Hemophilia A in a litter of Border Collies caused by a one base pair deletion in the F8 gene.

In dogs, hemophilia A is known to affect different breeds. This is a case report describing hemophilia A in a litter of Border Collies. A privately owned bitch and her puppies (n = 7) were presented to the referring veterinarian after acute hematoma formation in the male offspring (n = 3) following microchip implantation. Global coagulation testing, as well as determination of factor VIII and IX activity, were carried out. Based on the results, factor VIII deficiency was suspected. Two of the affected male puppies were euthanized within a few days. Genetic testing of the mother and the surviving male puppy resulted in the description of a deletion in exon 14 of the F8 gene. This c.3206delA variant leads to a frameshift in amino acid sequence and a premature stop codon (p.Asn1069IlefsTer7). The detection of the mutation and consequent testing of related dogs revealed that the deletion most likely had occurred spontaneously in the mother and had been transmitted to several of her offspring in different litters. Identified carriers were taken out of the breeding scheme. It is concluded that genetic testing in the context of suspected genetic disease can lead to preventive measures, including timely exclusion of carriers from breeding.

A frameshift deletion in F8 associated with hemophilia A in Labrador Retriever dogs.

Hemophilia A is the most common inherited coagulation factor disorder in dogs. It manifests as excessive bleeding resulting from pathogenic variants in the X-chromosomal F8 gene encoding coagulation factor VIII (FVIII) protein. In this study, we performed careful clinical phenotyping to confirm hemophilia A in two distinct Labrador Retriever (LR) pedigrees. Whole-genome sequencing on an affected dog from litter 1 identified a case-specific frameshift deletion variant in F8 predicted to cause a premature stop codon (c.2923_2924del, p.(E975Kfs*8)). This variant was hemizygous in all the affected males from litter 1 (n = 3), while all the unaffected LRs in the pedigree were heterozygous or wild-type (n = 22). Additionally, screened samples from 199 LRs were all found to be wild-type. As a result of this study, a gene test can now be developed to screen dogs before breeding to prevent further cases. However, it is important to note that the affected LR with decreased FVIII activity from litter 2 was wild-type for the identified deletion variant, and no segregating F8 variants were detected when this dog's DNA sample was whole-genome sequenced. Thus, the cause of decreased FVIII activity in this dog remains to be unraveled in future studies.

Transcutaneous ultrasound-mediated gene delivery into canine livers achieves therapeutic levels of factor VIII expression.

A safe, effective, and inclusive gene therapy will significantly benefit a large population of patients with hemophilia. We used a minimally invasive transcutaneous ultrasound-mediated gene delivery (UMGD) strategy combined with microbubbles (MBs) to enhance gene transfer into 4 canine livers. A mixture of high-expressing, liver-specific human factor VIII (hFVIII) plasmid and MBs was injected into the hepatic vein via balloon catheter under fluoroscopy guidance with simultaneous transcutaneous UMGD treatment targeting a specific liver lobe. Therapeutic levels of hFVIII expression were achieved in all 4 dogs, and hFVIII levels were maintained at a detectable level in 3 dogs throughout the 60-day experimental period. Plasmid copy numbers correlated with hFVIII antigen levels, and plasmid-derived messenger RNA (mRNA) was detected in treated livers. Liver transaminase levels and histology analysis indicated minimal liver damage and a rapid recovery after treatment. These results indicate that liver-targeted transcutaneous UMGD is promising as a clinically feasible therapy for hemophilia A and other diseases.

A SINE Insertion in F8 Gene Leads to Severe Form of Hemophilia A in a Family of Rhodesian Ridgebacks.

Hemophilia A is the most common coagulation factor disorder in humans and dogs. The disease is characterized by the lack or diminished activity of Factor VIII (FVIII), caused by variants in the F8 gene and inherited as an X chromosomal trait. Two related male Rhodesian Ridgebacks were diagnosed with Hemophilia A due to reduced FVIII activity. The purpose of the study was to determine the genetic cause and give breeding advice for the remaining family members in order to eradicate the variant. By Sanger sequencing a short interspersed nuclear element (SINE) insertion in exon 14 of the F8 gene was found. Perfect correlation of this genetic variant with clinical signs of hemophilia A in the family tree, and the lack of this genetic variant in more than 500 unrelated dogs of the same and other breeds, confirms the hypothesis of this SINE being the underlying genetic cause of Hemophilia A in this family. The identification of clinically unaffected female carriers allows subsequent exclusion of these animals from breeding, to avoid future production of clinically affected male offspring and more subclinical female carriers.

