GENYOi005-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) carrying a p.Thr196Ala variant.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients.

Low endoscopy bleeding risk in patients with congenital bleeding disorders.

INTRODUCTION: Haemophilia A and haemophilia B, von Willebrand disease (VWD), factor VII deficiency and factor XI deficiency are congenital bleeding disorders predisposing to bleeding during invasive procedures. The ageing population of people with congenital bleeding disorders will likely increasingly require gastrointestinal endoscopy. The bleeding risk postgastrointestinal endoscopy and optimal prophylactic treatment regimens are not well described. METHODS: We performed a retrospective chart review at the McGill University Health Centre. Adult patients with haemophilia A or B, VWD, FVII deficiency and FXI deficiency who underwent gastrointestinal endoscopic procedures were included. Bleeding prophylaxis included combinations of plasma-derived factor (VWD) or recombinant factor (haemophilia A and haemophilia B), desmopressin and/or tranexamic acid. Our primary outcome was the 72-hour postendoscopy bleeding rate. RESULTS: One hundred and four endoscopies were performed in 48 patients. Haemophilia A (45.3% of endoscopies) was the most common bleeding disorder, followed by VWD (38.5%), FXI deficiency (8.7%), haemophilia B (4.8%) and FVII deficiency (2.9%). All patients were reviewed by the Haemophilia Treatment Center with peri-procedure treatment protocols put in place as required. The overall 72-hour bleeding rate was 0.96%, confidence interval (CI) 95% (0.17%-5.25%). The colonoscopic postpolypectomy bleeding rate was 1/21 (4.8%, CI 95% (0.9%-22.7%)) in comparison with the general population rate of 0.3%-10% for high-risk endoscopy (including colonoscopic polypectomy). CONCLUSION: To the best of our knowledge, this is the largest study describing patients with inherited bleeding disorders undergoing gastrointestinal endoscopy. The bleeding risk is not significantly higher to the general population when haemostatically managed by a team experienced in bleeding disorders.

Parent's report on oral health-related quality of life of children with haemophilia.

INTRODUCTION: Among children with haemophilia and their caregivers; problems arising from the teeth and the surrounding tissues have an important role in the treatment of this disease and it affects the quality of life of children and their parents. AIM: Aim of this study is to evaluate the oral health-related quality of life of children with haemophilia from the perspective of their parents. METHODS: Paediatric oral health-related quality of life (POQL) instrument was used in this cross-sectional study for quality of life measurement. The research data collected by the questionnaire form were coded for scale items and personal information questions and transferred to SPSS, a multivariate statistical analysis program for social sciences. SPSS 23.0 (IBM Corp, Armonk, NY) package program was used for statistical analysis of the data. RESULTS: Primary dentition dmft scores of patients with haemophilia are higher than the control group; mean value of haemophilic group is 3.5 vs control group are 2.6, respectively (P = 0.034). In spite of higher dmft scores, the haemophilia and control groups have shown no significant difference in oral health-related quality of life scores; median scores were 63.9 in haemophilic group and 85.3 in control group (P = 0.336), respectively. CONCLUSION: In spite of lower oral health measures, children with haemophilia and their parents reported no difference in oral health-related quality of life from their healthy counterparts.

Bone Marrow Morphology Associated With Germline RUNX1 Mutations in Patients With Familial Platelet Disorder With Associated Myeloid Malignancy.

Germline mutations in RUNX1 result in autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM). To characterize the hematopathologic features associated with a germline RUNX1 mutation, we reviewed a total of 42 bone marrow aspirates from 14 FPDMM patients, including 24 cases with no cytogenetic clonal abnormalities, and 18 with clonal karyotypes or leukemia. We found that all aspirate smears had ≥10% atypical megakaryocytes, predominantly characterized by small forms with hypolobated and eccentric nuclei, and forms with high nuclear-to-cytoplasmic ratios. Core biopsies showed variable cellularity and variable numbers of megakaryocytes with similar features to those in the aspirates. Granulocytic and/or erythroid dysplasia (≥10% cells per lineage) were present infrequently. Megakaryocytes with separate nuclear lobes were increased in patients with myelodysplastic syndrome (MDS) and acute leukemia. Comparison to an immune thrombocytopenic purpura cohort confirms increased megakaryocytes with hypolobated eccentric nuclei in FPDMM patients. As such, patients with FPDMM often have atypical megakaryocytes with small hypolobated and eccentric nuclei even in the absence of clonal cytogenetic abnormalities; these findings are related to the underlying RUNX1 germline mutation and not diagnostic of MDS. Isolated megakaryocytic dysplasia in patients with unexplained thrombocytopenia should raise the possibility of an underlying germline RUNX1 mutation.

