DNA Sanger sequencing of the coding portions of FGA/FGB/FGG has been the gold standard in the last 20 years for the identification of molecular defects underlying afibrinogenemia and hypofibrinogenemia. This is due to the fact that these disorders show extreme allelic heterogeneity (with most mutations being “private” defects and only few being recurrent). Hence, genetic screenings have traditionally been performed by polymerase chain reaction (PCR) amplifications of exons, splice sites, and promoter regions of the FGA/FGB/FGG genes, followed by direct sequencing of the amplified products. This approach has been coupled in some laboratories with a screening based on the Multiplex Ligation Probe Amplification (MLPA) method to search essentially for large deletions otherwise missed by Sanger sequencing.

The molecular screening based on Sanger sequencing has, of course, its pros and cons. In fact, despite its standardization, the ease of execution, and the relative ease of analyzing the results, Sanger sequencing can sometimes be costly, time consuming, and quite complicated (like in the case of large and multi-exonic genes; in the case of fibrinogen, there is the necessity to screen the entire cluster). In addition, it is possible to miss mutations lying deep in introns unless the entire gene is sequenced.

The advent of next-generation sequencing (NGS) techniques is, of course, changing the overall picture of genetic diagnosis, and “inherited bleeding, thrombotic, and platelet disorders” (collectively called BPDs and including a- and hypo-fibrinogenemia), are not exceptions. In this frame, a first seminal paper appeared in the literature in 2016: Simeoni and colleagues [78] developed a high-throughput sequencing platform targeting a total of 63 genes (the ThromboGenomics platform) and applied their design to 137 individuals with a suspect of BPD. The diagnostic yield was 46%, underlying that there is still a need for identifying novel molecular causes of BPDs. Similar results were later obtained by Downes et al. [79], who applied the same platformto screen 2396 BPD patients: they reached a diagnostic yield of 50%, identified hundreds of mutations (half of which novel), and also proposed an oligogenic model of inheritance for some patients.

Specifically concerning fibrinogen disorders, the largest study based on NGS screening of affected patients appeared in 2019 [80]. Here, a total of 17 Spanish patients (suffering from a-, hypo, or dys-fibrinogenemia) were screened by using a NGS approach based on sequencing the complete FGA, FGB and FGG genes (i.e., also including introns). All patients were associated with one/two mutations, thus underlying the overall good performance of the adopted strategy [80].

For the future, it is conceivable that—also thanks to the significant drop in costs—genetic diagnosis will be carried out by using more comprehensive NGS strategies, i.e., whole-exome sequencing (WES) or whole-genome sequencing (WGS). These approaches will offer novel intriguing possibilities: (i) to highlight the oligogenic nature of specific conditions/patients; (ii) as for WGS, to identify “elusive” mutations (large rearrangements, large deletions, deep-intonic mutations, mutations in enhancers, or other regulatory regions); and (iii) to identify modulators of the phenotype. In this last case, it could be possible to answer to open questions, such as the differences in the severity of hemorrhagic manifestations in individuals carrying the same mutation or the reason why some afibrinogenemic patients are susceptible to develop thrombosis. Of course, we are aware that these are currently speculations (and hopes). However, it should be noted that some encouraging examples related to congenital fibrinogen disorders come from the literature. For instance, dysfibrinogenemias are often related to pathogenic variants affecting residues p.Gly17, p.Pro18, p.Arg19, and p.Val20 in the amino-terminal region of the fibrinogen Aα-chain. However, while mutations at residues p.Gly17, p.Pro18, and p.Val20 are exclusively linked to bleeding tendency, the clinical phenotype of patients with mutations at amino acid p.Arg19 can vary from bleeding to thrombotic tendency [81]. In this frame, the case described by Bor and colleagues [81] is intriguing: they exome sequenced a Danish family with thrombotic episodes, revealing from one side the dysfibrinogenemia-causing mutation Aα-p.Arg19Gly and from the other a series of single-nucleotide polymorphisms located in FGA, FGB, and FGG. These polymorphisms are possibly responsible for an increased fibrin fiber thickness and fibrin mass-to-length ratio, thus suggesting that the combination of genotypes may contribute to the thrombogenic phenotype of these patients

A final thought concerns the management of recessively inherited coagulation disorders, which depends on two fundamental steps: genetic counseling in consanguineous marriages and prenatal diagnosis in families at risk for having members with severe form of the disorder. These approaches are not easily realizable in the praxis [31].

Pregnant women with a family history, predominantly those with a history of consanguinity, ought to be properly counseled with regard to risk of having a child with the disorder. If we know the mutation, prenatal analysis could be planned: in fact, for a disease such as afibrinogenemia, where bleeding after loss of the umbilical cord stump is frequent and, in some cases, lethal, the prenatal diagnosis of an affected infant can allow treatment immediately after birth and before the first bleeding manifestation. However, the issue of prenatal diagnosis in rare bleeding disorders is still under debate [82], especially considering that, in most cases, it is performed using invasive procedures (such as withdrawal of chorionic villi) that can have dramatic consequences on the fetus. The first prenatal diagnosis for afibrinogenemia was done for a Palestinian family with two affected daughters by Neerman-Arbez et al. in 2003 [83]. In babies of known/suspected carrier couples, cord blood detection of genetic mutations can be done. Indirect prenatal testing by the use of linkage analyses might be an option in rare inherited bleeding disorders, too [82].