A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.

Haemophilia A in a female mixed-breed dog.

A spayed female mixed-breed dog was presented with excessive bleeding from a wound in the mouth. The dog had a history of self-limiting bleeding following ovariohysterectomy. A coagulation test revealed prolongation of the activated partial thromboplastin time (20.2 seconds; reference interval: 11.0-15.0 seconds), prothrombin time was normal and factor VIII activity was markedly decreased (1.9%; reference interval: >50%). The von Willebrand factor antigen concentration was 158% (reference interval: >50%). A cross-mixing test indicated that the diminished factor VIII activity was due to deficiency or dysfunction of factor VIII rather than inhibition of factor VIII activity. Based on these results, the dog was diagnosed with haemophilia A. Haemophilia A should be considered in the differential diagnosis of bleeding disorders also in female mixed-breeds dogs.

Impact of gene therapy for canine monogenic diseases on the progress of preclinical studies.

Rapid progress in knowledge of the organization of the dog genome has facilitated the identification of the mutations responsible for numerous monogenic diseases, which usually present a breed-specific distribution. The majority of these diseases have clinical and molecular counterparts in humans. The affected dogs have thus become valuable models for preclinical studies of gene therapy for problems such as eye diseases, immunodeficiency, lysosomal storage diseases, hemophilia, and muscular dystrophy. Successful gene therapies in dogs have significantly contributed to decisions to run clinical trials for several human diseases, such as Leber's congenital amaurosis 2-LCA2 (caused by a mutation of RPE65), X-linked retinitis pigmentosa-XLRP (caused by mutation RPGR), and achromatopsia (caused by mutation of CNGB3). Promising results were also obtained for canine as follows: hemophilia (A and B), mucopolysaccharidoses (MPS I, MPS IIIB, MPS VII), leukocyte adhesion deficiency (CLAD), and muscular dystrophy (a counterpart of human Duchenne dystrophy). Present knowledge on molecular background of canine monogenic diseases and their successful gene therapies prove that dogs have an important contribution to preclinical studies.

Absence of functional compensation between coagulation factor VIII and plasminogen in double-knockout mice.

Plasminogen deficiency is associated with severely compromised fibrinolysis and extravascular deposition of fibrin. In contrast, coagulation factor VIII (FVIII) deficiency leads to prolonged and excessive bleeding. Based on opposing biological functions of plasminogen and FVIII deficiencies, we hypothesized that genetic elimination of FVIII would alleviate the systemic formation of fibrin deposits associated with plasminogen deficiency and, in turn, elimination of plasminogen would limit bleeding symptoms associated with FVIII deficiency. Mice with single and combined deficiencies of FVIII (F8-/-) and plasminogen (Plg-/-) were evaluated for phenotypic characteristics of plasminogen deficiency, including wasting disease, shortened lifespan, rectal prolapse, and multiorgan fibrin deposition. Conversely, to specifically examine the role of plasmin-mediated fibrinolysis on bleeding caused by FVIII deficiency, F8-/- and F8-/-/Plg-/- mice were subjected to a bleeding challenge. Mice with a combined deficiency in FVIII and plasminogen displayed no phenotypic differences relative to mice with single FVIII or plasminogen deficiency. Plg-/- and F8-/-/Plg-/- mice exhibited the same penetrance and severity of wasting disease, rectal prolapse, extravascular fibrin deposits, and reduced viability. Furthermore, following a tail vein-bleeding challenge, no significant differences in bleeding times or total blood loss could be detected between F8-/- and F8-/-/Plg-/- mice. Moreover, F8-/- and F8-/-/Plg-/- mice responded similarly to recombinant FVIII (rFVIII) therapy. In summary, the pathological phenotype of Plg-/- mice developed independently of FVIII-dependent coagulation, and elimination of plasmin-driven fibrinolysis did not play a significant role in a nonmucosal bleeding model in hemophilia A mice.

