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

Nouv Rev Fr Hematol (1978). 1992;34(1):85-91.


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)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Mutational Analysis*
  • Electrophoresis, Polyacrylamide Gel
  • Exons
  • Factor VIII / genetics*
  • Genes
  • Hemophilia A / genetics*
  • Humans
  • Male
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Denaturation
  • Restriction Mapping


  • Factor VIII