Two novel mutations in the human coagulation factor VII promoter

The factor VII genes of five unrelated Finnish female patients, F1-F5, with moderate bleeding tendency, were screened for mutations using single strand conformational polymorphisms and DNA sequencing. Heterozygous shifts were detected in exons 5 and 8 for patient F1, and sequencing confirmed the presence of the silent dimorphism H115H, the polymorphism R353Q and the mutation A294V. The patient F1 was also heterozygous for a novel -59T/G transversion mutation in the Hepatocyte nuclear factor 4-binding site. The remaining four patients carried a -32A/C transversion mutation located in a footprint (-51 to -32) covering the major transcription initiation start site -51). There was also a consensus sequence match to an initiator response-like binding element covering -51. Two patients were homozygous and two heterozygous for this mutation. Plasma FVII:Ag and FVII:C levels were reduced in parallel. A strong reduction in binding affinity of a specific nuclear protein to the -32C-containing oligonucleotide was found by electrophoretic mobility shift assays on nuclear extracts from HepG2 cells. EDTA caused no reduced binding. A minimal promoter (-191 to +15) containing the wild-type sequence or the -32A/C or -59T/G mutations was cloned in front of the firefly luciferase reporter gene and transiently transfected into Hep3B cells. Reduced activities [23.0 +/- 3.1% (-32C), 55.4 +/- 6.3% (-59G), 100% (wild-type construct)] were found for the mutated promoters. Southwestern blotting and UV crosslinking analysis showed binding of three proteins (20, 20 and 50 kDa) to the putative initiator response element. The -32A/C mutant oligonucleotide bound two proteins.