INTRODUCTION:Congenital fibrinogen disorders (CFDs), encompassing quantitative (hypo-/afibrinogenemia) and qualitative (dysfibrinogenemia) defects, can result in bleeding or thrombotic events. This study aimed to enhance understanding of the clinical and genetic characteristics of CFD patients. METHODS:The Dutch cross-sectional RBiN study included 47 CFD patients (median age 38, 55 % women), categorized into (hypo)dysfibrinogenemia, severe (<500 mg/L), moderate (500-1000 mg/L) and mild hypofibrinogenemia (1000-1800 mg/L) as well as carriers with pathogenic variants but normal fibrinogen levels (>1800 mg/L). Clinical assessments included bleeding phenotype, thrombosis history, fibrinogen activity and antigen levels, thrombin and plasmin generation assays and genotypic analysis. RESULTS:Patients with severe hypofibrinogenemia displayed the highest median ISTH-BAT score (16), followed by moderate hypofibrinogenemia (11), (hypo)dysfibrinogenemia (6), mild hypofibrinogenemia (4) and carriers (0). Female-specific bleeding (postpartum hemorrhage, heavy menstrual bleeding) was prevalent across all CFD subtypes, with moderate hypofibrinogenemia showing high average scores on these ISTH-BAT items (3.0 and 2.3). Postoperative bleeding was common in moderate and severe hypofibrinogenemia (average ISTH-BAT item scores of 2.5 and 2.8, respectively). Patients with biallelic variants had lower fibrinogen activity levels (median 200 mg/L) than those with monoallelic variants (935 mg/L, p < 0.001). Fibrinogen activity levels correlated positively with plasmin peak height (R = 0.74, p < 0.001) and inversely with thrombin potential (R = -0.55, p = 0.002). Thrombin potential was 1.77-fold higher in patients with a venous thrombosis history (n = 5, p = 0.03) than in healthy controls. CONCLUSIONS:In patients with CFDs, postoperative bleeding correlates with fibrinogen activity, while female-specific bleeding affects all CFD subtypes. Elevated thrombin generation might explain thrombosis risk in these patients.

Background: Rare coagulation factor deficiencies (RCFDs) can cause significant bleeding, but activated partial thromboplastin time (APTT) and prothrombin time (PT) may not detect abnormalities, and factor activity measurements do not predict bleeding severity. Thrombin generation assays may better reflect overall hemostatic capacity. Objectives: This study investigated whether thrombin generation can detect RCFDs and correlate with bleeding severity in patients with congenital deficiencies of factor (F)II, FV, FV/FVIII, FVII, FX, and FXI. Methods: Thrombin generation was measured using the Nijmegen hemostasis assay in patients from the cross-sectional Dutch Rare Bleeding disorders in the Netherlands (RBiN) study (2017-2019). Parameters analyzed included thrombin potential, lag time, and thrombin potential-to-lag time (TP/LT) ratio, normalized using pooled normal plasma and expressed as percentages of the mean from 37 healthy controls. Nijmegen hemostasis assay data were correlated with bleeding severity and compared with APTT and PT. Results: We included 106 patients, mostly with mild deficiencies (median factor activity 5%-53%). Overall, thrombin generation in patients was significantly reduced compared with controls, with decreased thrombin potentials (median 58%) and prolonged lag times (150%), resulting in reduced TP/LT ratios (45%). These parameters correlated with bleeding severity; across RCFDs, median TP/LT ratios ranged from 30% to 104% in patients without spontaneous bleeding (bleeding severity grade 0-1), 26% to 49% in mild spontaneous bleeding (grade 2), and 0% to 22% in severe spontaneous bleeding (grade 3). At 95% specificity, TP/LT ratio showed 68% to 100% sensitivity, outperforming APTT and PT (14%-80%) in all RCFDs except FVII and FV/ FVIII deficiency. Conclusion: Thrombin generation profiling correlated with bleeding severity and showed higher sensitivity than conventional screening assays in detecting RCFDs, supporting its potential in screening and clinical evaluation of RFCDs.

