Hereditary Bleeding Disorder Research Articles

Laboratory Monitoring in Emicizumab-Treated Persons with Hemophilia A.

Hemophilia A (HA) is an X-linked hereditary bleeding disorder caused by deficiency of coagulation factor (F) VIII activity. One of the greatest complications in the treatment of HA is the development of neutralizing alloantibodies, known as FVIII inhibitors. HA patients who develop FVIII inhibitors have limited treatment options available to them and experience greater disease- and treatment-related burdens than HA patients without FVIII inhibitors. Emicizumab, a recently approved bispecific monoclonal antibody, mimics the function of FVIIIa by bridging FIXa and FX to restore effective hemostasis. Although emicizumab and FVIII show some functional similarities, several key differences influence the results of standard laboratory assays when conducted in the presence of emicizumab, and can result in a misleading interpretation of coagulation assays in emicizumab-treated patients. Here, we discuss current laboratory monitoring methods, including activated partial thromboplastin time, FVIII one-stage clotting assays, FVIII chromogenic assays, and global coagulations assays; address why these conventional methods may be inappropriate for monitoring of HA patients receiving emicizumab; and suggest alternative methods applicable to monitoring HA treatment in an evolving treatment landscape.

PS1106 A RETROSPECTIVE ANALYSIS OF IMMUNE THROMBOCYTOPENIA PATIENTS TREATMENT RESULTS AFTER SEQUENTIAL SWITCHING OF THROMBOPOIETIC RECEPTOR‐AGONISTS

Background:Immune thrombocytopenia (ITP) is an immune‐mediated disease characterized by increased platelet destruction and impaired platelet production with transient or persistent decrease in the platelet count and increased risk of bleeding. Romiplostim (R) and Eltrombopag (E) are the first thrombopoietic receptor‐agonists (TPO‐ra) with demonstrated efficacy against ITP. So far, very few data are available about the potential contribution of switching from romiplostim to eltrombopag or vice versa in terms of efficacy or tolerance.Aims:To evaluate the reasons for and result of TPO‐ra switching in adult ITP patientsMethods:We retrospectively analyzed all patients who received sequential treatment with both TPO‐RA between 2014 and 2018 in Moscow city hematology center (Botkin hospital).Results:Thirty‐three pts with primary ITP were included; 19 received first Romiplostim and 15 received first Eltrombopag. Reasons for switching were inefficacy (n = 30) and adverse events (n = 3). All patients (33) had previously received multiple courses of corticosteroids and IV Ig infusions, four (10%) received immunosuppression (rituximab), and five (14%) performed splenectomy. The median platelet count at the administration of the second TPO‐ra was 17x109/l (range 3–25x109/l). Twenty‐five (76%) patients had bleeding symptoms before the second TPO‐ra administration.From 33 included patients partial response (> 50x109/l without platelet transfusions and bleeding) was achieved in 16 (70%). PR was seen in sixteen from 19 (84%) in E to R group and seven from 15 (47%) in R to E group. Complete response (>100x109/l without pl. transfusions and bleeding) were achieve in 17 from 33 (52%). Twelve pts from 19 (63%) achieve CR in E to R group and five from 15 (30%) in R to E group. Two of 17 (10%) pts achieved a CR within 1–3 months, 11 (67%) pts in 3–12 months, and four (23%) pts later than 1 year from the start of switching to second TPO‐ra therapy.Three pts (2 in E group due to repeated thrombosis, and 1 in R group due to persistent increased liver enzymes) with recommendation of switching to second TPO‐ra due to adverse events resolved rapidly and maintained remission on alternative drug.In nine pts (28%) the switching to second TPO‐ra therapy was ineffective (blood platelet level was less than 30x109/l pl). All of them were resistant to first TPO‐ra.Summary/Conclusion:Our results confirmed that switching from one thrombopoietic receptor‐agonist to another could be beneficial for patients with chronic immune thrombopenia who failed to respond. Yet longer follow‐up and larger number of pts enrolled are required to fully assess the potential benefits of such therapy change.

