University dissertation from Karin Knobe, Dept. of Pediatrics, University Hospital, Malmö, SE- 20502

Abstract: The general objective of the research underlying this dissertation was to conduct clinical and basic scientific studies to further elucidate the hemophilia and the coagulation process, with the goal of improving the care of families with these disorders. More specific aims were to evaluate epidemiological, genetic, clinical, and biochemical aspects of the development of inhibitors (antibodies) against FVIII and FIX in Swedish patients with hemophilia A and B (papers I, II, and III). Furthermore, to investigate the relationship between genotype and structure/function by characterizing specific mutations in the EGF-domains of FIX (papers IV and VI) and, more generally, by determining the impact of genotype on FIXC levels in hemophilia B carriers (paper V). The results: The incidence of inhibitors was 19% in the subjects with severe hemophilia A and 37% in those with severe hemophilia B born 1980–1999 in Sweden. Inhibitors were first detected when the patients were 12–18 months old and after a median of 15 days of exposure to factor concentrate. Genotype was found to have a general impact on the development of inhibitors. The hemophilia A patients exhibited no significant increase in incidence of inhibitors in the 1990s, when they were treated mainly with recombinant products. Forty percent of the inhibitor patients had repeated bleeds, and the outcome was fatal in two. The results also indicate that breastfeeding does not protect against the development of FVIII/FIX inhibitors. Moreover, the two mutations Arg593Cys (in the A2 domain) and Tyr2105Cys (in the C1 domain) cause mild hemophilia A, probably by interfering with secretion of FVIII from the cells to the circulation, and, in the case of Tyr2105Cys, possibly also by obstructing interactions with vWF. Furthermore, our computer model suggests that these mutations will induce local structural changes in the secreted protein that will predispose to production of antibodies against the wtFVIII, which in turn will cross-react with the mutant FVIII. Recombinant systems were established to produce large quantities of FIX proteins for use in functional experiments. This demonstrated that the mutation Pro55Ser causes hemophilia B primarily by impairing the ability to activate FX, regardless of whether FVIIIa is included in the assay, which supports the notion that EGF1 does not interact directly with FVIIIa. FIXPro55Leu is, at least in vitro, degraded soon after activation; cleavage occurs in the serine protease domain at Arg318, indicating that there is intramolecular communication between EGF1 and the serine protease domain of FIXa. It seems that all hemophilia B carriers, notwithstanding the severity of the disease in the family, can be detected with the same reference values of FIX coagulant activity (FIXC), since neither genetic anomaly nor severity of the disorder affected the FIXC levels in the carriers studied. The mutation G17736A, Val107Val (silent), was found in five out of a total of 79 families with hemophilia B in Sweden. Analyzing FIX mRNA from lymphocytes, we found no signs of exon skipping or retention of introns, possibly because erroneously spliced mRNA is degraded or that the mutation interferes with a splicing enhancer site.

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