Factor IX is a vitamin K dependent plasma protein synthesized in the liver and required for normal coagulation. It occupies a central position in the clotting cascade, where it undergoes critical interactions with Factors XI, VII, VIII and X. A deficiency of Factor IX results in the clinical syndrome hemophilia B. The major thrust of this project is to make use of the cloned Factor IX gene to study the structure, function and expression of Factor IX. Sources of material for study will include the large numbers of patients with hemophilia B followed at this institution, and the colony of dogs with hemophilia B here. An initial goal will be to isolate and characterize at the nucleotide level selected human Factor IX mutations. The patients selected for study include: 1) patients with a variant designated hemophilia BM. These variants are known to have a defect in interaction with Factors VII and/or X. 2) Patients with a subtype of CRM-reduced hemophilia B (those with equal reductions in antigen and activity). Through examination of these variants, regions of the Factor IX DNA sequence which control or are necessary for expression will be identified. Methods to be used include construction and screening of genomic libraries, and M13/dideoxy sequence analysis. The goal of the second group of experiments is to determine the level at which changes in Factor IX protein levels are regulated. Utilizing an animal model, it will be possible to examine rates of transcription, transcript stability, transcript size, translational efficiency, post translational modifications and protein secretion; and to determine at which of these levels changes in Factor IX expression are regulated. Finally, taking advantage of Factor IX's location on the X chromosome, experiments will be carried out to determine whether differences in the chromatin configuration can be detected between the active and inactive X chromosomes in the case of Factor IX. Specifically, use will be made of liver tissue from dogs who are carriers of hemophilia B (and in whom, therefore, the two X alleles can be differentiated with the use of appropriate probes), to determine whether differences in DNAse hypersensitive sites and cytosine methylation exist between the expressed and the non-expressed allele.
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