Haemophilia-A (HA), which results from deficiencies of coagulation Factor VIII (FVIII) is the most common inherited bleeding disorder. The development of inhibitory antibodies to plasma-derived or recombinant FVIII in these patients makes the management of the disease extremely expensive and results in higher levels of morbidity and mortality and a poor quality of life. Consistent with the precept that synthesis of the FVIII polypeptide is necessary for inducing central tolerance, large alterations in the F8 gene and absence of circulating antigenically cross-reactive material (CRM) in the plasma are associated with the development of inhibitory antibodies. HA patients with missense mutations in the F8 gene develop inhibitors with a frequency of about 5% whereas the rate of inhibitor development for patients with large gene deletions has been reported to be as high as 88%. Based on these criteria, the most common mutation in severe HA patients, an intron-22 inversion (I22I) in the F8 gene, should be a high risk-factor for developing inhibitors. However, an analysis of clinical data demonstrates that this is not so, presenting a conundrum. The F8 gene structure suggests that it may be possible for the entire primary sequence of the FVIII protein to be synthesized by patients with the I22I, but as two polypeptide chains. A larger transcript would synthesize a polypeptide representing exons 1–22 and a smaller transcript synthesizing the so-called FVIIIB protein derived from exons 23–26. If these polypeptides were indeed synthesized it would provide an explanation for the lower than expected frequency of inhibitory antibodies in patients with the I22I. To explore this possibility we studied the FVIII protein levels in lymphoblast's cells derived from a normal individual (NL) and a HA patient with I22I mutation (IL). Gene expression analysis using Taqman probe demonstrated the presence of both mRNA transcripts in IL cells. Moreover, FVIII was detected in lysates prepared from both NL and IL cells using a polyclonal anti-FVIII antibody in an immunoblot. A larger set of monoclonal anti-FVIII antibodies directed to specific regions of FVIII was used to characterize the different polypeptides synthesized in these cells using flow cytometry and confocal micropcopy. Permeabilized cells (both NL and IL) showed a 5–25 fold increase in fluorescence intensity when anti-FVIII antibodies are used compared to the isotype control antibodies. We used the monoclonal antibodies ESH5, Ab-41188 and ESH8 which target the A1, A3 and C2 domains of the FVIII protein. As the antibody ESH8 can identify only the C2 domain of FVIII, in the normal individual this antibody can detect either the full-length FVIII or the FVIIIB but in the I22I patient, the ESH8 antibody detects the FVIIIB polypeptide alone. Furthermore we used this panel of antibodies to detect these two polypeptides in histological sections of a liver explanted from a HA patient with the I22I, due to hepatitis C induced cirrhosis. Finally, we evaluated the intracellular and extracellular levels of FVIII in the two cell lines. FVIII could be detected in both the cell lysate as well as extracellular medium of NL cells but only in the cell lysate of IL cells. Taken together, our results suggest HA patients with the I22I synthesize the entire FVIII sequence, albeit as two polypeptides and that these are not secreted. These findings provide plausible explanations for the CRM negative status and lower than expected immunogenicity of HA patients with the I22I.
Disclaimer: The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.