title: Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients.
abstract: The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversion of factor VIII occurred in 13 of 21 patients affected by severe hemophilia A. One patient showed a large extra band in addition to the three bands observed after Southern blotting with the F8A probe. An abnormal electrophoretic pattern of SSCP was detected in 85% and 50% of the patients affected by mild-to-moderate and severe disease, respectively. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This study shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemophilia A patients.
authors.affiliation: Department of Internal Medicine, State University of Campinas, UNICAMP, SP, Brazil.
authors.forename: V R
authors.initials: VR
authors.lastname: Arruda
authors.forename: W C
authors.initials: WC
authors.lastname: Pieneman
authors.forename: P H
authors.initials: PH
authors.lastname: Reitsma
authors.forename: P P
authors.initials: PP
authors.lastname: Deutz-Terlouw
authors.forename: J M
authors.initials: JM
authors.lastname: Annichino-Bizzacchi
authors.forename: E
authors.initials: E
authors.lastname: Briët
authors.forename: F F
authors.initials: FF
authors.lastname: Costa
mesh_terms: D001483:Base Sequence; D001938:Brazil; D005091:Exons / Q000235:genetics; D005169:Factor VIII / Q000235:genetics*; D006467:Hemophilia A / Q000208:ethnology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017384:Sequence Deletion; D016622:Silver Staining
chemical_list: D005169:Factor VIII