Inhibitors of factor VIII in black patients with hemophilia

Abstract

Background: Black patients with hemophilia A (factor VIII deficiency) are twice as likely as white patients to produce inhibitors against factor VIII proteins given as replacement therapy. There are six wild-type factor VIII proteins, designated H1 through H6, but only two (H1 and H2) match the recombinant factor VIII products used clinically. H1 and H2 are found in all racial groups and are the only factor VIII proteins found in the white population to date. H3, H4, and H5 have been found only in blacks. We hypothesized that mismatched factor VIII transfusions contribute to the high incidence of inhibitors among black patients.

Methods: We sequenced the factor VIII gene (F8) in black patients with hemophilia A to identify causative mutations and the background haplotypes on which they reside. Results from previous Bethesda assays and information on the baseline severity of hemophilia, age at enrollment, and biologic relationships among study patients were obtained from review of the patients' medical charts. We used multivariable logistic regression to control for these potential confounders while testing for associations between F8 haplotype and the development of inhibitors.

Results: Of the 78 black patients with hemophilia enrolled, 24% had an H3 or H4 background haplotype. The prevalence of inhibitors was higher among patients with either of these haplotypes than among patients with haplotype H1 or H2 (odds ratio, 3.6; 95% confidence interval, 1.1 to 12.3; P=0.04), despite a similar spectrum of hemophilic mutations and degree of severity of illness in these two subgroups.

Conclusions: These preliminary results suggest that mismatched factor VIII replacement therapy may be a risk factor for the development of anti-factor VIII alloantibodies.

Conflict of interest statement

Dr. Abshire reports serving on the advisory boards for CSL Behring, Novo Nordisk, and Bayer; Dr. Kasper, serving on a data and safety monitoring board for Wyeth and receiving grant support from CSL Behring; and Dr. Thompson, participating as a site investigator for Baxter, serving as a consultant for Ipsen, and receiving grant support from Bayer. Dr. Howard is the co-founder of Haplomics, which owns a patent application with claims for novel proteins and diagnostic methods that may be useful in treating patients with hemophilia A. No other potential conflict of interest relevant to this article was reported.

2009 Massachusetts Medical Society

Figures

Figure 1. Four Nonsynonymous Single-Nucleotide Polymorphisms (SNPs) Whose Haplotypes Encode Six Distinct Factor VIII Proteins, Designated H1 through H6

Human F8 contains four common nonsynonymous SNPs whose allelic combinations encode six distinct wild-type factor VIII proteins, only two of which have the amino acid sequences found in the recombinant factor VIII molecules used clinically. Panel A shows a schematic illustration of both F8, with its 26 exons and 25 introns indicated by red triangles and intervening lines, respectively, and factor VIII, with highlighting of its three A domains (A1, A2, and A3, shown in gray), single B domain (B, in blue), two C domains (C1 and C2, yellow), three acidic connecting peptides (a1, a2, and ap, black), and two immunodominant-inhibitor epitopes located in the A2 domain (red oval) and the C2 domain (blue oval). By sequencing all 26 exons of the F8 genes in 137 unrelated healthy persons from seven groups of diverse geographic origins, we identified four nonsynonymous SNPs: one in exon 10 (G1679A), two in exon 14 (A2554G and C3951G), and one in exon 25 (A6940G). These polymorphisms encode the following amino acid substitutions, respectively: histidine for arginine at position 484 (R484H), glycine for arginine at position 776 (R776G), glutamic acid for aspartic acid at position 1241 (D1241E), and valine for methionine at position 2238 (M2238V). The numbering systems used to designate the four nonsynonymous SNPs and the amino acid substitutions they encode are based on their nucleotide and residue locations, respectively, in the full-length F8 complementary DNA (with the use of the transcription start site found by Mansvelt et al.20) and the mature circulating form of factor VIII. Whereas R776G and D1241E are located in the B domain, R484H and M2238V are components of the A2 and C2 immunodominant epitopes, respectively, which have been mapped to residues located at epitopes R484 to I508 (isoleucine at position 508) and E2181 to V2243. Panel B shows the six structurally distinct wild-type factor VIII proteins encoded by the naturally occurring allelic combinations (haplotypes) of the F8 nonsynonymous SNPs G1679A, A2554G, C3951G, and A6940G. The amino acid residue at positions 484 (R or H), 776 (R or G), 1241 (D or E), and 2238 (M or V) are shown. The haplotype frequencies (f) listed for the six factor VIII proteins (H1 through H6) are based on their occurrence in 86 white (fwhite), 67 black (fblack), and 10 Chinese (fchinese) subjects., In Panel C, the two full-length recombinant factor VIII proteins used in replacement therapy, Kogenate and Recombinate, contain the same amino acid sequences found in H1 (R–R–D–M) and H2 (R–R–E–M), respectively.-

Figure 2. Hemophilic Mutations and the Four Wild-Type Factor VIII Proteins Predicted to Be Encoded by the Background F8 Haplotypes on Which They Were Identified

For factor VIII, the two immunodominant-inhibitor epitopes located in the A2 domain (red oval) and the C2 domain (blue oval) are shown. Mutations found in patients with either an H1 or an H2 haplotype (H1+H2) are shown in Panel A, and mutations found in patients with either an H3 or an H4 haplotype (H3+H4) are shown in Panel B. For all haplotypes, missense mutations are shown above the appropriate factor VIII protein, and the other mutation types are shown below. Missense and nonsense mutations are indicated by their residue positions in the mature factor VIII protein. The point mutation T38039C, which occurs at position +2 of the 5′ splice site (SS) of intron 6, is designated according to the genomic nucleotide numbering system used for the F8 reference sequence. The positions of four frameshift (FS)-inducing small deletions and insertions are numbered according to their locations in the full-length F8 complementary DNA (c) with respect to the transcription start site. Specifically, one deletion (c.4292ΔTAGA) and three insertions (c.3809InsA, c.4551InsA, and c.4291InsATAGA) are indicated by the number of the wild-type nucleotide positioned immediately 5′ of the mutation site. ΔEx13 indicates an in-frame deletion of the 210-bp exon 13 sequence and an unknown amount of flanking nonexonic sequences from introns 12 and 13. For those mutations that occurred in more than one patient, whether or not the patients were related, the number of times any given abnormality was observed (N) is indicated in parentheses. All previously unknown mutations are indicated with an asterisk. The baseline severity of hemophilia for each patient is shown by the color of the text defining his mutation, with black, blue, and red indicating severe, moderate, and mild disease, respectively. For those mutations found in at least one inhibitor-positive (Inh[+]) patient, the number of patients with a given abnormality in whom inhibitors developed is also indicated in parentheses. A 3′-terminal partial gene deletion involving exons 24, 25, and 26 in two inhibitor-positive brothers is not shown. D, E, H, M, R, and V denote the amino acids aspartic acid, glutamic acid, histidine, methionine, arginine, and valine, respectively.