A possible mechanism for Inv22‐related F8 large deletions in severe hemophilia A patients with high responding factor VIII inhibitors

Summary. Background: Intron 22 inversion (Inv22) of the coagulation factor (F)VIII gene (F8) is a frequent cause of severe hemophilia A. In addition to Inv22, a variety of F8 mutations (1492 unique mutations) causing hemophilia A have been reported, of which 171 involve deletions of over 50 bp (HAMSTeRs database; http://hadb.org.uk/ ). However, only 10% of these large deletions have been fully characterized at the nucleotide level. Patients and methods: We investigated gene abnormalities in three unrelated severe hemophilia A patients with high titer FVIII inhibitors. They had previously been shown to carry large deletions of the F8, but the precise gene abnormalities remain to be elucidated. Results: Inverse shifting‐PCR (IS‐PCR) Inv22 diagnostic tests revealed that these patients carried either type I or II Inv22. However, they showed a wild‐type (WT) pattern in the IS‐PCR Inv22 complementary tests. We further analyzed their X chromosomes to account for the puzzling results, and found that they had different centromeric breakpoints in the Inv22 X chromosomes, adjacent to the palindromic regions containing int22h‐2 or ‐3, and their spacer region, respectively. The connections appeared to be shifted towards the telomere of the WT F8 Xq28, resulting in a new telomere with an additional intact int22h copy. Conclusions: These gene rearrangements might result from double‐strand breaks in the most distal regions of the long arms of the Inv22 X chromosomes, followed by DNA restorations using the WT F8 Xq28 by non‐homologous end joining or break‐induced replication; thus leading to large F8 deletions in severe hemophilia A patients.