Self-conducted sonographic monitoring of the knee in patients with haemophilia-A feasibility study.

INTRODUCTION/AIM: To evaluate whether patients with haemophilia (PwH) can be enabled to perform ultrasonography (US) of their knees without supervision according to the Haemophilia Early Arthropathy Detection with Ultrasound (HEAD-US) protocol and whether they would be able to recognize pathologies. METHODS: Five PwH (mean age 29.6 years, range 20-48 years) were taught the use of a portable US device and the HEAD-US protocol. Subsequently, the patients performed US unsupervised at home three times a week for a total of 6 weeks with a reteaching after 2 weeks. All images were checked for mapping of the landmarks defined in the HEAD-US protocol by a radiologist. In a final test after the completion of the self-sonography period, participants were asked to identify scanning plane and potential pathology from US images of other PwH. RESULTS: On the images of the self-performed scans, 82.7% of the possible anatomic landmarks could be identified and 67.5% of the requested images were unobjectionable, depicting 100% of the required landmarks. There was a highly significant improvement in image quality following reteaching after 2 weeks (74.80 ± 36.88% vs. 88.31 ± 19.87%, p < .001). In the final test, the participants identified the right scanning plane in 85.0% and they correctly identified pathology in 90.0% of images. CONCLUSION: Appropriately trained PwH can perform the HEAD-US protocol of their knee with high quality and are capable to identify pathologic findings on these standardized images. Asynchronous tele-sonography could enable early therapy adjustment and thereby possibly reduce costs.

Antidrug antibodies against the polyethylene glycol moiety inhibit the procoagulant activity of therapeutic polyethylene glycolated factor VIII.

BACKGROUND: The standard therapy for patients with hemophilia A (HA) is the replacement with factor VIII (FVIII) therapeutics. To overcome the limitation of short half-life of wild-type FVIII protein, polyethylene glycol (PEG) can be coupled to therapeutic FVIII to improve pharmacokinetics. OBJECTIVES: We aimed to characterize antibodies developed against a FVIII therapeutic PEGylated with a 40-kDa PEG (40PEG-BDDFVIII) in 2 patients with mild HA. METHODS: An inhouse bead-based immunoassay was developed to characterize and confirm the specificity of the detected antibodies. The neutralizing nature of the antibodies toward PEGylated therapeutics was determined by a modified Nijmegen-Bethesda assay. RESULTS: Two out of 46 patients treated with 40PEG-BDDFVIII developed inhibitory antibodies toward the drug. Switching to a non-PEGylated FVIII successfully increased the FVIII activity in both patients. In patient 1, antibodies were raised against FVIII and PEG. Anti-FVIII antibodies were of the immunoglobulin (Ig)G isotype, whereas anti-PEG antibodies were of IgG, IgM, and IgA isotypes. In patient 2, antibodies of IgG and IgA isotypes were directed only against the PEG moiety. Competitive assays confirmed the specificity of the antibodies against PEG. The applied Nijmegen-Bethesda assay revealed that patients' anti-PEG antibodies and AGP3, an antibody against the backbone of PEG, can inhibit all currently available PEGylated therapeutics but to different degrees. No inhibitory FVIII antibodies were detected. CONCLUSION: Antibodies against the PEG moiety of 40PEG-BDDFVIII abolished the efficacy of the drug. This is the first report on real-world experiences with the development of neutralizing anti-PEG antibodies after treatment with PEGylated FVIII therapeutics in mild HA.

Applicability of the European Society of Cardiology Guidelines on the management of acute coronary syndromes to older people with haemophilia A - A modified Delphi consensus by the ADVANCE Working Group.

