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Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5 untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) ϳ300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates ϳ1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide ؊44 results in a significant but small increase in promoter activity. The activity of each of the various 5 deletion constructs is significantly enhanced by cotransfection of C/EBP␣ and D-site-binding protein expression plasmids, while cotransfection of both C/EBP␣ and C/EBP␤ plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides ؊740 and ؊1002. Since the minimal promoter sequence (؊44 to ؉148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5 untranslated region.

The fifth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of < 30bp) identified in haemophilia B patients. The 1,142 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.

Studies have shown that hemophilia B (Christmas disease; factor IX deficiency) results from many different mutations in the factor IX gene, of which >95% are single nucleotide substitutions. This study has identified a previously unreported form of hemophilia B in a patient who was a somatic mosaic for a guanine-to-cysine transversion at nucleotide 31,170 in the factor IX gene. This point mutation changes the codon for residue 350 in the catalytic domain of factor IX from a cysteine to a serine. We used differential termination of primer extension to confirm and measure the degree of mosaicism. Our study shows that a varying proportion of cells from hepatic, renal, smooth muscle, and hematopoietic populations possessed normal as well as mutant factor IX sequences. These results indicate that the mutation in this patient occurred either as an uncorrected half-chromatid mutation in the female gamete or as a replication or postreplication error in the initial mitotic divisions of the zygote preceding implantation. In addition, this kindred also contains two females in successive generations who have moderately severe factor IX deficiency. The molecular pathogenesis of this latter phenomenon has been studied and seems to relate to the unaccompanied expression of the mutant factor IX gene consequent upon a second, as yet undefined, genetic event that has prevented inactivation ofsequences including the mutant factor IX gene on the X chromosome inherited from the affected male.Hemophilia B (Christmas disease) is a chromosome X-linked recessive inherited bleeding disorder that has an incidence of approximately 1 in 30,000 males (1). The disorder is a result ofeither a deficient or defective factor IX molecule, a vitamin K-dependent serine protease that participates in the intrinsic pathway of hemostasis (2). The variability of the clinical and laboratory manifestations of hemophilia B suggests that the disease is the result of many different mutations within the factor IX gene, and studies have shown that >95% of these mutations are single nucleotide substitutions (3-5).The genetic pathology responsible for hemophilia B has been the focus of intense investigation since the cloning and characterization of the factor IX gene in 1982 (6-8). Study of the patterns ofthese mutations suggests that certain recurring mild mutations appear to have arisen from a common ancestor (9), whereas other severe phenotypes result from repeated mutations often at sequences involving CpG dinucleotides (5). The origin of these latter mutations has until recently been assumed to involve replication or postreplication DNA repair errors occurring during meiosis. There is growing evidence, however, to suggest that some (perhaps many) of these previously unreported mutations arise as a result of errors in replication during mitosis in early embryogenesis (10).The studies described here were prompted by two unusual observations in a family with hemophilia B. Elucidation of the causative mutation in the one affected male in the family revea...

The data base below lists known point mutations and short deletions and additions in the factor IX gene, causing the bleeding disorder haemophilia B or Christmas disease (for reviews, see Brownlee 1988, Giannelli 1989, Thompson 1990, Green et al 1991a These mutations result in a defective clotting factor IX-a glycoprotein of 415 amino acid residues normally present in plasma and an essential component of the clotting cascade. The disease is an X-linked inherited recessive disorder affecting 1 in about 30,000 males and only very rarely females.The purpose of this database is to update last year's one (Giannelli et al, 1991) by collecting in an accessible, summary form, molecular data on the causative mutations of haemophilia B patients worldwide. It is not intended to replace primary publications although it does contain a significant amount of unpublished work. As in previous years, we have included r observations of the same mutation, as well as molecularly unique mutations. We have continued our database numbering system (Giannelli et al (1991) giving all patients a unique Patient Identity Number (PIN or ID number).The factor IX gene lies on the long arm of the x chromosome at Xq27 and its entire sequence of 33 kb is known (Yoshitake et al, 1985). It contains 8 exons (a-h) encoding 6 major domains of factor IX. These are: (1) exon a-a hydrophobic signal peptide which targets the protein for secretion from the hepatocyte into the blood stream. (2) exons b and c-a propeptide and gla domain,-the latter containing 12 -y-carboxyglutamyl residues. This post-translational modification is required for the correct folding and calcium binding of factor IX. (3) exon d-a type B, or first epidermal growth factor-like domain, which shows homology to epidermal growth factor (EGF) and, in addition, contains conserved carboxylate residues including a ,hydroxyaspartate at amino acid 64. This domain binds an additional Ca2+ with high affinity (Handford et al, 1991). ( 4) exon e-a type A, or second epidermal growthfactor-like (EGF) domain which lacks the conserved carboxylate residues of the EGF type B domain. (5) exon f-an activation domain, within which factor XIa cleaves twice, converting factor IX to IXa; (6) exons g and h-the serine protease or catalytic domain, responsible for the proteolysis of factorxto Xa. This region is homologous to other well studied serine proteases (e.g. chymotrypsin) and it is thought likely that his (221), asp (269) and ser (365), all participate in the classical catalytic mechanism.Factor IX is initially synthesised in the liver as a precursor molecule, either 46, 41 or 39 amino acids (it is not known which, although 39 is probable (Pang et al, 1990)) longer at its Nterminus than the 415-long mature factor IX found in plasma. Processing steps occur in the hepatocyte prior to secretion and sequentially remove the hydrophobic signal peptide and the propeptide. In addition to the -y-carboxylation of the 12 N-terminal glutamyl residues carried out by a vitamin K-dependent carboxylase, and the parti...