HEMOPHILIA-B LEYDEN - THE EFFECT OF MUTATIONS AT POSITION -SPECIFIC TRANSCRIPTION OF THE FACTOR-IX GENE(13 ON THE LIVER)

Haemophilia B Leyden is characterized by low plasma levels (less-than- or-equal-to 1-13% of normal) of blood coagulation factor IX during chi ldhood. After the onset of puberty, plasma factor IX levels gradually rise, probably under the influence of androgens. Single point mutation s have been detected in the factor IX promoter at -21, -20, -6, -5, +6 , +8 and +13. This paper examines how two of these mutations (a deleti on of an A, and an A-->G substitution at +13) interfere with normal fa ctor IX gene transcription. It is shown that both mutations do impair factor IX promoter activity in transiently transfected HepG2 cells. Th e mutations at +13 lie in a region (+1 to +18) that is considered to c ontain a binding site for the CCAAT/enhancer binding protein. Transact ivation by the CCAAT/enhancer binding protein a of the wild-type and m utated factor IX promoters (-192 to +38) resulted in an approximately four-fold and approximately two-fold, respectively, increase of CAT ac tivity. Gel mobility shift assays revealed that the binding of the CCA AT/enhancer binding protein alpha is disrupted by both the deletion of an A, and the A-->G substitution at +13. The role of two additional b ZIP factors, D-site binding protein and liver-enriched transcriptional activator protein, in the binding and activation of the + 13 factor I X promoter region was examined. The D-site binding protein binds to th e factor IX promoter region (+1 to +18) in gel mobility shift assays. The deletion of an A at +13 does not interfere with the binding of the D-site binding protein. Transactivation by D-site binding protein of the wild-type promoter resulted in an approximately three-fold increas e of CAT activity in HepG2 cells. Neither the deletion of an A at +13 nor the G-->C mutation at -6 (also associated with the haemophilia B L eyden phenotype) reduced the responsiveness of the factor IX promoter to the D-site binding protein. The liver-enriched transcriptional acti vator protein could not activate the wild-type factor IX promoter nor was it capable of increasing the responsiveness of the promoter to CCA AT/enhancer binding protein alpha.