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.

Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500,000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.

Factor VIII C domains contain key binding sites for von Willebrand factor (vWF) and phospholipid membranes. Hemophilic patients were screened for factor VIII C-domain mutations to provide a well-characterized series. Mutated residues were localized to the high-resolution C2 structure and to a homology model of C1. Of 30 families found with mutations in the C domains, there were 14 missense changes, and 9 of these were novel. Of the missense mutations, 10 were associated with reduced vWF binding and 8 were at residues with surface-exposed side chains. Six of the 10 mutants had nearly equivalent factor VIII clotting activity and antigen level, suggesting that reduced vWF binding could cause hemophilia by reducing factor VIII stability in circulation. When the present series was combined with previously described mutations from an online international database, 11 C1 and C2 mutations in patients with mild or moderately severe hemophilia A were associated with antibody-inhibitor development in at least one affected individual. Of these substitutions, 6 occurred at surface-exposed residues. As further details of the C1 structure and its interface with C2 become available, and as binding studies are performed on the plasma of more patients with hemophilic C-domain mutations, prediction of surface binding sites should improve, allowing confirmation by site-specific mutagenesis of surface-exposed residues.

Summary. Factor VIII's exon 14 codes for its B domain that includes nearly one-third of its amino acid sequence that is not necessary for function. Frameshift mutations appear to occur more frequently within exon 14 than in other exons. To assess exon 14 frameshift mutations and their clinical correlates, a series of unrelated, severe or moderately severe haemophilia A patients were screened for heteroduplex formation in amplified exon 14 fragments. In 25 families, a frameshift mutation was identified by sequencing. Occurrence of haemophilia was isolated in 18 of these families. Moderate severity was noted in at least six out of 13 families with an A insertion or deletion at one of two sequences where the frameshift resulted in a sequence of 8±10 As. Inhibitors occurred in five of the other 12 families including one with an A insertion within a sequence of six As. Recurrent insertions into an A 8 (codons 1439±1441) or an A 9 (codons 1191±1194) sequence or of an A deletion from the A 9 sequence are common, recurrent causes of haemophilia A that may have a moderately severe phenotype.

SummaryHeteroduplex screening identified 74 small mutations in the factor VIII genes of 72 families with hemophilia A. In addition, patients from 3 families with high titer inhibitors had partial gene deletions and 5 unrelated families that were negative for heteroduplex formation had a mutation on direct sequencing. The latter had mild hemophilia A with an inhibitor, and sequencing their exon 23 fragments found a transition predicting a recurrent Arg2150 to His. Of 69 distinct mutations (including the 3 partial gene deletions), 47 are novel. Of small mutations, 51 were missense (one possibly a normal variant and two that could also alter splicing) at 39 sites, 13 were small deletions or insertions (3 inframe and one a normal variant in an intron), 13 were nonsense at 12 sites and 2 altered intron splice junctions. In 24 families, at least one affected member had evidence for an alloimmune response to factor VIII; of these, 11 were associated with missense mutations. In 14 families, de novo origin was demonstrated.

In genotyping a severe hemophilia B subject, exons 1-3 and 5-8 were normal. Exon 4 did not amplify, suggesting a partial gene deletion. Previously, a French family with an exon 4 deletion had severe haemophilia B with a circulating, dysfunctional factor IX protein missing its first growth factor-like domain; breakpoints were not analyzed. Using a 5' primer for exon 3 and a 3' primer for exon 5 fragments, the subject's factor IX gene amplified a 5 kb fragment whereas 11 kb was predicted, indicating a 6 kb deletion. Restriction endonucleases localized the 3' intron 4 deletion breakpoint to 1.2 kb 5' to exon 5. Sequencing through the breakpoints revealed a 5,969 bp deletion that included exon 4 and was accompanied by a 13 bp duplication inserted near the 3' breakpoint site. Haemophilia was familial; on testing, his mother was confirmed as a heterozygous carrier, whereas his sister was homozygous for the normal, larger fragments. As exons 4 and 5 of the factor IX gene are in frame, this deletion should produce a shortened transcript, missing 114 bp (38 codons from the first growth factor-like domain). Reverse transcription of mRNA prepared from whole blood and PCR identified the shorter cDNA fragment. Western blotting demonstrated a smaller factor IX protein.

Factor VIII C domains contain key binding sites for von Willebrand factor (vWF) and phospholipid membranes. Hemophilic patients were screened for factor VIII C-domain mutations to provide a well-characterized series. Mutated residues were localized to the high-resolution C2 structure and to a homology model of C1. Of 30 families found with mutations in the C domains, there were 14 missense changes, and 9 of these were novel. Of the missense mutations, 10 were associated with reduced vWF binding and 8 were at residues with surface-exposed side chains. Six of the 10 mutants had nearly equivalent factor VIII clotting activity and antigen level, suggesting that reduced vWF binding could cause hemophilia by reducing factor VIII stability in circulation. When the present series was combined with previously described mutations from an online international database, 11 C1 and C2 mutations in patients with mild or moderately severe hemophilia A were associated with antibody-inhibitor development in at least one affected individual. Of these substitutions, 6 occurred at surface-exposed residues. As further details of the C1 structure and its interface with C2 become available, and as binding studies are performed on the plasma of more patients with hemophilic C-domain mutations, prediction of surface binding sites should improve, allowing confirmation by site-specific mutagenesis of surface-exposed residues.

Haemophilia A (HA) is a bleeding disorder caused by mutations within the X-linked F8 gene. A series of 42 unrelated Moldovan patients with HA had their disease-causative mutation determined to provide clinically valuable genotyping information for a historically underserved population and to utilize factor VIII (FVIII) structural information to analyse the effects of haemophilic missense substitutions. DNA samples were analysed to detect intron 22 and intron 1 inversions followed by heteroduplex analysis of PCR-amplified fragments containing all exonic sequences. Missense sites identified by DNA sequencing were visualized in the recently described crystal structures of human FVIII. Of the 26 different point mutations, 12 were novel. Gel electrophoresis identified samples with a second major DNA band that migrated abnormally; these amplified products were sequenced. Thirteen intron 22 inversions and one intron 1 inversion were found. Two patients had large, partial gene deletions and there were six frameshift, two non-sense, two splicing and 16 missense genotypes. Two subjects with an intron 22 inversion and one with a large, partial gene deletion developed an alloimmune inhibitor. Their localization suggests intra- and possibly inter-molecular interactions that are important for the structural integrity and/or procoagulant function of FVIII.