Coagulation Characterization of Prothrombin 20209C > T Variant: About 27 New Cases

Prothrombin (F2) 20210G > A gene polymorphism is a well-established moderate risk factor of first venous thromboembolism (VTE) event.[1] [2] [3] It enhances messenger ribonucleic acid (mRNA) processing, leading to elevated prothrombin levels.[4] Beside, many rare substitutions have been reported. They are mostly mutually exclusive of 20210G > A.[5] [6] [7] [8] [9] Given their low allelic frequency, their impact on coagulation has been poorly investigated, and their association with VTE is still a matter of debate. Here, we report 27 cases of F2 20209C > T variant located within the 3′ untranslated region of F2 gene and assess their coagulation profile using prothrombin activity measurement and thrombin generation assay (TGA).

Our retrospective descriptive study was conducted in accordance with the ethical principles of the Declaration of Helsinki. Patients consented for genetic studies. From January 2001 to April 2020, 42,500 patients referred to the outpatient clinics at Cochin and Hotel-Dieu university hospitals (AP-HP, Centre-Université de Paris, Paris, France) for thrombophilia screening following personal or familial VTE history. Thrombophilia screening tests were performed on STAR Evolution analyzer (Stago) and included prothrombin activity (Siemens), prothrombin time, activated partial thromboplastin time, antithrombin activity, and proteins C (PC) and S (PS) anticoagulant activities, all from Stago. Dilute Russell viper venom time (Siemens) was performed as part of lupus anticoagulant screening. Anticardiolipin (Theradiag) and anti-β2-glycoprotein antibodies (Werfen) levels were measured using enzyme-linked immunosorbent assay. F2 20210G > A and F5 Leiden (506R > Q) determination used commercial kits (Roche Diagnostics) on LightCycler. Detection of F2 20210G > A polymorphism was based on the difference of melting temperatures (Tm) between 20210G (mean ± standard deviation, 59 ± 2.5°C) and 20210A (48 ± 2.5°C) alleles. Based on the presence of abnormal peak with a Tm between 51 and 54°C, atypical variants were identified by Sanger sequencing of F2 gene from exons 13 to 14.[10] TGA was performed on calibrated automated thrombogram (CAT) system[11] using microplate fluorometer (Fluoroskan Ascent) and Thrombinoscope software (Thrombinoscope BV, version 5.0.0.742). It was triggered with 5 pM tissue factor and 4 μM phospholipid vesicles (Stago). Parameters recorded were peak height (PH) and endogenous thrombin potential (ETP). Samples were run in duplicate and averages were analyzed. Intra-assay coefficient of variation values were 3.1% for PH and 2.9% for ETP. Additional details are provided in [Supplementary Material] (available in the online version).

F2 20210G > A screening resulted in abnormal melting curves in 27 out of the 42,500 subjects. Sanger sequencing identified heterozygous C > T substitution at nucleotide (nt) 20209, with an allelic frequency of 0.06%, consistent with previous report.[12] Demographic, clinical, and laboratory characteristics of F2 20209C > T subjects are detailed in [Table 1]. Median age was 42 years (interquartile range [IQR]: 36–53) and male-to-female ratio was 4/23. Twelve subjects were Caucasian, 5 were African, and 10 were Afro-Caribbean. Neither geographical nor ethnic distribution of F2 20209C > T variant has been established with certainty, some case–control studies suggesting higher prevalence in black populations.[12] [13] [14] [15] [16] However, most of them included African-Americans or sub-Saharan African subjects. Here, we included a significant proportion of Caucasian subjects. No restriction to a country of origin or to a particular ethnic group was noticed.

Table 1 Detailed characteristics of 27 F2 20209C > T subjects

No.	Sex	Age	Ethnicity	1st degree familial VTE	Personal VTE	Age of 1st VTE	Clinical risk factors at 1st VTE	Site of thrombosis	Recurrent TE	Other laboratory abnormalities	Previous OCC
1	F	23	African	No	Yes	13	Immobilization	Proximal VTE			No
2	F	27	African	No	Yes	17		Cerebrovascular TE		PC deficiency	No
3[a]	F	36	Afro-Caribbean	No	Yes	24	Pregnancy interruption	Distal VTE			Yes
4	F	52	Afro-Caribbean	No	Yes	26	Postpartum	Distal VTE	Distal VTE		Yes
5	F	30	African	Yes	Yes	28	Postpartum	Proximal VTE + PE			No
6	F	50	Caucasian	Yes	Yes	35	OCC	Distal VTE	Distal VTE + PE		Yes
7	F	43	Caucasian	No	Yes	37	Cushing syndrome	PE	Proximal VTE		No
8	F	39	Afro-Caribbean	No	Yes	37	OCC	Proximal VTE		PC deficiency	Yes
9	F	46	Afro-Caribbean	No	Yes	38	Postpartum	Superficial TE			Yes
10	M	37	Caucasian	Yes	Yes	39		Proximal VTE	Proximal VTE + PE		–
11	M	50	Caucasian	Yes	Yes	45		PE		F5 Leiden +/−	–
12[b]	F	54	Caucasian	Yes	Yes	46	Trauma + OCC	Distal VTE			Yes
13	F	69	Afro-Caribbean	No	Yes	50	Postsurgery + OCC	Proximal VTE + PE		PS deficiency	Yes
14	M	56	Caucasian	No	Yes	57	Postsurgery	Proximal VTE + PE			–
15	F	72	Afro-Caribbean	No	Yes	66	Postsurgery	Distal VTE	Distal VTE		No
16	F	20	Afro-Caribbean	Yes	No					PC deficiency	Yes
17[a]	F	28	Afro-Caribbean	Yes	No						No
18	F	31	Afro-Caribbean	Yes	No						No
19	F	35	African	No	No						No
20	F	38	Caucasian	Yes	No						Yes
21	F	41	Afro-Caribbean	Yes	No						Yes
22[c]	F	42	Caucasian[d]	Yes	No					F5 Leiden +/−	Yes
23[b]	F	42	Caucasian	Yes	No					F5 Leiden +/−	Yes
24	F	42	Caucasian[d]	Yes	No					PC deficiency	No
25[c]	F	44	Caucasian[d]	Yes	No					F5 Leiden +/−	No
26[c]	F	71	Caucasian[d]	Yes	No						Yes
27	M	75	African	Yes	No						–

