Diagnosis and classification of hereditary fibrinogen disorders

A fibrinogen disorder is usually suspected in patient addressed for a bleeding tendency with decreased fibrinogen level. However, incidental finding is frequent, especially in patients with mild fibrinogen deficiencies. Before starting investigations of HFD, it is important to rule out an acquired fibrinogen disorder. Liver disease, drugs and cancer, the most common causes, can affect the synthesis, the secretion, or the proteolysis of fibrinogen resulting in decreased levels of fibrinogen activity^{9, 10}. The familial history is also crucial, often helping to distinguish between a hereditary and acquired fibrinogen disorder.

The first step in diagnosis of HFDs relies on measurement of fibrinogen activity by the Clauss method¹¹. As second step, if Clauss assay is decreased, fibrinogen antigen has to be measured. As indicated in table 1, if both activity and antigen are undetectable, afibrinogenemia can be diagnosed. If fibrinogen activity and antigen are proportionally decreased, a hypofibrinogenemia should be considered. Discordance between decreased fibrinogen activity and normal antigen is highly predictive of dysfibrinogenemia. A ratio fibrinogen activity / antigen with a cut-off of 0.7 has historically be proposed to distinguish between quantitative and qualitative fibrinogen disorders, but not fully validated¹². Recently, a cut-off of 0.55 has also been tested¹³. If fibrinogen activity and antigen are disproportionally decreased, a hypodysfibrinogenemia should be considered.

Thrombin time and reptilase time are not mandatory for the diagnosis of HFDs, even though they provide useful information on the degree of defect in fibrin polymerisation¹⁴. Derived fibrinogen from the prothrombin time (PT-der) is widely used to assess the fibrinogen concentration¹⁵. The PT-der is an indirect estimation of fibrinogen concentration. In dysfibrinogenemia, PT-der overestimated the fibrinogen activity but has a very good correlation with antigen value¹⁶. A ratio PT-der / Clauss with a cut-off of 1.43 seems to provide an excellent specificity and sensitivity for the diagnosis of dysfibrinogenemia¹⁷. In addition, recent data promotes novel approaches for diagnosis of HFDs. Clot wave form from Clauss could have a similar performance to detect dysfibrinogenemia¹³.

It should be noted that in dysfibrinogenemia several variables such as clot detection methods, reagents, and type of fibrinogen variant, influence the measurement of routine hemostasis assays¹⁸. For instance, Fibrinogen Longmont is better detected by photo-optical endpoint^{19, 20}. In case of very low values of fibrinogen activity, due the limit of quantification of analysers, it could be difficult to distinguish between afibrinogenemia and hypofibrinogenemia or between hypofibrinogenemia and hypodysfibrinogenemia. In these cases, genotype is necessary to confirm the diagnosis²¹. In the setting of research, analyses of fibrin clots, may complete the fibrinogen work-up in order to better determine the patient’s clinical phenotype²².

HFDs result from monoallelic or biallelic mutations in FGA, FGB and FGG genes in chromosome 423. Besides the confirmation of the diagnosis, genotype may help for familial screening, prenatal testing, and prediction of the clinical phenotype. The development of next generation sequencing allows complete analysis of FGA, FGB and FGG exons enhancing the identification of a causative mutation²⁴. However, in developing country, Sanger sequencing is still the best option²⁵. Usually, afibrinogenemia results by homozygosity for a null mutation²⁶. Thus, patients with hypofibrinogenemia are heterozygous carriers of afibrinogenemic allele. These mutations affect either the synthesis or the assembly or the secretion of fibrinogen into circulation. Some mutations are more common, such as the large deletion 11Kb of FGA and the splice site mutation FGA c.510+1G>T^{27, 28}. Most of dysfibrinogenemia are due to missenses mutation located in exon 2 of FGA or exon 8 of FGG²⁹. Overall, two hotspot mutations (i.e, FGA c.103C>A or c.104G>A and FGG c.901C>T or c.902G>A) represent more than 80% of causative mutations in dysfibrinogenemia. These mutations yield to a delay in thrombin-mediated fibrinopeptide cleavage or defective fibrin polymerisation^{30, 31}. In hypodysfibrinogenemia, a single mutation can affect both the secretion and the function of the fibrinogen molecule. In alternative, a compound mutation can confer a concomitant “hypofibrinogenemia” and “dysfibrinogenemia” trait⁴.