Advances in the Treatment of Hemophilia: Implications for Laboratory Testing.

BACKGROUND: Until recently, clinical laboratories have monitored hemophilia treatment by measuring coagulation factors before/after infusion of human-derived or recombinant factors. Substantial changes are expected in the near future based on new therapeutic approaches that have been or are being developed. CONTENT: Hemophilia treatment includes replacement therapy with human-derived/recombinant factors or treatment with bypassing agents for patients without or with inhibitors, respectively. Accordingly, laboratory methods for monitoring include one-stage clotting or chromogenic assays meant to measure either factor VIII/IX or global coagulation tests to measure the effect of bypassing agents. Recently, modified long-acting coagulation factors have been introduced for which discrepant results may be expected when measurement is performed with one-stage clotting or chromogenic assays. Currently, novel drugs not based on coagulation factors are under development and are being tested in clinical studies. These drugs do require new methods and therefore laboratory evaluation of hemophilia will undergo dramatic changes in the near future. SUMMARY: From the analysis of the current practice and literature, we draw the following conclusions: (a) Thrombin generation or thromboelastometry are the logical candidate assays to monitor bypassing agents. (b) Considerable differences are expected when measuring modified long-acting coagulation factors, depending on whether one-stage or chromogenic assays are used. Although no definitive conclusions can presently be drawn, chromogenic assays are probably more suitable than one-stage clotting. (c) Novel drugs not based on coagulation factors such as emicizumab, fitusiran, or concizumab that are entering the market do require alternative methods that are not yet well established.

Utility valuation of health states for haemophilia and related complications in Europe and in the United States.

INTRODUCTION: There is currently a paucity of health utility data describing the consequences of haemophilia and related complications. AIM: To quantify the impact of distinct stages of severity of haemophilia and disease-related complications on health-related quality of life, expressed as health utilities in Europe and the United States. METHODS: Nine health state descriptions were developed based on literature review and interviews with haematologists and haemophilia patients. Three descriptions characterized the impact of mild, moderate and severe haemophilia without inhibitors. Six descriptions characterized disease-related complications added to the moderate haemophilia description (arthroscopic synovectomy, prosthetic joint replacement, chronic pain, spontaneous bleed, traumatic bleed and end-stage joint disease). Time trade-off (TTO) interviews were conducted with 100 adults from the general public in the UK, France, Germany, Italy, Sweden and the United States. Mean TTO-derived utility values were expressed on a scale from 0 (death) to 1 (full health). RESULTS: Utility values obtained for the health states corresponding to mild (0.73-0.86), moderate (0.68-0.76) and severe (0.64-0.71) haemophilia followed the increase in severity. The addition of a complication to the "moderate" state leads to a decrease in the associated utility value. The most severe disutility (0.23-0.36) across all countries was associated with the burden of end-stage joint disease. CONCLUSIONS: This study underlines the value that the French, Italian, German, Swedish, United States and UK populations ascribe to the avoidance of disease progression in haemophilia without inhibitors. Improved treatment options hold a potential for important benefits to haemophilia patients.

Over two decades of orthopaedic surgery in patients with inhibitors-Quantifying the complication of bleeding.

Patients with haemophilia who have developed inhibitors against factor VIII (FVIII) or factor IX present a significant concern to those surgeons who operate on them. The evidence base for bypassing agents such as recombinant factor VIIa and activated prothrombin complex concentrate has amassed over several decades. The literature is open to positive interpretation on the successful use of these agents in the treatment of inhibitor-positive patients. However, there are equally persistent concerns amongst surgeons, in particular orthopaedic surgeons, regarding the high complication rate of bleeding. To explore and quantify this concern, we present a literature review spanning two decades of publications on haemophilia patients with inhibitors undergoing orthopaedic surgery. Irrespective of the progress made with haemostatic protocols, trepidation on embarking on surgery is valid. The high risk of bleeding is a function of the inherent complexity of the disease and rightfully translates into difficulties in its management. Combined with the prospect of orthopaedic surgery, those involved in the care of such patients are justified in their continued anxiety and diligence when considering the benefits in quality of life against the prevalent complications.