Abbreviations: +/− , heterozygous; F5, factor V; OCC, oral combined contraception; PC, protein C; PE, pulmonary embolism; PS, protein S; VTE, venous thromboembolic event.

^a Sisters.

^b Aunt and niece.

^c Mother and daughters.

^d Jewish-Moroccan patients.

Thrombophilia screening revealed PC deficiency, PS deficiency, and heterozygous F5 Leiden in 4, 1, and 4 subjects, respectively ([Table 1]). No other laboratory abnormality was found in the F2 20209C > T cohort. F2 20209C > T variant was previously described in patients suffering from obstetrical complications and/or venous thrombosis but the causality and the mechanism were not firmly established.[12] [13] [14] [15] [16] [17] [18] [19] Here, 15 out of 27 subjects had previous VTE ([Table 1]), 5 of which suffered from recurrent events. VTE were documented clinically as well as by medical imaging. Median age at the time of first VTE was 37 years (IQR: 27–48 years). Interestingly, 14 out of the 15 subjects exhibited either clinical or laboratory well-established risk factors of thrombosis. A well-designed linkage analysis is required to adjudicate on the presence or absence of any association between F2 20209C > T and VTE occurrence but it would require hundreds of patients and controls, which is not foreseeable due to this variant rarity.

Prothrombin activity was measured in 25 subjects, the two remaining patients being under vitamin K antagonists. Despite a slight tendency toward an increased prothrombin activity levels, all values were within the normal range. They did not differ between F2 20209C > T subjects and 34 age- and sex-matched control individuals with homozygous wild-type F2 gene (106% ± 12 vs. 99% ± 14, respectively, p = 0.085; [Fig. 1A]). Excluding siblings from propositi data showed similar results. Previous in vitro gene expression experiments showed contradictory results. While thymine introduction at nt 20209 decreased F2 gene expression due to reduced efficiency of RNA polyadenylation,[20] others reported an upregulation of mRNA and consequently of prothrombin level.[21] Unlike in F2 20209C > T subjects, prothrombin activity levels (127% ± 20) were significantly increased (p < 0.0001) in 25 age- and sex-matched heterozygous F2 20210G > A subjects in comparison to controls ([Fig. 1A]).

Fig. 1 Prothombin activity levels and thrombin generation assay (TGA) parameters in F2 20209C > T individuals. Prothrombin activity level (A), peak height (B), and endogenous thrombin potential (C) were pairwise compared between F2 20209C > T, controls, and F2 20210G > A age- and sex-matched subjects using t-test. Dashed lines correspond to the normal range of prothrombin levels (70–140%). Unlike F2 20210G > A, F2 20209C > T was not associated with increased levels of prothrombin activity or high TGA parameters.

To refine the characterization of the coagulation profile of F2 20209C > T variant, we hypothesized of the added value of TGA that could be performed in 17/27 samples for which sufficient plasma volume was available. TGA parameters were 315 nM ± 87 and 325 nM ± 107 for PH, and 1,772 nM.min ± 434 and 1,679 nM.min ± 388 for ETP in F2 20209C > T and controls samples, respectively. Neither PH (p = 0.730) nor ETP (p = 0.440) significantly differed, contrary to what was observed with F2 20210G > A samples: PH (385 nM ± 85) and ETP (2278 nM.min ± 542), which were increased compared with F2 20209C > T (p = 0.013 and p = 0.003) and controls (p = 0.025 and p < 0.0001) samples, respectively ([Fig. 1B] and [C]). As for prothrombin activity, excluding siblings from propositi data showed similar results. Differences between F2 20210G > A and F2 20209C > T or controls samples were more evident considering ETP than PH. Of note, TGA was performed in the absence of thrombomodulin to evaluate the presence of any procoagulant state independently of the presence of F5 Leiden or PC and PS deficiencies.

In conclusion, we describe the largest cohort of individuals with rare F2 20209C > T variant reported so far. Coagulation study revealed the absence of association of this variant with a hypercoagulant profile.

Authors' Contributions

G.J., M.F., C.F., and O.K. designed the study. G.J., S.L., O.K., and C.F. performed experiments and analyzed data. J.C., E.M., M.F., and M.H.H. recorded patients. G.J. and M.F. wrote the manuscript. M.F. supervised the study. S.L., O.K., C.F., J.C., E.M., E.A., M.M.S., M.H.H., I.G.T., and J.D. critically read the manuscript and gave final approval.

Publication History

Received: 08 June 2020

Accepted: 05 August 2020

Article published online:
15 September 2020

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