Abnormal secretion and function of recombinant human factor VII as the result of modification to a calcium binding site caused by a 15-base pair insertion in the F7 gene

A case of a novel mutation in the F7 gene that results in factor VII coagul ant activity (VII:c) of less than 1% and VII antigen (VII:Ag) levels of 10% is presented. DNA analysis revealed a homozygous 15-base pair (bp) in-fram e insertion-type mutation at nucleotide 10554, This insertion consisted of a duplication of residues leucine (L)213 to aspartic acid (D)217 (leucine, serine, glutamic acid, histidine, and aspartic acid), probably arising by s lipped mispairing between 2 copies of a direct repeat (GCGAGCACGAC) separat ed by 4 bp, Molecular graphic analyses showed that the insertion is located at the surface of the catalytic domain in an exposed loop stabilized by ex tensive salt-bridge and hydrogen bond formation at which the calcium bindin g site is located. The mutation probably interferes with protein folding du ring VII biosynthesis and/or diminishes functional activity through the los s of calcium binding, In vitro expression studies demonstrated that the lev els of VII:Ag in lysates of cells transfected with wild type VII (VIIWT) we re equivalent to those with mutant type VII (VIIMT), but the level of secre ted VIIMT was 5% to 10% that of VIIWT. Pulse chase studies demonstrated tha t VIIMT did not accumulate intracellularly, and studies with inhibitors of protein degradation showed that recombinant VIIMT was partially degraded in the pre-Golgi compartment, Accordingly, only small amounts of VIIMT with u ndetectable procoagulant activity were secreted into conditioned media. The se results demonstrate that a combination of secretion and functional defec ts is the mechanism whereby this insertion causes VII deficiency. (C) 2001 by The American Society of Hematology.