PS1105 CONGENITAL DEFICIENCY OF COAGULATION FACTOR VII IN THE SOUTH OF TUNISIA: ABOUT 88 CASES

Background:Congenital factor VII deficiency (FVII) is the least rare among uncommon hereditary bleeding disorders (1:500 000). This coagulopathy, inherited in an autosomal recessive pattern, is characterized by a bleeding tendency, varied prognosis and a lack of correlation between clinical phenotypes and biological analysis.Aims:The objective of our study was to describe and to explore the clinical and the biological features of FVII deficiency in the South of Tunisia.Methods:Our FVII‐deficient cohort consisted of 88 patients whose FVII activity was less than 50% and who were referred to our laboratory from January 2000 to December 2018 because of a prolonged prothrombin time (PT) or hemorrhagic manifestations. The FVII deficiency was considered as severe for FVII coagulant activities (FVII:C) <2%, moderate for rates between 2 and 19% and minor for values between 20 and 49%. The statistical analysis was based on Chi‐squared test.Results:The mean age of the patients was 22 years old ranging between 21 months and 63 years old and the sex ratio was about 0.83. Among 88 patients, belonging to 58 unrelated Tunisian families, 45 cases (51.2%) were diagnosed accidentally during presurgical screening, 28 cases (31.8%) were noticed following family screening and 15 patients (17%) were discovered due to bleeding accidents. Clinically, 64 patients (72.7%) were asymptomatic whereas the bleeding frequency was about 27.3%. Clinical examination revealed several bleeding manifestations as follows : gingivorrhagia noticed in 12 patients (50%), followed by epistaxis in nine patients (37.5%). Four cases had hematemesis (16.6%), hematoma was found in three (12.5%) and one of the patients had a cerebral haemorrhage (4.2%). Added to that, some bleeding signs, not commonly related to the FVII deficiency, had been noticed such as: menorrhagia, haemoperitoneum, hematuria and bruising. The laboratory severity of each patient was also determined : FVII deficiency was classified as severe in 3.4%, moderate in 14.8% and minor in 81.8% of cases. With reference to statistics, results showed a weak correlation between clinical data and biological levels of FVII:C (p = 0.004). In fact, 15 patients with a minor FVII deficiency had presented haemorrhages with a variable bleeding severity; while one of the patients was asymptomatic despite the severity of his defect.Summary/Conclusion:Our study, in accordance with the literature, revealed a great interindividual heterogeneity of the clinical phenotype of FVII deficiency. In addition, bleeding severity did not necessarily correlate with FVII:C and varies markedly between individuals with a similar FVII:C. This would explain the delay and the underestimation of the diagnosis. In addition, the family screening had a prominent effect in improving the earliness of the diagnosis and in ensuring an adequate monitoring of these Tunisian patients.

Strategies for Individualized Dosing of Clotting Factor Concentrates and Desmopressin in Hemophilia A and B.

Hemophilia A and hemophilia B are hereditary bleeding disorders, caused by a deficiency of clotting factor VIII or clotting factor IX, respectively. To treat and prevent bleedings, patients can administer clotting factor concentrates (hemophilia A and B) or desmopressin (hemophilia A). Both clotting factor concentrates and desmopressin are currently dosed according to the patients' body weight. However, clotting factor concentrates exhibit considerable pharmacokinetic (PK) variability. Therefore, several alternative dosing strategies to individualize dosing of clotting factor concentrates and desmopressin in hemophilia A and B have been proposed. In this study, a review of the existing literature on the individualization of dosing based on PK guidance was performed. In total, 79 articles were included. The methods to individualize dosing were divided into 3 categories: (1) methods using clinical parameters, (2) empirical individual PK-guided methods, and (3) maximum a posteriori (MAP) Bayesian estimation methods. The clinical parameter mainly used to individualize dosing is bleeding phenotype. Dosing based on bleeding phenotype may decrease clotting factor consumption. However, with this method, it is not possible to individualize on-demand dosing during bleeding events or in the perioperative setting. Empirical individual PK-guided methods can be used both for prevention and treatment of bleedings. These methods include dose individualization using a nomogram and individualized in vivo recovery. In the perioperative setting, adjustment of the rate of continuous infusion can be applied to obtain a specific target level. The final category, MAP Bayesian estimation methods, relies on the availability of a population PK model. In total, 22 population PK models describing clotting factor concentrate or desmopressin dosing are currently available in literature. MAP Bayesian estimates can be used to calculate the individualized doses required to achieve or maintain a target level in every setting. The application of PK-guided and pharmacodynamic-guided dosing of clotting factor concentrates and desmopressin seems promising, although further investigation is warranted. Prospective studies analyzing its potential benefit are on the way.

