A homozygous is bp insertion in the human factor vu gene results in modification of a calcium binding site and reduced secretion/function

We have previously reported the first homozygous 15 bp in-frame insertion type mutation at nucleotide 10554, within the catalytic domain of the human factor VII (FVII) gene. This mutation arises as a result of a duplication of residues Leu213 to Asp217 (Leu, Ser, GIu, His, Asp), probably by slipped mispairing between 2 copies of a direct repeat (GCGAGCACGAC) separated by 4 bp. The mutation was identified in an Arabic child and results in a severe type I deficiency (FVII:C<1 % ,FVII:Ag 10%.). The crystal structure for FVIIa-TF shows that the loop formed by residues Leu213-Asp217 is exposed at the surface of the catalytic domain and is stabilized by extensive salt-bridge and hydrogen bond formation. The insertion includes part of a calcium binding site formed by contacts with the carboxyls of Glu210 and Glu220 and the mainchain oxygen atoms of Asp212 and Glu215. The insertion of five residues at this location is therefore expected to disrupt the correct formation of the loop and may lead to a misfolded protein that may be degraded or not secreted. Furthermore, the correct mainchain conformation of the Asp212-Glu215 loop is critical for calcium-binding and the insertion will directly affect this. To explore these hypotheses, wildtype FVII (FVIIWT) and mutant FVII (FVHMT) cDNAs were expressed transiently in COS 1 cells and stably in CHO cells. In lysates of cells transfected with either the FVIIWT or FVHMT constructs, the FVII:Ag levels were equivalent. However, the amount of FVILAg secreted by cells transfected with FVIIMT was 5-10% of that secreted by cells transfected with FVIIWT. Using stably transfected CHO cells, pulse chase studies demonstrated that FVIIMT did not accumulate intracellularly. The use of various inhibits of protein synthesis reveals that part of the recombinant protein is degraded in the pre-Golgi compartment and only small amounts of proteolytically inactive FVII are secreted into conditioned media. These results verify both hypotheses derived from inspection of the FVIIa-TF crystal structure and demonstrate that both a secretion and a functional defect are the mechanisms through which the 15bp insertion results in FVII deficiency.