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Hemophilia B caused by five different nondeletion mutations in the protease
domain of factor IX
M Ludwig, AK Sabharwal, HH Brackmann, K Olek, KJ Smith, JJ Birktoft and SP Bajaj
Institute of Experimental Haematology and Blood Transfusion, Bonn, Germany.
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, Ser365Ile; 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
IXDreihacken (Arg248Gln), in which case it was approximately 4% of normal.
The Ser365Gly and Ser365Ile 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 alpha-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 approximately 41% and Glu245Val had
approximately 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 VIIIa with factor IXaN. We have recently shown that a
high-affinity calcium binding site exists in the protease domain of IXN;
among the proposed Ca(2+)-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.(ABSTRACT
TRUNCATED AT 400 WORDS)
Volume 79,
Issue 5,
pp. 1225-1232,
03/01/1992
Copyright © 1992 by The American Society of Hematology
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D J Bowen
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A. Mathur and S. P. Bajaj
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A. Mathur, D. Zhong, A. K. Sabharwal, K. J. Smith, and S. P. Bajaj
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