INTRODUCTION: As people with haemophilia (PWH) receive better treatment and live longer they are more likely to encounter cardiovascular disease (CVD) and other comorbidities. ESC guidelines for the acute management of patients presenting with acute coronary syndrome (ACS) are based on the non-haemophilia population. AIM: To review the guidelines and propose relevant adaptations for PWHA without inhibitors who are treated with prophylaxis and present with ACS. METHODS: As part of the ADVANCE Group, 20 European haemophilia experts used a modified Delphi approach to develop and gain consensus on proposed adaptations of the ESC guidelines for PWHA without inhibitors. RESULTS: Of the 32 Class I recommendations across both guidelines, adaptions were considered necessary and proposed for 15. The adaptions highlight the need to provide sufficient FVIII trough levels at the time of antithrombotic treatment in people with haemophilia A (HA) without inhibitors. Patients receiving emicizumab prophylaxis and requiring oral anticoagulation therapy or combined single antiplatelet plus oral anticoagulation therapy will require additional FVIII replacement therapy. CONCLUSION: In the absence of high-quality clinical evidence, the combined expert opinion used to develop these adaptions to the current ESC guidelines may help to guide clinicians in their treatment decisions when a PWHA presents with ACS.

A Homozygous Deep Intronic Variant Causes Von Willebrand Factor Deficiency and Lack of Endothelial-Specific Secretory Organelles, Weibel-Palade Bodies.

A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of the conventional diagnostic analysis, including multiplex ligation-dependent probe amplification and sequencing of the promoter, exons, and flanking intronic regions of the VWF gene (VWF). In this study, we intended to elucidate causative mutation through next-generation sequencing (NGS) of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The NGS revealed a variant in the intronic region of VWF (997 + 118 T > G in intron 8), for the first time. The bioinformatics assessments (e.g., SpliceAl) predicted this variant creates a new donor splice site (ss), which could outcompete the consensus 5' donor ss at exon/intron 8. This would lead to an aberrant mRNA that contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The subsequent quantitative real-time PCR confirmed the virtual absence of VWF mRNA in IP ECFCs. Additionally, the IP ECFCs demonstrated a considerable reduction in VWF secretion (~6% of healthy donors), and they were devoid of endothelial-specific secretory organelles, Weibel−Palade bodies. Our findings underline the potential of NGS in conjunction with RNA analysis and patient-derived cell studies for genetic diagnosis of mutation-negative type 3 VWD patients.

Peak pressure during gait in patients with severe haemophilia: A controlled cross-sectional study.

BACKGROUND: Patients with severe haemophilia suffer from bleeding-related joint changes in which the ankle joint is most frequently affected. In the resulting gait changes, the forefoot is involved by reducing the foot pressure. However, it is unclear which changes in foot pressure are present in the individual's foot zones. RESEARCH QUESTION: The aim of the study was to determine whether compensation mechanisms are present in the foot zones regarding the peak pressure under dynamic conditions and to identify possible underlying mechanisms for gait changes. METHODS: In a controlled cross-sectional study, a pedobarography was performed during gait with a standardized speed (3 km/h) in patients with haemophilia (PwH;n = 40) and healthy controls (Con;n = 40). Pressure pain thresholds (PPT) were detected, and Haemophilia Joint Health Score (HJHS) was performed to determine the current joint status. RESULTS: PwH showed a decreased peak pressure in metatarsals II-IV and heel compared to Con. Patients with major-affected ankle joints (determined with the HJHS) showed a decreased single-step length, stride-length and stride-time. Accordingly, the cadence was increased by 10 ± 11 steps/min in PwH compared to Con. Furthermore, PwH showed decreased ankle range of motion (ROM) in HJHS and an altered pain perception due to reduced PPT. SIGNIFICANCE: PwH showed a changed gait pattern in peak pressure compared to Con. A restricted rolling behavior, which might be caused by movement restrictions and pain sensation, leads to reduced pressure in the center forefoot, resulting in a shorter stride-length. Future therapies should focus on maintaining joint mobility for better rolling behavior and improving ankle joints' stability to achieve a balanced load between the midfoot, heel, and forefoot. The use of insoles adapted to our data, based on group differences between PwH and Con, could be supportive in this case.

