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.
Ruan
C
C
Jiangsu Institute of Hematology, Suzhou Medical College, China.
Gu
J
J
Lavergne
J M
JM
Meyer
D
D
D000293:Adolescent; D001120:Arginine / Q000235:genetics*; D003545:Cysteine / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D005169:Factor VIII / Q000235:genetics*; D006467:Hemophilia A / Q000235:genetics*; D006801:Humans; D008297:Male; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D005169:Factor VIII; D001120:Arginine; D003545:Cysteine