Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding

Introduction

Congenital factor (F) VII deficiency is an inherited bleeding disorder with an autosomal recessive inheritance pattern. We report on two probands who had intracranial bleedings as toddlers. One of them had concomitant high titer FVII inhibitor that developed soon after initiation of treatment with recombinant FVII (eptacog alfa, NovoSeven). The underlying molecular mechanisms of these variants by recombinant overexpression in human cell lines were characterized.

Methods

All nine F7 exons, including exon/intron boundaries, were sequenced, and the sequences were aligned with the NCBI reference sequence for F7. Clotting factor activity, FVII antigen (FVII:Ag) and thrombin generation (TG) were measured in patient plasma. Structural analysis was based on the previously determined crystal structure of the complex of FVII with soluble tissue factor (TF) (PDB ID: 1DAN) and the molecular visualization system PyMOL. The mutations were created by site-directed mutagenesis and human embryonic kidney 293 or Chinese hamster ovary K1 cells were transiently transfected with lipofectamine. FVII:Ag was measured in cells and in supernatants. Endoplasmic reticulum (ER) stress was evaluated by a luciferase reporter assay, and by quantitative RT-PCR. Intracellular localization of FVII was assessed using confocal immunofluorescence microscopy.

Results

Proband 1 (P1) was homozygous for a deletion of two nucleotides in exon 1, c.27_28delCT, leading to a frame shift and a premature stop codon at amino acid 16 (p.C10Pfs*16), causing nonsense-mediated mRNA decay (NMD). Proband 2 (P2) was homozygous for a missense mutation in exon 8, c.718G>C, resulting in a glycine to arginine substitution at amino acid 240 (p.G240R). The latter variant has previously been described in the literature, but caused by a c.718G>A transition. Both probands had FVII activity < 1 IU/dL and FVII:Ag was concomitantly reduced, i.e. a type I deficiency. P1 developed a high-titer FVII inhibitor (>50 Bethesda Units/mL) shortly after initiation of treatment with NovoSeven. In such plasma with a high-titer of FVII inhibitor, thrombin was not generated within 90 min after the addition of TF and phospholipids to the re-calcified plasma. In P2, TG, especially the parameter lag time, was severely reduced. The amino acid G240 is located in the hydrophobic core within the catalytic domain. Our structure analysis suggests that a glycine to arginine substitution at this position likely will cause steric interactions and destabilization of the catalytic domain. The intracellular levels of FVII-c.27-28delCT were non-detectable and the levels of FVII-c.718 was reduced by 35%, relative to FVIIwt. FVII:Ag secreted to the conditioned medium of both variants was non-detectable. The ER stress luciferase assay and analysis of spliced (s) X-box binding protein (XBP) 1 mRNA demonstrated significantly increased ER stress levels in cells expressing FVII-c.718 compared to FVIIwt. Confocal immunofluorescence microscopy showed that the FVII-c.718 was localized in the ER and not in the Golgi, whereas FVIIwt could be found in both compartments.

Conclusions

The deficient secretion of the FVII-c.27_28delCT variant was caused by a lack of synthesis of the FVII protein, as a consequence of NMD. The inhibitor development in P1 was likely linked to the complete absence of circulating FVII. The molecular mechanism underlying the FVII-c.718 mutation could be reduced secretion caused by protein destabilization and misfolding.