Identification and functional analysis of a novel missense mutation Ser250Phe underlying congenital coagulation factor Ⅶ deficiency / 中华医学遗传学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To identify and characterize a missence mutation Ser250 Phe underlying coagulation factor Ⅶ (FⅦ) deficiency in a Chinese patient and his family.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>