10.4081/btvb.2023.75
Congenital fibrinogen disorders (CFD) include several types and subtypes of fibrinogen deficiency, resulting from monoallelic or biallelic mutations in one of the three fibrinogen genes. While it is relatively easy to make an accurate diagnosis based on activity and antigen levels of fibrinogen and genotype, prediction of the clinical phenotype is challenging. Even among patients with the same genotype, the clinical features are heterogeneous and unpredictable. The development of next-generation sequencing rises the possibility to integrate genetic modifiers to explain the subtle relationship between genotype and clinical phenotype. A recent development in integrative hemostasis assays can also help in the determination of patients at risk of bleeding or thrombosis. In this short review, we go through these topics and explain why CFD could be considered an oligogenic rather than a monogenic disease.
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Alessandro Casini
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Bleeding, Thrombosis and Vascular Biology
fibrinogen
Addressing some challenges of congenital fibrinogen disorders in 2023 and beyond
Text.Serial.Journal
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2023-03-22
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Bleed Thromb Vascul Biol
Alessandro Casini
2023/08/03
10.4081/btvb.2023.75
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Casini A, Duval C, Pan X, et al. Fibrin clot structure in patients with congenital dysfibrinogenaemia. Thromb Res 2016;137:189-95.
Addressing some challenges of congenital fibrinogen disorders in 2023 and beyond
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                                                                                                                             REVIEW

Addressing some challenges of congenital fibrinogen disorders in 2023
and beyond
Cristina Santoro,1 Alessandro Casini2
1
  Hematology Unit, Azienda Ospedaliera Universitaria Policlinico Umberto I, Rome, Italy; 2Division of Angiology and Hemostasis,
University Hospitals of Geneva, Faculty of Medicine, Geneva, Switzerland



ABSTRACT
    Congenital fibrinogen disorders (CFD) include several types and subtypes of fibrinogen deficiency, resulting from monoallelic or
biallelic mutations in one of the three fibrinogen genes. While it is relatively easy to make an accurate diagnosis based on activity and
antigen levels of fibrinogen and genotype, prediction of the clinical phenotype is challenging. Even among patients with the same
genotype, the clinical features are heterogeneous and unpredictable. The development of next-generation sequencing rises the possibility
to integrate genetic modifiers to explain the subtle relationship between genotype and clinical phenotype. A recent development in
integrative hemostasis assays can also help in the determination of patients at risk of bleeding or thrombosis. In this short review, we
                                                                          go through these topics and explain why CFD could be




                                                                                                     ly
                                                                          considered an oligogenic rather than a monogenic disease.




                                                                                              on
  Correspondence: Dr. Alessandro Casini, Division of Angiology and
  Hemostasis, University Hospitals of Geneva, 1211 Geneva 14,




                                                                                        e
  Switzerland.
  Tel.: +41.223729751 - Fax: +41.223729891.
  E-mail: alessandro.casini@hcuge.ch

  Citation: Santoro C, Casini A. Addressing some challenges of con-
                                                                                 us
                                                                            Introduction
                                                                                 Congenital fibrinogen disorders (CFD) encompass a het-
                                                                            al
  genital fibrinogen disorders in 2023 and beyond. Bleeding, Throm-         erogeneous group of fibrinogen deficiencies.1 Diagnosis of
  bosis, and Vascular Biology 2023;2:75.                                    CFD is based on the assessment of functional and antigenic
                                                                  ci

                                                                            fibrinogen levels, allowing to differentiate afibrinogenemia
  Key words: afibrinogenemia, hypofibrinogenemia, dysfibrinogen-
                                                                er


                                                                            (complete absence of fibrinogen), hypofibrinogenemia (de-
  emia, fibrinogen.
                                                                            creased levels of functional fibrinogen), dysfibrinogenemia
                                                               m



  Contributions: CS and AC made the literature review and wrote             (normal levels of dysfunctional fibrinogen) and hypodysfib-
  the manuscript.                                                           rinogenemia (decreased levels of dysfunctional fibrinogen).
                                                    om




                                                                            Assessment of fibrinogen antigen is mandatory to distinguish
  Conflict of interest: AC reports Speaker at satellite symposia or-        dysfibrinogenemia from hypofibrinogenemia. However, fib-
  ganized by LFB, participation at advisory board of LFB, Oc-               rinogen immunological assays are not widely available in he-
  tapharma, Takeda, consulting for LFB, Pentapharma, travel support
                                             -c




