The messenger RNA (mRNA) from 5 of 69 patients with severe hemophilia A did not support amplification of complementary DNA containing the first few exons of the factor VIII (F8) gene but supported amplification of mRNA containing exon 1 of F8 plus exons of the VBP1 gene. This chimeric mRNA signals an inversion breaking intron 1 of the F8 gene. Using an inversion patient, one deleted for F8 exons 1 to 6, and cosmids mapped 70 to 100 kb telomeric of the F8 gene, this study shows that this break strictly affects a sequence (int1h-1) repeated (int1h-2) about 140 kb more telomerically, between the C6.1A and VBP1 genes. The 1041-base pair repeats differ at a single nucleotide (although int1h-2 also showed one polymorphism) and are in opposite orientation. The results demonstrate that they cause inversions by intrachromosome or intrachromatid homologous recombination. The genomic structure of the inversion region shows that transcription traverses intergenic spaces to produce the 2 chimeric mRNAs containing the F8 sequences and characteristic of the inversion. This observation prompts the suggestion that nature may use such extended transcription to test whether the addition of novel domains from neighboring genes creates desirable new genes. A rapid polymerase chain reaction test was developed for the inversion in both patients and carriers. This has identified 10 inversions, affecting F8 genes with 5 different haplotypes for the BclI, IntroductionHiguchi et al 1,2 observed that thorough screening of all the exons of the factor VIII (F8) gene was efficient in detecting the mutations of patients with mild and moderate hemophilia A and yet failed in 50% of patients with severe disease. Traces of factor VIII messenger RNA (mRNA) from peripheral lymphocytes soon revealed that this high failure rate was largely due to mutations affecting internal regions of intron 22 of the F8 gene. 3 These mutations were later shown to be inversions resulting from homologous intrachromatid or intrachromosome (ie, intranemic) recombination between a 9503-base pair (bp) sequence (int22h-1) in intron 22 of the F8 gene and one or other of 2 inverted copies of this sequence (int22h-2, int22h-3) located, respectively, 500 and 600 kb more telomeric. [4][5][6] The int22h-related inversions appeared to be sufficiently frequent to account for the shortfall in mutation detection experienced using methods based on the screening of all exons of the F8 gene. However, analysis of factor VIII mRNA continued to detect further mutations that escape detection by this approach. Base substitutions deep inside introns were found that generate novel exons disrupting the factor VIII coding sequence 7 (R. D. Bagnall, unpublished observations, January 2001). Moreover, an inversion was identified that broke the F8 gene at intron 1 and resulted in the production of 2 chimeric mRNAs. 8 One of these mRNAs, presumably under the control of the factor VIII promoter, contains the first exon of the F8 gene followed by facultative exons and then exons 2 to 6 of a gene (VBP...
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.
The mRNA sequence of the human intrinsic clotting factor IX (Christmas factor) has been completed and is 2802 residues long, including a 29 residue long 5′ non‐coding and a 1390 residue long 3′ non‐coding region, but excluding the poly(A) tail. The factor IX gene is approximately 34 kb long and we define, by the sequencing of 5280 residues, the presumed promoter region, all eight exons, and some intron and flanking sequence. Introns account for 92% of the gene length and the longest is estimated to be 10 100 residues. Exons conform roughly to previously designated protein regions, but the catalytic region of the protein is coded by two separate exons. This differs from the arrangement in the other characterized serine protease genes which are further subdivided in this region.
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