[A novel missense mutation in the B domain of factor VIII causes severe hemophilia A]

To identify the genetic defect of a patient with severe hemophilia A (SH9).

PCR, denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to screen mutations in the factor VIII gene. Intron 22 inversion was excluded previously by Southern blotting with F8A probe. PCR primers were designed to cover all the coding regions and flanking intron sequences. Amplified products were analysed with DGGE, and bands of abnormal mobility were directly sequenced.

PCR fragment 14-2 showed slower mobility than normal. A single nucleotide substitution C2535A causing a missense mutation in the B domain, 826Asp(GAC)-->Glu(GAA) was identified by DNA sequencing.

This nucleotide substitution may be the molecular etiology of SH9. The possible molecular mechanisms underlying this candidate mutation is yet to be clarified.