Background: Clinical bleeding phenotype varies substantially among patients with rare bleeding disorders (RBDs). Patient-reported outcomes may provide valuable insights into health-related quality of life (HRQoL) and disease burden. Objectives: To evaluate HRQoL in patients with rare coagulation factor deficiencies and fibrinolytic disorders included in the nationwide, cross-sectional Rare Bleeding Disorders in the Netherlands (RBiN) study. Methods: Bleeding scores (ie, the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool [ISTH-BAT]) were assessed during a single study visit, and electronic questionnaires captured demographic and HRQoL data (36-item Short Form survey [SF-36], Patient-Reported Outcomes Measurement Information System, Profile 29 [PROMIS-29]). Only differences exceeding the minimally important difference were considered clinically relevant and reported. Results: HRQoL data from 167 adults and 34 children were available. HRQoL of patients with RBDs measured by SF-36 was not significantly different compared to the Dutch reference population. PROMIS-29 scores indicated significantly better sleep, social participation, and pain-related outcomes in patients with RBDs than the reference populations. Subgroup analyses within the RBiN population showed worse physical health in patients with a severe bleeding phenotype than in those with a mild-to-moderate phenotype. Women with a history of heavy menstrual bleeding reported worse physical health and pain-related outcomes than those without. Patients reporting severe disease had worse pain interference and mental health scores (PROMIS-29) than those reporting nonsevere disease. ISTH-BAT scores were negatively associated with physical functioning. Conclusions: Overall HRQoL in patients with RBDs was comparable to the Dutch reference population. Within the RBiN population, a history of heavy menstrual bleeding, clinical bleeding phenotype, patient-reported disease severity, and ISTH-BAT scores were associated with impaired HRQoL, reflecting disease burden in patients living with RBDs.

Background Limited data exist on persons with rare bleeding disorders possessing a heterozygous genotype, as most studies focus on biallelic genotypes and more severe coagulation factor deficiencies. A growing body of evidence suggests that persons with a heterozygous genotype experience clinically relevant bleeding symptoms. Objectives This study aimed to explore the incidence of bleeding symptoms and postoperative bleeding in persons with a heterozygous genotype. Methods This cross-sectional substudy of the Rare Bleeding Disorders in the Netherlands study (2017-2019) included persons with rare coagulation factor deficiencies and disorders of fibrinolysis with a heterozygous or biallelic genotype. Clinical data and laboratory samples were collected during a single study visit along with questionnaires. Results In total, 86 persons with a heterozygous genotype and 55 with a biallelic genotype were included. Median factor activity levels in persons with a heterozygous genotype approached 50% with considerable heterogeneity (range, 11%-93%). In 75%, persons with a heterozygous genotype reported bleeding severity of grade II or III. Female-specific bleeding was common. In total, 425 surgical procedures were performed. Persons with a heterozygous genotype were less likely to receive periprocedural treatment, and omission of periprocedural treatment was associated with postoperative bleeding in procedures with intermediate-high bleeding risk. Postoperative bleeding was comparable for persons with a heterozygous genotype (35%; 59/171) and a biallelic genotype (35%; 86/247; P = .926). Conclusion In our population with rare bleeding disorders, the majority of persons possessing a heterozygous genotype exhibited spontaneous bleeding symptoms. Especially in intermediate-high risk procedures, a proactive approach to periprocedural hemostatic treatment in persons with a heterozygous genotype seems beneficial.

Background: Between 2002 and 2011, the incidence of severe primary postpartum hemorrhage (PPH) in Dutch women with von Willebrand disease (VWD) and hemophilia carriers (HCs) was 8% vs 4.5% in the general population. Objectives: To determine the contemporary incidence of severe primary PPH in women with VWD and HCs. Methods: All women with VWD or HCs who delivered between 2012 and 2017 were selected from all 6 Dutch hemophilia treatment centers. Data on patient and disease characteristics, peripartum hematologic and obstetric management, and outcomes were retrospectively collected. Incidence of severe primary (>= 1000 mL of blood loss <= 24 hours after childbirth) and primary (>= 500 mL within <= 24 hours after childbirth) PPH was compared with the (1) previous cohort and (2) general Dutch population and between (3) women with VWD and HCs with third-trimester coagulation activity levels <50 international units (IU)/dL vs >= 50 IU/dL and (4) women treated with vs without peripartum hemostatic prophylaxis. Results: Three-hundred forty-eight deliveries (151 VWD, 167 hemophilia A, and 30 hemophilia B carriers) were included. The severe primary PPH incidence was 10% (36/ 348) and remained stable over time, whereas this incidence has increased in the general population (to 8%), leading to a similar risk (P = .17). Severe primary PPH risk was comparable between women with coagulation activity levels <50 and >= 50 IU/dL (11% [7/66] vs 10% [29/279]; odds ratio, 1.02; 95% CI, 0.43-2.44) and comparable between those with and those without prophylaxis (12% [11/91] vs 10% [25/254]; odds ratio, 1.26; 95% CI, 0.59-2.68). Conclusion: Severe primary PPH in women with VWD and HCs remained stable and is comparable with the increasing prevalence in the general population. More research is needed to find the optimal pregnancy management strategy for safe delivery in VWD and HC.

