A directed search for mutations in hemophilia A using restriction enzyme analysis and denaturing gradient gel electrophoresis.  A study of seven exons in the factor VIII gene of 170 cases. 
Genomic DNA from 170 unrelated hemophilia A patients was examined for gene defects in the coding region of the Factor VIII gene.  Exons 18, 22-24 and 26 contain a CGA codon for arginine within the recognition sequence for the restriction enzyme Taq I. These five sites were amplified by the polymerase chain reaction and tested for abnormal Taq I restriction patterns.  In five cases, the enzyme Taq I failed to digest the amplified fragments.  Direct sequencing of the amplified products demonstrated a C to T transition in the coding strand of exons 18, 22 and 24 in three severe hemophilia A patients resulting in TGA termination codons.  Two patients showed G to A transition in exons 24 and 26 reflecting a C to T transition in the non-coding strand substituting a glutamine for an arginine.  Three deletions involving exon 26 and one exons 23-26 were found in severe hemophiliac patients.  In contrast, exons 23 and 24 failed to amplify in one patient with a moderate form of the disease suggesting an in-frame splicing of exons 22 and 25.  Exon 8 and the 3' end of exon 14 were analyzed by denaturing gradient gel electrophoresis (DGGE).  Two patients with a moderate form of the disease demonstrated an abnormal electrophoretic pattern in exon 8 and sequencing demonstrated missense mutations at codon 372 for arginine within a thrombin activation site.  One missense mutation was a C to T transition substituting cysteine for arginine and the other was an infrequent G to C transversion at an adjacent nucleotide changing the same arginine to proline.(ABSTRACT TRUNCATED AT 250 WORDS)