DISRUPTION OF A BINDING-SITE FOR HEPATOCYTE NUCLEAR FACTOR-1 IN THE PROTEIN-C GENE PROMOTER IS ASSOCIATED WITH HEREDITARY THROMBOPHILIA

A heterozygous T --> C transition was detected in the putative promote r region of the protein C (PROC) gene in a patient with type I protein C deficiency and a history of recurrent venous thrombosis. This mutat ion occurred 14 bp upstream of the transcription initiation site and w ithin a sequence strongly homologous to the consensus binding site for the liver-enriched transcription factor, hepatocyte nuclear factor 1 (HNF-1. Transfection experiments demonstrated that a CAT reporter gene construct containing 626 bp of the putative PROC gene promoter was ca pable of driving CAT expression in HepGP hepatoma cells. Levels of CAT expression from constructs bearing the mutation were found to be dras tically reduced by comparison with the wild-type, consistent with the reduced plasma protein C antigen levels observed in the patient. Gel r etardation and cotransfection experiments demonstrated that the mutati on abolished both the binding and the transactivating ability of HNF-1 observed with the wild-type PROC gene promoter. Further, the ability of the mutation to disrupt HNF-1 binding appears to be a function not only of the nature of the nucleotide substitution and its position wit hin the recognition sequence, but also of the relative affinity of the wild-type binding site for HNF-1. This analysis is therefore indicati ve of a vital role for HNF-1 in the expression of the PROC gene in viv o. Taken together with the identification of a human hepatoma cell lin e which contains HNF-1 but which does not express protein C, these fin dings are consistent with the view that HNF-1 is necessary although no t sufficient for PROC gene expression in the liver.