Good Care and Long-Term Follow-up in Young Hemophiliacs

Introduction: Hemophilia is a hereditary bleeding disorder that presents with primarily hemarthrosis and hematoma. The most common and frequent complication, especially in severe hemophilia, where factor VIII or IX is not administered prophylactically, is the hemophilic arthropathy that develops as a result of recurrent bleeding. In this study, the relationship between arthropathy development and timing of the prohylaxis and orthopedic interventions in hemophiliacs was presented.Materials and Methods: The data of 99 haemophilia (HA) and hemophilia B (HB) patients under the age of 25 years at our center were reviewed retrospectively. Age, type of disease, factor level, follow-up duration, prophylaxis dose and frequency, annual bleeding rate (ABR), target joints, arthropathic joints, applied radioactive synovectomy and orthopedic interventions were recorded.Results: Seventy-six HA and 23 HB were included. The median age was 14 (range 1-25) years old. Ten of them (10.1%) were mild, 15 (15.2%) were moderate and 74 (74.7%) were severe. Severe hemophiliacs were most of the patients in both types. Eighty-nine (89.9%) patients including 17 of mild-to-moderate type with target joint and / or surgical intervention were under prophylaxis and 10 (10.1%) were receiving on-demand therapy. The annual bleeding rate was 1.38. Twelve (12%) patients (8 HA, 4 HB) with degenerative hemophilic arthropathy had a mean age of 22 years old and a mean age of application was 12.3 years, a mean annual bleeding rate was 1.54 and a mean follow up and prophylaxis time was 9.5 years. Six patients (4 HA, 2 HB) with median age 21.8 years (range: 18-25) had 9 major orthopedic surgeries (arthrodesis (2), radial head resection (2), TEP, contracture releasing, acyloplasty, deformity correction, arthroscopic sinovectomy) and 22 patients with median age of 17.8 years had 29 minor orthopedic interventions (radiosynovectomy (28), closed reduction to femur fracture). Orthopedic interventions were performed at an advanced age, often who met with prophylaxis too late.Discussion: In our study, it has been shown that who had prophylaxis at an early age and prophylaxis for a longer period had less arthropathies which is the most important complication and additionally less arthroplasties. The fact that, the prevalence of permanent musclosceletal deformity was 59% before year 2000 (historical data) which reduced to 12% nowadays shows the sensitivity of hemophilia care in our center. Early and regular prophylaxis remains the most current treatment modality for the prevention of hemophilia complications. DisclosuresNo relevant conflicts of interest to declare.

The Tissue Factor Pathway Inhibitor Antibody, PF-06741086, Increases Thrombin Generation in Rare Bleeding Disorder and Von Willebrand Factor Deficient Plasmas

Hemophilia A and B are hereditary bleeding disorders that result from deficiencies in the intrinsic coagulation pathway leading to insufficient generation of Factor Xa (FXa) and thrombin to promote stable hemostasis. Coagulation defects are also observed in other inherited rare factor bleeding disorders. The extrinsic pathway of coagulation in these disorders cannot generate sufficient levels of FXa due to the regulation by Tissue Factor Pathway Inhibitor (TFPI). TFPI is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation by inhibiting the FXa/tissue factor (TF)/Factor VIIa (FVIIa) complex. PF-06741086, a fully human inhibitory monoclonal antibody, binds the Kunitz-2 domain and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. The addition of PF-06741086 in vitro to donor plasma from both healthy normal volunteers and hemophilia patients promoted thrombin generation and restored hemostasis in vivo in murine hemophilia bleeding models. Pharmacological effects of PF-06741086 on thrombin generation were also observed in a healthy volunteer Phase 1 study. Other rare disease coagulopathies also result in the insufficient generation of thrombin. In this study, the potential of PF-06741086 to restore thrombin generation in rare disease plasma was explored.Thrombin generation was measured in citrated platelet poor Factor XI (FXI), Factor V (FV), FVII, von Willebrand Factor (vWF) deficient (Type 1, 2A, 2B and 3) congenital donor plasma following the in vitro addition of PF-06741086 (0, 1, 10 or 100 nM) or a human IgG1 antibody; initiated with 1 pM TF and 4 µM phospholipid. FXI, FV, and FVII donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone was also included in the analysis. In FXI deficient plasmas, a concentration-dependent increase in peak thrombin and a shortening of the lag time was observed with the addition of PF-06741086 normalizing and restoring levels to those observed in the non-hemophilic plasma. A similar response was also observed in all of the vWF deficient plasmas. In one FVII deficient plasma, an increase in peak thrombin was observed at dose of 100 nM PF-06741086, however, the lag time (20 minutes) was significantly extended, relative to healthy volunteer non-hemophilic plasma. As expected, the addition of PF-06741086 to FV deficient plasma did not increase thrombin generation at any concentration.The in vitro addition of the TFPI antibody, PF-06741086, improved thrombin generation in selected coagulation factor deficient plasmas, including vWF deficiency, to the levels observed in normal plasma. This data suggestion that the inhibition of TFPI may promote hemostasis in rare bleeding disorders such as FXI deficiency and vWF deficiencies. DisclosuresRakhe:Pfizer: Employment. Patel-Hett:Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

