CHARACTERISTIC MESSENGER-RNA ABNORMALITY FOUND IN HALF THE PATIENTS WITH SEVERE HEMOPHILIA-A IS DUE TO LARGE DNA INVERSIONS

Surprisingly half of all severe haemophilia A patients have no mutatio n in the promoter, coding sequences and normal RNA processing signals of the factor VIII gene. Instead they manifest a unique mRNA defect th at prevents the amplification of the message across the boundary betwe en exon 22 and 23. This locates the defect to internal regions of intr on 22. Novel sequences 3' to exon 22 were isolated from the 9 availiab le patients with the above abnormality by combining RACE and vectorett e amplifications on trace amounts of mRNA. This showed that exons 1 - 22 of the factor VIII mRNA had become part of a hybrid message contain ing new multi exonic sequences expressed in normal cells. The novel se quences were not located in a YAC covering the whole factor VIII gene. Southern blots from patients probed by novel sequences and clones cov ering intron 22 showed no obvious abnormalities. This suggested invers ions involving intron 22 repeated sequences. Screening of 3 YAC librar ies with the novel sequences located them at least 200 kb telomeric (5 ') to factor VIII and pulsed field gel analysis detected abnormal band s in patients. This demonstrates that the mutations in the patients ar e inversions of long DNA regions possibly involving the repeated seque nces and occurring at the surprising rate of approximately 4 x 10(-6) per gene per gamete per generation.