Multifaceted pathomolecular mechanism of a VWF large deletion involved in the pathogenesis of severe VWD.

An in-frame heterozygous large deletion of exons 4 through 34 of the von Willebrand factor (VWF) gene was identified in a type 3 von Willebrand disease (VWD) index patient (IP), as the only VWF variant. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/factor VIII concentrates. Transcript analysis confirmed transcription of normal VWF messenger RNA besides an aberrant deleted transcript. The IP endothelial colony-forming cells (ECFCs) exhibited a defect in the VWF multimers and Weibel-Palade bodies (WPBs) biogenesis, although demonstrating normal VWF secretion compared with healthy cells. Immunostaining of IP-ECFCs revealed subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2, nuclear accumulation) and P-selectin. Besides, the RNA-sequencing (RNA-seq) analysis showed upregulation of pro-inflammatory and proangiogenic genes, P-selectin, interleukin 8 (IL-8), IL-6, and GROα, copackaged with VWF into WPBs. Further, whole-transcriptome RNA-seq and subsequent gene ontology (GO) enrichment analysis indicated the most enriched GO-biological process terms among the differentially expressed genes in IP-ECFCs were regulation of cell differentiation, cell adhesion, leukocyte adhesion to vascular endothelial, blood vessel morphogenesis, and angiogenesis, which resemble downstream signaling pathways associated with inflammatory stimuli and Ang2 priming. Accordingly, our functional experiments exhibited an increased endothelial cell adhesiveness and interruption in endothelial cell-cell junctions of the IP-ECFCs. In conclusion, the deleted VWF has a dominant-negative impact on multimer assembly and the biogenesis of WPBs, leading to altered trafficking of their pro-inflammatory cargos uniquely, which, in turn, causes changes in cellular signaling pathways, phenotype, and function of the endothelial cells.

New Inhibitors in the Ageing Population: A Retrospective, Observational, Cohort Study of New Inhibitors in Older People with Hemophilia.

INTRODUCTION: A second peak of inhibitors has been reported in patients with severe hemophilia A (HA) aged >50 years in the United Kingdom. The reason for this suggested breakdown of tolerance in the aging population is unclear, as is the potential impact of regular exposure to the deficient factor by prophylaxis at higher age. No data on hemophilia B (HB) have ever been reported. AIM: The ADVANCE Working Group investigated the incidence of late-onset inhibitors and the use of prophylaxis in patients with HA and HB aged ≥40 years. METHODS: A retrospective, observational, cohort, survey-based study of all patients aged ≥40 years with HA or HB treated at an ADVANCE hemophilia treatment center. RESULTS: Information on 3,095 people aged ≥40 years with HA or HB was collected. Of the 2,562 patients with severe HA, the majority (73% across all age groups) received prophylaxis. In patients with severe HA, the inhibitor incidence per 1,000 treatment years was 2.37 (age 40-49), 1.25 (age 50-59), and 1.45 (age 60 + ). Overall, the inhibitor incidence was greatest in those with moderate HA (5.77 [age 40-49], 6.59 [age 50-59], and 4.69 [age 60 + ]) and the majority of inhibitor cases were preceded by a potential immune system challenge. No inhibitors in patients with HB were reported. CONCLUSION: Our data do not identify a second peak of inhibitor development in older patients with hemophilia. Prophylaxis may be beneficial in older patients with severe, and possibly moderate HA, to retain a tolerant state at a higher age.

Extended Half-Life Factor VIII/Factor IX Products: Assay Discrepancies and Implications for Hemophilia Management.

