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Background: Congenital fibrinogen disorders are classified based on both fibrinogen levels and the clinical phenotype. For dysfibrinogenemia, normal fibrinogen levels are typical., Key Clinical Question: We highlight the importance of comprehensive thrombotic risk assessment, including lipoprotein a (Lp[a]) and hypertriglyceridemia in association with severe thrombosis and poor wound healing in dysfibrinogenemia., Clinical Approach: We report the case of a 42-year-old male patient with a rare congenital thrombotic-related dysfibrinogenemia (fibrinogen Naples) and multiple thrombotic episodes throughout his life and an unhealing ankle wound. Despite all thrombotic episodes and surgery, the patient had undetectable D-dimer, suggestive of fibrinolytic defect, further supported by over 4-fold elevated Lp(a) levels. The last arterial thrombosis was preoperatively managed by plasma exchange, antithrombotics, and thereafter continued fibrinogen replacement therapy, under which the chronic wound has healed., Conclusion: The combination of thrombogenesis, abnormal fibrinogen, and high Lp(a) levels is a clinical and research topic deserving more attention., (© 2024 The Authors.)

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., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person. An added feature of the program is that talks streamed at the designated times will then be available on-line for asynchronous viewing. The program included 77 State of the Art (SOA) talks, thematically grouped in 28 sessions, given by internationally recognized leaders in the field. The SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. The topics, across the main scientific themes of the congress, include Arterial Thromboembolism, Coagulation and Natural Anticoagulants, COVID-19 and Coagulation, Diagnostics and Omics, Fibrinogen, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostasis in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Angiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the congress., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

The outcome of congenital fibrinogen defects (CFD) is often unpredictable. Standard coagulation assays fail to predict the clinical phenotype. We aimed to assess the pheno- and genotypic associations of thrombin generation (TG) and ROTEM in CFD. We measured fibrinogen (Fg) activity and antigen, prothrombin fragments F1+2, and TG by ST Genesia ® with both Bleed- and ThromboScreen in 22 patients. ROTEM was available for 11 patients. All patients were genotyped for fibrinogen mutations. Ten patients were diagnosed with hypofibrinogenemia, nine with dysfibrinogenemia, and three with hypodysfibrinogenemia. Among the 17 mutations, eight were affecting the Fg γ chain, four the Fg Bβ chain, and five the Fg Aα chain. No statistical difference according to the clinical phenotypes was observed among FGG and FGA mutations. Median F1+2 and TG levels were normal among the different groups. Fg levels correlated negatively with F1+2 and peak height, and positively with lag time and time to peak. The pheno- and genotypes of the patients did not associate with TG. FIBTEM by ROTEM detected hypofibrinogenemia. Our study suggests an inverse link between low fibrinogen activity levels and enhanced TG, which could modify the structure-function relationship of fibrin to support hemostasis.

Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga -/- larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga +/- larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding Aα Δ 19-56 . This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα +/ Δ 19-56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga -/- mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα +/ Δ 19-56 . We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by a proportional decrease of functional and antigenic fibrinogen levels. Hypofibrinogenemia can be considered the phenotypic expression of heterozygous loss of function mutations occurring within one of the three fibrinogen genes ( FGA , FGB , and FGG ). Clinical manifestations are highly variable; most patients are usually asymptomatic, but may appear with mild to severe bleeding or thrombotic complications. We have sequenced all exons of the FGA , FGB , and FGG genes using the DNA isolated from the peripheral blood in two unrelated probands with mild hypofibrinogenemia. Coagulation screening, global hemostasis, and functional analysis tests were performed. Molecular modeling was used to predict the defect of synthesis and structural changes of the identified mutation. DNA sequencing revealed a novel heterozygous variant c.1421G>A in exon 8 of the FGB gene encoding a Bβ chain (p.Trp474Ter) in both patients. Clinical data from patients showed bleeding episodes. Protein modelling confirmed changes in the secondary structure of the molecule, with the loss of three β sheet arrangements. As expected by the low fibrinogen levels, turbidity analyses showed a reduced fibrin polymerisation and imaging difference in thickness fibrin fibers. We have to emphasize that our patients have a quantitative fibrinogen disorder; therefore, the reduced function is due to the reduced concentration of fibrinogen, since the Bβ chains carrying the mutation predicted to be retained inside the cell. The study of fibrinogen molecules using protein modelling may help us to understand causality and effect of novel genetic mutations.

