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BACKGROUND: Inversions involving intron 22 (Inv22) of F8 are detected in approximately 45% of all severe hemophilia A patients. Diagnosis is complicated by the large size of the ~9.5 kb int22h repeated sequence which generates the inversions. Methods such as long-range PCR and inverse-shifting PCR are currently used diagnostically, but suffer from low PCR efficiencies and are difficult to standardize.OBJECTIVES: To design and validate a sensitive and robust assay for the detection of F8 int22h inversions.METHODS: Digital droplet PCR using mile-post assays was used to investigate archival DNA samples.RESULTS: The detection of linkage as a function of physical distance between loci was investigated using an anchor locus and mile-post loci located at 1, 6, 12 and 15 kb distances from the anchor locus. The proportion of linked molecules decreased with increasing distance between loci and showed 30-40% linked molecules for loci 12-15 kb apart. Mile-post assays specific for wild type and Inv22 type 1 and 2 chromosomes were then designed and optimized. All three assays showed high specificities and sensitivities, with coefficients of variation < 5% for all assays. Analysis of 106 patients and 20 carrier mothers showed complete concordance with previously known mutation status. The analysis demonstrated the robustness of the assays versus input DNA concentration (6 ng and higher) and level of fragmentation.CONCLUSIONS: Digital droplet PCR and mile-post assays can be used to detect F8 int22h inversions. The assay systems are technically simple to perform, highly efficient and robust. (Less)

Research has been lacking on the natural history, complications, and treatment of haemophilia B, which is less common than haemophilia A and was recognized as a distinct clinical entity in 1947. Although the two diseases share the same clinical manifestations, they differ in causative mutation, risk of inhibitor development, and patient quality of life. Frequently debated is whether haemophilia B is as clinically severe as haemophilia A, with much of the published data on overall and haemophilia-specific health outcomes suggesting that haemophilia B may have a less severe clinical phenotype. However, although fewer haemophilia B than haemophilia A patients appear to experience bleeding, bleeds are just as severe. We review distinguishing characteristics of haemophilia B and its treatment, including management strategies for neonates, therapeutic approaches for patients who develop inhibitors, pharmacokinetics of factor IX concentrates administered as replacement therapy, and potential future treatments.

Although effective therapies for haemophilia have been available for decades, the prevention and treatment of joint disease remain major clinical concerns for all haemophilia patients. Early identification of joint disease is vital to initiate or modify treatment, and prevent arthropathy. However, there remains a need for more sensitive and accurate methods, which may also detect improvement in patient outcome with new therapies or different prophylaxis regimens. These topics were explored at the Ninth Zürich Haemophilia Forum. A summary of our shared views on the limitations of current assessment methods, and the potential advantages of more recently developed tools, is provided. Ultrasonography enables more frequent routine monitoring and the early detection of joint disease. In addition, serological markers may provide suitable biomarkers of early arthropathy. To prevent arthropathy, in our opinion, prophylaxis is key to prevent joint bleeds and subsequent initiation of the 'vicious circle of joint disease'. However, issues remain, including when prophylaxis should be started, stopped, and if it is efficacious for inhibitor patients. Once joint bleeding has occurred, enhanced on-demand treatment should be considered. For more advanced stages of joint disease, the issues regarding the treatment options available are explored. Radiosynovectomy should be performed to treat chronic synovitis, and may prevent the need for elective orthopaedic surgery (EOS). Ultimately, however, EOS can be considered once all other treatment options have been explored. While, bypassing agents have facilitated the use of EOS in inhibitor patients, a multidisciplinary approach and careful surveillance is required for good patient outcome.

Aims: To study the number, outcome and reasons for prenatal diagnosis (PND) and how it has affected the incidence of haemophilia, the number of siblings and the number of potential carriers in the families. Study Group: Women in SWE, compromising over 95% of total, who underwent PND of haemophilia during the years 1970-2010. A total of 46 women were identified who together underwent 79 PND procedures. Method: Structured personal interview and registry of laboratory analysis. So far, 27 women have been interviewed. Preliminary Results: 19/79 PND had been performed by analysis of foetal blood, 55/79 by genetic analysis of chorionic villi sampling and 5/79 by analysis of amniotic fluid. A total of 24 foetuses were found to be affected with haemophilia and 13/24 were aborted. The 24 foetuses affected with haemophilia were carried by 20 women. Twelve of these 20 women chose to end their pregnancy because of the findings. The preliminary results suggest that the drop in incidence of haemophilia due to PND in the 1990s no longer exists since almost half of the women today use PND to prepare themselves and their families psychologically to have a child with haemophilia and not to terminate the pregnancy. Furthermore, there seem to be more women becoming mothers since the improvement of care, thus giving birth to more children, both boys and girls, the latter being possible carriers. Conclusions: PND of haemophilia is still requested, but the number of affected foetuses aborted has diminished. Furthermore, the number of siblings seems to have increased, so the number of future carriers will also increase. (Less)