Comparison of albumin, colloid osmotic pressure, von Willebrand factor, and coagulation factors in canine cryopoor plasma, cryoprecipitate, and fresh frozen plasma.

OBJECTIVE: To compare albumin concentrations, coagulation factors activity, and colloid osmotic pressure (COP) of cryoprecipitate (CRYO) and cryopoor plasma (CPP) to that of source fresh frozen plasma (FFP). DESIGN: Prospective in vitro study. SETTING: University teaching hospital. ANIMALS: Ten healthy, non-Greyhound dogs enrolled in an academic teaching hospital blood donor program. INTERVENTIONS: Fresh blood was obtained from canine blood donors and separated into FFP and packed red blood cells. The source FFP was further separated into CRYO and CPP. Albumin and fibrinogen concentrations, COP, activities of coagulation factors II, V, VII, VIII, IX, X, and von Willebrand factor (vWf) were assessed for each FFP, CRYO, and CPP. MEASUREMENTS AND MAIN RESULTS: The mean albumin concentration and COP in CPP were significantly higher compared with those found in FFP, with 31.7 g/L (±6) in CPP compared to 28.9 g/L (±0.5) in FFP (P < 0.001) and 14.5 mm Hg (±0.7) in CPP compared to 12.7 mm Hg (±0.3) in FFP (P = 0.03), respectively. CRYO had significantly higher concentrations of fibrinogen (median 3.46 g/L, 95% CI 2.65-4.27), and higher activities of factor VIII (mean activity 427.0%, ±95.4) and vWf (mean activity 504.7%, ±41.39) as compared to the other products. The activities of vitamin K dependent factors II, VII, and X were similar in CPP compared to FFP, although factor IX activity was lower in CPP. There was no significant difference in factor II or VII activities between the 3 products. CONCLUSIONS: The mean albumin concentration and COP were highest in CPP, suggesting that CPP may be a potential alternative to FFP for oncotic support and albumin replacement. CRYO contained higher activities of vWf and factor VIII than other products and could be used to treat vWf deficiency and hemophilia A. As vitamin K dependent coagulation factors II, VII, and X in CPP were similar to FFP, CPP may be an option for replacement of most of vitamin K dependent factors.

Bone haemophilic pseudotumour of the ulna: A rare complication of haemophilia in a dog.

OBJECTIVES: This case report describes for the first time a bone haemophilic pseudotumour in a dog. CASE DESCRIPTION: A seven-month-old German Shepherd male dog was presented with the complaint of a forelimb weight-bearing lameness with major swelling that expanded dramatically after fine needle aspiration. Radiographs showed a large, well-defined ulnar diaphyseal cystic-like osteolytic lesion. Based on prolonged activated partial thromboplastin time (aPTT) and low factor VIII activity, haemophilia A was diagnosed. Bone scintigraphy, computed tomography, magnetic resonance imaging, and histological findings definitely ruled out malignant neoplasia or inflammation and strongly supported a bone haemophilic pseudotumour over an aneurysmal bone cyst. Segmental ulnar resection  and replacement by a polymethylmethacrylate spacer combined with perioperative bleeding management resulted in a successful outcome. DISCUSSION: This case provided evidence that a bone haemophilic pseudotumour may be the sole presenting clinical sign of haemophilia A in dogs. Early diagnosis, based on history and magnetic resonance imaging findings, is imperative for prompt treatment leading to successful outcome. It is challenging as fine needle aspiration or biopsy is contraindicated. As described in humans, surgical excision of the lesion combined with management of severe postoperative bleeding was associated with successful outcome in the present case. CLINICAL SIGNIFICANCE: A bone haemophilic pseudotumour should be considered in the differential diagnosis of expanding mass associated with osteolysis, especially in young male dogs. Perioperative monitoring of the bleeding disorder and subsequent FVIII replacement therapy was of paramount importance in the present case.

Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test - Demonstrated in vitro and ex vivo.