                                                                            mostasis laboratories. The prothrombin-derived fibrinogen
  from Sobi, unrestricted grant from NovoNordisk and CSL Behring.
                                                                            assay is an accurate alternative in view of its good correlation
                                    on




  Funding: none.                                                            with the fibrinogen antigen level in dysfibrinogenemia.2 In ad-
                                                                            dition, a recent communication from the Subcommittee FXIII
                                                                            and Fibrinogen of the International Society of Thrombosis and
                              N




  Ethical approval: not applicable.
                                                                            Hemostasis proposes a classification in several subtypes ac-
  Availability of data and material: not applicable.                        cording to the genotype and the clinical manifestations.3
                                                                                 Since the first description of afibrinogenemia in 1920 by
  Received: 22 March 2023.                                                  Rabe and Salomon and the first report on dysfibrinogenemia
  Accepted: 21 June 2023.
                                                                            in 1958 by Imperato and Dettori,4,5 hundred families with CFD
  Publisher’s note: all claims expressed in this article are solely those
                                                                            have been identified in the last decades. Gathering and study-
  of the authors and do not necessarily represent those of their affili-    ing CFD cases has improved our knowledge of fibrinogen mol-
  ated organizations, or those of the publisher, the editors and the re-    ecules and enhanced our understanding of the multiple
  viewers. Any product that may be evaluated in this article or claim       functions of fibrin(ogen).6 Nevertheless, several aspects re-
  that may be made by its manufacturer is not guaranteed or endorsed        garding the diagnosis, the prediction of outcomes, and the clin-
  by the publisher.                                                         ical management still require to be solved. The emergence of
                                                                            next-generation sequencing (NGS) rises the possibility to in-
  ©
   Copyright: the Author(s), 2023                                           tegrate genetic modifiers to explain the subtle relationship be-
  Licensee PAGEPress, Italy                                                 tween genotype and clinical phenotype. Recent development
  Bleeding, Thrombosis and Vascular Biology 2023; 2:75
                                                                            in integrative hemostasis assays can help in the determination
  doi:10.4081/btvb.2023.75
                                                                            of patients at risk of bleeding or thrombosis. In this short re-
  This work is licensed under a Creative Commons Attribution-               view, we provide some insights into these two selected aspects
  NonCommercial 4.0 International License (CC BY-NC 4.0).                   and give some perspectives for further clinical and fundamen-
                                                                            tal research.


                                              Bleeding, Thrombosis and Vascular Biology 2023; 2:75
                               Addressing some challenges of congenital fibrinogen disorders in 2023 and beyond                                           3



                                                                                        stability of the fibrin network; iii) decreased fibrinolysis result-
Correlation between genotype                                                            ing from impaired binding of plasminogen or tissue-type plas-
and phenotype                                                                           minogen activator to abnormal fibrin.24 In addition, many defects
                                                                                        in dysfibrinogenemia affect fibrinogen clottability to different
     Genotype is necessary to confirm the diagnosis of CFD, it                          degrees and can lead to a mild bleeding phenotype. In a recent
distinguishes between hypofibrinogenemia and hypodysfibrino-                            review, Li et al. identified several fibrinogen variants that could
genemia or severe hypofibrinogenemia and afibrinogenemia and                            be related to bleeding. These included: i) mutations in the NH2-
simplifies family screening and prenatal diagnosis. Of note,                            terminal portion of the Aα chain that hamper fibrinogen fitting
proper experimental validation at the protein or RNA level is                           into the active site cleft of thrombin and drastically slow the con-
important to confirm the pathogenic effect of new fibrinogen                            version of fibrinogen into monomeric fibrin; ii) mutations
variants, for instance by protein modeling, mass spectrometry,                          adding new N-linked glycosylation sites introduce bulky and
or protein expression.7,8                                                               negatively charged carbohydrate side chains and undermine the
     A few rare fibrinogen variants are strongly associated with                        alignment of fibrin monomers during polymerization; iii) muta-
a clinical phenotype. This is the case of the fibrinogen storage                        tions generating unpaired cysteine form extra disulfide bonds
disease (hypofibrinogenemia type 2D according to the ISTH                               between the abnormal fibrinogen chains and produce highly
classification) and the thrombotic-related fibrinogen variants                          branched and fragile fibrin networks; iv) truncation mutations
(dysfibrinogenemia type 3B) (Table 1).9-22 Fibrinogen storage                           in the fibrinogen αC regions impair the lateral fibril aggregation,
diseases are usually suspected in familial history of cryptogenic                       as well as factor XIII crosslinking, endothelial cell, and platelet




