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Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at an early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Probands 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C < 1 IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding., Competing Interests: E.A. and M.E.C. report grants from South-Eastern Norway Regional Health Authority, during the conduct of the study. P.H.B. reports grants from Research Council of Norway, during the conduct of the study., (Thieme. All rights reserved.)

Congenital factor (F) VII deficiency is a bleeding disorder caused by a heterogeneous pattern of mutations in the F7 gene. Protein misfolding due to mutations is a strong candidate mechanism to produce the highly represented type I FVII deficiency forms, characterized by a concomitant deficiency of FVII antigen and activity. Misfolded proteins can accumulate within the endoplasmic reticulum (ER) causing ER stress with subsequent activation of the unfolded protein response (UPR). So far, there are limited data on this important issue in FVII deficiency. In this study, we chose as candidate FVII model mutations, the p.Q160R, p.I289del and p.A354V-p.P464Hfs, which are all associated with severe to moderate type I FVII deficiency. In vitro expression of the recombinant (r) mutants rFVII-160R, rFVII-289del or rFVII-354V-464Hfs, which are characterized by either amino acid substitution, deletion, or by an extended carboxyl terminus, demonstrated inefficient secretion of the mutant proteins, probably caused by intracellular retention and association with ER chaperones. Both ER stress and UPR were activated following expression of all FVII mutants, with the highest response for rFVII-289del and rFVII-354V-464Hfs. These data unravel new knowledge on pathogenic mechanisms leading to FVII deficiency, and support the investigation of pharmaceutical modulators of ER stress and UPR as therapeutic agents., Competing Interests: The authors state that they have no conflict of interests., (Schattauer GmbH Stuttgart.)