African-Americans Express Multiple Haplotypic Forms of the Wildtype Factor VIII (FVIII) Protein: A Possible Role for Pharmacogenetics in FVIII Inhibitor Development?.

Recombinant factor VIII (r-fVIII) is the most widely-used and effective therapy for hemophilia A (hA). Many patients unfortunately become refractory when the wildtype(wt) protein is seen as foreign and is targeted by functionally neutralizing anti-fVIII antibodies termed inhibitors. While inhibitors occur most often in severe hemophiliacs with complex fVIII mutations and a complete circulating absence of the fVIII protein, recent studies show patients with missense-mutations (mMt) have a higher incidence than previously thought and demonstrate r-fVIII can be immunologically targeted even when differing by only a single amino acid. Because mMt represent the most frequent overall etiology of hA (~39% of cases), common non-hemophilic protein variants of fVIII may represent an important novel modulator of inhibitor development in this setting. To determine the extent of such variation, we resequenced all coding regions of the fVIII loci (F8) in 137 healthy subjects from 7 ethnic groups, including 86 Caucasians and 16 African-Americans (AA). We identified 5 common nonsynonymous single nucleotide polymorphisms (nsSNPs). Surprisingly, whereas 4 were polymorphic in AA only 2 were variable in Caucasians, despite having examined 6x the number AA X-chromosomes. Since recent studies show AA patients have a 2-fold higher inhibitor incidence than Caucasians, thus establishing ethnicity as a risk factor in this complication, we were intrigued to find that minor alleles for 2 nsSNPs are restricted to AA and substitute amino acids in major B-cell inhibitor epitopes located in the A2 and C2 domains. To confirm these findings and accurately define the number and frequency of human haplotypes (H) (eg. distinct combinations in which the alleles of these 5 nsSNPs are linked in vivo) we resequenced F8 in a second study group that included 75 additional healthy AA. Here we show there are at least 7 distinct wt forms of the human fVIII protein by defining 7 haplotypes (H) from the 5 nsSNPs: H1, H2, …, H7. H1 exists in all ethnic groups, is the most common overall form of fVIII and represents 2 of the 3 r-fVIII concentrates used clinically. While H2 is the most common form in AA (44%) and possibly represents the other therapeutic r-fVIII molecule, we found that at least 20% will have AA-restricted fVIII proteins; H4 (4%), H5 (12%) and H7 (4%). Only 2 forms of fVIII were found in Caucasians, H1 (90%) and H2 (10%), in contrast, and neither were ethnically restricted. In summary, when combined with reports of at least 3 other nsSNPs, our findings establish as inaccurate the long held view that fVIII is basically a monomorphic protein in non-hemophiliacs. We hypothesize that greater immunologic barriers exist when r-fVIII is infused into patients with mMt in endogenous fVIII molecules containing one or more minor alleles of these nsSNPs. Moreover, due to the number and frequency of AA-restricted wt fVIII variants, we predict these nsSNPs contribute pharmacogenetically to the higher incidence of inhibitors in this ethnic group. As such, we have established a 6-site multi-center study to test this hypothesis using AA hA patients as the optimal group. So far we have enrolled 34 of 223 total AA hA patients at the participating centers and are obtaining blood samples from each for fVIII:C and fVIII:Ag measurements and F8 mutation detection. Because the allelic basis of hA has not been studied in AA, this mutation scan is essential to rule out the plausible alternative hypothesis that their higher inhibitor incidence is due to a distinct spectrum of molecular abnormalities. Ultimately, all patients with mMt will be tested to determine the presence and titer of anti-fVIII, using both Bethesda and ELISA assays, and the H of their mutant fVIII will be defined. The number of patients with inhibitors and AA-restricted forms (H4, H5 or H7) will then be compared to those with inhibitors but whose mMt are in haplotypes found in r-fVIII molecules (H1 or H2).