Substitution therapy is effective in the treatment of bleeding episodes in congenital fibrinogen disorders [55,75]. If possible, specific plasma-derived factor concentrate deprived of active viruses ought to be administered preferentially in rare bleeding diseases. Fresh frozen plasma (FFP) or cryoprecipitate should be administered in the unavailability of plasma-derived factor concentrate [3]. FFP has several disadvantages: the decreased amount of fibrinogen that is infused leads to the need to repeat the administration more times to achieve the appropriate fibrinogen level; in addition, there are transfusion-related risks (e.g., transfusion-related acute lung injury (TRALI) and transmission of virus infections) [75,76]. In addition, cryoprecipitate has many of these handicaps; it also needs compatibility testing, thawing, and it has a complicated application [76]. It is not provided in many Western European countries, but it is still administered in the United States and the United Kingdom [66]. A standard dose of 10–20 units (500–1000 mL) of methylene blue-cryoprecipitate is awaited to raise fibrinogen activity by 0.6–1.2 g/L in a 70 kg adult [84,85]. It may continue daily or every other day in the absence of consumption with frequent monitoring of the activity, according to the indication and reaction [76].

To date, we know six fibrinogen concentrates: RiaSTAP /Haemocomplettan^® P (CSL Behring, Marburg, Germany), Fibryga^® (Octapharma, Lachen, Switzerland), FibCLOT^®/CLOTTAFACT^® (LFB, Les Ullis, France), Fibrinogen HT^® (Benesis, Osaka, Japan), FibroRAAS^® (Shangai RAAS, Shangai, China), and FIB Grifols^® (Grifols, Barcelona, Spain) [2,10,37,69,86,87].

Fibrinogen replacement treatment, especially the most frequently administered concentrate Haemocomplettan P/Riastap, is suggested as therapy for spontaneous bleeding events and as prophylaxis before surgical interventions or against unprovoked bleeds in individuals with congenital and acquired fibrinogen deficiency [56,62].

Safety (less frequent allergic reactions), accuracy, simple dosing in small amounts, and rapidity of administration are the primary reasons for the paramount use of fibrinogen concentrates [62,87]. On the other hand, venous or arterial thrombotic events were present in 30% of subjects with fibrinogen deficiency treated by fibrinogen concentrates, mostly in afibrinogenemics [3,63]. Moreover, the potential risk of prion-transmission or venous access complications are other side effects of their usage [2]. As the pharmacokinetic properties of fibrinogen after substitution show a large among-patients variability, tailoring of the prophylactic regimen to the pharmacokinetics of the individuals can be a possibility [87]. This is the reason why the individualized management is considered to be “a job of mastery".

According to the pharmacokinetics of fibrinogen described above, a standard dosage of fibrinogen concentrate of 4–6 g should raise plasma fibrinogen activity by 1.0–1.5 g/L in a 70 kg adult [3,63]. For a better calculation of the dosage, the following formula may be helpful: dose (g) = awaited increase in g/L x plasma volume. Plasma volume calculate: 0.07 × (1 − hematocrit) × weight (kg) [55]. Moreover, the fibrinogen dose using the FIBTEM assay can be calculated as follows: Fibrinogen concentrate dose (g) = (target FIBTEM MCF (mm) − actual FIBTEM MCF (mm)) × (body weight (kg)/70) × 0.5 g/mm [68].

For unprovoked hemorrhage, suggested fibrinogen concentrations are >1 g/L until hemostasis is normalized and >0.5 g/L until the bleeding surface is entirely restored [55]. Fibrinogen concentrate of 50–100 mg/kg every 2–4 days with resultant fibrinogen activity >1.0–1.5 g/L was normally needed to treat or prevent spontaneous or surgical hemorrhage [37,63]. Therefore, for these situations in afibrinogenemia, hypofibrinogenemia, or hemorrhagic dysfibrinogenemia, the United Kingdom Hemophilia Centre Doctors’ Organization guidelines suggest assessment of fibrinogen concentrate in the dose 50–100 mg/kg, with smaller doses repeated if needed at 2–4 day intervals to maintain fibrinogen activity >1.0 g/L [3].

Antifibrinolytics can be helpful in the cases of mucosal bleeding, but they ought to be used very carefully in subjects with a personal or family history of thrombotic episode. Estrogen/progesterone derivatives have been given in menorrhagia [3,76].

Moreover, in prevention, the trough fibrinogen level to be targeted is unknown because fibrinogen concentrate has been potentially linked to a risk of thrombosis.

For women with fibrinogen activity <0.5 g/L or with previous poor pregnancy outcomes, the prophylaxis during pregnancy with fibrinogen concentrate at firstly 50–100 mg/kg twice per week, tailored to retain trough fibrinogen activity >1 g/L is suggested [3,76].