A Neutralizing Monoclonal Antibody (PF-06741086) Against Tissue Factor Pathway Inhibitor in Combination with Activated Prothrombin Complex Concentrate Increases Hemostasis in Inhibitor Hemophilia Plasmas without Excessive Thrombin Generation

Hemophilia A and B are hereditary bleeding disorders caused by intrinsic coagulation pathway deficiencies of Factor VIII or Factor IX, respectively. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation. PF-06741086 is a fully human monoclonal antibody which binds the Kunitz-2 domain and neutralizes the inhibitory activity of human tissue factor pathway inhibitor and is currently under development as a potential prophylactic treatment to prevent bleeding episodes in hemophilia A and hemophilia B patients with and without inhibitors. Activated prothrombin complex concentrate (aPCC) is used as bypass treatment for the resolution of bleeding in some hemophilia patients with inhibitors. Hemophilia inhibitor patients receiving PF-06741086 have a possibility to also receive treatment with aPCC. The aim of the current study was to assess the potential additive effect of PF-06741086 with aPCC added in vitro to Hemophilia A and B inhibitor plasmas using a thrombin generation assay (TGA).Thrombin generation in the presence of 1 pM tissue factor and 4 µM phospholipid, was measured using the calibrated automated thrombogram (CAT) system in citrated platelet poor hemophilia A inhibitor (88-160 Bethesda Units) donor plasma or hemophilia B inhibitor (FIX immune-depleted and spiked with FIX neutralizing antibody, 14 Bethesda Units) plasma following the addition of PF-06741086 or aPCC (FEIBA) either alone or in combination. All donors had less than 1% coagulation factor activity. Non-hemophilic plasma from healthy donors alone or spiked in vitro with 16 µg/mL of PF-06741086 was also included in the analysis. Non-hemophilic plasma would have the full complement of coagulation factors. Dose-dependent increases in peak thrombin were observed with the addition of aPCC alone or PF-06741086 alone to the hemophilia plasmas. For combination studies, the aPCC concentration of 1 Unit/mL was selected to correspond to plasma levels that could be achieved clinically post-dosing. The concentration of PF-06741086 at 16µg/mL in these studies was chosen to approximate the Cmax concentration following a single 300 mg subcutaneous dose. Both PF-06741086 (16 µg/mL) and aPCC (1 Unit/mL) decreased the lag time in hemophilia plasma, however, there was not an additive decrease in the lag time with the combination of PF-06741086 and aPCC. The addition of PF-06741086 in combination with aPCC to hemophilia plasma resulted in an increase in thrombin generation including a higher peak thrombin concentration compared to the addition of either alone, but was within the range reported in studies for non-hemophilic normal plasma. To summarize, the addition of aPCC (1 Unit/mL) in combination with PF-06741086 (16µg/mL) in vitro resulted in increased thrombin generation in hemophilia A and hemophilia B inhibitor plasmas without inducing excessive coagulation. DisclosuresRakhe:Pfizer: Employment. Bowley:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Development of a Pegylated Dimeric Recombinant Factor VIII-Fc Fusion Protein for the Treatment for Hemophilia a Patients