Background Rare coagulation factor deficiencies and disorders of fibrinolysis (defined as rare bleeding disorders [RBDs]) present with a heterogeneous bleeding phenotype, and bleeding severity is difficult to predict. Objectives Describe underlying rare genetic variants in the Dutch RBD population and investigate the relationship between genotype, laboratory phenotype, and clinical phenotype. Methods The Rare Bleeding Disorders in the Netherlands is a cross-sectional, nationwide study conducted between October 1, 2017, and November 30, 2019. Bleeding scores and blood samples were collected during a single study visit. Coagulation factor levels were measured centrally, and targeted exome analysis was performed on 156 genes involved in thrombosis and hemostasis. Pathogenicity was assigned according to the Association for Clinical Genetic Science guidelines. Results Rare genetic variants specific to the diagnosed RBD were found in 132 of 156 patients (85%). Of the 214 rare genetic variants identified, 57% (n = 123) were clearly pathogenic, 19% (n = 40) were likely pathogenic, and 24% (n = 51) were variants of unknown significance. No explanatory genetic variants were found in patients with plasminogen activator inhibitor type 1 deficiency or hyperfibrinolysis. A correlation existed between factor activity levels and the presence of a genetic variant in the corresponding gene in patients with rare coagulation factor deficiencies and alpha-2-antiplasmin deficiency. Co-occurrence of multiple genetic variants was present in a quarter of patients, but effect on phenotype remains unclear. Conclusion Targeted exome analysis may offer advantages over single-gene analysis, emphasized by a number of combined deficiencies in this study. Further studies are required to determine the role of co-occurring hemostasis gene variants on the bleeding phenotype in RBDs.

Background: Spleen rupture is described as a rare hemorrhagic manifestation of afibrinogenemia.Pathogenesis and best management are still not well defined.Aims: We report the clinical characteristics and the management of spleen rupture in the Lebanese cohort with afibrinogenemia.Methods: It is a retrospective observational descriptive study in a welldefined cohort, "the Lebanese patient with afibrinogenemia," during a follow up period of 21 years.Variables studied were sex, age, gene mutation, bleeding score and thrombotic event, occurrence of spleen rupture, radiological finding of spleen, management of spleen rupture, and outcome.All patients provided written informed consent and university ethic committee give his approval.Results: 17 (23%) out of 73 patients had spontaneous spleen rupture with no obvious trigger factor.There was 5/31 (16%) women and 12/42 (28.5%) men.Mean age of occurrence was 16.3 years [6;42].Hom FGG R134X and hom FGA L212X were the 2 gene mutation.Mean bleeding score was 22.7 [12;34].Ten had previous history of thromboembolic event while 17 out of the 73 in the studied population Presenting symptoms were sudden abdominal pain, hypotension and hemoperitoneum.Abdominal ultrasound and CT scan identifies patches of spleen parenchyma necrosis with hemoperitoneum Splenectomy was performed in 11 patients, 4 had conservative treatment.Two died from hemorrhagic shock before any treatment.Splenectomy was complicated in 4 patients by a portal thrombosis.The four patients were treated with Low-Molecular-Weight-Heparin with no recanalization.Fibrinogen substitution was given as conservative treatment for 1 to 3 weeks than a prophylaxis regimen every 15 days.2 patient received Low-Molecular-Weight-Heparin.Spleen returned to normal in one to 3 weeks.Recurrence was observed in one patient who stopped prophylaxis with a follow up of 3 years. Conclusion(s):Spleen rupture is significantly associated with thromboembolic events ( p < 0,001) and could be an expression of thromboembolic event.Conservative treatment is effective.

