Diagnostic challenges in von Willebrand disease. Report of two cases with emphasis on multimeric and molecular analysis.

Identification of qualitative variants of von Willebrand disease (VWD) can be a diagnostic challenge because of discrepant results obtained in the multiple laboratory tests available for its appropriate classification. We report two cases of infrequent inherited variants of VWD with unclear preliminary results with the test panel available at the time of first consultation and that were finally diagnosed as a VWD type 2A/IID with a c.8318 G > C, p.Cys2773Ser mutation and a VWD type 2M with c.4225 T > G, p.Val1409Phe mutation, respectively. The description of these two cases highlights that despite the limited diagnostic panel for the evaluation of von Willebrand Factor (VWF) functionality, the multimeric analysis and genetic family studies were fundamental tools to achieve the final diagnosis.

Immunogenicity, efficacy and safety of Nuwiq® (human-cl rhFVIII) in previously untreated patients with severe haemophilia A-Interim results from the NuProtect Study.

INTRODUCTION: Nuwiq® (Human-cl rhFVIII) is a fourth generation recombinant FVIII, produced in a human cell line, without chemical modification or protein fusion. No inhibitors developed in studies with Nuwiq® in 201 previously treated patients with haemophilia A (HA). The immunogenicity, efficacy and safety of Nuwiq® in previously untreated patients (PUPs) with severe HA are being assessed in the ongoing NuProtect study. METHODS: The study, conducted across 38 centres worldwide, is evaluating 110 true PUPs of all ages and ethnicities enrolled for study up to 100 exposure days (EDs) or 5 years maximum. The primary objective is to assess the immunogenicity of Nuwiq® (inhibitor activity ≥0.6 BU) using the Nijmegen-modified Bethesda assay at a central laboratory. RESULTS: Data for 66 PUPs with ≥20 EDs from a preplanned interim analysis were analysed. High-titre (HT) inhibitors developed in 8 of 66 patients after a median of 11.5 EDs (range 6-24). Five patients developed low-titre inhibitors (4 transient). The cumulative incidence (95% confidence interval) was 12.8% (4.5%, 21.2%) for HT inhibitors and 20.8% (10.7%, 31.0%) for all inhibitors. During inhibitor-free periods, median annualized bleeding rates during prophylaxis were 0 for spontaneous bleeds and 2.40 for all bleeds. Efficacy was rated as "excellent" or "good" in treating 91.8% of bleeds. Efficacy of surgical prophylaxis was "excellent" or "good" for 8 (89%) procedures and "moderate" for 1 (11%). No tolerability concerns were evident. CONCLUSION: These interim data show a cumulative incidence of 12.8% for HT inhibitors and convincing efficacy and tolerability in PUPs treated with Nuwiq® .

Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. SUMMARY: Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level.

Practical aspects of factor concentrate use in patients with von Willebrand disease undergoing invasive procedures: a European survey.

INTRODUCTION: The bleeding propensity in von Willebrand disease (VWD) is usually moderate or mild and patients with VWD do not need continuous treatment, but do require extra increased haemostatic cover when undergoing dental or surgical procedures. Desmopressin can be effective in certain patient groups and this has been considered in a previous publication. AIM: This paper now seeks to evaluate current knowledge and practice in the use of factor concentrate in the management of VWD patients undergoing invasive procedures. METHODS: A literature search was performed on the use of factor concentrates to cover invasive procedures and a survey of current practice in a number of specialist haematology centres across Europe represented by the European Haemophilia Strategy Board was conducted. RESULTS: Our review of the literature and the results of the survey showed considerable heterogeneity in treatment regimens, and a lack of consistency in reporting of the variables that determine factor concentrate dosing and monitoring. CONCLUSION: By analysing the literature, examining guidelines and using consensus deliberation, this survey allowed the group to develop recommendations for management of VWD patients undergoing invasive procedures.

Early prophylaxis in children with severe haemophilia A: clinical and ultrasound imaging outcomes.

