Lack of FVIII detection in humans and dogs with an intron-22 inversion challenges hypothesis regarding inhibitor risk

BACKGROUND: Almost half of severe hemophilia A (HA) cases are caused by an intron-22 inversion mutation (Int22Inv), which truncates the 26-exon F8 mRNA after exon 22. Another F8 transcript, F8 B, is initiated from within F8-intron-22. F8 B mRNA consists of a short exon spliced to exons 23-26 and is expressed in multiple human cell types. It has been hypothesized that Int22Inv patients have self-tolerance to partial FVIII proteins expressed from these two transcripts. FVIII is expressed in endothelial cells, primarily in liver and lung. Several studies have reported FVIII expression in other cell types, although this has been controversial.

OBJECTIVES: To determine if partial FVIII proteins are expressed from intron 22-inverted and/or F8 B mRNA and if FVIII is expressed in non-endothelial cells.

METHODS: A panel of FVIII-specific antibodies was validated and employed to label FVIII in cells and tissues, and for immunoprecipitation followed by western blots and mass spectrometry-proteomics analysis.

RESULTS: Immunofluorescent (IF) staining localized FVIII to endothelial cells in liver sections from non-HA but not HA-Int22Inv dogs. Neither FVIII nor FVIII B was detected in human PBMCs, B-cell or T-cell lines, or in cell lines expanded from PBMCs, whereas FVIII antigen and activity were readily detected in primary non-hemophilic liver sinusoidal endothelial cells.

CONCLUSIONS: If FVIII is expressed in non-endothelial cells, or if partial FVIII proteins are expressed in HA-Int22Inv, the concentrations are below the detection limits of these sensitive assays. Our results argue against promotion of immune tolerance through expression of partial FVIII proteins in Int-22Inv patients.