Screening for mutations in haemophilia A patients by multiplex PCR-SSCP, Southern blotting and RNA analysis: the detection of a genetic abnormality in the factor VIII gene in 30 out of 35 patients.

The molecular characterization of mutations in haemophilia A patients in this study was carried out by PCR-SSCP, Southern blotting, and reverse transcribed-PCR. A multiplex PCR in which four to eight exons were co-amplified was developed to reduce the time needed for screening the coding region of the factor VIII gene. PCR-SSCP was used to screen for small molecular defects, and reverse transcriptase PCR combined with Southern blotting was used to screen DNA for the inversions that occur frequently in intron 22 of the factor VIII gene. A group of 35 haemophilia A patients was analysed by these methods and 31 mutations were detected. In one patient two mutations were identified. The cases of mild and moderate haemophilia A showed changes in single nucleotides which predicted amino acid changes. The patients affected by severe haemophilia A showed two types of mutations. First, deletions or insertions that result in a frameshift in the coding DNA sequence were observed. Second, inversions were found which result in a disruption of the gene. With the screening strategies used we succeeded in elucidating an abnormality in the factor VIII gene in 30/35 haemophilia A patients.