▪Hemophilia A is a hereditary bleeding disorder resulting from reduced factor FVIII activity. It occurred in 1/5000 male. Currently, the treatment option is with the factor FVIII replacement therapy. A long-acting recombinant monomeric FVIII-Fc fusion protein product (Eloctate®) has been approved in 2014 by the US FDA, it requires to infuse the drug for every 3 days or twice a week. There is a clinical need to develop longer half-life product to extend the treatment option to once a week infuse for hemophilia A patients. Recently, we have developed a dimeric recombinant factor VIII-Fc (drFVIII-Fc) fusion protein therapeutic candidate, which is entering the clinical development in China. To generate a longer half-life recombinant FVIII product, we have developed a PEGylation method to PEGylated this drFVIII-Fc fusion protein to PEGdrFVIII-Fc. We have analyzed and characterized the fusion protein by various analytic methods, as well as in vivo animal tests. It was shown that PEGdrFVIII-Fc fusion protein has been modified with about five Y type of 40kd PEG; the remaining activity is around 700 IU/mg, and the in vivo tests in cynomolgus monkey demonstrated that the fusion protein has a half-life of about 37 hours. The data also showed that there was no detectable affinity binding activity of vWF to a PEGdrFVIII-Fc fusion protein, as compared with the binding activity of 5.16X10-4M for the molecule of a drFVIII-Fc fusion protein. In conclusion, we are able to generate a PEGylated form of a drFVIII-Fc molecule with the relevant specific activity and has been shown the molecule with the prolonger half-life in the in vivo tests. The further biochemical analysis demonstrated PEGdrFVIII-Fc fusion protein with no detectable vWF binding activity, which might explain why its half-life is longer than vWF's ~15hours half-life in vivo. This molecule is likely to be used as a once-weekly treatment option for hemophilia A patients. Currently, we are in the development stage of an IND filing in China. DisclosuresNo relevant conflicts of interest to declare.

The unusual pattern of hereditary bleeding disorders in the province of Newfoundland and Labrador—Canada’s most Eastern Province

Newfoundland and Labrador (NL), the most eastern province of Canada, is characterized by a unique topography and pattern of settlement. The current population is descended from a small founding population of indigenous Innu, Inuit and Mi’kmaq and an estimated 28,000 settlers. These settlers originated from Southwest England and Southeast Ireland and came to invest and work in one of the world’s richest fisheries. They settled in bays, coves and islands off the coast, in small settlements called out-ports. These isolated communities developed unusual patterns of genetic disease including an unusual prevalence of some extremely rare Hereditary Bleeding Disorders (HBD). This study was designed to document the prevalence of these rare disorders, at a snapshot in time, using our provincial HBD registry. These diagnoses were verified by reviewing the original initial diagnostic coagulation results to confirm or refute each diagnosis. When available, we also recorded the underlying mutation. Population based prevalence rates were then compared with data published from the World Federation of Hemophilia (WFH) Global Registry. The results are striking. Using the WFH data the per capita prevalence in NL of Hemophilia A, Factors V, XI, and XIII Deficiency are higher than that of mainland Canada minus Labrador by a factor of 2.89, 4.54, 5.44 and 9.22, respectively. The increased prevalence of mild Hemophilia A is explained by a founder effect of the Val 2016 Ala mutation. All the severe FXIII deficient patients are homozygotes for c.691-1 G > A mutation. These results show that NL’s unique geography and population distribution led to a genetic drift that increased the prevalence of some rare factor deficiencies. This comparatively high prevalence provides a potential pool of patients for genotype/phenotype research.

Optimized Adeno-Associated Viral-Mediated Human Factor VIII Gene Therapy in Cynomolgus Macaques.

Hemophilia A is a common hereditary bleeding disorder that is characterized by a deficiency of human blood coagulation factor VIII (hFVIII). Previous studies with adeno-associated viral (AAV) vectors identified two liver-specific promoter and enhancer combinations (E03.TTR and E12.A1AT) that drove high level expression of a codon-optimized, B-domain-deleted hFVIII transgene in a mouse model of the disease. This study further evaluated these enhancer/promoter combinations in cynomolgus macaques using two different AAV capsids (AAVrh10 and AAVhu37). Each of the four vector combinations was administered intravenously at a dose of 1.2 × 1013 genome copy/kg into five macaques per group. Delivery of the hFVIII transgene via the AAVhu37 capsid resulted in a substantial increase in hFVIII expression compared to animals administered with AAVrh10 vectors. Two weeks after administration of E03.TTR packaged within the AAVhu37 capsid, average hFVIII expression was 20.2 ± 5.0% of normal, with one animal exhibiting peak expression of 37.1% of normal hFVIII levels. The majority of animals generated an anti-hFVIII antibody response by week 8-10 post vector delivery. However, two of the five macaques administered with AAVhu37.E03.TTR were free of a detectable antibody response for 30 weeks post vector administration. Overall, the study supports the continued development of AAV-based gene therapeutics for hemophilia A using the AAVhu37 capsid.

