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F. Bernardi and G. Mariani et al.
However, the therapeutic thresholds conferring protec- tion from traumatic or post-operative bleeds are still a matter of debate.44,45 Surgical bleeding has been reported in 15-24% of cases42,45 with FVIIc <7%.42 Bleeds in the first post-operative day46 are particularly frequent if the bleed- ing disorder was not diagnosed before, and are mostly reported during orthopedic procedures,46 a major hemo- static challenge. Presenting symptoms as post-circumci- sion bleeds and hemorrhage from the umbilical stump frequently herald a severe disease form.
Diagnosis
The spectrum of bleeding symptoms is wide, involves both males and females, can start after birth and may occur at any age. The most important predictors of bleed- ing risk, familial and personal histories, can be silent as observed in diseases with recessive inheritance. The rele- vance of the gynecological and obstetric histories are dis- cussed in a dedicated paragraph.
The laboratory workup47 performed after a bleeding episode or during a family screening includes routine screening assays (prothrombin time [PT], activated partial thromboplastin time [aPTT]), followed by FVIIc measure- ment, which is necessary to confirm the diagnosis. Evaluation of FVIIc is determined with a one-stage assay adding diluted plasma samples to FVII-deficient plasma and thromboplastin as the source of TF. The accuracy of FVIIc assays is related to the sensitivity of the thrombo- plastin reagent and to the quality of the FVII-deficient plasma and of the calibrators. The most suitable activa- tors are the human placenta-derived and the recombinant preparations,47 that are also recommended to monitor the treatment with rFVIIa.25 New chromogenic or fluorogenic assays for FVIIa are available for research purposes and to monitor treatment with rFVIIa.48
The sensitivity of the FVIIc assay to levels below 2% may be poor, with negative implications for the accurate definition of the relationship between FVII levels and bleeding phenotypes (Table 1), as well as between geno- type and phenotype. A low assay sensitivity may explain some inconsistencies such as the paradoxical observation of asymptomatic cases with low FVII levels. Furthermore, severe bleeding is sporadically observed in individuals with moderately reduced levels of FVII. The combination of the low assay sensitivity with the minimal amount of FVIIa-TF complex needed to trigger coagulation does not permit one to efficiently differentiate clinical phenotypes. As a consequence, in FVII deficiency a variety of bleeding phenotypes are observed in the presence of apparently modest differences in FVIIc levels (Table 1). In summary, the clotting tests do not always predict the degree of bleeding tendency in the single individual.
The presence of the FVII protein (FVII antigen [FVIIag]) can be determined by enzyme-linked immunosorbent assays or immune-turbidimetric assays, using monoclon- al epitope-specific antibodies.47 The FVIIag assay47 does not predict the bleeding tendency, but does allow one to distinguish between type I (quantitative defects, with decrease of both FVIIc and FVIIAg), and type II defects (qualitative defects, with low FVIIc and normal or reduced levels of FVIIAg), and may help to clarify the dif- ferent mutational mechanisms.
Molecular genetics
The key findings are summarized on a historical time
scale in Figure 1B, and the main findings in individuals with FVII deficiency are shown in Table 3. The function- al characterization of protein variants in plasma was car- ried out in seminal studies49 well before the genetic char- acterization of FVII deficiencies. The first F7 gene muta- tions in FVII deficiency were reported in the UK50 and Italian populations, and more than 1,000 genetic diag- noses in several countries are reported in the International Registry on FVII deficiency (IRF7)10 and the Greifswald Registry.11 Mutations and residual FVII levels have been presented in databases.51 The recent EHAD database12 (http://www.umd.be/F7/W_F7/index.html) includes 221 unique variants identified in 728 individu- als, of great help for the evaluation of F7 mutations found during genetic counseling and diagnosis, including prenatal diagnosis.5
F7 mutations have been detected by sequencing coding, splicing and in promoter regions in the vast majority of patients. High throughput genomic sequencing will help in the case no mutations are found.52 Cases with severe symptoms are virtually all homozygous or doubly het- erozygous for F7 mutations. Symptomatic mutations impairing vitamin K metabolism, and thus posttransla- tional modification of GLA residues, have been found to cause combined vitamin K-dependent clotting factor defi- ciency that includes an abnormal FVII biosynthesis and low FVIIc levels.
As reported in registries11 and databases,12 single nucleotide variants are the most common type of gene defect (around 90%), and include a large proportion (between two thirds and three quarters) of missense mutations.
Numerous missense mutations have been found in the homozygous condition, related to geographical, ethnic and mutational pattern features. Clusters of homozygotes often indicate genetic isolates or ethnic customs favoring consanguinity (see below).
Since they have been well presented in databases,12 we shall briefly comment on a few of them.
The severe Gln100(160)Arg53,54 and the moderate/mild Ala294(354)Val55,56 are prevalent in Northern Europe. The frameshift mutation in the last carboxyl terminal codons (Pro404(464)Hfs) is very prevalent in cis with the Ala294(354)Val missense change and causes a 28 residues- elongated FVII molecule. This moderate to severe and combined variant is frequent in Central Europe and espe- cially in Slovakia.10 The mild Gly331(391)Ser mutation is prevalent in the gulf of Naples, Italy, as well as in other countries.57 Other mild/asymptomatic changes, the Arg304(364)Gln50, Cys310(370)Phe/Ser and Thr359(419)Met substitutions, have been found in several populations, suggesting recurrent nucleotide changes at a CpG site. The frequent Arg304(364)Gln is also character- ized by discrepant FVII activity findings depending on the thromboplastin reagent.49
Splicing mutations are not rare,52 and the homozygous splicing mutation IVS7+5 g>a, relatively frequent in Italy, has been used for cellular58,59 and animal60 models of gene therapy.
F7 deletions are also not rare and together with duplica- tions, insertions and indel rearrangements represent about 10% of all genetic lesions.12 However, large and extended deletions have not been described in homozygous individ- uals. Differently from hemophilia A and B, individuals with complete FVII deficiency might die perinatally or
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haematologica | 2021; 106(2)