AIM: This observational study was undertaken with the aim to describe the characteristics and evaluate the outcomes of prophylactic treatment in children with severe haemophilia A (HA) treated at our centre. METHODS: Twenty-five patients aged 4-19 years with severe HA, no history of inhibitors and treated with at least two infusions of factor VIII (FVIII) per week were studied. Prophylactic doses and annual joint bleeding rate (AJBR) were retrospectively evaluated over the last 5 years. Current joint status was assessed using the Haemophilia Joint Health Score (HJHS) (136 joints of 23 patients) and the Haemophilia Early Arthropathy Detection with Ultrasound (HEAD-US) procedure (124 joints of 21 patients). RESULTS: Median AJBR was 0.2 and median prophylaxis dose 65.4 IU-1  kg-1  week-1 . Median total HJHS was 0 (range 0-13) and total HEAD-US 1 (0-8). At the joint level, 85.3% of joints were normal on HJHS and 79.0% on US. The ankle was the joint most commonly affected, considering bleeding and ultrasound results. Correlation was found between HEAD-US scores and bleeding scores but not between HEAD-US and HJHS scores. HJHS and HEAD-US scores were concordant in 91/124 (73.4%) joints (86 joints normal and five abnormal). Ultrasound detected minimal changes in 19.6% of joints with normal physical function, whereas 12.2% of joints considered normal on ultrasound showed changes at HJHS. CONCLUSION: A well-preserved joint status was found in our cohort. High-resolution US detected a higher percentage of abnormalities than the physical evaluation, but the clinical implications of these findings still need to be ascertained.

Practical aspects of DDAVP use in patients with von Willebrand Disease undergoing invasive procedures: a European survey.

INTRODUCTION: Desamino D-arginine vasopressin (DDAVP or desmopressin) is a useful and effective haemostatic treatment for patients with von Willebrand Disease (VWD). However, there are still issues regarding in which subtypes of VWD DDAVP is appropriate and little consensus on its use in different surgical settings. We also lack information concerning the appropriate laboratory parameters that should be monitored. AIM: The European Haemophilia Therapy Strategy Board (EHTSB) wished to investigate published information and clinical use of DDAVP in VWD patients. METHODS: We conducted a literature survey on management of VWD during surgical interventions and undertook a survey of specialist haematologist centres across Europe to assess current management of VWD patients. RESULTS: DDAVP is ineffective in type 3 VWD and its use in type 2B remains controversial due to the possibility of thrombocytopenia. It can, however, be used effectively to cover minor surgery and dental procedures in most other VWD patients. For major surgery there is wider use of factor concentrate in preference to DDAVP depending on the subtype of VWD. We give consensus recommendations on the use of DDAVP for surgical interventions in VWD including laboratory parameters that denote an adequate response and contraindications to its use. CONCLUSIONS: DDAVP can be recommended to cover invasive procedure in selected patients with VWD, however, we need more information and systematic recording of adverse events associated with DDAVP use in VWD. A companion paper will be published covering the use of factor concentrates in VWD patients.

Adherence to prophylaxis and quality of life in children and adolescents with severe haemophilia A.

Treatment adherence in adolescents with chronic diseases is around 50%, and failure is more common in preventive therapy. In haemophilia, contradictory results are reported by the published studies. The objective of this study was to evaluate adherence with factor VIII (FVIII) prophylaxis in Spanish patients with severe haemophilia A between age 6 and 20 years. Data were collected retrosp-ectively in the previous 2 years. The primary endpoint was the absolute adherence index (AAI), and the endpoints were related to clinical status, age, prophylaxis regimen, responsibility for factor administration and quality of life (QoL), assessed by the Haemo-QoL questionnaires. A total of 78 patients from 14 Spanish hospitals were recruited. Adherence ranged between -64.4 and 66.7 (mean -3.08). No differences were observed between children and adolescents (7.11 vs. 6.39; P = 0.809). A statistically significant association (P < 0.010) between infra adherent group and target joint was found, as was a statistically significant difference (P < 0.010) between the number of bleeding episodes experienced by the adherent group (mean 1.4) and by infra adherents (mean 4.5). There was no significant difference between AAI and prophylactic regimen (6.35 vs. 6.96, P = 0.848), neither between AAI and the person responsible for factor administration (5.57 vs. 8.79, P = 0.326). The Haemo-QoL scores (8-12 years) were related to adherence level (P < 0.05). Adherence was approximately ideal and patients perceived a high QoL. Because of the repercussions for compliance, it is essential to work during puberty on emotional and self-acceptance aspects of the disease, as well as coping, and the patient's family, school and health team relationships.