Principles of haemophilia care: The Asia‐Pacific perspective

Optimal haemophilia care is best established and implemented through a well-coordinated plan guided by clearly defined principles and priorities. A document which enunciates those details is therefore important. A successful example of this approach is the definition of principles of haemophilia care (PHC) outlined by the European Association for Haemophilia and Associated Disorders (EAHAD) and also the World Federation of Hemophilia. A similar document applicable to the Asia-Pacific region must take into account not only the highly varied healthcare systems but also the tremendous socio-economic and cultural diversities which impact provision of such care. The Asia-Pacific Haemophilia Working Group (APHWG), representing the countries in this region, has prepared this perspective of the PHC. While endorsing the overall framework outlined by EAHAD, this APHWG document emphasizes regional priorities on education and training of healthcare personnel in the diagnosis and management of hereditary bleeding disorders. Central coordinating agencies with wide stakeholder input, networks of haemophilia treatment centres and national registries as well as robust processes for procurement and distribution of safe and effective clotting factor concentrates (CFCs), implementation of prophylaxis programmes and management of patients with inhibitors should also be developed. The implementation of these strategies should lead to establishment of good comprehensive care programmes. This document should also be an advocacy tool to lobby for improved care for people with haemophilia (PWH) in the region. We urge national healthcare policy makers to consider these principles and initiate strong and decisive action to reach these goals.

No. 163-Gynaecological and Obstetric Management of Women With Inherited Bleeding Disorders.

The prevalence of bleeding disorders, notably von Willebrand disease (vWD), among adult women with objectively documented menorrhagia is consistently reported to be 10% to 20% and is even higher in adolescents presenting with menorrhagia. Diagnostic tools and specific medical and, where appropriate, surgical alternatives to management are reviewed and evidence-based recommendations presented. A MEDLINE search of the English literature between January 1975 and November 2003 was performed using the following key words: menorrhagia, uterine bleeding, pregnancy, von Willebrand, congenital bleeding disorder, desmopressin/DDAVP, tranexamic acid, oral contraceptives, medroxyprogesterone, therapy, hysterectomy, anesthesia, epidural, spinal. Recommendations from other society guidelines were reviewed. The quality of evidence reported in this document has been described USing the Evaluation of Evidence criteria outlined in the Report of the Canadian Task Force on the Preventive Health Exam (Table1).13 RECOMMENDATIONS.

A 12.3-kb Duplication Within the VWF Gene in Pigs Affected by Von Willebrand Disease Type 3.

Von Willebrand Disease (VWD) type 3 is a serious and sometimes fatal hereditary bleeding disorder. In pigs, the disease has been known for decades, and affected animals are used as models for the human disease. Due to the recessive mode of inheritance of VWD type 3, severe bleeding is typically seen in homozygous individuals. We sequenced the complete porcine VWF (Von Willebrand Factor) complementary DNA (cDNA) and detected a tandem duplication of exons 17 and 18, causing a frameshift and a premature termination codon (p.Val814LeufsTer3) in the affected pig. Subsequent next generation sequencing on genomic DNA proved the existence of a 12.3-kb tandem duplication associated with VWD. This duplication putatively originates from porcine Short Interspersed Nuclear Elements (SINEs) located within VWF introns 16 and 18 with high identity. The premature termination truncates the VWF open reading frame by a large part, resulting in an almost entire loss of the mature peptide. It is therefore supposed to account for the severe VWD type 3. Our results further indicate the presence of strong, nonsense-mediated decay in VWF messenger RNA (mRNA) containing the duplication, which was supported by the almost complete absence of the complete VWF protein in immunohistochemistry analysis of the VWD-affected pig. In the past, differentiation of wild-type and heterozygous pigs in this VWD colony had to rely on clinical examinations and additional laboratory methods. The present study provides the basis to distinguish both genotypes by performing a rapid and simple genetic analysis.

Prevalence of von willebrand disease among university students in Riyadh, Saudi Arabia