INTRODUCTION: Canine models of severe haemophilia resemble their human equivalents both regarding clinical bleeding phenotype and response to treatment. Therefore pre-clinical studies in haemophilia dogs have allowed researchers to make valuable translational predictions regarding the potency and efficacy of new anti-haemophilia drugs (AHDs) in humans. To refine in vivo experiments and reduce number of animals, such translational studies are ideally preceded by in vitro prediction of compound efficacy using a plasma based global coagulation method. One such widely used method is the thrombin generation test (TGT). Unfortunately, commercially available TGTs are incapable of distinguishing between normal and haemophilia canine plasma, and therefore in vitro prediction using TGT has so far not been possible in canine plasma material. AIM: Establish a modified TGT capable of: 1) distinguishing between normal and haemophilia canine plasma, 2) monitoring correlation between canine plasma levels of coagulation factor VIII (FVIII) and IX (FIX) and thrombin generation, 3) assessing for agreement between compound activity and thrombin generation in ex vivo samples. METHODS: A modified TGT assay was established where coagulation was triggered using a commercially available activated partial thromboplastin time reagent. RESULTS: With the modified TGT a significant difference was observed in thrombin generation between normal and haemophilia canine plasma. A dose dependent thrombin generation was observed when assessing haemophilia A and B plasma spiked with dilution series of FVIII and FIX, respectively. Correlation between FVIII activity and thrombin generation was observed when analyzing samples from haemophilia A dogs dosed with canine FVIII. Limit of detection was 0.1% (v/v) FVIII or FIX. CONCLUSION: A novel modified TGT suitable for monitoring and prediction of replacement therapy efficacy in plasma from haemophilia A and B dogs was established.

Severe Hemophilia A in a Male Old English Sheep Dog with a C→T Transition that Created a Premature Stop Codon in Factor VIII.

Animals with hemophilia are models for gene therapy, factor replacement, and inhibitor development in humans. We have actively sought dogs with severe hemophilia A that have novel factor VIII mutations unlike the previously described factor VIII intron 22 inversion. A male Old English Sheepdog with recurrent soft-tissue hemorrhage and hemarthrosis was diagnosed with severe hemophilia A (factor VIII activity less than 1% of normal). We purified genomic DNA from this dog and ruled out the common intron 22 inversion; we then sequenced all 26 exons. Comparing the results with the normal canine factor VIII sequence revealed a C→T transition in exon 12 of the factor VIII gene that created a premature stop codon at amino acid 577 in the A2 domain of the protein. In addition, 2 previously described polymorphisms that do not cause hemophilia were present at amino acids 909 and 1184. The hemophilia mutation creates a new TaqI site that facilitates rapid genotyping of affected offspring by PCR and restriction endonuclease analyses. This mutation is analogous to the previously described human factor VIII mutation at Arg583, which likewise is a CpG dinucleotide transition causing a premature stop codon in exon 12. Thus far, despite extensive treatment with factor VIII, this dog has not developed neutralizing antibodies ('inhibitors') to the protein. This novel mutation in a dog gives rise to severe hemophilia A analogous to a mutation seen in humans. This model will be useful for studies of the treatment of hemophilia.

[Acquired von-Willebrand factor and factor-VIII deficiencies caused by angiostrongylosis in a dog]. / Erworbener Von-Willebrand-Faktor- und Faktor-VIII-Mangel durch eine Angiostrongylose bei einem Hund.

An 8-year-old male Australian Shepherd was presented with bleeding from the lip and hemoabdomen. Hematology demonstrated marked thrombocytopenia and anemia. Abdominal ultrasound followed by laparotomy did not detect the cause of bleeding. Plasma transfusion resulted in temporary stabilization. Severe von-Willebrand factor deficiency and factor-VIII deficiency were diagnosed. After subsequent bleeding episodes, infection with Angiostrongylus vasorum was found in both a fecal parasitological examination and by PCR from EDTA-blood. Following successful therapy with fenbendazole, von Willebrand factor and factor VIII were within the normal range. This is the second case report of an acquired von-Willebrand-factor deficiency associated with Angiostrongylus-vasorum infection in a dog, and the first case report with concurrent factor-VIII deficiency.

Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs.

Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1-2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs.

Abnormal coagulation factor VIII transcript in a Tennessee Walking Horse colt with hemophilia A.

