Lack of factor VIII detection in humans and dogs with an intron 22 inversion challenges hypothesis regarding...

Almost half of severe hemophilia A (HA) cases are caused by an intron 22 inversion (Int22Inv) mutation, which truncates the 26-exon F8 messenger RNA (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 to 26 and is expressed in multiple human cell types. It has been hypothesized that Int22Inv patients have self-tolerance to partial factor (F)VIII proteins expressed from these 2 transcripts. FVIII is expressed in endothelial cells, primarily in the liver and lungs. Several studies have reported FVIII expression in other cell types, although this has been controversial.

To determine if partial FVIII proteins are expressed from intron 22-inverted and/or F8_B mRNA and if FVIII is expressed in nonendothelial cells.

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.

Immunofluorescent 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 peripheral blood mononuclear cells, B cell or T cell lines, or cell lines expanded from peripheral blood mononuclear cells, whereas FVIII antigen and activity were readily detected in primary nonhemophilic liver sinusoidal endothelial cells.

If FVIII is expressed in nonendothelial 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.