BACKGROUND: von Willebrand disease (vWD) is the most common hereditary bleeding disorder, affecting up to 1% of the general population. OBJECTIVES: Estimating the prevalence of vWD among adolescents. DESIGN: This study was conducted between February 2014 and January 2016 on Saudi students in Riyadh. SETTINGS: We conducted an epidemiological survey on university students, using the standardized questionnaire based on molecular and clinical markers for the diagnosis and management of type 1 VWD. MATERIALS AND METHODS: All blood samples were tested for complete blood count, prothrombin time, partial thromboplastin time (PTT), and platelet function analyzer (PFA-100). MAIN OUTCOME MEASURES: Samples had an abnormal result of PTT and/or PFA-100 were tested for von Willebrand factor (vWF) antigen and factor VIII (FVIII) activity. SAMPLE SIZE: 2000 university students aged between 17 and 22 years were included. RESULTS: Of these students, 730 (36.5%) had reported bleeding symptoms, 326 (44.6%) had agreed to give blood samples, 116 (35.5%) samples had prolonged PTT (>41 s), 48 (14.7%) had prolonged PFA-100 adenosine diphosphate, 39 (11.9%) had prolonged PFA-100 epinephrine, and 72 (22.0%) had abnormal results in both PTT and PFA-100. Out of 275 samples tested for vWF (Ag and activity) and FVIII, 13 (3.9%) had reduced levels or nonfunction of vWF and 5 (1.6%) had reduced FVIII levels. After correlation with ABO blood group, only 5 (1.6%) cases were confirmed for vWD. The prevalence of vWD among Saudi adolescents in the selected student population was 1.5%. CONCLUSION: In this study, we report for the first time epidemiological survey of bleeding disorders in Arab ethnicity. LIMITATIONS: As this is a prevalence study, we have no limitations to discuss.

Research progress of hereditary blood diseases

Hereditary blood diseases are disorders due to genetic factors, including hereditary red blood cell disorders, hereditary white blood disorders and hereditary bleeding disorders. These diseases can be diagnosed via gene analysis, but about half of pathogenic gene mutations of these kinds of diseases are still unknown. Currently, replacement therapies are the only effective management for the hereditary blood diseases and gene therapy may be the curative options. Recent years have found encouraging progress in gene therapy, while carcinogenesis and long-term expressions are the major concerns. In this paper, we review the recent progress and unmet needs in hereditary blood diseases.

Epidemiology of Hereditary Coagulation Bleeding Disorders: A 15-Year Experience From Southern Iran

Background: Data on the frequency of hereditary bleeding disorders (HBDs) and associated mortality and morbidities during a long-term follow-up from Iran are scarce. Objective: This study evaluated the epidemiologic features among patients with HBD in one of the largest referral centers in southern Iran. Methods: In this cross-sectional study, 619 patients with HBD were evaluated during the period 1996 to 2011. Aside from baseline characteristics and type of factor deficiency, associated morbidities including viral infections, neurological disorders, asthma, thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, diabetes, hypertension, cardiac and renal diseases were evaluated. Furthermore, among patients who died, the underlying disease and etiology of death were also evaluated. Results: Patients’ mean age was 24.4 ± 13.5 years. Factor VIII deficiency was the most prevalent type (50.4%) of HBD, and combined Von–Willebrand and factor XIII deficiency (2.3%) was the most prevalent type of combined factor deficiency. A total of 0.5% had hepatitis B and 11.5% had hepatitis C. Cardiac disease was seen in 1.5%, hypertension in 0.2%, renal disease in 0.2%, and diabetes in 1.3% of patients. Overall, 5.2% had intracranial hemorrhage, 2.1% had epilepsy, and 0.8% had mental retardation. During the 15-year follow-up, 22 patients died; car accident was the leading cause of death in this population. Conclusion: Associated morbidities were seen in 24.3% of patients with HBD. Most prevalent morbidities were HCV infections (11.5%) and neurological disease (7.3%). The mortality rate among patients with HBD was 3.4%, and the most common cause of death was accident, which is similar to that of normal Iranian populations.

Factor XIII deficiency diagnosis: Challenges and tools.

Factor XIII deficiency (FXIIID) is a rare hereditary bleeding disorder arising from heterogeneous mutations, which can lead to life-threatening hemorrhage. The diagnosis of FXIIID is challenging due to normal standard coagulation assays requiring specific FXIII assays for diagnosis, which is especially difficult in developing countries. This report presents an overview of FXIIID diagnosis and laboratory methods and suggests an algorithm to improve diagnostic efficiency and prevent missed or delayed FXIIID diagnosis. Assays measuring FXIII activity: The currently available assays utilized to diagnose FXIIID, including an overview of their complexity, reliability, sensitivity, and specificity, as well as mutational analysis are reviewed. The use of a FXIII inhibitor assay is described. Diagnostic tools in FXIIID: Many laboratories are not equipped with quantitative FXIII activity assays, and if available, limitations in lower activity ranges are important to consider. Clot solubility tests are not standardized, have a low sensitivity, and are therefore not recommended as routine screening test; however, they are the first screening test in almost all coagulation laboratories in developing countries. To minimize the number of patients with undiagnosed FXIIID, test quality should be improved in less well-equipped laboratories. Common country-specific mutations may facilitate diagnosis through targeted genetic analysis in reference laboratories in suspected cases. However, genetic analysis may not be feasible in every country and may miss spontaneous mutations. Centralized FXIII activity measurements should also be considered. An algorithm for diagnosis of FXIIID including different approaches dependent upon laboratory capability is proposed.