BACKGROUND:Heavy menstrual bleeding (HMB) is associated with a reduced quality of life and limitations in social and physical functioning. Data on HMB in women with rare bleeding disorders (RBDs), including coagulation factor deficiencies and fibrinolytic disorders, are scarce. OBJECTIVES:To analyze the prevalence, severity, and treatment of HMB in Dutch women with an RBD. METHODS:The Rare Bleeding Disorders in the Netherlands (RBiN) study included 263 patients with an RBD from all 6 hemophilia treatment centers (October 2017-November 2019). In this analysis, data of 111 women aged ≥16 years were studied. According to the International Society on Thrombosis and Haemostasis bleeding assessment tool, HMB symptoms were scored from 0 (no/trivial) to 4 (severe symptoms requiring medical intervention). HMB was defined as a score ≥1. Age at RBD diagnosis was extracted from patient files. RESULTS:HMB was reported by 80% of women (89/111) and was more prevalent in women with a fibrinolytic disorder (33/35; 94%) than in women with a coagulation factor deficiency (56/76; 74%) (P = .011). Of the 89 women with HMB, 82% (n = 73) ever required treatment. Multiple treatment modalities were frequently used, both in severe and mild deficiencies. Hormonal treatment was mostly used (n = 64; 88%), while antifibrinolytics were prescribed less frequently (n = 18; 25%). In women with HMB since menarche (n = 61; 69%), median age at RBD diagnosis was 28 years (IQR, 14-41). CONCLUSION:HMB is common in women with RBDs. Women with mild deficiencies also frequently reported HMB. Only a minority of women were treated with hemostatic agents. A significant diagnostic delay was observed after the onset of HMB symptoms.

BACKGROUND:Patients with rare inherited bleeding disorders (RBDs) exhibit hemorrhagic symptoms, varying in type and severity, often requiring only on-demand treatment. Prolonged bleeding after invasive procedures is common. Adequate peri-procedural therapy may reduce this bleeding risk. OBJECTIVE:To describe general treatment plans of RBD patients and evaluate the use of peri-procedural hemostatic therapy. METHODS:In the Rare Bleeding Disorders in the Netherlands (RBiN) study, RBD patients from all six Dutch Hemophilia Treatment Centers were included. General treatment plans were extracted from patient files. Patients with a dental or surgical procedure in their history were interviewed about use of peri-procedural treatment and bleeding complications. RESULTS:Two-hundred sixty-three patients with a rare coagulation factor deficiency or fibrinolytic disorder were included. Eighty-four percent had a documented general treatment plan. General treatment plans of patients with the same RBD were heterogeneous, particularly in factor XI deficiency. Overall, 308 dental and 408 surgical procedures were reported. Bleeding occurred in 50% of dental and 53% of surgical procedures performed without hemostatic treatment and in 28% of dental and 19% of surgical procedures performed with hemostatic treatment. Not only patients with severe RBDs, but also patients with mild deficiencies, experienced increased bleeding without proper hemostatic treatment. CONCLUSION:Large heterogeneity in general treatment plans of RBD patients was found. Bleeding after invasive procedures was reported frequently, both before and after RBD diagnosis, irrespective of factor activity levels and particularly when peri-procedural treatment was omitted. Improved guidelines should include uniform recommendations for most appropriate hemostatic products per RBD and emphasize the relevance of individual bleeding history.

BACKGROUND:Women with rare bleeding disorders (RBDs), including coagulation factor deficiencies and fibrinolytic disorders, may have a higher risk of postpartum hemorrhage (PPH). Information on this patient category is lacking in the existing PPH guidelines because data on PPH in patients with RBDs are scarce.OBJECTIVE:To describe the prevalence of PPH in women with an RBD and evaluate the use of peripartum hemostatic prophylaxis.METHODS:In the Rare Bleeding Disorders in the Netherlands (RBiN) study, patients with RBDs (n = 263) were included from all 6 Dutch hemophilia treatment centers. Patient-reported information on delivery, peripartum hemostatic prophylaxis, and occurrence of PPH was collected retrospectively. If available, information about the precise volume of postpartum blood loss was extracted from electronic patient files. PPH was defined as blood loss ≥500 mL (World Health Organization guideline).RESULTS:A total of 244 pregnancies, including 193 livebirths, were reported by 85 women. A considerable proportion of these women experienced PPH, ranging from 30% in factor V deficiency to 100% in hyperfibrinolysis. Overall, PPH was reported in 44% of deliveries performed with and 53% of deliveries performed without administration of peripartum hemostatic prophylaxis. Blood loss was significantly higher in deliveries without administration of hemostatic prophylaxis (median 1000 mL) compared to deliveries with administration of prophylaxis (median 400 mL) (p = 0.011). Patients with relatively mild deficiencies also frequently experienced PPH when peripartum hemostatic prophylaxis was omitted.CONCLUSION:PPH is common in rare coagulation factor deficiencies, both severe and mild, and fibrinolytic disorders, especially when peripartum prophylactic hemostatic treatment was not administered. The use of prophylactic hemostatic treatment was associated with less postpartum blood loss.

