Factor VIII Point Mutations with Higher Risk for Inhibitor Development in Mild/Moderate Hemophilia A: A Rapid, Specific Test Panel.
Authors: Buzby Jeffrey S. , McDaniel Marianne , Tucker Jeralyn C. , Williams Shirley A. , Soni Amit , Nugent Diane J. 
Published: October 2019

Abstract 2264

The conversion of mild or moderate Factor VIII-deficient patients into those with a severe bleeding disorder can be an alarming consequence of inhibitor development, which is not typically anticipated in these patients. Cross-reactivity of the inhibitor antibodies with the patient's own circulating FVIII can reduce their baseline FVIII activity from that of 5–10% to 0–1%, with severe bleeding diathesis. Immune tolerance can be effective, as with more severely deficient cases. But patients must undergo daily desensitization and use costly bypassing agents. While cross-reactivity of the inhibitor antibodies with the patient's circulating FVIII remains poorly understood, there is growing evidence that mild/moderate hemophilia A patients with specific FVIII point mutations are at greater risk than others for developing this life-threatening complication. Current hypotheses suggest that certain mutations result in greater conformational changes in the patient's circulating FVIII, so that the infused FVIII becomes immunogenic.

Based on review of previously published reports and the Haemophilia A Mutation, Structure, Test, and Resource Site (HAMSTeRS) database, we have selected nine point mutations, associated with multiple cases of mild/moderate hemophilia A, where at least 10% of those patients reported have developed inhibitors. Six of the nine are in the FVIII C-domain (Arg2150His, Trp2229Cys, Tyr2105Cys, Asn2286Lys, Pro2300Leu, and Arg2307Gln) and the other three are in the A-domain (Arg593Cys, Arg1997Trp, and Glu1999Gly).

As an alternative to complete DNA sequencing or heteroduplex analysis, we have developed a more straightforward panel to rapidly test for these point mutations directly. Specific restriction endonucleases (New England BioLabs) were identified with overlapping recognition sites for each of the nine point mutations. For seven of the mutations, the restriction site is lost if a mutation occurs. For the other two, the restriction site is gained when the mutation is present. We evaluated this panel by digesting PCR amplicons from synthetic, control DNA templates (IDT), representing the wild-type and mutated FVIII exons encompassing each of the nine mutations. Differences between the resulting restriction enzyme digestion patterns were successfully detected with a BioAnalyzer DNA 1000 Kit (Agilent). A pilot study has further tested genomic DNA from eleven pediatric hemophilia A patients, all of whom were negative for each of the nine mutations. The results from testing of a larger bank of pediatric hemophilia A genomic DNA will also be presented.

A more rapid, specific molecular screen that could be performed at the initial diagnosis of mild/moderate FVIII deficiency would allow providers to more readily identify those patients at risk for developing a FVIII inhibitor. This form of “personalized medicine” could help to prevent the potentially tragic outcome of inhibitor development, which can last from months to many years, in those patients that unexpectedly convert into a severe bleeding phenotype.

Disclosures:

No relevant conflicts of interest to declare.