Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

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Summary. Background: As the publication of the sequence of the factor VIII gene (FVIII) in 1984, a large number of mutations that cause hemophilia A (HA) have been identified. Thanks to the advances in the detection of mutations, it is now possible to identify a putative FVIII sequence alteration in the vast majority of patients with HA. Objectives: Our main objective was to report on the spectrum of FVIII mutations and their distribution throughout the gene in 120 patients with HA. Methods: Screening of FVIII mutations was performed using direct sequencing. Newly described missense mutations were further studied by molecular modeling. Results: A total of 47 different HA causative FVIII mutations have been identified, 26 of which are described for the first time. These novel mutations include 14 missense and six nonsense mutations, two small deletions, one large deletion and three splice-site mutations. We further investigated the development of FVIIIspecific inhibitors in all patients with HA. We found that four novel mutations (Ser882X, Tyr1786Ser, Ala2218Thr and a splice-site defect in intron 22) were associated with inhibitor development. Conclusion: These data extend our insight into the mechanisms by which novel amino acid substitutions may lead to HA, and how HA patient genotypes influence the risk of FVIII inhibitor development.

The appearance of inhibitory antibodies against factor VIII (FVIII) is the most severe and costly complication of replacement therapy in patients with haemophilia A (HA). To determine the relationship between FVIII genotype and inhibitor development, baseline FVIII activity, genotype and inhibitor development were reviewed in 1104 patients with HA. In patients with severe HA, splicing errors present the highest frequency of inhibitors, ahead of inversion of intron 1 and of intron 22, nonsense mutations and large deletions. The lowest inhibitor frequency in severe HA is found in patients with missense mutations and small deletions/insertions. Subanalyses indicate that nonsense mutations and small deletions/insertions leading to a frameshift in the light chain are associated with a significant higher risk of inhibitor formation than similar mutations occurring in the heavy chain (27% vs. 14%). These mutation types also have a higher frequency of inhibitors when occurring in exons 23-26, where a second FVIII transcript originates, compared with similar mutations in exons 1-22 (28% vs. 17%). These results suggest that complete absence of FVIII because of null mutations, including splice site mutations, or the absence of a second transcript result in an increased risk of inhibitor development.