Identification of mutations in two families with sporadic hemophilia A

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 (E1704----K) 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 (E1704----K) 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 (P2300----S) 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 P2300----S 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.