Factor IX Denver, ASN 346→ASP Mutation Resulting in a Dysfunctional Protein with Defective Factor VIIIa Interaction

Abstract

SummaryHemophilia B is a sex-linked recessive bleeding disorder characterized by the presence of either a decreased amount of normal factor IX (FIX) or the presence of a dysfunctional FIX. We have identified a unique mutation in a family with mild hemophilia B. DNA analysis of family members revealed a single base transition in the 8th exon of the FIX gene predicting an amino acid change of Asn 346→Asp in the catalytic domain. The FIX variant, named FIX Denver, was purified from proband plasma. Kinetic studies of factor X (FX) interactions with normal FIXa or FIXa Denver and phospholipid (PL) showed little difference in kcat, but a significant difference when factor VIIIa (FVIIIa) was included in the reaction. Using kinetic assays to infer the Kd of FIXa for FVIIIa, normal FIXa had a Kd of 0.095 nM while that of FIXa Denver was 9.85 nM. The major defect caused by this point mutation is a marked decrease in the affinity of FIXa Denver for factor VIIIa.

Molecular pathogenesis of a novel Met394Thr variant causing hemophilia B

“…The FIX‐Met394Thr variant is immediately adjacent to loop 386–393, which may affect this interaction. A previous study reported that the FIX‐Asn393 (346) Asp variant caused a significant decrease in FIXa and FVIIa affinity (Lefkowitz et al, 2001). The FIX‐Met394Thr variant is adjacent to this variant site and may cause HB due to impaired interaction with FVIIa.…”