Due to structural differences between extended half-life (EHL) factor VIII (FVIII) or FIX products and equivalent plasma wild-type molecules used for assay calibration, reagent-dependent discrepancies during monitoring of FVIII- and FIX-replacement therapies with EHL products have been described. To assess the performance of available one-stage clotting and chromogenic substrate assays on the Siemens Atellica COAG 360 analyzer, an in vitro study using spiked plasma samples was performed. The described results confirm previously described findings and allowed allocation of each EHL product to an appropriate assay. In addition, corresponding EHL product-specific analytes were defined within the order entry system of the University Hospital Bonn. The requirement of product-specific FVIII and FIX assays complicates patient monitoring and demonstrates the need for both continuous education and communication between treating physicians and the coagulation laboratory.

Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5' splice site.

Disease-associated silent mutations are considered to affect the accurate pre-messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 5' splice site (5'ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 5'ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 5'ss. This hairpin sequesters the 5'ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA.

Immunoadsorption for pregnancy-associated severe acquired hemophilia.

Postpartum hemorrhage is a common cause of maternal mortality. Acquired hemophilia (AH) is a rare, life-threatening bleeding disorder induced by autoantibodies against coagulation factors (inhibitors). We report about eight patients with postpartum AH (out of 82). Seven AH patients with severe bleeding complications were treated by the "Modified Bonn-Malmö Protocol (MBMP)" which consists of inhibitor elimination via immunoadsorption (IA) in combination with immunosuppression and high-dose Factor VIII substitution. One patient was treated only by immunosuppression. Seven out of eight patients with severe AH and mean inhibitor titers (IT) of 118 BU/mL were referred to our center. They were severe cases with a median delay of diagnosis of 30.5 days (range 7-278 days). After a median of 3 IA sessions (range 3-5 days), no inhibitor was detectable. The factor substitution was discontinued after a median of 13 IA sessions (range 8-24 days) and IA was terminated after a median of 15 sessions (range 9-27 days). One less severe affected patient (IT: 2.1 BU/mL) received prednisolone (1.5 mg/kg BW) for 120 days. Complete remission was achieved in all patients with a median follow-up of 100 months (range 56-126 m). The delayed diagnosis of pregnancy-associated AH leads to a high bleeding risk with bleeding associated complications. Immunoadsorption offers an important treatment option in severe AH, enabling a fast reconstitution of the blood coagulation with a reduced time for the Factor VIII substitution and for immunosuppressive treatment. In cases of postpartum bleeding the diagnosis of AH should be routinely considered.

A common F13A1 intron 1 variant IVS1+12(A) is associated with mild FXIII deficiency in Caucasian population.

Mild factor XIII deficiency is an underdiagnosed coagulation disorder. Considering the large number of coding and non-coding polymorphisms identified in the F13A1 gene, there is a possibility that some of these might result in alterations of plasma FXIII levels and cause mild FXIII deficiency. Recently, a homozygous F13A1 gene intron 1 variant (IVS1+12C>A) was found in a patient with FXIII deficiency. In vitro expression studies for this variant demonstrated its lowering effect on FXIII levels. In order to determine the impact of this variant on a population level, we analysed the prevalence of this variant in three clinically and genetically defined population cohorts: an apparently healthy control cohort C1 (n = 102), a mild FXIII deficiency cohort C2 with no detectable F13A1 or F13B gene mutations (n = 183) and a mild FXIII-A deficiency cohort C3 exhibiting heterozygous F13A1 mutations (n = 37). FXIII activity was determined using photometric assay on plasma samples. The F13A1 gene intron 1 variant was analysed by direct sequencing. The C1 cohort showed a normal distribution of FXIII activity (mean 114.1 ± 20.86%). Mean FXIII activity levels for the C2 and C3 cohorts were 54.45 ± 11.12% and 44.21 ± 10.16%, respectively. The frequencies of minor allele (A) were 0.07 in C1 cohort, 0.19 in C2 cohort and 0.11 in C3 cohort. The difference in minor allele frequencies for the C1 and C2 cohorts were highly significant (p < 0.001). The greater frequency of the IVS1+12(A) variant among C2 cohort patients suggests that this polymorphism is associated with mild FXIII deficiency.