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Anita Kavlie, Leena Hiltunen, Vesa Rasi, Hans Prydz
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.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 90, 08/2003
Pages: 194 - 205