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%]).

Surprisingly half of all severe haemophilia A patients have no mutation in the promoter, coding sequences and normal RNA processing signals of the factor VIII gene. Instead they manifest a unique mRNA defect that prevents the amplification of the message across the boundary between exon 22 and 23. This locates the defect to internal regions of intron 22. Novel sequences 3' to exon 22 were isolated from the 9 available patients with the above abnormality by combining RACE and vectorette amplifications on trace amounts of mRNA. This showed that exons 1-22 of the factor VIII mRNA had become part of a hybrid message containing new multi exonic sequences expressed in normal cells. The novel sequences were not located in a YAC covering the whole factor VIII gene. Southern blots from patients probed by novel sequences and clones covering intron 22 showed no obvious abnormalities. This suggested inversions involving intron 22 repeated sequences. Screening of 3 YAC libraries with the novel sequences located them at least 200 kb telomeric (5') to factor VIII and pulsed field gel analysis detected abnormal bands in patients. This demonstrates that the mutations in the patients are inversions of long DNA regions possibly involving the repeated sequences and occurring at the surprising rate of approximately 4 x 10(-6) per gene per gamete per generation.

Haemophilia A is a mutationally heterogeneous disease caused by defects in the large and complex factor VIII gene. Recent studies examining the putative promoter, all exons and most intron/exon boundaries have failed to detect mutations in half the patients with severe disease leading to hypotheses such as mutations in remote controlling regions or even in genes other than factor VIII. We have amplified the factor VIII gene (putative promotor, coding region and polyadenylation/cleavage signal region) in 8 fragments from reverse transcribed mRNA and genomic DNA. Any mutation is then located by chemical mismatch detection and characterised by direct sequencing. This rapid and efficient method has been fully successful and has revealed an unusual cluster of mutations causing severe disease. Of the 28 patients we have reported, 5 had mild or moderate disease and all had a missense mutation. Twenty-three patients were severely affected and 13 of these had different detrimental mutations that were fully characterised at the genomic DNA level. The remaining 10 patients all had mRNA with exon 22 not contiguous to exon 23. Since all exons were normal and so were the splice sites of intron 22, the mutation in these patients should be in the regions of intron 22 that were not screened. These results prove that all haemophilia A cases are due to mutations of the factor VIII gene where, unexpectedly, intron 22 seems to be the target of approximately 40% of the mutations causing severe haemophilia A.

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