BackgroundIn recent years, more awareness is raised about sex-specific dilemmas in inherited bleeding disorders. However, no large studies have been performed to assess differences in diagnosis, bleeding phenotype and management of men and women with bleeding disorders. Therefore, we investigated sex differences in a large cohort of well-defined patients with autosomal inherited bleeding disorders (von Willebrand disease (VWD), rare bleeding disorders (RBDs) and congenital platelet defects (CPDs)).MethodsWe included patients from three nationwide cross-sectional studies on VWD, RBDs and CPDs in the Netherlands, respectively the WiN, RBiN and TiN study. In all studies a bleeding score (BS) was obtained, and patients filled in an extensive questionnaire on the management and burden of their disorder.FindingsWe included 1092 patients (834 VWD; 196 RBD; 62 CPD), of whom 665 (60.9%) were women. Women were more often referred because of a bleeding diathesis than men (47.9% vs 36.6%, p = 0.002). Age of first bleeding was similar between men and women, respectively 8.9 ± 13.6 (mean ±sd) years and 10.6 ± 11.3 years (p = 0.075). However, the diagnostic delay, which was defined as time from first bleeding to diagnosis, was longer in women (11.6 ± 16.4 years) than men (7.7 ± 16.6 years, p = 0.002). Similar results were found when patients referred for bleeding were analyzed separately. Of women aging 12 years or older, 469 (77.1%) had received treatment because of sex-specific bleeding.InterpretationWomen with autosomal inherited bleeding disorders are more often referred for bleeding, have a longer diagnostic delay, and often require treatment because of sex-specific bleeding.FundingThe WiN study was supported (in part) by research funding from the Dutch Hemophilia Foundation (Stichting Haemophilia), Shire (Takeda), and CSL Behring (unrestricted grant).

Introduction The diagnostic trajectory of patients with increased bleeding tendency can be very costly and time-consuming. In addition, previous studies have shown that half of these patients remain without final diagnosis despite all efforts. Aim This study aimed to improve insight into the current diagnostic process of these patients. Methods A total of 117 adult patients, referred to an academic hospital because of being suspected to have an increased bleeding tendency, were included. Different parameters were compared between patients receiving final diagnosis, patients without final diagnosis but a high Tosetto bleeding assessment tool (BAT) score (classified as bleeding of unknown cause, or BUC) and a control group consisting of patients without final diagnosis and a low BAT score. Results The BAT score was significantly higher in patients in the BUC group as compared to patients reaching final diagnosis (8.1 vs 4.9). Interestingly, the two subcategories most prevalently increased were surgery and post-partum haemorrhage-associated bleeding (surgery: 2.1 vs 1.1; post-partum haemorrhage: 0.7 vs 0.0). Laboratory screening results were more often abnormal in patients reaching final diagnosis compared to patients remaining without diagnosis and a high BAT score (n = 32 (78%) vs n = 14 (46%), 95% CI 1.5-12), especially concerning the PFA (=27 (66%) vs n = 10 (33%), 95% CI 1.4-10) and von Willebrand factor activity levels (n = 11 (27%) vs n = 1 (3%), 95% CI 1.3-91). Conclusion Isolated high bleeding score on surgical or post-partum bleeding correlates with a lower chance of receiving final diagnosis. Withholding extensive haemostatic testing should be considered. Better screening and confirmative haemostatic assays are still needed.