Plasma fibrinogen molecules comprise 2 copies of Aα, Bβ, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting., (© 2020 by The American Society of Hematology.)

Fibrinogen is an abundant protein synthesized in the liver, present in human blood plasma at concentrations ranging from 1.5-4 g/L in healthy individuals with a normal half-life of 3-5 days. With fibrin, produced by thrombin-mediated cleavage, fibrinogen plays important roles in many physiological processes. Indeed, the formation of a stable blood clot, containing polymerized and cross-linked fibrin, is crucial to prevent blood loss and drive wound healing upon vascular injury. A balance between clotting, notably the conversion of fibrinogen to fibrin, and fibrinolysis, the proteolytic degradation of the fibrin mesh, is essential. Disruption of this equilibrium can cause disease in distinct manners. While some pathological conditions are the consequence of altered levels of fibrinogen, others are related to structural properties of the molecule. The source of fibrinogen expression and the localization of fibrin(ogen) protein also have clinical implications. Low levels of fibrinogen expression have been detected in extra-hepatic tissues, including carcinomas, potentially contributing to disease. Fibrin(ogen) deposits at aberrant sites including the central nervous system or kidney, can also be pathological. In this review, we discuss disorders in which fibrinogen and fibrin are implicated, highlighting mechanisms that may contribute to disease., (Copyright© 2020 Ferrata Storti Foundation.)

The study of inherited fibrinogen disorders, characterized by extensive allelic heterogeneity, allows the association of defined mutations with specific defects providing significant insight into the location of functionally important sites in fibrinogen and fibrin. Since the identification of the first causative mutation for congenital afibrinogenemia, studies have elucidated the underlying molecular pathophysiology of numerous causative mutations leading to fibrinogen deficiency, developed cell-based and animal models to study human fibrinogen disorders, and further explored the clinical consequences of absent, low, or dysfunctional fibrinogen. Since qualitative disorders are addressed by another review in this special issue, this review will focus on quantitative disorders and will discuss their diagnosis, clinical features, molecular bases, and introduce new models to study the phenotypic consequences of fibrinogen deficiency., Competing Interests: The authors declare no conflict of interest.

Whole-exome and targeted sequencing of 13 individuals from 10 unrelated families with overlapping clinical manifestations identified loss-of-function and missense variants in KIAA1109 allowing delineation of an autosomal-recessive multi-system syndrome, which we suggest to name Alkuraya-Kučinskas syndrome (MIM 617822). Shared phenotypic features representing the cardinal characteristics of this syndrome combine brain atrophy with clubfoot and arthrogryposis. Affected individuals present with cerebral parenchymal underdevelopment, ranging from major cerebral parenchymal thinning with lissencephalic aspect to moderate parenchymal rarefaction, severe to mild ventriculomegaly, cerebellar hypoplasia with brainstem dysgenesis, and cardiac and ophthalmologic anomalies, such as microphthalmia and cataract. Severe loss-of-function cases were incompatible with life, whereas those individuals with milder missense variants presented with severe global developmental delay, syndactyly of 2 nd and 3 rd toes, and severe muscle hypotonia resulting in incapacity to stand without support. Consistent with a causative role for KIAA1109 loss-of-function/hypomorphic variants in this syndrome, knockdowns of the zebrafish orthologous gene resulted in embryos with hydrocephaly and abnormally curved notochords and overall body shape, whereas published knockouts of the fruit fly and mouse orthologous genes resulted in lethality or severe neurological defects reminiscent of the probands' features., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