International cross-cultural validation study of the Canadian haemophilia outcomes: kids' life assessment tool.

Health-related quality of life (HRQoL) assessment is recognized as an important outcome in the evaluation of different therapeutic regimens for persons with haemophilia. The Canadian Haemophilia Outcomes-Kids' Life Assessment Tool (CHO-KLAT) is a disease-specific measure of HRQoL for 4 to 18-year-old boys with haemophilia. The purpose of this study was to extend this disease-specific, child-centric, outcome measure for use in international clinical trials. We adapted the North American English CHO-KLAT version for use in five countries: France, Germany, the Netherlands, Spain and the United Kingdom (UK). The process included four stages: (i) translation; (ii) cognitive debriefing; (iii) validity assessment relative to the PedsQL (generic) and the Haemo-QoL (disease-specific) and (iv) assessment of inter and intra-rater reliability. Cognitive debriefing was performed in 57 boys (mean age 11.4 years), validation was performed in 144 boys (mean age 11.0 years) and reliability was assessed for a subgroup of 64 boys (mean age 12.0 years). Parents also participated. The mean scores reported by the boys were high: CHO-KLAT 77.0 (SD = 11.2); PedsQL 83.8 (SD = 11.9) and Haemo-QoL 79.6 (SD = 11.5). Correlations between the CHO-KLAT and PedsQL ranged from 0.63 in Germany to 0.39 in the Netherlands and Spain. Test-retest reliability (concordance) for child self-report was 0.67. Child-parent concordance was slightly lower at 0.57. The CHO-KLAT has been fully culturally adapted and validated for use in five different languages and cultures (in England, the Netherlands, France, Germany and Spain) where treatment is readily available either on demand or as prophylaxis.

Proteomic analysis of platelet N-glycoproteins in PMM2-CDG patients.

UNLABELLED: PMM2-CDG, the most frequent congenital disorder of N-glycosylation, is an autosomal recessive disease with a multisystem presentation. PMM2-CDG patients show an increased risk for thrombosis, which might be in part due to spontaneous platelet aggregations as previously described. A potential hypoglycosylation of platelet proteins in these patients might explain this increased reactivity, as removal of sialic acid from platelets, particularly of GPIbα, leads to enhance platelet aggregation and clearance from the circulation. This study is the first one that has evaluated the glycosylation status of platelet proteins in 6 PMM2-CDG patients using different approaches including immunoblot, RCA120 lectin binding to platelets and expression of different membrane platelet N-glycoproteins by flow cytometry, as well as by platelet N-glycoproteome analysis. RCA120 lectin binding to the platelet membrane of PMM2-CDG patients showed evidence for decreased sialic acid content. However, immunoblot and flow cytometric analysis of different platelet N-glycoproteins, together with the more sensitive 2D-DIGE analysis, suggest that platelet N-glycoproteins, including GPIbα, seem to be neither quantitatively nor qualitatively significantly affected. The increased binding of RCA120 lectin could be explained by the abnormal glycosylation of hepatic proteins being attached to the platelets. CONCLUSIONS: This is the first study that has evaluated the platelet N-glycoproteome. Our findings suggest that platelet proteins are not significantly affected in PMM2-CDG patients. Further studies are still warranted to unravel the mechanism(s) that increase(s) the risk of thrombosis in these patients.

