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Hemophilia B Caused By Five Different Nondeletion Mutations in the Protease Domain of Factor IX

Factor IX is a multidomain protein and is the proenzyme of a serine protease, factor IXa, essential for hemostasis. In this report, we describe the molecular basis of hemophilia B (deficiency of factor IX activity) in five patients who have neither deletions nor rearrangements of the factor IX gene....

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Bibliographic Details
Published in:Blood 1992-03, Vol.79 (5), p.1225-1232
Main Authors: Ludwig, M., Sabharwal, A.K., Brackmann, H.H., Olek, K., Smith, K.J., Birktoft, J.J., Bajaj, S.P.
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Language:English
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Summary:Factor IX is a multidomain protein and is the proenzyme of a serine protease, factor IXa, essential for hemostasis. In this report, we describe the molecular basis of hemophilia B (deficiency of factor IX activity) in five patients who have neither deletions nor rearrangements of the factor IX gene. By enzymatic amplification and sequencing of all exons and promoter regions, the following causative mutation in the protease domain of factor IX was identified in each patient: IXSchmallenberg: nucleotide 31,215G → T, Ser365lle; IXVarel: nucleotide 31,214A → G, Ser365Gly; IXMechtal: nucleotide 31,211G → C, Asp364His; IXDreihacken: nucleotide 30,864G → A, Arg248Gln; and IXMonschau: nucleotide 30,855A T, Glu245Val. In IXVarel, nucleotide 31,213T was also replaced by C, which results in a silent mutation (GAT → GAC) at Asp-364. Thus, this patient has a double base-pair substitution of TA to CG at nucleotides 31,213 and 31,214 but only a single amino acid change of Ser-365 to Gly. This patient also developed an antibody to factor IX during replacement therapy, which suggests that deletion of the factor IX gene is not necessary for development of the antibody in hemophilia B patients. The levels of plasma factor IX antigen in the patients ranged from 40% to 100% except for IXDrelhackan (Arg248Gln), in which case it was ~4% of normal. The Ser365Gly and Ser365lle mutants are nonfunctional because of lack of the active site serine residue. Mutant Asp364His is inactive because it cannot form the hydrogen bond between the carboxylate group of Asp-364 and the a-amino group of Val-181 generated after activation. As observed in other homologous serine proteases, this hydrogen bond is essential for maintaining the correct active site conformation in normal factor IXa (IXaN). Purified Arg248Gln had ~41% and Glu245Val had ~17% of the activity of normal factor IX (IXN) in a partial thromboplastin time (aPTT) assay. In immunodot blot experiments, the isolated Glu245Val mutant did and the Arg248Gln mutant did not bind to an anti-IXN monoclonal antibody that has been shown previously to inhibit the interaction of factor Villa with factor IXaN. We have recently shown that a high-affinity calcium binding site exists in the protease domain of IXN; among the proposed Ca2+-binding ligands is the carboxyl group of Glu-245. Further, a part of the epitope for the above antibody was shown to be contained in the 231 to 265 residue segment of factor IX. Now, from the mutant data, we conclud
ISSN:0006-4971
1528-0020
DOI:10.1182/blood.V79.5.1225.1225