                                                                                                               ly
liver disease associated with hypofibrinogenemia.23 Diagnosis                           binding.25 However, in a multicentric study of 101 patients with




                                                                                                     on
relies on histological observation of fibrinogen inclusion in he-                       dysfibrinogenemia with a mean follow-up of 8.8 years the most
patocytes and genotype.13 The molecular reasons for fibrinogen                          frequent mutations (i.e., FGA Arg35His/Cys and FGG
inclusions are still not understood. Among others, it has been re-                      Arg301His/Cys) were statistically associated neither with major




                                                                                              e
ported that conformational changes in the region of the globular                        bleeding (HR 0.8, 95% CI 0.1-4.1, P=0.79) nor with thrombosis
domain of fibrinogen involved in the “end-to-end” interaction                           (HR 0.8, 95% CI 0.3-2.4, P=0.68).26
can cause an abnormal exposure of hydrophobic patches in the
fibrinogen γ chain, which becomes available for interactions
                                                                                     us      One of the most striking clinical features of CFD is the het-
                                                                                        erogeneity of symptoms even among patients with the same
with lipids enhancing the accumulation of fibrinogen and
                                                                             al
                                                                                        genotype. Based on historical records on dysfibrinogenemia and
apolipoprotein B.12                                                                     more recent cohort studies, it is accepted that at diagnosis about
                                                           ci

     Thrombotic-related dysfibrinogenemia variants were first                           55% of patients are asymptomatic, 25% have a tendency to bleed
described in communication by SSC ISTH in 1995. An associ-                              and 20% have a thrombotic phenotype (in venous and arterial
                                                         er


ation between thrombophilia and the fibrinogen variant was                              territories).26-30 Similarly, while most patients with afibrinogen-
clearly observed in 26 probands with a history of personal and                          emia suffer from severe, sometimes life-threatening, bleeding,
                                                        m




familial thrombosis at a young age without other biological risk                        about 20% also experience thrombotic events and a few are even
factors. Different mechanisms, often overlapping, may account                           asymptomatic.31 The thrombotic occurrence in afibrinogenemia
                                            om




for the increased risk of thrombosis in these fibrinogen variants:                      is a clinical conundrum. Among others, concomitant throm-
i) elevated levels of circulating thrombin resulting from the fail-                     bophilia and fibrinogen supplementation especially with fresh
ure in fibrin(ogen) binding; ii) altered strength, architecture, and                    frozen plasma or cryoprecipitates, may increase the risk of
                                     -c
                            on




Table 1. Variants of hypofibrinogenemia associated with fibrinogen storage disease and variants of dysfibrinogenemia associated with
thrombosis.
                      N




Mutant fibrinogen                            Gene                     Exon                    Native protein                  Reference
Fibrinogen storage disease
 Aguadilla                                    FGG                        9                      p.Arg401Trp                         9
 AI duPont                                    FGG                        8                      p.Thr340Pro                         10
 Angers                                       FGG                        8                 p.delGVYYQ372-376                        11
 Ankara                                       FGG                        8                      p.His366Asp                         12
 Beograd                                      FGG                        9                      p.Gly392Ser                         13
 Brescia                                      FGG                        8                     p.Gly310Arg                          14
 Pisa                                         FGG                        8                     p.Asp342Asn                          13
 Trabzon                                      FGG                        9                       p.Thr397Ile                        15
Thrombotic-related dysfibrinogenemia
 Caracas V                                    FGA                        5                      p.Ser551Cys                         16
 Dusart*                                      FGA                        5                      p.Arg573Cys                         17
 Ijmuiden**                                   FGB                        2                      p.Arg44Cys                          18
 Melun                                        FGG                        8                      p.Asp390Val                         19
 Naples***                                    FGB                        2                       p.Ala98Thr                         20
 New York                                     FGB                        2                       p.del39-102                        21
 Nijmegen                                     FGB                        2                      p.Arg74Cys                          22
*also named Paris V, Chapel Hill III; **also named Christchurch II, London VIII, St-Germain III, Vicenza III; ***at homozygous state.


