Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A

Abstract

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...

A recurrent F8 mutation (c.6046C>T) causing hemophilia A in 8% of northern Italian patients: evidence for a founder effect

“…Another common F8 gene defect is the intron 1 inversion that causes 1–6% of all severe cases of hemophilia A (Bagnall et al. 2002). …”

Heritable skewed X-chromosome inactivation leads to haemophilia A expression in heterozygous females

“…To screen for additional F8 gene mutations, samples were tested for the intron 1 inversion 14 and analysed by conformation sensitive gel electrophoresis (CSGE) 15 by Dr Lillicrap, National Program for Hemophilia Mutation Testing, Department of Pathology and Molecular Medicine, Kingston, Ontario. Together with the F8 intron 22 inversion test, these techniques detect 85 -90% of F8 gene mutations (Dr Lillicrap, personal communications).…”

Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A

“…Intron 1 inversions were detected by polymerase chain reaction (PCR), as reported earlier (Bagnall et al 2002). Long PCR for detecting the intron 22 inversion was performed as previously described (Liu et al 1998).…”