title: [Molecular analysis of a patient with hemophilia A caused by FVIII His99Arg mutation].
abstract: OBJECTIVE
  To investigate the molecular mechanism of a Chinese hemophilia A patient in whom there was a discrepancy between the clinical bleeding symptoms and laboratory assay of FVIII activity (FVIII: C).
  
  
  METHODS
  FVIII: C was detected by chromogenic and one-stage methods, and FVIII: Ag by ELISA. The APTT corrected test was used to screen the FVIII inhibitor and PCR amplification to analyze all the exons and flanking sequences of F8 gene of the proband, PCR products were purified and sequenced directly. The corresponding gene sites of family members were detected according to the gene mutation sites. Two B domain deleted human FVIII mutant expression plasmids His99Arg and His99Ala (pRC/RS V - BDhFVIIIcDNA) were constructed and transfected into HEK293T transiently. FVIII: Ag and FVIII: C of the expression products were assayed.
  
  
  RESULTS
  The proband APTT was prolonged, FVIII: Ag was 120% but FVIII: C <1% and no FVIII inhibitor in plasma. The results of anticoagulation and fibrinolytic functions were normal. The cross reacting material positive (CRM+) hemophilia A was diagnosed. Gene analysis revealed a A28828G substitution in exon 3 resulted in a H (His) to R (Arg) missense mutation and the same heterozygous was identified in his mother. In vitro expression of FVIII: Ag and FVIII: C of His99Arg were 180.0% and 5.8% , respectively, while FVIII: Ag and FVIII: C of His99Ala were 45.0% and 20.0% of that of wild type, respectively. His99Arg and His99Ala were diagnosed as CRM+ and CRM- mutations, respectively.
  
  
  CONCLUSIONS
  Both the two F VIII mutations could express FVIII protein. However, CRM His99Arg mutant protein has little FVIII procoagulant activity and His99Ala has reduced FVIII function by routine methods.
authors.affiliation: State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital of Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
authors.forename: Huan-huan
authors.initials: HH
authors.lastname: Qin
authors.forename: Xue-feng
authors.initials: XF
authors.lastname: Wang
authors.forename: Qiu-lan
authors.initials: QL
authors.lastname: Ding
authors.forename: Ye-ling
authors.initials: YL
authors.lastname: Lu
authors.forename: Jing
authors.initials: J
authors.lastname: Dai
authors.forename: Xiao-dong
authors.initials: XD
authors.lastname: Xi
authors.forename: Hong-li
authors.initials: HL
authors.lastname: Wang
mesh_terms: D000328:Adult; D004252:DNA Mutational Analysis; D005169:Factor VIII / Q000235:genetics*; D005838:Genotype; D006467:Hemophilia A / Q000209:etiology* / Q000235:genetics*; D006801:Humans; D008297:Male; D020125:Mutation, Missense
chemical_list: D005169:Factor VIII