GNB5 encodes the G protein β subunit 5 and is involved in inhibitory G protein signaling. Here, we report mutations in GNB5 that are associated with heart-rate disturbance, eye disease, intellectual disability, gastric problems, hypotonia, and seizures in nine individuals from six families. We observed an association between the nature of the variants and clinical severity; individuals with loss-of-function alleles had more severe symptoms, including substantial developmental delay, speech defects, severe hypotonia, pathological gastro-esophageal reflux, retinal disease, and sinus-node dysfunction, whereas related heterozygotes harboring missense variants presented with a clinically milder phenotype. Zebrafish gnb5 knockouts recapitulated the phenotypic spectrum of affected individuals, including cardiac, neurological, and ophthalmological abnormalities, supporting a direct role of GNB5 in the control of heart rate, hypotonia, and vision., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD., (© 2016 by The American Society of Hematology.)

The expression of plasma proteins changes dramatically as a result of cytokine induction, particularly interleukin-6, and their levels are used as clinical markers of inflammation. miRNAs are important regulators of gene expression and play significant roles in many inflammatory diseases and processes. The interactions between miRNAs and the genes that they regulate during the acute phase response have not been investigated. We examined the effects of IL-6 stimulation on the transcriptome and miRNome of human and mouse primary hepatocytes and the HepG2 cell line. Using an integrated analysis, we identified differentially expressed miRNAs whose seed sequences are significantly enriched in the 3' untranslated regions of differentially expressed genes, many of which are involved in inflammation-related pathways. Our finding that certain miRNAs may de-repress critical acute phase proteins within acute timeframes has important biological and clinical implications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We conducted a multicenter study of 101 patients with congenital dysfibrinogenemia (CD) to characterize the incidence of hemorrhagic and thrombotic events as well as complications of pregnancy and surgery. At the time of diagnosis, 10.9% and 13.9% had experienced major bleeding and thrombotic events, respectively. During a mean follow-up of 8.8 years after CD diagnosis, the incidence of major bleeding and thrombotic events was 2.5 and 18.7 per 1000 patient-years, respectively, with estimated cumulative incidences at age 50 years of 19.2% and 30.1%. We identified 111 pregnancies with an overall incidence of spontaneous abortions and postpartum hemorrhage of 19.8% and 21.4%, respectively. The risk of postpartum hemorrhage was associated with a previously identified bleeding phenotype (odds ratio, 5.8; 95% CI, 1.2 to 28.0). Among 137 surgical procedures analyzed, 9 (6.5%) were complicated by abnormal bleeding. Propositi vs relatives, sex, mutation hotspots, fibrinogen levels, and activity:antigen ratios were not associated with the risk of thrombotic or bleeding outcomes. In conclusion, the results of our study, the largest in genotyped CD and the first including long-term history, indicate that propositi with CD and their relatives carry not only a high risk of major bleeding, including postpartum hemorrhage, but also of thrombotic event., (© 2015 by The American Society of Hematology.)

Mutations in the human fibrinogen genes can lead to the absence of circulating fibrinogen and cause congenital afibrinogenemia. This rare bleeding disorder is associated with a variable phenotype, which may be influenced by environment and genotype. Here, we present a zebrafish model of afibrinogenemia. We introduced targeted mutations into the zebrafish fga gene using zinc finger nuclease technology. Animals carrying 3 distinct frameshift mutations in fga were raised and bred to produce homozygous mutants. Using a panel of anti-zebrafish fibrinogen antibodies, fibrinogen was undetectable in plasma preparations from homozygous mutant fish. We observed hemorrhaging in fga mutants and reduced survival compared with control animals. This model will now serve in the search for afibrinogenemia modifying genes or agents and, to our knowledge, is the first transmissible zebrafish model of a defined human bleeding disorder.