Coagulation and Clinical Features Associated with the Arg304gln Mutation (Factor VII Padua) in the Irf7 Study Group Response to Letter to the Editor Regarding “Factor VII Deficiency (Semin Thromb Hemost 2009;35(4):400–406)”

The comments by Dr. Girolami and collegues[1] on our article[2] are interesting and regard a very well-known mutation of the factor VII (FVII) gene, the Arg304Gln mutation (R304Q), first described as a clotting phenotype by Girolami et al (identified with the toponym “FVII Padua”),[3] and subsequently characterized at the genotype level by our group and others.[4] [5] To clarify Dr. Girolami’s comments on the variation of FVII levels, it is essential to consider the source of our data (Table [1]),[2] which are not our own observations but stem from the International FVII Registry (515 cases retrospectively reported from 61 centers worldwide).[6] This database is full of information that we believe is of interest for the readers of Seminar of Thrombosis and Hemostasis, given the features and the rarity of this specific clotting defect.

Table 1 Distribution of Patients Carrying the FVII 304 RQ Mutation for Gender, Zygosity, and Symptomaticity Zygosity Age at Enrollment, Mean (Range) Male Female Total Symptomatic/Asymptomatic Symptomatic/Asymptomatic Symptomatic/ Asymptomatic Heterozygotes 44.1 (37–50) 0/3 0/3 0/6 Homozygotes 44.6 (33–64) 0/3 4/4 4/7 Compound heterozygotes 28.4 (5–74) 1/2 2/2 3/4 Total 40 (5–74) 1/8 6/9 7/17

FVII Padua mutation is recurrent in different populations because it is caused by the ¹⁰⁸²⁸g/a transition in a CpG site. To help clarify Dr. Girolami’s comments[1] we have retrospectively analyzed data pertaining to 11 homozygotes or compound heterozygotes (6 females, 5 males) and 6 heterozygotes (3 for each gender) for the R304Q mutation. These patients were observed in five centers in Europe and South America. FVII levels reported in these subjects showed an ample variation that was clearly related to the thromboplastin used (Fig. [1A]). Unfortunately, hog-brain thromboplastin was not used (or at least not reported) in any of these cases; therefore data on this relationship are unavailable. Nevertheless, in line with the comments of Girolami et al,[1] the highest FVII coagulant (FVII:C) values were obtained when human tissue factor was employed for the specific assay, and conversely, the lowest procoagulant activity was observed when rabbit tissue extracts were used. Also worth noting is that, even using human tissue factor, we observed an ample variation in FVII:C levels among individuals with the R304Q mutation, which may be ascribed to the presence of other elements, including biological and genetic components influencing FVII expression and laboratory confounding effects. However, the key issue is the same as noted by Girolami et al[1] (i.e., differences in FVII assay outcome when tissue extracts from different species are used).

Figure 1 Relationship between carriership of the 304RQ mutation and plasma Factor (F)VII levels. (A) Distribution of FVII activity levels in (left) heterozygotes and in (right) homozygotes and compound heterozygotes (circled). The thromboplastin preparation used in the assays is reported. Note: Thromborel, human placenta; Roche, rabbit brain; Neoplastin, rabbit brain. (B) Relationship between FVII antigen and FVII activity in homozygotes (empty circles), compound heterozygotes (gray squares), and heterozygotes (black triangles).

To further clarify the clotting phenotype in this condition, we analyzed the relationship between the clotting activities (FVII:C) and FVII antigen levels (FVII:Ag) (Fig. [1B]), which indicates, with the limitation of the thromboplastin used and of the different enzyme-linked immunosorbent assays, a good correlation in the homozygotes (r = 0.93) and compound heterozygotes (r = 0.77). When both categories are collapsed, the correlation is excellent (r = 0.95). In the heterozygous condition, however, the presence of the wild-type allele causes a lack of relationship (r = 0.07) between the two parameters, as expected from an admixture of FVII molecules with different specific activities, including the FVII-304Q variant (a dysfunctional molecule lowering the activity/antigen ratio to ~0.25).

All the heterozygotes were asymptomatic, whereas 4 of 7 homozygotes and 3 of four compound heterozygotes were symptomatic displaying epistaxis, postoperative bleeding, and menorrhagia as the most frequent symptoms (~50% of the cases), and with easy bruising and gum bleeding representing the other, less frequent bleeds (~25% of the cases). In no cases were severe hemorrhagic symptoms (central nervous system, gastrointestinal, or hemarthroses) reported, a fact that confirms this variant is a mild one, as supported by the median age of diagnosis (48 years in symptomatic individuals).

The take-home message from our data is that these subjects have an apparently variable coagulation phenotype, heavily modulated by the thromboplastins and laboratory protocols used, and a mild clinical phenotype with a not negligible proportion of individuals diagnosed during family studies or occasionally. Taken together, the genetic (recurrent mutation) and clinical (mild to asymptomatic condition) features suggest that many more asymptomatic and undetected subjects homozygous for the R304Q mutation should be present around the world.

As concerns Dr. Girolami’s comments on the FVIIa assay,[1] one has to consider that FVIIa is the active form of FVII, and its evaluation is in principle important for the treatment monitoring of recombinant activated FVIIa (rFVIIa) in both FVII deficiency and hemophilia patients with inhibitor.[7] In fact, the laboratory response to infused rFVIIa is rather variable, and FVII:C assays, which are not specific for the infused factor, might contribute to this variability.[8] In addition, the FVIIa assay can be easily standardized on coagulometers as pointed out by several groups, including ours,[9] [10] in population-based studies.

Because the journal Seminars in Thrombosis and Hemostasis is intended both for clinical and laboratory experts, we thought these comments could be of some practical interest.

REFERENCES

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• 2 Mariani G, Bernardi F. Factor VII deficiency.  Semin Thromb Hemost. 2009;  35(4) 400-406
• 3 Girolami A, Fabris F, Dal Bo Zanon R, Ghiotto G, Burul A. Factor VII Padua: a congenital coagulation disorder due to an abnormal factor VII with a peculiar activation pattern.  J Lab Clin Med. 1978;  91(3) 387-395
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• 10 Mariani G, Conard J, Bernardi F et al.. Oral contraceptives highlight the genotype-specific association between serum phospholipids and activated factor VII.  Arterioscler Thromb Vasc Biol. 1999;  19(8) 2024-2028