Molecular genetic analysis of haemophilia A and B in several dog breeds

The objective of this study was to identify the underlying genetic defect responsible for the haemophilic phenotype in different dogs. Animals: The study included a Fila Brasileiro suffering from haemophilia B and three haemophilic A dogs: a Wire Haired Dachshund, a Great Dane, and a Poodle mix. The genetic study also included two related Wire-Haired Dachshund dogs, which were suspected to be carriers of haemophilia A, four related healthy Wire-Haired Dachshund dogs and further healthy control dogs. Materials and methods: DNA was isolated from EDTA blood from all these dogs. In the haemophilic Fila Brasileiro, the coding region and exon intron boundaries of the canine FIX gene were amplified and sequenced by using polymerase chain reaction (PCR) and electrophoresis. The obtained nucleotide sequences of the FIX gene were compared with the wild type canine FIX mRNA. Furthermore, intron sequences around exon 4 were also analysed. Genomic DNA from three healthy Fila Brasileiros was used to validate the results. In patients suffering from haemophilia A and related dogs, the coding region and exon intron boundaries of the canine FVIII gene were also analysed. The obtained nucleotide sequences were compared with the wild type canine FVIII mRNA. To confirm the results, specific exon screenings were carried out with new primer pairs. The results were validated with genomic DNA from ten random dog breeds and ten Great Danes. In addition, several protein structure modelling were carried out to evaluate the structural and functional relevance of the identified mutation in the Great Dane. Results: The failure of the amplification of exon 4 and intron sequences around it from genomic DNA of the haemophilic Fila Brasileiro and not from the control dog, as well as the correct amplification of exon 4 from genomic DNA of three normal Fila Brasileiros, revealed that a mutational event involving exon 4 and approximately 900 nucleotides upstream and at least 1000 nucleotides downstream of exon 4 is responsible for the haemophilic phenotype in this dog. Sequence analysis of the coding region and exon intron boundaries of the canine FVIII gene of a suspected carrier (dog 4) and five healthy Wire-haired Dachshunds (dogs 5-9) revealed five single nucleotide polymorphisms (SNPs): a c.141C>T in exon 1, a c.2782A>G, a c.2943G>A and a c.3608C>T in exon 14, and c.5292C>T in exon 15. Three of these SNPs (c.141C>T, c.2943G>A, and c.5292C>T) were synonymous polymorphisms and the two remaining nucleotide changes (c.2782A>G and c.3608C>T), which were also identified in exon 14 of some of ten random control dogs, were characterised as non-synonymous polymorphisms. Interestingly, sequence analysis of the canine FVIII gene of the haemophilic Wire-haired Dachshund did not reveal differences between the patient and the wild type canine FVIII cDNA, which suggested that the genetic defect responsible for haemophilia A in this dog is not located in the coding region and exon intron boundaries of the canine FVIII gene. Unfortunately, analysis of the FVIII gene of the second suspected carrier, which could have contributed to identifying the underlying defect of this dog family, was not possible due to the fact that the DNA sample was contaminated with human genomic DNA. The genetic defect which was responsible for haemophilia A in a Great Dane was identified as a c.6217T>C in exon 21 of canine FVIII gene, which leads to the replacement of tryptophan by arginine at amino acid position 2073 in the C1 domain of the canine FVIII protein. The relevance of this amino acid replacement is related to the great differences in charge, polarity and structure between these amino acids. This mutation was confirmed by the fact that screening of genomic DNA of ten healthy Great Danes did not show this missense mutation. In addition, elaborated protein models suggested that a possible active site is close to the altered amino acid, and confirmed a change in the structure of the truncated FVIII molecule. In contrast, no changes were observed in the FVIII molecule of healthy dogs. Results of the elaborated genetic study in the Poodle mix demonstrated with sufficient clarity that a 40-46 bp insertion in exon 14 of canine FVIII gene was the causative mutation responsible for the haemophilic phenotype in this dog. The identified insertion contained a short sequence (TAAAG), followed by a poly-A tail (20-26 bp), and was flanked by a DNA duplication (15-bp). Analysis of the insertion revealed that a transcriptional frameshift and a premature stop codon “TAA” was located at the beginning of the insertion, c.3217-3219, which probably results in a 1072 amino acids translation product. This truncated protein would probably lack 59 % of amino acids of the B domain, as well as complete A3, C1 and C2 domains. In addition, four known SNPs were identified: c.141C>T in exon 1, c.2943G>A and c.3608C>T in exon 14, as well as c.5292C>T in exon 15. Conclusion: The results of the present study underline the fact that the molecular bases of canine haemophilia B and A are diverse in different breeds , which has also been confirmed in human haemophilia B and A. The development of specific genetic screening tests based on the identified mutations would enable the identification of possible carriers and receive valuable data on the incidence of the defect in the analysed breeds, which would be useful for the control of these hereditary disorders of haemostasis.