Direct sequencing of segments of the factor VIII gene in 30 hemophiliacs with sporadic disease (32+ kb of sequence in total) revealed two missense transitions: glutamate 1704 to lysine (E17~ in a patient with severe hemophilia A and proline 2300 to serine (p2300__> S) in a patient with mild hemophilia. Both transitions are likely to be causative mutations because the amino acids affected were evolutionarily conserved. Haplotype and sequence analysis of the mother and grandparents of patient HA12 (E17~ indicate that the mutation arose in the grandfather who was 27 years old when his daughter was conceived. The origin of mutation in patient HA39 (p23~176 could not be determined. As mutations that cause mild disease can be found in seemingly unrelated families, 96 unrelated hemophiliacs were screened rapidly for the p23~176 mutation with polymerase chain reaction (PCR) amplification of specific alleles (PASA). None of these patients had the mutation. PASA was also used to conveniently assess a polymorphic site in intron 7. The polymorphism is estimated to be informative in 13% of Korean females and in 23% of Western European females. region, exon 1, the 3' portion of exon 14, exon 23, and the coding region of exon 26. The promoter region was chosen because of the possibility of also finding polymorphisms that could account for the threefold variation in protein expression that has been linked to the factor VIII gene (Filippi et al. 1984). Exon 1 was chosen because one of the patients with severe hemophilia A died in infancy of unexplained liver failure and we speculated that a defect in the signal peptide might produce cytotoxicity by retargeting factor VIII into an intracellular compartment. The remaining regions were chosen because they had clusters of CpG dinucleotides, known hotspots of mutation. The 3' region of exon 14 was also chosen because it contains a thrombin activation site and a putative yon Willebrand binding site (Eaton et al. 1986;Foster et al. 1988).
Two missense mutations were found: E17~ which is 15 residues carboxy to a thrombin activation site and p23~176 which is 32 residues from the carboxy terminus. In addition to reporting the two mutations, we define one origin of mutation and present a rapid method of detection of the recently defined polymorphism in intron 7 (Kogan and Gitschier 1990;Traystman et al. 1990).