Assessment and validation of a defined fluid restriction protocol in the use of subcutaneous desmopressin for children with inherited bleeding disorders.

INTRODUCTION: Despite the availability of subcutaneous desmopressin (1-deamino-8-d-arginine vasopressin, SC-DDAVP) as a haemostatic agent for children with mild bleeding disorders, few publications specifically address the safety or efficacy of this mode of administration. AIM: Our aim was to assess whether a defined fluid restriction protocol was effective in preventing hyponatremia in children receiving perioperative SC-DDAVP, and to document adequate biological and clinical response in this setting. METHODS: We retrospectively analysed a cohort of children with mild bleeding disorders prescribed SC-DDAVP over a 5-year period following institution of a 'two-thirds maintenance' fluid restriction protocol. RESULTS: Sixty-nine patients received SC-DDAVP following this protocol, including 15 with mild haemophilia A, 49 with von Willebrand disease (VWD) and five with platelet storage pool disorder. In patients who underwent formal preoperative assessment a complete or partial response was observed in 28/29 with type 1 VWD and 14/15 with mild haemophilia A. Perioperative SC-DDAVP provided excellent haemostasis in all patients, with no requirement for factor concentrate or blood products. Mild asymptomatic hyponatremia was detected in seven children who received multiple doses of DDAVP (lowest sodium 129 mmol L(-1) ); however, adherence to the prescribed fluid restriction protocol was questionable in six of these cases. Symptomatic hyponatremia was not observed. CONCLUSION: Subcutaneous desmopressin was well-tolerated, with no serious side-effects observed, and good biological responses in preoperative trials. A two-thirds maintenance fluid regimen was effective at preventing symptomatic hyponatremia in our cohort, and is now the standard protocol for fluid restriction post-DDAVP administration in our centre.

The incidence, risk and functional outcomes of intracranial haemorrhage in children with inherited bleeding disorders at one haemophilia center.

INTRODUCTION: Intracranial haemorrhage (ICH) is the most serious bleeding event for patients with inherited bleeding disorders (IBD). The risks and long-term consequences remain unknown. AIM: This single-centre service evaluation aimed to identify the incidence, risks and long-term outcomes following ICH in patients with IBD. METHODS: The IBD database and medical notes between 1987 and 2013 were reviewed. Children without apparent neurological deficit following ICH completed standardized assessments and supplementary information sheets. RESULTS: ICH was confirmed in 38/1111 children with IBD. The overall risk of ICH amongst children with IBD was 3.4% (95% CI: 2.5, 4.7%). However, 27/38 had an ICH in the first year of life, 18 of which were in the neonatal period. In children with IBD who had an ICH, the risks of ICH in the neonatal period or first year of life were 18/38 (47%) (95% CI: 32, 63%) and 27/38 (71%) (95% CI: 55, 83%) respectively. Mortality risk from ICH in children with an IBD was 5/38 (13%) (95% CI: 5.8, 27.3 %). Ten of 32 survivors had known neurological sequelae including motor disorder deficits (MDD) while 22 had no documented evidence of neurological impairment or MDD. Re-evaluation was possible in 17/22 children, 8 of whom demonstrated evidence of MDD. After re-evaluation, the risk of significant neurological MDD from ICH increased from 31% CI (95% CI: 18, 49%) to 56% CI (95% CI: 39, 72%). CONCLUSION: Risks and consequences of ICH in IBD were highest within the neonatal period and first year of life. MDD after ICH was not reliably identified in early life and ongoing monitoring in the first decade of life will facilitate educational support or physical rehabilitation.

Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders.

INTRODUCTION: Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype-phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost. AIM: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. METHODS: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. RESULTS: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. CONCLUSION: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.