Continuous infusion in haemophilia: current practice in Europe.

Summary. Continuous infusion (CI) of factor VIII (FVIII) is an effective method for replacement therapy in haemophilia. Recently, concerns have been raised regarding association of CI with the development of inhibitors. The aim of this study was to gain information on the current practices in Europe regarding CI and the true inhibitor incidence after this mode of therapy. In a cross sectional study performed in 22 Comprehensive Care Centres (CCCs), we evaluated CI techniques, treatment protocols, efficacy, safety and complications of CI including inhibitors. Thirteen (59%) CCCs reported a total of 1079 CI treatments, given peri-operatively or for major bleeds, in 742 patients. Most centres used 'adjusted dose' CI aimed at median target FVIII level of 0.8 IU mL(-1). CI was haemostatically very effective with a low incidence of complications: median incidence of postoperative bleeding was 1.8%, six centres observed phlebitis in 2-11% of CI treatments. Only nine (1.2%) patients developed inhibitors (0.45% of 659 severe and 7.2% of 83 mild haemophilia patients). Additional analysis of inhibitor patients revealed several confounding factors (low number of prior FVIII exposure days, high steady-state factor levels during CI, high-risk genotype). In this unprecedentedly large cohort, CI treatment appears to be an effective and safe treatment that does not increase the risk of inhibitor development in patients with severe haemophilia. Thus, previous small case series reports suggesting that CI may increase inhibitors cannot be confirmed. Inhibitor risk in mild haemophilia could not be evaluated as the influence of other, potentially confounding, risk factors could not be excluded.

Acquired haemophilia A. First line treatment with calcineurin inhibitors and steroid pulses: a 10-year follow-up study.

Acquired haemophilia A (AH) is defined as the presence of autoantibodies or inhibitors against factor VIII (FVIII) with a clinical bleeding onset that can be life-threatening. Immunosuppressant therapy must be initiated rapidly to eradicate the inhibitor. Current treatments based on steroids plus cyclophosphamide or rituximab are quite effective, but with significant side-effects. Based on previous described AH cases treated with cyclosporine, with a good side-effect profile, we aimed at assessing prospectively a first-line calcineurin inhibitor based immunosuppressive therapy. We included a total of 11 patients affected with AH. Once diagnosed, pulse steroids and calcineurin inhibitors were started. Time to achieve sustained response (SR), defined as testing negative for inhibitor and with stable FVIII level >50%, immunosuppressant side-effects, and relapse of AH were evaluated. Eight patients received cyclosporine and three patients received tacrolimus. SR was achieved in 10 of 11 patients (90.9%) in a median time of 3 weeks (range 2-8 weeks), and none of them relapsed during a median follow-up time of 14 months (range 4-120). One major side-effect appeared (posterior encephalopathy) that forced to discontinue cyclosporine. Overall 5-year survival rate was 54.5%, with a total of five patients dying during the follow-up (mortality rate of 45.5%). These five patients had achieved SR and died because of complications of basal morbidities and/or senescence, not related to AH (bleeding) or to immunosuppressant's (infection) side-effects. Combination therapy of calcineurin inhibitors and pulse steroids seems clinically effective as a first-line treatment of AH.

The study of the effect of splicing mutations in von Willebrand factor using RNA isolated from patients' platelets and leukocytes.

