Absence of Circulating Factor IX Antigen in Hemophilia B Dogs of the UNC-Chapel Hill Colony

The hemophilia B dog colony maintained at the Francis Owen Blood Laboratory at UNC-Chapel Hill serves as an invaluable model for coagulation factor and gene therapy studies.These animals have severe hemophilia B that closely mirrors the human disease, including spontaneous joint and soft tissue bleeds.Numerous published studies have reported absence of detectable canine factor IX (F.IX) antigen in the circulation of these animals using ELISA or RIA techniques (1-6).The genetic defect in these hemophilic dogs has been defined as a missense mutation within the sequence of the F.IX gene encoding the catalytic domain (1).Molecular modeling studies suggest that the change from a glycine to a glutamic acid residue does not allow the protein to fold properly which likely results in intracellular degradation of the protein and accounts for the absence of circulating protein (1).In a recently published letter by Chao and Walsh, the authors present evidence that the hemophilia B dogs at UNC have circulating F.IX antigen that can be detected by Western blot of dilute plasma samples (7).Moreover, F.IX antigen levels by this assay were found to be identical in normal and hemophilia B dogs.Western blot analysis on crude plasma samples generally must be viewed with caution, because of the enormous number of proteins in the sample and potential cross-reactivity of antibodies.Here, we demonstrate that Western blot results published by Chao and Walsh do not allow the conclusion that the hemophilic dogs have circulating F.IX antigen.Rather, Western blots performed in our laboratories strongly support previously published and unpublished results from several laboratories, i.e. absence of F.IX antigen in plasma of these animals.In Western blots summarized in Fig. 1, we tested pooled normal dog plasma (NDP) and plasma samples from two different hemophilia B dogs (canine hemophilia B plasma, HBP) for the presence of canine F.IX (cF.IX) antigen.Plasma samples where diluted 1:20 in PBS (that is twice as concentrated as the lowest dilution chosen by Chao and Walsh).The diluted samples were mixed with an equal volume of loading dye (10 μl of sample per lane), heatdenatured, and separated by SDS-PAGE under non-reducing or reducing conditions.We