Two cases of hereditary bleeding disorder diagnosed as haemophilia A were recently observed in Japanese Brown cattle. We sequenced the entire coding region of the factor VIII gene of the affected animals to find a causative mutation. A nucleotide substitution of T to A resulting in an amino acid substitution of leucine to histidine (p.Leu2153His) was identified in a highly conserved residue in the C1 domain of factor VIII. Genotyping of 254 normal animals including the pedigree of the affected animals and randomly sampled animals of different breeds confirmed that the substitution is the causative mutation of cattle haemophilia A.
ABSTRACT. A coagulopathy with subcutaneous bleeding and muscular or peritracheal/periesophageal bleeding occurred in two male Japanese Brown calves of the same dam. One of the affected calves died three days after the onset of bleeding and the other survived normally until being slaughtered despite once suffering from subcutaneous hematoma. Hemostatic tests of the latter case showed prolonged activated partial thromboplastin time (APTT), and severely reduced factor VIII activity. In addition, von Willebrand factor activity, determined by the human platelet aggregation test, was within the normal range; therefore, the calf was diagnosed with hemophilia A. These are the first bovine cases of hemophilia A definitely diagnosed clinicopathologically. KEY WORDS: factor VIII, hemophilia A, Japanese Brown cattle.
Hemophilia A is a severe congenital bleeding disorder characterized by subcutaneous hematoma and hemorrhage into muscles resulting from a deficiency of blood coagulation factor VIII. The authors have recently reported two cases of hemophilia A in Japanese Brown cattle and identified a nucleotide substitution in the factor VIII gene, resulting in an amino acid substitution of Leu to His, as a possible cause of the deficiency. In the present study, a simple and effective polymerase chain reaction (PCR)-based diagnostic method was developed to identify carriers of this disorder, using a mismatch primer in combination with restriction enzyme digestion. The PCR reaction amplified a 118 bp fragment, which was not digested by the BspT104I restriction enzyme in affected animals but was digested into two fragments in normal animals. Both digested and undigested fragments were observed in carrier animals. This method was applied to identify the carriers of hemophilia A in a population of Japanese Brown cattle. By screening 155 DNA samples from Japanese Brown cattle, except for the dam of the two probands, no carriers were identified. It was therefore concluded that the probands represent isolated cases of hemophilia A, and that the frequency of the mutant allele in the Japanese Brown cattle population is very low.
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