymphocytes, and monocytes were higher in affected dogs (10.7 ± 1.3, 4.4 ± 1.2 and 1.4 ± 0.5 × 109/l, respectively) than in normal control dogs (6.8 ± 1.4, 3.0 ± 0.3 and 0.5 ± 0.2 × 109/l, respectively). Affected dogs also had higher serum iron (58 ± 25 μmol/l) and ferritin (1081 ± 172 μg/l) concentrations than normal control dogs (24 ± 5 μmol/l and 512 ± 222 μg/l, respectively), providing evidence for increased body iron stores. Serum haptoglobin was very low in most affected dogs, indicating that some degree of intravascular haemolysis occurs even when haemoglobinuria is not observed. Serum chemical analysis revealed slightly higher potassium, magnesium, and calcium values in affected dogs compared to normal dogs. Consistent with the presence of a haemolytic anaemia, serum bilirubin from affected dogs was increased in most affected dogs (14 ± 8 μmol/l compared to 2 ± 1 μmol/l in normal control dogs). Serum urea was also higher (10.9 ± 2.8 v 6.2 ± 1.3 mmol/l), but creatinine was lower (53 ± 8 v 85 ± 12 μmol/l) in affected dogs, compared to normal control dogs. Serum creatine kinase, lactate dehydrogenase, and alkaline phosphatase from affected dogs were at least double that of normal control dogs. Serum protein and total globulins were slightly higher, but albumin was slightly lower in affected compared to normal control dogs.

Neutrophil Function in Dogs with Congenital Ciliary Dyskinesia

In vitro neutrophil function was assessed in two English Springer Spaniel dogs, two Bichon Frise dogs, and one Chow Chow dog with congenital ciliary dyskinesia; three clinically normal English Springer Spaniel dogs that were presumed heterozygous for congenital ciliary dyskinesia; and five control dogs. Chemotaxis and random migration in affected and heterozygous dogs were found to be comparable to those of control dogs. Increased (P less than or equal to 0.05) neutrophil adhesion, antibody dependent cell-mediated cytotoxicity, iodination of proteins, and oxygen radical production in neutrophils from affected dogs were probably the result of chronic bacterial infection in vivo. Bacterial ingestion by neutrophils from the three heterozygous English Springer Spaniel dogs was significantly increased compared to control dogs but was not different from affected English Springer Spaniel dogs, suggesting a breed-related phenomenon. Significant decreases in neutrophil function were not seen in any of the dogs with congenital ciliary dyskinesia, indicating that a defective microtubular system is not shared by respiratory cilia and neutrophils and that defective neutrophil function does not contribute to respiratory infection.

Dyserythropoiesis, Polymyopathy, and Cardiac Disease in Three Related English Springer Spaniels

A polysystemic disorder was observed in three related English Springer Spaniel dogs that demonstrated regurgitation from an early age, slowly progressive temporal muscle atrophy with partial trismus, and less pronounced generalized skeletal muscle atrophy. All dogs exhibited moderate dyserythropoietic anemia, polymyopathy with megaesophagus, and varying degrees of cardiomegaly. The prevalence, etiology, underlying pathomechanism, and possible mode of inheritance remain to be elucidated.

Reproductive abnormalities in canine fucosidosis

Male English springer spaniel dogs affected with fucosidosis, a lysosomal storage disorder, were found to be infertile while females with the disease reproduced successfully. Ejaculates of semen collected from affected dogs had reduced total sperm output and morphologically abnormal spermatozoa. A high proportion of ejaculated spermatozoa had midpiece droplets, bent tails and poor motility. Severely vacuolated epididymal epithelial cells were observed by light microscopy. Electron microscopic examination revealed membrane-bound vacuoles of variable size containing scanty amounts of granular to fibrillar material in epididymal epithelial cells, smooth muscle, myoid cells and Sertoli cells. Male infertility is believed to result from lysosomal storage of fucosyl-linked substrates in cells of the reproductive system. The extensive lesions in the epididymis may have interfered with maturation and transport of spermatozoa. Also, deficiency of alpha- l-fucosidase activity could have impaired the shedding of cytoplasmic droplets from spermatozoa and altered the surface glycoprotein composition of the sperm during epididymal transit.

Autosomal recessive inherited phosphofructokinase deficiency in English springer spaniel dogs

Three families of English springer spaniel dogs with phosphofructokinase (PFK) deficiency causing haemolysis were studied. Four male dogs and one female dog with chronic haemolysis and haemolytic crises were found to have markedly reduced PFK activity in erythrocytes (8-20% of control English springer spaniels). PFK-deficient erythrocytes exhibited an extreme alkaline and sucrose lysis. The oxygen dissociation curve of erythrocyte suspensions was shifted to the left with a 50% saturation of haemoglobin at a partial oxygen pressure of 16-17 mmHg (normal 26-31 mmHg). Muscle wasting and mildly increased serum creatine phosphokinase activity were also noted. Six clinically normal first degree relatives of affected dogs had erythrocyte PFK activities that were 38-51% of controls. In these family members, there was an erythrocytosis and mild reticulocytosis probably due to a mildly enhanced haemoglobin-oxygen affinity but no increase in serum creatine phosphokinase. These studies confirm the familial nature of muscle-type PFK deficiency in English springer spaniels and support the conclusion that this animal model of the human glycogen storage disease type VII is inherited as an autosomal recessive trait.

Characterization of the enzymatic lesion in inherited phosphofructokinase deficiency in the dog: an animal analogue of human glycogen storage disease type VII.

