title: 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.
abstract: 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)
authors.affiliation: INSERM U143, International Hemophilia Training Center, Hôpital Bicêtre, Le Kremlin Bicêtre, France.
authors.forename: J M
authors.initials: JM
authors.lastname: Lavergne
authors.forename: B R
authors.initials: BR
authors.lastname: Bahnak
authors.forename: M
authors.initials: M
authors.lastname: Vidaud
authors.forename: Y
authors.initials: Y
authors.lastname: Laurian
authors.forename: D
authors.initials: D
authors.lastname: Meyer
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005169:Factor VIII / Q000235:genetics*; D005796:Genes; D006467:Hemophilia A / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009691:Nucleic Acid Denaturation; D015183:Restriction Mapping
chemical_list: D005169:Factor VIII