GENE ANALYSIS IN HEMOPHILIA

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The hemophilias, FVIII:C and FIX deficiencies, are the second commonest X-linked, recessive disorders. They are characterized as being clinically heterogenous based on percent clotting activity, cross-reactive material (CRM), and the presence of inhibitors to the coagulation protein. The genes for FVIII:C and FIX have been recently cloned and their coding sequences determined. The objective of this proposal is to study the molecular mutations in hemophilia genes using the recently generated FVIII:C and FIX genomic and cDNA probes and to correlate molecular defects to the clinical hemophilia phenotype. Other objectives of this proposal are to detect the mechanisms and parental source of spontaneous mutations in some hemophilia pedigrees, detect new DNA polymorphisms in the FVIII:C and FIX gene, develop new restriction fragment-length polymorphism probes (RFLPs) for the Xq27-28 subchromosomal region and, finally, to analyze multigenerational hemophilia pedigrees for recombinational events using these new tools. Essential to this study is the development of new approaches using recombinant DNA technology to screen the 186 kb FVIII:C and 34 kb FIX genes for point mutations and deletions. Methods of restriction endonuclease analysis, synthetic oligonucleotide probe analysis, DNA:RNA hybrid duplex analysis with ribonuclease A and field inversion gel electrophoresis will be utilized as screening methods to decipher mutation sites in hemophilia genes. Further analyses of the DNA sequences at the mutation sites will be performed by molecular cloning of FVIII:C and FIX genes into the lambda phage vectors Charon 21A and 30, EMBL3, and gamma gt10. The DNA sequences of FVIII:C or FIX recombinants will be determined by subcloning into M13 phage and generation of single-stranded phage and inserts for DNA sequencing by the method of dideoxy chain termination. The origin of spontaneous hemophilia mutations and recombinational frequencies can be studied by the use of haplotype analysis using multiple DNA markers. DNA polymorphic sites in both the FVIII:C and FIX genes as well as in the flanking RFLPs probes, Dx13, St14 and 52A will be used for these analyses. In addition, the development of new linked RFLP probes, as well as the discovery of intragenic DNA polymorphs, will be essential to further define the recombination sites either within FVIII:C and FIX genes or their chromosomal locus. Finally, these studies should permit improved genetic diagnostic and carrier detection in the hemophilias.