A few fibrinogen variants are strongly correlated with a clinical phenotype³². Patients with thrombotic-related fibrinogen variants are a very strong risk of thrombosis³³. Thrombosiscan occur in all vascular territories, including in young patients^34-39. These mutations affect the fibrin clot structure leading to a procoagulant state⁴⁰. Other mutations, located in exons 8 and 9 of FGG cause accumulation of fibrinogen aggregates in hepatocytes resulting in chronic liver inflammation and eventually cirrhosis⁴¹. In dysfibrinogenemia, mutations modifying specific aspects of the fibrinogen molecule, can decrease the clottability and therefore increase the bleeding tendency. For instance, mutations in the NH2-terminal portion slow the conversion of fibrinogen into monomeric fibrin; mutations adding new N-linked glycosylation sites introduce negatively charged carbohydrate side chains affecting the alignment of fibrin monomers during polymerization; mutations generating unpaired cysteine form extra disulfide bonds and produce highly branched and fragile fibrin networks; truncation mutations in the fibrinogen αC regions impair the lateral fibril aggregation and factor XIII crosslinking³⁰.

As indicated in Table 1, the International Society Thrombosis and Hemostasis (ISTH) classification introduces several subtypes of HFDs according not only on the fibrinogen levels but also on the clinical pattern and the genotype. Although severe bleeding is the prominent symptom in afibrinogenemia, some patients experience recurrent thrombosis⁴². In a recent large series of patients (n=204), 18% reported a thrombotic event⁴³. Management of thrombosis in afibrinogenemia is particularly difficult as physician has to deal with both the thrombotic and the bleeding risk⁶. Patients with afibrinogenemia and a thrombotic phenotype are classified as type 2A.

The bleeding risk in hypofibrinogenemia is strongly correlated to the fibrinogen concentration. In a registry from the Netherlands, the correlation between baseline fibrinogen activity and ISTH bleeding assessment tool score was considered as moderate (r= -0.683)⁴⁴. It is generally well accepted that patients with fibrinogen activity levels >0.8 g/L do not suffer from spontaneous bleeding⁴⁵. Therefore, patients with hypofibrinogenemia are classified as severe (2A), moderate (2B), or mild (2C) according to the fibrinogen activity. A fourth subtype (2D) is hypofibrinogenemia associated with fibrinogen storage disease. This subtype is classically suspected in familial history of mild hypofibrinogenemia and cryptogenic liver disease. The physiopathology is not fully understood. Mutations in fibrinogen γ chain could provoke conformational changes resulting in abnormal exposure of hydrophobic patches that become available for interactions with APOB-lipoproteinemia causing their intracellular retention and impairment of fibrinogen secretion⁴⁶.

Most of patients with dysfibrinogenemia will be asymptomatic at the time of diagnosis^47-49 but the natural course of dysfibrinogenemia is nevertheless characterized by a risk of bleeding and thrombosis. In a series of 101 patients with dysfibrinogenemia with a median follow-up of 8.8 years, the cumulative 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%⁷. These patients are classified as subtype 3A. As previously mentioned, the thrombotic risk is remarkably more important in patients with thrombotic-related fibrinogen variants (subtype 3B). In such patients, thrombosis usually occur in young patients, including in unusual vascular sites.⁵⁰

In hypodysfibrinogenemia the bleeding risk is proportional to the fibrinogen level, but it is also exacerbated by the dysfunctional fibrinogen. Indeed, hypodysfibrinogenemia is characterized by reduced fibrinogen function but also a less marked reduction of plasma fibrinogen antigen⁸. Therefore, patients with hypodysfibrinogenemia are classified as severe (2A), moderate (2B), or mild (2C) according to the fibrinogen antigen.