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Almost half of severe hemophilia A (HA) cases are caused by an intron-22 inversion mutation (Int22Inv), which truncates the 26-exon F8mRNA after exon 22. Another F8transcript, F8B, is initiated from within F8-intron-22. F8BmRNA 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.

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene inversion, which together cause ∼50% of severe hemophilia A cases. We report a simple and accurate RNA-based assay to detect these mutations in patients and heterozygous carriers. The assays do not require specialized equipment or expensive reagents; therefore, they may provide useful and economic protocols that could be standardized for central laboratory testing. RNA is purified from a blood sample, and reverse transcription nested polymerase chain reaction (RT-NPCR) reactions amplify DNA fragments with the F8sequence spanning the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1 inversion test). These sequences will be amplified only from F8RNA without an intron 22 or intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out to amplify the inverted sequences extending from F8exon 19 to the first in-frame stop codon within intron 22 or a chimeric transcript containing F8exon 1 and the VBP1gene. These latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as type 1 or type 2, reflecting the specific homologous recombination sites) by the standard DNA-based “inverse-shifting” PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes that cleave and ligate DNA in minutes were used, which is a substantial improvement over previous protocols that required overnight incubations. These protocols can accurately detect F8inversion mutations via same-day testing of patient samples.

The most common complication in hemophilia A (HA) treatment, affecting 25-30% of severe HA patients, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8haplotypes H1-H5 are defined by nonsynonymous single-nucleotide polymorphisms encoding sequence variations at FVIII residues 1241, 2238 and 484. Haplotypes H2-H5 are more prevalent in individuals with black African ancestry, while 80-90% of the white population has the H1 haplotype. This study used an established multiplex fluorescence immunoassay to determine anti-FVIII antibody titers in plasma from 394 HA subjects (188 black, 206 white), measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3/H5 and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic assay and linear B-cell epitopes characterized using peptide microarrays. FVIII-reactive antibodies were readily detected in most HA subjects, with higher titers in those with a current inhibitor, as expected. Neither total nor inhibitory antibody titers correlated with F8haplotype mismatches, and peptides with D1241E and M2238V polymorphisms did not comprise linear B-cell epitopes. Interestingly, the BDD-FVIII proteins were markedly more reactive than the full-length FVIII products with plasma antibodies. The stronger immunoreactivity of BDD-FVIII suggests that B-domain removal may expose novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.

The existence of natural antibodies recognizing endogenous factor VIII (FVIII) and of FVIII-specific CD4+T-cell responses in some healthy, non-hemophilic blood donors has been appreciated for >20 years. The Conti-Fine group measured CD4+T-cell proliferation following in vitrostimulation with FVIII protein or synthetic FVIII peptides. More recently, FVIII-specific CD4+T-cell lines were expanded from PBMCs isolated from large blood volumes donated by healthy individuals, and estimates of specific precursor frequency (~2/million CD4+T cells) were calculated on the basis of interferon (IFN)-gamma ELISPOT assays of FVIII-stimulated cells (Meuniere et al., Blood Advances 1(21): 1842-7). Escape of these self-reactive precursor cells from thymic editing via deletion or anergy and their subsequent persistence in the periphery may contribute to the rare but potentially severe autoimmune reactions to FVIII (“acquired hemophilia A”) and to the unusual immunogenicity of therapeutic FVIII administered i.v. to hemophilia A patients.

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 F8gene inversion, which together result in ~50% of severe hemophilia A cases. We report simple and accurate RNA- and DNA-based assays to detect these mutations in patients and carrier mothers. They do not require specialized equipment or expensive reagents, and therefore they may provide useful and economic protocols that could be standardized for central laboratory testing.