Thromb Haemost
DOI: 10.1055/s-0040-1716541
Letter to the Editor

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

1  AP-HP Centre Université de Paris, Hôpital Cochin, Paris, France
2  Innovative Therapies in Haemostasis, INSERM U1140, Université de Paris, Paris, France
,
Sophie Lobies
1  AP-HP Centre Université de Paris, Hôpital Cochin, Paris, France
,
Olivier Kosmider
1  AP-HP Centre Université de Paris, Hôpital Cochin, Paris, France
3  Institut Cochin, CNRS UMR8104, INSERM U1016, Université de Paris, Paris, France
,
Jérôme Duchemin
1  AP-HP Centre Université de Paris, Hôpital Cochin, Paris, France
,
Etienne Audureau
4  Unité fonctionnelle d'épidémiologie et de biostatistiques, AP-HP, Centre-Université de Paris, Paris, France
5  Institut Cochin, Université de Paris, Paris, France
,
Jacqueline Conard
1  AP-HP Centre Université de Paris, Hôpital Cochin, Paris, France
,
Elisabeth Mazoyer
1  AP-HP Centre Université de Paris, Hôpital Cochin, Paris, France
,
Marie-Hélène Horellou
1  AP-HP Centre Université de Paris, Hôpital Cochin, Paris, France
,
Isabelle Gouin-Thibault
6  Laboratoire d'hématologie, CHU Pontchaillou, Rennes, France
7  Université de Rennes 1, CIC-INSERM 1414, Rennes, France
,
Claire Flaujac
8  Laboratoire d'Hématologie, Centre Hospitalier de Versailles, Le Chesnay, France
,
Michaëla Fontenay
1  AP-HP Centre Université de Paris, Hôpital Cochin, Paris, France
3  Institut Cochin, CNRS UMR8104, INSERM U1016, Université de Paris, Paris, France
› Author Affiliations

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]).

Zoom Image
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.


Supplementary Material



Publication History

Received: 08 June 2020

Accepted: 05 August 2020

Publication Date:
15 September 2020 (online)

Georg Thieme Verlag KG
Stuttgart · New York