Mammalian phosphofructokinase (PFK; ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) exists in multimolecular forms, which result from random tetramerization of three distinct subunits, M (muscle-type), L (liver-type), and P (platelet-type), each under a separate genetic control. Human muscle and liver contain homotetramers M4 and L4, respectively, whereas erythrocytes contain a mixture of M4, M3L, M2L2, ML3, and L4 isozymes. Homozygous deficiency of the M subunit in man results in glycogen storage disease (GSD) type VII, which is characterized by exertional muscle weakness and compensated hemolysis; the residual erythrocyte PFK consists of isolated L4 isozyme. Recently, PFK deficiency associated with isolated hemolytic anemia has been identified among English springer spaniel dogs. We investigated the genetic control of the dog PFK system and the nature of the enzymatic defect in two PFK-deficient animals, using chromatographic and immunological techniques. Our studies indicate the existence of a trilocus isozyme system for the dog, as is the case with other mammals. Muscle PFK consists of M4 isozyme, whereas the predominant species of liver and platelet consists, respectively, of the L4 and P4 isozyme; erythrocyte PFK consists of a three- or four-membered set composed of M and P subunits. PFK deficiency in the dogs was found to result from a total and universal lack of the M subunit, as is the case in man. However, the probands consistently exhibited L4 isozyme in their muscle; P4, L4, and hybrids thereof in their erythrocytes; and an increase in the L-containing isozymes in their platelets, indicating a generalized anomalous presence of the L subunit. The apparent absence of muscle disease in these animals is most likely accounted for by both the well-known high oxidative potential of the canine muscle in general and the presence of liver PFK in the M-deficient muscle in particular. In contrast, presence of hemolysis despite residual P4 and hybrids of P and L in the erythrocytes may be inferred to result in severe glycolytic handicap under existing intraerythrocytic conditions.

Inherited phosphofructokinase deficiency in dogs with hyperventilation- induced hemolysis: increased in vitro and in vivo alkaline fragility of erythrocytes

Two male English springer spaniel dogs with a chronic hemolytic anemia and sporadic hemolytic crises, historically related to “stress” situations, were studied. Although canine erythrocytes are in general known to be more alkaline fragile, erythrocytes from both patients began to lyse earlier, at significantly lower pH values (near pH 7.4 at 37 degrees C), than erythrocytes from control dogs. Hyperventilation induced by 30 minutes of exercise, placement in a 39 degrees C water bath, or intravenous doxapram increased venous blood pH in dog 1 and control dogs, but transient hemoglobinemia, hemoglobinuria, and severe bilirubinuria occurred only in the studied patient. The erythrocyte phosphofructokinase (PFK) activity was severely decreased in both dogs (10% of controls). The erythrocyte 2,3-diphosphoglycerate content was markedly reduced and the cell chloride content was consequently increased. This change in cell chloride content is related to an increase in the erythrocyte pH, which may partially explain the pathogenesis of hemolysis in canine PFK deficiency. Thus, these studies demonstrate a presumably inherited erythrocyte PFK deficiency in English springer spaniels, which causes an increased in vitro and in vivo erythrocyte alkaline fragility. Dogs with PFK deficiency and inducible hemolytic crises may become a valuable genetic animal model in which to study the pathophysiology of hemolysis.

Inherited Phosphofructokinase Deficiency in Dogs With Hyperventilation-Induced Hemolysis: Increased In Vitro and In Vivo Alkaline Fragility of Erythrocytes

Two male English springer spaniel dogs with a chronic hemolytic anemia and sporadic hemolytic crises, historically related to "stress" situations, were studied. Although canine erythrocytes are in general known to be more alkaline fragile, erythrocytes from both patients began to lyse earlier, at significantly lower pH values (near pH 7.4 at 37 degrees C), than erythrocytes from control dogs. Hyperventilation induced by 30 minutes of exercise, placement in a 39 degrees C water bath, or intravenous doxapram increased venous blood pH in dog 1 and control dogs, but transient hemoglobinemia, hemoglobinuria, and severe bilirubinuria occurred only in the studied patient. The erythrocyte phosphofructokinase (PFK) activity was severely decreased in both dogs (10% of controls). The erythrocyte 2,3-diphosphoglycerate content was markedly reduced and the cell chloride content was consequently increased. This change in cell chloride content is related to an increase in the erythrocyte pH, which may partially explain the pathogenesis of hemolysis in canine PFK deficiency. Thus, these studies demonstrate a presumably inherited erythrocyte PFK deficiency in English springer spaniels, which causes an increased in vitro and in vivo erythrocyte alkaline fragility. Dogs with PFK deficiency and inducible hemolytic crises may become a valuable genetic animal model in which to study the pathophysiology of hemolysis.

Retinal dysplasia of English springer spaniel dogs: light microscopy of the postnatal lesions.

The histologic features of retinal dysplasia were studied in 26 English springer spaniel dogs of one day to four years of age. Dysplastic lesions in neonatal pups occurred predominantly in the peripapillary tapetal area of sensory retina. They consisted of complex linear folds and rosettes which were composed of hyperplastic neuroblasts. Dysplasia usually was not associated with incomplete differentiation of the outer neuroblastic layer. The outer limiting membrane was absent locally and numerous patent subretinal capillaries existed in the region of folds. The dysplastic features may be related to aberrant development of Muller cells and of retinal capillaries. In adolescent dogs, dysplastic lesions were less complex and usually were not accompanied by aberrant capillaries. Focal or complete retinal separation was present in some adolescent dogs. In adult dogs, features of retinal atrophy were found within dysplastic areas. No significant histologic lesions were found in the extraocular tissues of 12 affected dogs which were surveyed extensively.