Objective: To detect gene mutations in hemophilia A in Shanghai.
Methods: PCR, denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used. Fifty Chinese cases of hemophilia A without intron 22 inversion, including 24 severe, 9 moderate and 17 mild cases, were screened. Genomic DNA was amplified using GC-clamped primers covering all the exons and flanking intron regions, excluding the middle portion of extron 14 encoding the B domain, but including the thrombin cleavage sites at AA740 and 1689. The amplified GC-clamped PCR fragments were then electrophoresed on DGGE. The abnormal bands were sequenced.
Results: Eleven different mutations were identified, including 5 nonsense mutations, 5 missense mutations and one small deletion. Among them, 3 mutations, 466Lys (AAG)-Thr (ACG), 719Tyr (TAC)-Stop (TAG) and 312Ile (ATC)-xxC have not been reported before.
Conclusion: Apart from intron 22 inversion, most gene mutations in hemophilia A were point mutations resulted from single base substitution. Generally the genetic defects correspond to the clinical manifestations.