Hemophilia A is an X-chromosome-linked disorder caused by a deficiency in factor VIII (FVIII). Although foals have been diagnosed with hemophilia A based on deficiency in FVIII activity, causative gene mutations have not been identified. The genomic DNA and cDNA encoding FVIII of a Tennesee Walking Horse colt affected with hemophilia A and the genomic DNA of his dam and a normal unrelated horse were analyzed with no splice site or coding sequence abnormalities identified in any of the horses. Polymerase chain reactions (PCR) were then performed on hepatic cDNA from the affected colt and an unrelated normal horse, and no product was obtained for the sequence between and including exon 1 and exon 2 in the affected colt. Based on these results, suspected mutations were identified in the noncoding region of FVIII (intron 1), and genomic sequencing of intron 1 in the dam and the affected colt suggested maternal inheritance.

Clinical outcome after diagnosis of hemophilia A in dogs.

OBJECTIVE: To evaluate the clinical course of dogs with hemophilia A (factor VIII deficiency) and to determine whether factor VIII coagulant activity (FVIII:C) was associated with severity of clinical signs and outcome. DESIGN: Survey study. SAMPLE: Respondent information for 39 client-owned dogs with FVIII deficiency. PROCEDURES: Information was obtained via a survey distributed to the American College of Veterinary Internal Medicine and American College of Veterinary Emergency and Critical Care email list serves and to the Veterinary Information Network community to identify dogs with hemophilia A (FVIII:C ≤ 20%). Severity of FVIII deficiency was classified as mild (FVIII:C, 6% to 20%), moderate (FVIII:C, 2% to 5%), or severe (FVIII:C, < 2%). RESULTS: Data for 39 dogs (38 males and 1 female) were compiled. Mixed-breed dogs, German Shepherd Dogs, and Labrador Retrievers were most commonly affected. In most (34/39) dogs, disease was diagnosed at < 1 year of age. Bleeding associated with teething, minor trauma, vaccination, and elective surgical procedures most commonly prompted FVIII:C testing. Affected dogs had similar signs of spontaneous hemorrhage regardless of the magnitude of FVIII deficiency. Four dogs were euthanized without treatment at the time of diagnosis. Thirty dogs received ≥ 1 blood transfusion; FVIII:C did not appear to influence transfusion requirements. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that dogs with hemophilia A have variations in clinical course of the disease and may have a good long-term prognosis. Residual FVIII:C may not be useful for predicting severity of clinical signs, transfusion needs, or long-term prognosis.

Two novel missense mutations associated with hemophilia A in a family of Boxers, and a German Shepherd dog.

BACKGROUND: Hemophilia A is an X-linked disorder caused by a deficiency in coagulation factor VIII. Over 2300 unique mutations in the gene-encoding factor VIII have been documented in people, but limited information is known in dogs. An 11-week-old male Boxer and a 5-year-old male German Shepherd were diagnosed with hemophilia A based on diminished factor VIII activity. OBJECTIVE: The purpose of the study was to identify genetic mutations associated with hemophilia A in both dogs. METHODS: Genomic DNA was isolated from EDTA blood samples from the affected German Shepherd and Boxer, the Boxer's dam, 3 female siblings, and one asymptomatic male sibling. Primers were designed in noncoding regions to amplify the 26 exons of the factor VIII gene via PCR. RESULTS: The affected Boxer sequence revealed a single nucleotide change, cytosine to guanine, at nucleotide position 1412 (1412C>G) in exon 10. The change is predicted to result in the substitution of arginine for proline at amino acid 471 (P471R) in the A2 domain of factor VIII. The dam and female siblings were carriers, the male sibling did not have the mutation. The German Shepherd dog had a single nucleotide change of a guanine to adenine at position 1643 (1643G>A) in exon 11, predicting the substitution of tyrosine for cysteine at amino acid 548 (C548Y) in the A2 domain. CONCLUSIONS: Here we document 2 mutations associated with canine hemophilia A associated with < 1% factor VIII activity, similar to that in people. Another related Boxer with the P471R mutation was later identified.

Hemophilia A in a Belgian Shepherd Malinois dog: case report.

This case report presents a Belgian Shepherd Malinois dog affected by hemophilia A recognized at the age of seven months. The clinical follow-up including all the diagnostic procedures leading to the final diagnosis and the course of this disorder are presented. This is a typical proband case demonstrating the appearance of this genetic disease in a breed never involved by this coagulation disorder so far documented that started an intensive and laborious plan to reduce the incidence of hemophilia A and the further appearance of new cases.