Detection by denaturing gradient gel electrophoresis of an Arg1689Cys mutation in a Chinese patient with mild hemophilia A.
OBJECTIVE: To detect gene defects of factor VIII (F VIII) in Chinese hemophilia A patients. METHODS: 3' end of exon 14 of F VIII gene from a mild hemophilia A patient of Chinese origin was amplified by polymerase chain reaction (PCR) and identified mutations by denaturing gradient gel electrophoresis (DGGE) combining with direct sequencing. RESULTS: An upward shift band was detected by DGGE in W381. Direct sequencing demonstrated a C to T transition resulting in substitution of Arg1689Cys within a thrombin activation site of mature F VIII protein, which created a unique a thrombin activation site of mature F VIII protein, which created a unique PstI site in amplified fragment of F VIII. CONCLUSIONS: The association of PCR and DGGE can detect a single base substitution; the Arg1689Cys mutation that inhibited activation of F VIII by thrombin is a molecular defect associated with hemophilia A in W381.