                                                Bleeding, Thrombosis and Vascular Biology 2023; 2:75
4                                                         C. Santoro and A. Casini




thrombosis in afibrinogenemia.32 Of note, when evaluating the            complex mechanism of thrombin generation or, on the contrary,
patient’s clinical phenotype, it is crucial to take into account the     on the fibrinolytic potential.44
family history and environmental risk factors that can contribute             Several lines of growing evidence have pointed out the im-
to the heterogeneity of symptoms, beyond the genotype.33 In hy-          portant role of fibrin structure in thrombosis and/or bleeding.45
pofibrinogenemia, the bleeding phenotype is strongly correlated          The structure of the fibrin clot is a major determinant of the me-
to the fibrinogen concentration.34 However, to date, no prospec-         chanical properties and of the clot resistance to fibrinolysis by
tive data have been published allowing us to determine the real          tissue plasminogen activator and the plasmin system.46 There-
incidence of bleeding in hypofibrinogenemia or leading to iden-          fore, many efforts have been done in determining the effect of
tify factors modulating their clinical phenotype.                        dysfunctional fibrinogen on the fibrin clot network and proper-
     So far, most studies on CFD employed traditional Sanger             ties.47 On one side, patients with a bleeding phenotype have ab-
sequencing to identify molecular defects, thus missing other mu-         normal polymerization profiles, increased lysis, and thick fibrin
tations that could influence the patient’s clinical phenotype.8          fiber with large pores.48 On the other side, patients with a throm-
NGS now provides information on the most known genetic mod-              botic phenotype have fibrin networks composed of thin fiber
ifiers of fibrinogen and the hemostatic balance.35,36 In addition,       strands that have small pores and are more rigid and less per-
NGS allows the development of a large data set and the identi-           meable.49 If these methods are often investigated to explain the
fication of mutations not detected by standard PCR amplifica-            clinical presentation of single cases, data on large series are lim-
tion. However, strong bioinformatic tools are required to analyze        ited. Recently, defective fibrin polymerization and prolonged
the amount of data and especially filtering variants against the




                                                                                                    ly
                                                                         fibrinolysis have been reported in 24 patients with dysfibrino-
allele frequencies. When considering NGS in the investigation            genemia compared to healthy individuals, even though without




                                                                                            on
of a rare bleeding disorder, it is important to highlight that: i)       statistical significance.50 Overall, a comprehensive functional
additional analyses may be required to determine whether a vari-         assessment of properties and ultrastructure of the fibrin clot in a
ant is pathogenic or not; ii) that physicians may have to deal with      large cohort of patients is lacking. Moreover, fibrin clot studies




                                                                                      e
incidental findings and ethical issues; iii) that NGS has a great        in dysfibrinogenemia rarely take into account other genetic vari-
accuracy for single nucleotide changes, but complex rearrange-
ments are not detected.37
     Increased fibrinogen concentrations are associated with de-
                                                                               us
                                                                         ations that might influence or modulate the fibrin clot (i.e., in-
                                                                         termediate phenotype),24 and no prospective data are available
                                                                         so far. Overall, such studies could allow us to better predict the
                                                                        al
creased fibrin clot permeability and tighter clots when associated       clinical outcomes and offer a more personalized therapeutic
with a polymorphism (rs5985) of the F13A gene encoding factor            strategy toward precision medicine.
                                                            ci

XIII subunit A Val34Leu.38 Coding polymorphisms in the fib-
rinogen genes also influence the fibrin clot structure. The com-
                                                          er


mon FGB BβArg478Lys polymorphism (rs4220) is associated
                                                                         Conclusions
with thin fibrin fibers, small pores, increased stiffness, and hy-
                                                         m




pofibrinolysis,39 while the FGA AαThr331Ala polymorphism                     We should start considering CFD as an oligogenic rather
(rs6050) alters FXIII-dependent fibrin cross-linking.40 In the fu-
                                               om




                                                                         than just a monogenic disease and focus research on the assess-
ture, the largest studies focusing on the significance of polymor-       ment of common variants that might contribute to clinical vari-
phisms and the impact of variants of other genes involved in             ations between patients with CDF. Today we are far from having
coagulation will provide a global view of the hemostatic balance         clinically relevant information on most genetic modifiers and
                                         -c




of patients with CFD.                                                    their impact on the hemostasis balance. Understanding the ge-
                                 on




                                                                         netic-related thrombin generation and fibrin clot mechanisms
                                                                         involved in a patient’s phenotype will help in understanding the
Global hemostasis assays                                                 pathophysiological mechanisms underlying the bleeding and/or
                            N




and fibrin clot study                                                    thrombosis in CFD.

    Integrative hemostatic assays, such as thrombin generation
assay and studies of fibrin clots may provide additional infor-          References
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