The hematopoietic stem and progenitor cell marker CD34 is implicated in cell-cell interactions in the bone marrow niche. Upon hematopoietic maturation its expression is switched off. Aberrant CD34 expression on mature platelets has been associated with monogenic bleeding disorders that are caused by mutations in the transcription factors GFI1B (Growth Factor Independence 1B) and RUNX1 (RUNT related transcription factor 1).1, 2 These familial disorders are characterized by a bleeding diathesis, caused by disturbed megakaryocyte development, which results in the formation of dysfunctional platelets. In addition, these disorders are accompanied by abnormal retention of CD34 on platelets, which is not lost during differentiation. In this letter, we report a patient with CD34 expression on platelets associated with a partial deletion of the RUNX1 binding partner CBFB (Core Binding Factor B). CBFB is part of a heterodimeric transcription factor complex with either RUNX1, −2 or − 3. CBFB enables the function of RUNX1-3 through stabilization of their interaction with DNA. Together with GFI1B and RUNX1, CBFB is now the third hematopoietic transcriptional regulator that associates with abnormal platelet CD34 expression. The patient we report here, presented to our outpatient clinic with multiple bleeding events. A Tosetto bleeding score of 11 was established, consisting of spontaneous epistaxis, prolonged bleeding from small wounds, spontaneous bruising and menorrhagia. In addition, she experienced prolonged bleeding after wisdom teeth extraction, and severe bleeding following hysterectomy requiring relaparotomy and blood transfusion. However, she did not experience postpartum bleeding after giving birth to a son, and correction of clavicle hypoplasia as a child was uncomplicated. Both the parents and the patient's son were not affected by a bleeding tendency (Figure 1A). Her medical history revealed mild asthma, migraines and a clinical depression for which she used a selective serotonin reuptake inhibitor (SSRI). Platelet counts, as well as mean platelet volume, ADP levels, β-thromboglobulin and platelet factor 4 expression, and the ADP/ATP ratio were within the normal range (Table S1). The platelets showed no abnormalities in a May-Grünwald Giemsa stained blood film (data not shown). The platelet function analyzer measured prolonged closure time after stimulation with the collagen/epinephrine membrane (>300 seconds). Platelet light transmission aggregometry revealed an aggregation defect upon stimulation with epinephrine (10 μM), ADP (10 μM) and low concentration of collagen (1 μg/mL) (Table S1). Stimulation with high concentrations of collagen or ADP resulted in normal aggregation responses, similar to the strong agonists arachidonic acid, thrombin receptor-activating peptide and ristocetin. The ATP release from δ-granules was measured following stimulation with a high concentration of collagen (5 μg/mL), epinephrine (5 μM) and the thromboxane analogue U46619 (1 μM) (Table S1). The ATP release was normal after stimulation with a high concentration of collagen, as expected. No ATP was released upon stimulation with epinephrine and low levels were measured after stimulation with U46619. Cessation of the SSRI for 8 weeks did not correct platelet aggregation defects. Thin section electron microscopy (EM) showed normal platelets, with occasional platelets carrying few α-granules (Figure 1B). Whole mount EM showed increased numbers of δ-granules at two independent time points, compared to controls (on average 5.84 δ-granules vs 3.66 per platelet, respectively) (Figure 1C, D).2 Together, these data suggest that the index patient suffers from a mild δ-granule secretion defect. Remarkably, the platelets from the son showed defects in the light transmission aggregometry tests upon stimulation with epinephrine and low concentrations of collagen and ADP, similar to the index patient (Table S1). The adult son did not experience hemostatic challenges and had no clinical history of bleedings, which may suggest that the bleeding disorder observed in the index patient is not solely caused by the δ-granule secretion defect. To identify a genetic cause for the bleeding tendency, a panel of thrombosis and hemostasis associated genes was screened following whole exome sequencing.3 No disease-causing variants were called. Exome-wide analysis detected a ~100 Kb heterozygous chromosome 16q22.1 deletion that was confirmed by single nucleotide polymorphism (SNP) array. This deletion covers nine genes, including the last exon of CBFB (Figure 1F). No disease-causing variants were observed in the remaining wild type CBFB allele. The mother and son of the index patient did not have the 16q deletion. The father was deceased and could therefore not be tested for the deletion, however, no clinical history of bleedings was reported. The expression of CBFB transcripts was studied by 3′-Rapid Amplification of cDNA Ends (RACE) and Sanger sequencing. Readthrough from exon 5 into intron 5 was observed, predicting