We studied the functional role of two mutations, Pro55Ser and Pro55Leu, located in the N-terminal Epidermal Growth Factor-like domain (EGF1) of coagulation factor (F) IX. Both mutations cause mild hemophilia B with habitual FIX coagulant activities of 10-12% and FIX antigen levels of 50%. We found that activation by FVIIa/TF and FXIa was normal for FIXPro55Ser, but resulted in proteolysis of FIXPro55Leu at Arg318-Ser319 with a concomitant loss of amidolytic activity, suggesting intramolecular communication between EGF1 and the serine protease domain in FIX. This was further supported by experiments using an anti-EGF1 monoclonal antibody. Activation of FX by FIXaPro55Ser was impaired in both the presence and the absence of phospholipid or FVIIIa, indicating that Pro55 is not directly involved in binding to FVIIIa. We also studied the effect of the two Pro55 mutations on Ca2+ affinity and found only small changes. Thus, the Pro55Ser mutation causes hemophilia primarily through to an impaired ability to activate FX whereas at least in vitro the Pro55Leu defect interferes with the activation of FIX.

Prenatal diagnosis of haemophilia A or B is possible by means of chorionic villus biopsy in the first trimester which traces the mutation or informative genetic markers. If possible, direct gene analysis of the mutation is preferred. The natural starting point in haemophilia A is to ascertain whether the disease is due to inversion in the X-chromosome, which is the case in almost half of the severe cases. In haemophilia B, most families carry a unique mutation which needs to be characterized. In the immediate future, much of the prenatal diagnosis will be based on indirect genetic markers, repeats or polymorphisms, of the F.VIII and IX genes. Today chorionic villus sampling is the most widely used method but amniotic fluid, fetal blood and pre-implantation genetic diagnostics can also be used in selected cases. Prenatal diagnosis must be preceded by adequate genetic counselling and risk assessment of the potential carrier and subsequent support during the diagnostic process.

Aim: To identify prothrombotic risk profiles in children and adolescents referred to a regional coagulation centre in southern Sweden for a first thrombotic event. Methods: One hundred and twenty-eight consecutive children and adolescents (newborn to 20 y) referred for evaluations of a first episode of venous thrombosis were investigated. Clinical data were collected retrospectively, and the following variables were investigated: protein C, protein S, antithrombin; resistance to activated protein C; the genotypes FV-G1691A, F II-G20210A, MTHFR-C677T, MTHFR- A1298C; coagulation factors VIII and XI. Results: 104/128 subjects (81%) had identifiable acquired risk factors, most often indwelling catheters and hormone therapy. Predisposing genetic factors related to thromboembolic events were revealed in 53/83 (64%) of subjects who agreed to follow-up blood sampling, and 17/83 (20%) had two or more inherited risk factors. Combinations of genetic and acquired risk factors were identified in 45/83 (54%) of the subjects, and 77/83 (93%) had at least one such risk factor. Both sexes had one peak in frequency at less than 1 y of age and then an increase during adolescence, more in females than in males. Plasma values for coagulation factors VIII and XI were age appropriate and showed a normal Gaussian distribution. Conclusion: This study identified prothrombotic risk profiles in almost all children and adolescents with venous thrombosis, which underlines the importance of careful evaluation of genetic and acquired risk factors.

Purification of F.VIII:C devoid of F.VIII:Ag was achieved by antigen-antibody chromatography. The antibody used neutralized VIIIR:Ag but not VIII:C in liquid phase but extracted both VIII:Ag and VIII:C from plasma when bound to Sepharose. VIII:C was eluted with calcium-containing buffer. When plasma was used as starting material VIII:C was obtained free from VIIIR:Ag but contaminated with some other proteins. When a well-defined pure F.VIII preparation was used as starting material the VIII:C active fractions contained no immunoradiometrically detectable VIIIR:Ag, no VIIIR:RCF and no detectable protein. When stabilized with albumin VIII:C could be frozen and thawed with retained activity and could be activated with thrombin.

The effects of various concentrations of plasmin and activated protein C on the factor VIII procoagulant activity (VIII:C) and coagulant antigen (VIII:CAg) were studied in factor VIII concentrates and normal plasma. Small amounts (0.1 CTA U/ml) of plasmin rapidly destroyed VIII:C, and affected, but did not destroy VIII:CAg, in factor VIII concentrates. In normal plasma larger amounts of plasmin (1.8 CTA U/ml) was required to inactivate VIII:C in order to exceed the neutralizing capacity of alpha 2-antiplasmin. VIII:CAg was unchanged indicating a limited proteolysis. The difference between VIII:C and VIII:CAg was found also in urokinase-activated plasma. Activated protein C (5 micrograms/ml), in the presence of Ca2+ and phospholipids, inactivated VIII:C without affecting VIII:CAg in a high purity factor VIII concentrate. Higher concentrations of activated protein C (25 micrograms/ml) caused a slight decrease of VIII:CAg, even in the absence of Ca2+ and phospholipids, but did not change VIII:CAg in normal plasma or serum.