Characteristic mRNA abnormality found in half the patients with severe haemophilia A is due to large DNA inversions

Surprisingly half of all severe haemophilia A patients haveno mutation in the promoter, coding sequences and normal RNAprocessing signals of the factor VIII gene. Instead they manifesta unique mRNA defect that prevents the amplification of themessage across the boundary between exon 22 and 23. This locatesthe defect to internal regions of intron 22. Novel sequences3' to exon 22 were isolated from the 9 availlable patients withthe above abnormality by combining RACE and vectorette amplificationson trace amounts of mRNA. This showed that exons 1 – 22of the factor VIII mRNA had become part of a hybrid messagecontaining new multi exonic sequences expressed In normal cells.The novel sequences were not located in a YAC covering the wholefactor VIII gene. Southern blots from patients probed by novelsequences 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 locatedthem at least 200 kb telomeric (5') to factor VIII and pulsedfield gel analysis detected abnormal bands in patients. Thisdemonstrates that the mutations in the patients are inversionsof long DNA regions possibly involving the repeated sequencesand occurring at the surprising rate of approximately 4 x 10^–6per gene per gamete per generation.