the formation of a protein that exceeds 11 amino acids from the splice junction. To quantify RNA expression of this transcript in leukocytes, a quantitative real-time polymerase chain reaction (RT-qPCR) was designed within intron 5 (Figure 1G). Next to the patient (II.1) and her son (III.1) five additional healthy controls were tested for expression of this transcript. This showed on average a 10-fold increase of this transcript in the index case compared to the controls and her son (Figure 1H). Based on findings in the son we concluded that the δ-granule defect was not caused by the 16q deletion. Yet, the 16q deletion in combination with the δ-granule defect might contribute to the bleedings in the index patient. To date, none of the 16q-deleted genes have been implicated in inherited bleeding or platelet disorders. However, conditional Cbfb knockout in mice resulted in disturbed megakaryopoiesis and thrombocytopenia.4 Because bleeding disorders caused by RUNX1 mutations result in platelet CD34 expression and an interaction with CBFB is required for the function of RUNX1, the platelets were tested for CD34 expression.2 Flow cytometry clearly detected platelet CD34 expression in the index patient while platelets from her son and 10 nonrelated healthy controls did not. Thus, the 16q deletion associates with platelet CD34 expression (Figure 1E). Entire heterozygous CBFB deletions have been reported in rare cases with congenital bone abnormalities and mutations in its binding partner RUNX2 may cause cleidocranial dysplasia, associated with clavicle hypoplasia.5 The medical record of the patient indicated that she had undergone surgery at the age of 13 for a congenital hypoplastic clavicle. The mother and son of the index patient had no signs of clavicle hypoplasia and the father was reported not to have bone abnormalities either. Thus, the 16q deletion associates with platelet CD34 expression, and a congenital clavicle hypoplasia but not with disturbed platelet δ-granule secretion. To our knowledge, the patient reported here is the first in whom a deletion of chromosome 16q22.1 partially encompassing CBFB associates with platelet CD34 expression. The deletion is not causal to the platelet δ-granule abnormalities, because the son who did not inherit the deletion showed similar δ-granule defects as the mother. Recently, we and others reported CD34 expression on platelets from patients with GFI1B variants that did not necessarily cause bleedings on their own. This suggests that platelet CD34 expression may be uncoupled from a bleeding tendency for other transcription factor mutations as well.6, 7 During endothelial to hematopoietic transition GFI1B is a crucial RUNX1/CBFB target gene, which may explain the similarities between RUNX1, CBFB and GFI1B mutants.8 As the son had a δ-granule secretion defect, similar to the index patient, but no clinical history of bleedings, the index patient's bleeding tendency cannot solely be explained by the δ-granule dysfunction. This case underscores that mutations in various hematopoietic transcription factors may result in abnormal platelet development associated with increased CD34 expression, which may aggravate but are not necessarily the sole cause of the clinically manifested bleeding tendency. This work was supported by the Landsteiner Foundation for Blood Transfusion Research (project 1531). M.G.J.M.v.B., J.L.S., B.A.P.L.-v.G., S.E.M.S., M.C.J.J., J.H.J., and B.A.v.d.R. designed and coordinated research; M.G.J.M.v.B., J.L.S., A.S., K.M.H., Y.M.C.H., W.B., E.H., and F.W.P. collected and analyzed data; B.A.P.L.-v.G., and M.C.J.J. arranged for obtaining patient informed consent; and M.G.J.M.v.B. and B.A.v.d.R. wrote the manuscript that was critically revised by all authors. Table S1. Platelet phenotyping index case II.1 and III.1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Congenital afibrinogenemia is a rare autosomal recessive disorder associated with an increased risk of hemorrhage, thrombosis, and obstetric complications. This case series of 4 pregnancies in 2 related patients seeks to address the key clinical question of the necessary doses of fibrinogen concentrate during pregnancy and puerperium. One pregnancy without the prophylactic use of fibrinogen concentrate resulted in spontaneous abortion. The second pregnancy was complicated by a subchorionic hematoma despite the prophylactic administration of fibrinogen concentrate to maintain the plasma trough levels at ≥0.6 g/L. Labor was complicated by postpartum hemorrhage with a blood loss volume of 1480 cc. Two weeks later, the patient presented with postpartum thrombosis. The other 2 pregnancies were uncomplicated with fibrinogen trough levels ≥1.0 g/L during pregnancy and ≥1.5 g/L during labor. These cases illustrate that during pregnancy, patients may benefit from fibrinogen trough levels ≥1.0 g/L. In addition, the increased risk of postpartum thrombosis with prolonged fibrinogen supplementation warrants personalized postpartum advice that is guided by postpartum blood loss.