Canine hemophilia B resulting from a point mutation with unusual consequences

We have used the polymerase chain reaction to amplify the entire coding region of canine factor IX from a hemophilia B animal. When the sequence was compared to that which codes for normal canine factor IX, a single missense mutation was identified. This mutation (G----A at nucleotide 1477) results in the substitution of glutamic acid for glycine-379 in the catalytic domain of the molecule. The mutation creates a new restriction site that allowed confirmation of the abnormal sequence in both hemophilic and carrier animals. Amino acid 379 in canine factor IX corresponds to position 381 in human factor IX, a location at which no human mutations have been described. Moreover, it occurs at one of the few amino acids that have been rigorously conserved among the trypsin-like serine proteases throughout evolution. The mutation responsible for canine hemophilia B results in a complete lack of circulating factor IX in the affected animals. As it is unusual for a missense mutation to result in a complete absence of protein product, structural modeling of the mutant and normal proteins was pursued. These studies suggest that the observed mutation would have major adverse effects on the tertiary structure of the aberrant factor IX molecule. The elucidation of this mutation sheds light on structure-function relationships in factor IX and should facilitate future experiments directed toward gene therapy of this disease.