In silico analysis of Glanzmann variants of Calf-1 domain of \u03b1IIb\u03b23 integrin revealed dynamic allosteric effect

Integrin αIIbβ3 mediates platelet aggregation and thrombus formation. In a rare hereditary bleeding disorder, Glanzmann thrombasthenia (GT), αIIbβ3 expression / function are impaired. The impact of deleterious missense mutations on the complex structure remains unclear. Long independent molecular dynamics (MD) simulations were performed for 7 GT variants and reference structure of the Calf-1 domain of αIIb. Simulations were analysed using a structural alphabet to describe local protein conformations. Common and flexible regions as well as deformable zones were observed in all the structures. The most flexible region of Calf-1 (with highest B-factor) is rather a rigid region encompassed into two deformable zones. Each mutated structure barely showed any modifications at the mutation sites while distant conformational changes were observed. These unexpected results question the relationship between molecular dynamics and allostery; and the role of these long-range effects in the impaired αIIbβ3 expression. This method is aimed at studying all αIIbβ3 sub-domains and impact of missense mutations at local and global structural level.

Occult hepatitis B virus infection in Greek patients with congenital bleeding disorders

Occult Hepatitis B Infection (OBI) is a form of chronic HBV infection characterized by low level HBV DNA, without detectable HBV surface antigen (HBsAg). OBI is frequently associated with the presence of anti-HBc and in some cases also with anti-HBs. Patients, who formerly received non-inactivated factor concentrates, can potentially be considered at high risk for OBI, especially since these patients usually are HIV or HCV co-infected. This study aimed to assess the prevalence of occult HBV infection in Greek patients with hereditary bleeding disorders. The study sample comprised of 114 patients from a single haemophilia center. All patients were screened for HBV serum markers and individually tested for HBV DNA using a qualitative PCR. Presence of HBV DNA was further confirmed by quantification of viral load with an ultrasensitive in-house real time PCR. 88 and 21 patients with haemophilia A and B, respectively, 4 patients with von Willebrand Disease and 1 patient with severe factor VII deficiency were screened for the presence of OBI. Anti-HBc were detected in 53 (46.5%) subjects; 18 of them were anti-HBs(−) and 35 anti-HBs(+). Anti-HBe were present in 26 subjects. Two out of 114 patients were HBsAg(+). Of the remaining 112 HBsAg(−) patients tested, two (1.8%) were found HBsAg(−), HBV DNA(+), anti-HBc(+) and anti-HBs(−) and were identified as potential OBI cases. Both cases exhibited very low DNA levels; 38.2IU/mL in patient A and 14.2IU/mL in patient B. Both patients were HBeAg(−), but patient A had HBe antibodies. Patient B was also HIV/HCV co-infected. In conclusion, two cases of OBI with low HBV viraemia were identified among patients with congenital bleeding disorders. Although the incidence in our sample is moderately low (1.8%), close monitoring of these infections is of great clinical significance, especially in patients with co-infections and concomitant immunosuppression.

High‐throughput sequencing approaches for diagnosing hereditary bleeding and platelet disorders

Hereditary bleeding and platelet disorders (BPDs) are characterized by marked genetic heterogeneity, far greater than previously appreciated. The list of genes involved in the regulation of megakaryopoiesis, platelet formation, platelet function and bleeding has been growing rapidly since the introduction of high-throughput sequencing (HTS) approaches in research. Thanks to the gradual adoption of HTS in diagnostic practice, these discoveries are improving the diagnostic yield for BPD patients, who may or may not present with bleeding problems and often have other clinical symptoms unrelated to the blood system. However, it was previously found that screening for all known etiologies gives a diagnostic yield of over 90% when the phenotype closely matches a known BPD but drops to 10% when the phenotype is indicative of a novel disorder. Thus, further research is needed to identify currently unknown etiologies for BPDs. Novel genes are likely to be found to be implicated in BPDs. New modes of inheritance, including digenic inheritance, are likely to play a role in some cases. Additionally, identifying and interpreting pathogenic variants outside exons is a looming challenge that can only be tackled with an improved understanding of the regulatory landscape of relevant cell types and with the transition from targeted sequencing to whole-genome sequencing in the clinic.