Spectrum of clinical and genetic features of patients with inherited platelet disorder with suspected predisposition to hematological malignancies: a nationwide survey in Japan.

BACKGROUND: Inherited thrombocytopenia (IT) contains several forms of familial thrombocytopenia and some of them have propensity to hematological malignancies. The etiological and genetic features of this heterogeneous syndrome have not yet been elucidated. PATIENTS AND METHODS: We conducted a nationwide survey to collect clinical information and samples from patients with familial thrombocytopenia and/or hematological malignancies in order to obtain a comprehensive understanding of IT. RESULTS: Among the 43 pedigrees with clinical samples, RUNX1 mutations were identified in 8 pedigrees (18.6%). While MYH9 and ANKRD26 mutations were identified in 2 and 1 pedigrees, respectively, no gene mutations were detected in the remaining 32 pedigrees from a panel of previously reported pathogenetic mutations. Clinical data were comparable between FPD/AML and non-FPD/AML probands. CONCLUSIONS: Our study clarified that it is unexpectedly difficult to diagnose FPD/AML based on clinical information alone, and thus, genetic testing is strongly recommended. Our survey also identified some pedigrees with a strong family history of myelodysplastic syndromes of unknown origin. Additionally, there were 14 pedigrees in which three or more members were affected by immune thrombocytopenia (ITP), and a computer-aided simulation suggested that such a distribution almost never happens by coincidence, which implicates a genetic predisposition to ITP.

Utilization Patterns of Coagulation Factor Consumption for Patients with Hemophilia.

Hemophilia is a serious rare disease that requires continuous management and treatment for which the medicine is costly at the annual average of 100 million KRW for an individual. The aim of this study was to investigate trends in the utilization of coagulation factor (CF) used for hemophilia treatment using the National Health Insurance database from 2010 to 2013 in Korea and compare the utilization of CF with other countries. The consumption of CF per capita (IU) in Korea was not more than other countries with similar income to Korea. However, CF usage per patient IU was higher because the prevalence rate of hemophilia in Korea was lower than in other countries while the number of serious patients was much more. Therefore, it is difficult to say that the consumption of hemophilia medicine in Korea is higher than that in other countries. The consumption and cost of hemophilia medicine in Korea is likely to increase due to the increased utilization of expensive bypassing agents and the widespread use of prophylaxis for severe hemophilia. Even during the research period, it increased slightly and other countries show a similar trend. Thus, hemophilia patient management should accompany active monitoring on the health and cost outcomes of pharmaceutical treatment in the future. This study is expected to contribute to further insight into drug policies for other countries that face similar challenges with high price pharmaceuticals.

Beyond hemostasis: the challenge of treating plasminogen deficiency. A report of three cases.

Congenital plasminogen deficiency is a rare autosomal recessive disorder, characterized by chronic mucosal membranous lesions. Although the most common clinical manifestation is eye involvement as ligneous conjunctivitis, extra-ocular lesions affecting other mucosal surfaces indicates a systemic disease. In this report we describe two cases with atypical extra-ocular involvement that includes pericarditis and recurrent hematocolpos, and one with paradoxical correlation between ocular lesions and plasminogen levels. In ligneous conjunctivitis, although different treatment strategies have been tried with mild success, the only effective therapy is topical or systemic plasminogen concentrates that are not commercially available. Unfortunately there is not either effective management for cases with multisystemic disease. Hence, treatment for plasminogen deficiency is still a challenge and the variability of the clinical spectrum in this pathology makes necessary a multidisciplinary approach.

Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis.

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.

Targeted correction of RUNX1 mutation in FPD patient-specific induced pluripotent stem cells rescues megakaryopoietic defects.

Familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease of the hematopoietic system that is caused by heterozygous mutations in RUNX1. FPD/AML patients have a bleeding disorder characterized by thrombocytopenia with reduced platelet numbers and functions, and a tendency to develop AML. No suitable animal models exist for FPD/AML, as Runx11/2 mice and zebra fish do not develop bleeding disorders or leukemia. Here we derived induced pluripotent stem cells (iPSCs) from 2 patients in a family with FPD/AML, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in 1 FPD iPSC line through gene targeting, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients.