Diagnostic utility of bleeding assessment tools in congenital fibrinogen deficiencies.

BACKGROUND: The assessment of clinical history is crucial before referring a patient for further laboratory testing. Bleeding assessment tools (BAT) are developed to standardize clinical evaluation. A small number of patients with congenital fibrinogen deficiencies (CFDs) have been evaluated with these tools without definitive results. AIMS: We compared the adequacy of the ISTH-BAT and the European network of rare bleeding disorders bleeding score system (EN-RBD-BSS) to identify patients with CFDs. The correlation between the two BATs and fibrinogen levels and patient clinical grade severity was further analyzed. METHODS: We included 100 Iranian patients with CFDs. Routine coagulation and fibrinogen-specific tests (fibrinogen antigen [Fg:Ag] and activity [Fg:C]) were performed. The ISTH-BAT and EN-RBD-BSS were used to assess the bleeding score (BS) of all patients. RESULTS: The ISTH-BAT and EN-RBD-BSS median (range) were 4 (0-16) and 2.21 (-1.49 to 6.71), with a statistically significant moderate correlation between the two systems (r = .597, P < .001). In patients with quantitative deficiencies (afibrinogenemia and hypofibrinogenemia), the correlation between Fg:C and the ISTH-BAT was moderately negative (r = -.4, P < .001), while the correlation between Fg:C and the EN-RBD-BSS was weakly negative (r = -.38, P < .001). Overall, 70% and 72% of patients with fibrinogen deficiencies were correctly identified by both the ISTH-BAT and EN-RBD-BSS, respectively. CONCLUSION: These results suggest that in addition to the ISTH-BAT, the EN-RBD-BSS may also be useful in identifying CFD patients. We found a significant level of sensitivity for detecting fibrinogen deficiency in the two BATs, and bleeding severity classification correctly identified severity grades in almost two-thirds of patients.

A homozygous duplication of the FGG exon 8-intron 8 junction causes congenital afibrinogenemia. Lessons learned from the study of a large consanguineous Turkish family.

Congenital afibrinogenemia is the most severe congenital fibrinogen disorder, characterized by undetectable fibrinogen in circulation. Causative mutations can be divided into two main classes: null mutations with no protein production at all and missense mutations producing abnormal protein chains that are retained inside the cell. The vast majority of cases are due to single base pair mutations or small insertions or deletions in the coding regions or intron-exon junctions of FGB, FGA and FGG. Only a few large rearrangements have been described, all deletions involving FGA. Here we report the characterization of a 403 bp duplication of the FGG exon 8-intron 8 junction accounting for congenital afibrinogenemia in a large consanguineous family from Turkey. This mutation, which had escaped detection by Sanger sequencing of short polymerase chain reaction (PCR) amplicons of coding sequences and splice sites, was identified by studying multiple alignments of reads obtained from whole exome sequencing of a heterozygous individual followed by PCR amplification and sequencing of a larger portion of FGG. Because the mutation duplicates the donor splice site of intron 8, we predicted that the impact of the mutation would be on FGG transcript splicing. Analysis of mRNA produced by cells transiently transfected with normal or mutant minigene constructs showed that the duplication causes production of several aberrant FGG transcripts generating premature truncating codons.

Treatment of rare factor deficiencies other than hemophilia.

The deficiency of fibrinogen, prothrombin, factor V (FV), FVII, FVIII, FIX, FX, FXI, and FXIII, called rare coagulation disorders (RCDs), may result in coagulopathies leading to spontaneous or posttrauma and postsurgery hemorrhages. RCDs are characterized by a wide variety of symptoms, from mild to severe, which can vary significantly from 1 disease to another and from 1 patient to another. The most typical symptoms of all RCDs are mucosal bleedings and bleeding at the time of invasive procedures, whereas other life-threatening symptoms such as central nervous system bleeding and hemarthroses are mostly present only in some deficiencies (afibrinogenemia, FX, and FXIII). At variance with hemophilia A and B and von Willebrand disease, RCDs are much less prevalent, ranging from 1 case in 500 000 to 1 in 2 million in the general population. Their clinical heterogeneity associated with the low number of patients has led to a delay in the development of appropriate therapies. Indeed, a similar heterogeneity can also be found in the treatment products available, ranging from the specific recombinant proteins to treat FVII- and FXIII-deficient patients to the complete absence of specific products to treat patients with FII or FV deficiencies, for whom prothrombin complex concentrates or fresh frozen plasma are, to date, the only option. The recent development of novel hemostatic approaches for hemophilia, such as the use of nonsubstitutive therapy as RNA interference, anti-tissue factor pathway inhibitor, and the gene therapy aimed at improving the patient's quality of life may also have an important role in the treatment of patients with RCDs in the future.

An international registry of patients with plasminogen deficiency (HISTORY).

Plasminogen deficiency is an ultra-rare multisystem disorder characterized by the development of fibrin-rich pseudomembranes on mucous membranes. Ligneous conjunctivitis, which can result in vision impairment or loss, is the most frequent symptom reported. Affected systems may also include the respiratory tract, oropharynx, female reproductive tract, gingiva, middle ear, renal collecting system, skin and central nervous system. Untreated, plasminogen deficiency may result in significant reduction in quality of life and morbidity with potential life-threatening complications. Non-specific therapies are inadequate and plasminogen concentrates are not commercially available. The current understanding of plasminogen deficiency and management of disease symptoms and its progression are based on case reports/series and two small clinical trials. To date there has never been a comprehensive, international study to examine the natural history or optimal therapeutic intervention; knowledge gaps include identification of contributing factors and triggers of disease manifestations, inability to predict disease course, and insufficient real-world data for use of therapeutics. We have created an international, observational study (HISTORY) in a large cohort of persons with plasminogen deficiency and first-degree family members to address these gaps and to advance knowledge and care. HISTORY will build upon the established relationship between the Indiana Hemophilia and Thrombosis Center and the Fondazione Angelo Bianchi Bonomi, IRCCS Ca' Granda Ospedale Maggiore Policlinico - University of Milan and will utilize a modified version of the Prospective Rare Bleeding Disorders Database (PRO-RBDD). A biorepository containing samples from subjects with plasminogen deficiency will be established. This article describes the rationale behind the study and efforts towards its goals.

Exploring the global landscape of genetic variation in coagulation factor XI deficiency.

Factor XI (FXI) deficiency is an autosomal bleeding disorder, usually posttrauma or postsurgery, characterized by reduced levels of coagulation FXI in plasma. The disease is highly prevalent in Ashkenazi Jews (heterozygote frequency, ∼9%), whereas it is considered a rare condition in most populations (prevalence of the severe deficiency, 1 in 106 in the white population). So far, >190 causative mutations have been identified throughout the F11 gene. To have a global landscape of genetic variation of F11, we explored publicly available exome-based data obtained from >60 000 individuals belonging to different ethnicities (Exome Aggregation Consortium resource). This analysis revealed profound differences in heterozygote frequencies among populations (allele frequencies: African = 0.0016; East Asian = 0.0045; European = 0.0036; Finnish = 0.00030; Latino = 0.0021; South Asian = 0.0015), and a prevalence significantly higher than that reported so far (eg, the calculated prevalence of the severe deficiency in Europeans would be: 12.9 in 106). In addition, this analysis allowed us to evidence recurrent and ethnic-specific mutations: p.Phe223Leu in Africans (23.5% of all mutated alleles), p.Gln263X and p.Leu424CysfsX in East Asians (28.2% and 20.5%, respectively), and p.Ala412Thr in Latinos (25%).

Genome editing of factor X in zebrafish reveals unexpected tolerance of severe defects in the common pathway.

Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish f10 by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use f10-/- zebrafish to confirm 5 novel human F10 variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of F10 variants. These findings as well as the prolonged survival of f10-/- mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders.

Understanding the Impact of Aberrant Splicing in Coagulation Factor V Deficiency.

Rare inherited coagulation disorders (RICDs) are congenital deficiencies of the plasma proteins that are involved in blood coagulation, which generally lead to lifelong bleeding manifestations. These diseases are generally qualitative and/or quantitative defects that are associated with monoallelic or biallelic mutations in the relevant gene. Among RICDs, factor V (FV) deficiency is one of the least characterized at the molecular level. Here, we investigated four unrelated patients with reduced plasma FV levels (three severe, one mild), which were associated with a moderately severe bleeding tendency. Sequence analysis of the FV gene identified seven different variants, five hitherto unknown (p.D1669G, c.5789-11C>A, c.5789-12C>A, c.5789-5T>G, and c.6528G>C), and two previously reported (c.158+1G>A and c.5789G>A). The possible pathogenic role of the newly identified missense variant was studied by in silico approaches. The remaining six genetic defects (all putative splicing mutations) were investigated for their possible effects on pre-mRNA splicing by transient transfection experiments in HeLa cells with plasmids expressing appropriate hybrid minigenes. The preparation of minigene constructs was instrumental to demonstrate that the two adjacent variants c.5789-11C>A and c.5789-12C>A are indeed present in cis in the analyzed FV-deficient patient (thus leading to the c.5789-11_12CC>AA mutation). Ex vivo experiments demonstrated that each variant causes either a skipping of the relevant exon or the activation of cryptic splice sites (exonic or intronic), eventually leading to the introduction of a premature termination codon.

Frequency of the p.Gly262Asp mutation in congenital Factor X deficiency.

INTRODUCTION: Congenital factor X (FX) deficiency is a rare bleeding disorder inherited as an autosomal recessive trait with an incidence of 1 : 500 000-1 000 000. A total or partial deficiency of FX causes an impairment of clot formation, leading to a haemorrhagic disease, which manifests with bleeding symptoms of different severity, also unprovoked. AIM: We analysed the clinical manifestations, laboratory phenotype and genotype in 12 patients from Turkey affected with severe FX deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT), FX activity (FX:C) and FX antigen level (FX:Ag) were measured, and mutation analysis was performed for all patients. RESULTS: The most frequent bleeding episodes in patients were epistaxis and easy bruising (11/12, 91%), followed by haemarthroses (10/12, 83%). FX:C was <1% in 11 patients, and 4% in one. FX:Ag was reduced in all patients, consistent with type II deficiency. Direct sequencing of the factor X gene (F10) identified two different mutations: the novel 33 bp in-frame deletion p.Thr176_Gln186, c.526_558del, which seems to be associated with milder bleeding symptoms and the c.785G>A, p.Gly262Asp missense mutation (previously reported as Gly222Asp), which is associated with severe bleeding symptoms. CONCLUSION: The p.Gly262Asp missense mutation was identified in 11 of the 12 patients in this study. Previously published cases on the same p.Gly262Asp mutation were Iranian patients originating from the border between Turkey and Iran suggesting that this mutation may be candidate as a good tool for mutational screening analysis in this area.

Congenital fibrinogen disorders: a retrospective clinical and genetic analysis of the Prospective Rare Bleeding Disorders Database.

ABSTRACT: Congenital fibrinogen deficiency (CFD) is a rare bleeding disorder caused by mutations in FGA, FGB, and FGG. We sought to comprehensively characterize patients with CFD using PRO-RBDD (Prospective Rare Bleeding Disorders Database). Clinical phenotypes, laboratory, and genetic features were investigated using retrospective data from the PRO-RBDD. Patients were classified from asymptomatic to grade 3 based on their bleeding severity. In addition, FGA, FGB, and FGG were sequenced to find causative variants. A total of 166 CFD cases from 16 countries were included, of whom 123 (30 afibrinogenemia, 33 hypofibrinogenemia, 55 dysfibrinogenemia, and 5 hypodysfibrinogenemia) were well characterized. Considering the previously established factor activity and antigen level thresholds, bleeding severity was correctly identified in 58% of the cases. The rates of thrombotic events among afibrinogenemic and hypofibrinogenemic patients were relatively similar (11% and 10%, respectively) and surprisingly higher than in dysfibrinogenemic cases. The rate of spontaneous abortions among 68 pregnancies was 31%, including 86% in dysfibrinogenemic women and 14% with hypofibrinogenemia. Eighty-six patients received treatment (69 on-demand and/or 17 on prophylaxis), with fibrinogen concentrates being the most frequently used product. Genetic analysis was available for 91 cases and 41 distinct variants were identified. Hotspot variants (FGG, p.Arg301Cys/His and FGA, p.Arg35Cys/His) were present in 51% of dysfibrinogenemia. Obstetric complications were commonly observed in dysfibrinogenemia. This large multicenter study provided a comprehensive insight into the clinical, laboratory, and genetic history of patients with CFDs. We conclude that bleeding severity grades were in agreement with the established factor activity threshold in nearly half of the cases with quantitative defects.

Rare bleeding disorders: worldwide efforts for classification, diagnosis, and management.

Rare bleeding disorders (RBDs) comprise the inherited deficiencies of coagulation factors such as fibrinogen, factor (F)II, FV, FV + FVIII, FVII, FX, FXI, and FXIII, and are usually transmitted as autosomal recessive disorders. RBDs are characterized by a wide variety of symptoms from mild to severe; however, due to their rarity, only little information is available on the adequate management of patients affected with these deficiencies. Moreover, the limitations of laboratory assays and the lack of a definitive consensus concerning their classification have prevented adoption of optimal approaches to their individual management. To overcome these limitations, new strategies are therefore necessary, such as the establishment of global collaborations and networks among treatment centers, as well as increasing support provided by public health organizations.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: research priorities for ultra-rare inherited bleeding disorders.

BACKGROUND: Ultra-rare inherited bleeding disorders (BDs) present important challenges for generating a strong evidence foundation for optimal diagnosis and management. Without disorder-appropriate treatment, affected individuals potentially face life-threatening bleeding, delayed diagnosis, suboptimal management of invasive procedures, psychosocial distress, pain, and decreased quality-of-life. RESEARCH DESIGN AND METHODS: The National Hemophilia Foundation (NHF) and the American Thrombosis and Hemostasis Network identified the priorities of people with inherited BDs and their caregivers, through extensive inclusive community consultations, to inform a blueprint for future decades of research. Multidisciplinary expert Working Group (WG) 3 distilled highly feasible transformative ultra-rare inherited BD research opportunities from the community-identified priorities. RESULTS: WG3 identified three focus areas with the potential to advance the needs of all people with ultra-rare inherited BDs and scored the feasibility, impact, and risk of priority initiatives, including 13 in systems biology and mechanistic science; 2 in clinical research, data collection, and research infrastructure; and 5 in the regulatory process for novel therapeutics and required data collection. CONCLUSIONS: Centralization and expansion of expertise and resources, flexible innovative research and regulatory approaches, and inclusion of all people with ultra-rare inherited BDs and their health care professionals will be essential to capitalize on the opportunities outlined herein.

Living with an ultra-rare inherited bleeding disorder is challenging. Patients can feel alone and unsure of where to find support because their disorder is so rare. In this paper, a group of ultra-rare bleeding disorder experts, including doctors, researchers, regulators, patient advocates, and patients, identify the research that could best improve the lives of people with these disorders. They propose a national network of specialists who can help doctors, who may never have seen these disorders before, to find the right diagnosis faster. A centralized laboratory specialized in ultra-rare bleeding disorders could also improve diagnosis and do research studies. This would help us learn, for example, how symptoms change throughout a patient's life, how effective different treatments are, and what it is like for patients to live with these disorders. A second research priority is to better understand each individual disorder so that the best treatments can be chosen or developed. A pathway showing doctors which treatment options to try, in which order, would help them help their patients. The third research priority is to make it easier to study new treatments for ultra-rare bleeding disorders. This requires designing studies with very small numbers of participants, identifying meaningful outcomes to measure, and convincing pharmaceutical companies to invest in these studies. International agreement on these requirements would allow more patients to participate and benefit from the research. These top-priority research goals should greatly improve knowledge about, and diagnosis and treatment of, ultra-rare inherited bleeding disorders.

Safety and effectiveness of recombinant factor XIII-A2 in congenital factor XIII deficiency: Real-world evidence.

BACKGROUND: Regular factor XIII (FXIII) prophylaxis is standard treatment for congenital FXIII A-subunit deficiency (FXIII-A CD). Recombinant factor XIII-A2 (rFXIII-A2) was extensively evaluated in the mentor trials. OBJECTIVE: To assess real-world safety and treatment effectiveness of rFXIII-A2 prophylaxis from the mentor 6 trial. PATIENTS/METHODS: mentor 6 was a noninterventional, postauthorization safety study investigating rFXIII-A2 prophylaxis in FXIII-A CD. rFXIII-A2 treatment was observed for 2 to 6 years per patient. The primary end point was documentation of adverse drug reactions (including anti-FXIII antibody development). Secondary end points were serious adverse events (SAEs), medical events of special interest (MESIs), and annualized bleeding rate (ABR). RESULTS: Among 30 patients (mean age, 25.5 years), there were 44 adverse events (AEs) (30 mild, 13 moderate, 1 severe). Eleven AEs were possibly/probably related to rFXIII-A2. Of four MESIs, two were unlikely related to rFXIII-A2 (accidental overdose, deep vein thrombosis), and two were possibly/probably related (nonneutralizing anti-FXIII antibody, decreased therapeutic response). All 10 SAEs were unlikely related to rFXIII-A2. Over a follow-up of 75.4 patient-years, there were six treatment-requiring bleeds (all trauma-related with no spontaneous bleeds), giving a treatment-requiring ABR of 0.066; five bleeds were treated successfully with rFXIII-A2. Eight of nine minor surgeries performed during rFXIII-A2 prophylaxis reported successful hemostatic outcomes (one missing evaluation). CONCLUSIONS: These data confirm that rFXIII-A2 prophylaxis is well tolerated as long-term care. There were no spontaneous bleeds, ABR was low, and rFXIII-A2 successfully treated bleeds in patients receiving rFXIII-A2 prophylaxis.

Clinical and laboratory diagnosis of rare coagulation disorders (RCDs).

Rare coagulation disorders (RCDs) are a group of diseases due to coagulation factors deficiency leading to life-long bleeding diathesis. The diagnosis of RCDs is challenging due to the limited knowledge of these disorders and the large heterogeneity of their bleeding patterns. The clinical symptoms of RCDs are extremely diverse in terms of bleeding type, site, severity, age at onset, and duration. The strength of the association between clotting factor activity level in plasma and clinical symptoms is also variable within each RCD. The clinical evaluation of RCDs starts with a detailed collection of clinical history and has been facilitated by bleeding assessment tools, however their effectiveness in diagnosing RCDs requires further investigation. The following laboratory diagnosis of RCDs involves coagulation screening tests, including activated partial thromboplastin time, prothrombin time, and thrombin time. After ruling out the presence of an inhibitor by mixing studies, in case of abnormal results, the specific deficiency is identified by performing one-stage clotting assays using the specific factor-depleted plasmas as substrate. In fibrinogen and FXIII deficiencies coagulation screening tests are not informative, therefore additional tests are needed. Global assays have been developed and are thought to aid in patient management, however, they are not well standardized yet. In addition to outlining the principles of clinical and laboratory diagnosis, this review explores molecular basis of RCDs and laboratory techniques for genetic analysis, and discusses the importance and effectiveness of quality control programs to ensure standardized laboratory results.

Factor X deficiency.

Factor X (FX) deficiency is a rare, recessively inherited bleeding disorder representing 10% of all rare bleeding diseases and affecting 1 in every 1,000,000 people. Its clinical presentation places FX deficiency among the most severe of the rare coagulation defects, typically including hemarthroses, hematomas, and umbilical cord, gastrointestinal, and central nervous system bleeding. Phenotype diagnosis is based on the concomitant prolongation of the prothrombin time and activated partial thromboplastin time. Through the measurement of plasma level of FX antigen and its coagulant activity, two main types of deficiency can be distinguished: type I (concomitantly low levels of activity and antigen) and type II (low coagulant activity, but normal or borderline antigen levels). FX protein is mainly synthesized by the liver and is encoded by a gene ( F10) of 27 kb located on chromosome 13, containing 8 exons. One hundred five mutations on F10 have been identified to date, 78% being missense mutations, with no hot-spot regions. There is no specific FX concentrate available, and current treatment includes the administration of fresh-frozen plasma or prothrombin complex concentrates (PCCs) containing FX in addition to other vitamin K-dependent factors. Administration of PCCs is associated with the risk of thromboembolic complication due to the unknown concentrations of other coagulant factors; however, to overcome this problem, a concentrate containing well-defined amounts of FX (and FIX) has recently been developed.

Introduction. Rare bleeding disorders: general aspects of clinical features, diagnosis, and management.

Rare bleeding disorders (RBDs) are autosomal recessive diseases including the inherited deficiencies of coagulation factors such as fibrinogen, factor (F) II, FV, FV + FVIII, FVII, FX, FXI, FXIII, and multiple deficiency of vitamin K-dependent factors, with clinical manifestations ranging from mild to severe. They represent 3 to 5% of all the inherited coagulation deficiencies with a prevalence in the general population varying between 1 in 500,000 and 1 in 2 million, being higher in areas where consanguineous marriages are diffuse. Despite the progress made in past years, as a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, the actual management of bleeding episodes and particularly the prophylactic treatment in patients affected with RBDs are not well established. In this introductory article, the main features, diagnosis, available treatment options, and treatment complications of RBDs will be discussed.

Management of rare acquired bleeding disorders.

Autoantibodies toward clotting factors may develop in people suffering from autoimmune or neoplastic diseases, after drug intake or even in subjects without apparent conditions. They are more commonly directed against factor VIII (FVIII) or von Willebrand factor leading to acquired hemophilia A or acquired von Willebrand syndrome, respectively. Rarely, autoantibodies develop against other clotting factors, such as fibrinogen, FII, FV, FVII, FX, FXI, and FXIII. The clinical picture of an acquired bleeding disorder includes a wide spectrum of clinical manifestations ranging from minimal or no bleeding to life-threatening events. Patients with no previous personal or family history of bleeding may have sudden-onset hemorrhagic manifestations, sometimes fatal, especially if an early diagnosis is not made. On the other hand, some patients may not have hemorrhagic symptoms at onset, and their diagnosis can therefore be delayed. The laboratory diagnostic assessment is performed by screening coagulation tests followed by specific factor-level measurement and inhibitor-titrating assays. An early diagnosis of acquired coagulopathies is mandatory for starting the appropriate treatment aimed at both controlling the acute bleeding episode mainly using the bypassing agents, and eradicating the anticlotting factor autoantibody, using immunosuppressive treatment. Therefore, prompt intervention by an expert and a specialized center is needed for immediate recognition and treatment of the disease.

Analysis of factor V in zebrafish demonstrates minimal levels needed for early hemostasis.

In humans, coagulation factor V (FV) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Zebrafish possess many distinct advantages including high fecundity, optical clarity, external development, and homology with the mammalian hemostatic system, features that make it ideal for genetic studies. Our aim was to study the role of FV in zebrafish through targeted mutagenesis and apply the model to the study of human F5 variants. CRISPR-mediated genome editing of the zebrafish f5 locus was performed, generating mutants homozygous for a 49 base pair deletion in exon 4. Thrombus formation secondary to vascular endothelial injury was absent in f5 -/- mutant embryos and larvae. Despite this severe hemostatic defect, homozygous mutants survived before succumbing to severe hemorrhage in adulthood. Human F5 variants of uncertain significance from patients with FV deficiency were evaluated, and the causative mutations identified and stratified by their ability to restore thrombus formation in larvae. Analysis of these novel mutations demonstrates variable residual FV function, with minimal activity being required to restore hemostasis in response to laser-induced endothelial injury. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology, although limitations exist. Furthermore, homozygous mutant embryos tolerate what is a severe and lethal defect in mammals, suggesting the possibility of species-specific factors enabling survival, and allowing further study not possible in the mouse. Identification of these factors or other genetic modifiers could lead to novel therapeutic modalities.

Phenotype and genotype report on homozygous and heterozygous patients with congenital factor X deficiency.

Factor X deficiency is a severe rare hemorrhagic condition inherited as an autosomal recessive trait. It is one of the most severe recessive inherited coagulation disorders. We analyzed the clinical manifestations, laboratory phenotype and genotype in 10 patients with severe Factor X deficiency and in their heterozygous relatives. The most frequent bleeding episodes were hematomas (70%) and gum bleeding (60%). Fifty percent of the homozygous patients required blood transfusion and one-third of heterozygotes required treatment after surgery or delivery. The genetic characterization revealed six different missense mutations, two of which were novel: p.Glu69Lys and p.Asp103His. Haplotype analysis, performed with intra- and extra- FX gene polymorphic markers in Indian, Iranian and Italian patients with the same mutations failed to establish identity by descent, despite the same Caucasian origin. In conclusion, factor X deficiency was confirmed to be one of the most serious among rare bleeding disorders and genetically heterogeneous in different populations.