title: Two novel mutations in the human coagulation factor VII promoter.
abstract: 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.
authors.affiliation: Biotechnology Centre of Oslo, Gaustadalléen 21, N-0349 Oslo, Norway.
authors.forename: Anita
authors.initials: A
authors.lastname: Kavlie
authors.forename: Leena
authors.initials: L
authors.lastname: Hiltunen
authors.forename: Vesa
authors.initials: V
authors.lastname: Rasi
authors.forename: Hans
authors.initials: H
authors.lastname: Prydz
mesh_terms: D000328:Adult; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins; D004492:Edetic Acid / Q000494:pharmacology; D004586:Electrophoresis; D005167:Factor VII / Q000235:genetics*; D005168:Factor VII Deficiency / Q000235:genetics*; D005260:Female; D051557:Hepatocyte Nuclear Factor 4; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008875:Middle Aged; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D018807:Polymorphism, Single-Stranded Conformational; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017186:Titrimetry; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002614:Chelating Agents; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D014157:Transcription Factors; D005167:Factor VII; D004247:DNA; D004492:Edetic Acid