BACKGROUND: In von Willebrand factor (VWF) the effect of mutations potentially affecting mRNA processing or splicing is less predictable than that of other mutations (e.g. nonsense or missense substitutions). Bioinformatic tools can provide a valuable means to determine the consequences of potential splice site mutations (PSSM), but functional studies are mandatory to elucidate the true effect of the variation detected. OBJECTIVES, PATIENTS AND METHODS: After identification of PSSM in VWD patients, we began a systematic study of their in vivo effect in RNA extracted from the patients' platelets and leukocytes. RESULTS AND CONCLUSIONS: Thirteen pairs of primers were designed for full amplification of VWF mRNA by RT-PCR that, after sequencing of aberrant products, enabled elucidation of the PSSM consequences for mRNA processing. This procedure was used to study seven different PSSM identified in four patients demonstrating diverse molecular mechanisms such as exon skipping (c.533-2A>G and c.8155+3G>C) and the activation of a cryptic splice site (c.7730-1G>C). No visible effect was evident for c.1533+15G>A and c.5170+10C>T and the consequence of c.[546G>A;7082-2A>G] was hidden by nonsense-mediated mRNA decay (NMD). Results were compared with in silico predictions of four splice-site analysis tools. We demonstrate selective degradation of VWF mRNA bearing PSSM by NMD for several mutations, which suggests that NMD represents a general mechanism for truncating mutations in VWF. Furthermore, because NMD efficiency varies between cell types, use of RNA from both platelets and leukocytes for in vivo study of VWF PSSM offers complementary results, particularly in cases in which NMD occurs in the allele carrying the mutation.

Non-genetic risk factors and the development of inhibitors in haemophilia: a comprehensive review and consensus report.

SUMMARY: The development of inhibitors to the infused factor in patients with haemophilia is a serious clinical problem. Recent evidence suggests that alongside the strong genetic contribution to inhibitor formation, there are a number of non-genetic factors--perceived by the immune system as danger signals--which promote formation of inhibitors. This study provides a comprehensive review of clinical studies relating to these factors and also presents a survey of opinion concerning their importance and clinical influence, conducted among the members of the European Haemophilia Treatment Standardisation Board (EHTSB). Taken together, this information highlights the lack of robust data concerning the influence of several non-genetic risk factors on inhibitor development, and an urgent need for prospective, well-conducted studies that adhere to recommendations made by the European Medicines Agency (EMEA) for studying inhibitors. Based on current literature, the EHTSB formulated consensus recommendations. It is desirable to minimize intensive treatment wherever possible, given the clinical situation. Prophylaxis should be offered to all children, although we still need to determine optimal dosing with respect to inhibitor development, and age for starting treatment. Vaccinations should be given subcutaneously and concomitant factor concentrate infusions avoided. According to the board, there is no evidence in the literature supporting suggestions that the type of concentrate influences inhibitor risk; but all patients should be monitored during their first exposures. Furthermore, there is no evidence to support an association between pregnancy-related issues, breast feeding and treatment-related factors (e.g. route of administration, or use of blood components) and inhibitor development.

Replacement therapy for invasive procedures in patients with haemophilia: literature review, European survey and recommendations.

Although most surgical and invasive procedures can be performed safely in patients with haemophilia, the optimal level and duration of replacement therapy required to prevent bleeding complications have not been established conclusively. For providing more insight into optimal therapy during invasive procedures, a literature review of surgical procedures in patients with haemophilia was conducted. Concomitantly, current practice was surveyed in 26 European Haemophilia Comprehensive Care Centres, representing 15 different countries. The review identified 110 original papers published between 1965 and 2007. Of these, only two studies were randomized controlled trials. Target levels and the duration of replacement therapy in the published studies were as follows. For major orthopaedic surgery: preoperative targets were 80-90%; postoperative targets showed a high degree of variation, with trough levels ranging from 20% to 80%, duration 10-14 days; for liver biopsy, 70-100%, 1-7 days; tonsillectomy: 90-100%, 5-11 days; indwelling venous access device insertion: 100%, 3-10 days; circumcision: 50-60%, 2-4 days; dental surgery: 30-50%, single treatment. With the exception of dental surgery, current practice in Europe, as assessed by the survey, was largely in agreement with published data. In conclusion, this study provides both a comprehensive review and a large survey of replacement therapy in patients with haemophilia undergoing invasive procedures; these data have informed the consensus practical treatment recommendations made in this paper. This study highlights the need for better-designed studies in order to better define minimal haemostatic levels of replacement therapy and optimal treatment duration.