title: [Identification and functional analysis of a novel missense mutation Ser250Phe underlying congenital coagulation factor Ⅶ deficiency].
abstract: OBJECTIVE
  To identify and characterize a missence mutation Ser250 Phe underlying coagulation factor Ⅶ (FⅦ) deficiency in a Chinese patient and his family.
  
  
  METHODS
  The FⅦ gene (F7) was analyzed by DNA sequencing, and the FⅦ levels (including antigen and activity) in patient's plasma were determined with enzyme-linked immunoabsorbent assay (ELISA) and one stage prothrombin time based method. In addition, a FⅦ-250 Phe mutant corresponding to the identified mutation was expressed in HEK293 cells, and a subcellular localization experiment in CHO cells was performed to clarify the molecular mechanism of FⅦ deficiency caused by the FⅦ-250 Phe mutation.
  
  
  RESULTS
  The patient had a prolonged prothrombin time (PT: 36.5 s) and low levels of both FⅦ antigen and activity (130.2 ng/mL and 4.0%, respectively). Two heterozygous mutations were identified in the F7 gene (NG-009262.1), which included a g.15975 G>A mutation at the splice receptor site of intron 6 (IVS6-1G>A) and a novel g.16750 C>T mutation in exon 8, which resulted in replacement of Ser (TCC) 250 with Phe (TTC)250 in the vicinity of a charge-stablizing system. By gene expression experiments, the antigen and activity levels of FⅦ-250 Ser and FⅦ-250 Phe in the culture medium were (37.77 ± 2.30) ng/mL and (4.02 ± 0.52) ng/mL, respectively. ELISA and Western blotting analyses indicated that expression of the mutant FⅦ-250 Phe and wild type FⅦ-250 Ser was (130.51 ± 2.32) ng/mL and (172.45 ± 2.25) ng/mL, respectively. FⅦ-250 Phe was found in endoplasmic reticulum and Golgi apparatus, suggesting that the mutant FⅦ-250 Phe could be normally synthesized in the cells but was inefficiently secreted.
  
  
  CONCLUSIONS
  Compound heterozygous mutations in F7 gene (g.15975G>A and g.16750C>T) may be responsible for the FⅦ deficiency in this patient. The novel FⅦ 250 Phe can be transported from endoplasmic reticulum to Golgi apparatus, but may be degraded or inefficient.
authors.affiliation: Department of Laboratory Medicine, Second Affiliated Hospital, Wenzhou Medical College, Zhejiang 325027, P.R. China.
authors.forename: Ming-hua
authors.initials: MH
authors.lastname: Jiang
authors.forename: Zhao-yue
authors.initials: ZY
authors.lastname: Wang
authors.forename: Zi-qiang
authors.initials: ZQ
authors.lastname: Yu
authors.forename: Jian
authors.initials: J
authors.lastname: Su
authors.forename: Li-juan
authors.initials: LJ
authors.lastname: Cao
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000328:Adult; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D005167:Factor VII / Q000235:genetics* / Q000502:physiology; D005168:Factor VII Deficiency / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D020125:Mutation, Missense
chemical_list: D005167:Factor VII