11 NOVEL MUTATIONS IN THE FACTOR-VIII GENE FROM BRAZILIAN HEMOPHILIA-A PATIENTS

The molecular characterization of the mutations in hemophilia A patien ts is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protoc ol involving multiplex polymerase chain reaction in which 19 exons wer e amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutatio ns. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) l ocated in intron 22 of the factor VIII gene and two copies close telom eric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversi on of factor VIII occurred in 13 of 21 patients affected by severe hem ophilia A. One patient showed a large extra band in addition to the th ree bands observed after Southern blotting with the F8A probe. An abno rmal electrophoretic pattern of SSCP was detected in 85% and 50% of th e patients affected by mild-to-moderate and severe disease, respective ly. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This stu dy shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemoph ilia A patients. (C) 1995 by The American Society of Hematology.