To the Editor:

Since the discovery of the factor (F) V Arg 506 to Gln mutation (FV:R506Q) as the most common inherited disorder associated to venous thrombophilia1-6 and its apparent cosegregation with other well-established inherited prothrombotic risk factors,7-12evidence is accumulating that the association of double or multiple hemostatic defects greatly increase the penetrance of thrombotic disease. This finding raises the question whether the novel sequence variation in the prothrombin gene (20210 G to A variant),13which has been identified as a common but probably mild risk factor for venous thromboembolism (VTE),13-16 may also cosegregate with the FV:R506Q mutation and contribute to the thrombotic tendency in subjects being affected by activated protein C (APC)-resistance.

Therefore, we read with interest the recent report by Alhenc-Gelas et al17 about the rare association between the prothrombin 20210 A allele and FV:Q506 in thrombophilic families. These investigators looked for an association of the two risk alleles in 288 subjects belonging to 26 families; 151 carried the FV:R506Q mutation and 66 had had thromboses. However, no probands or family members had the 20210 A allele. Thus they concluded that the prothrombin variant does not frequently contribute to thrombosis in individuals with the FV mutation. The question is this: Are the findings reported by Alhenc-Gelas et al17 affected by the high precentage of asymptomatic subjects studied or by the selection of patients, respectively? Furthermore, because no separated and detailed data about age or clinical setings were given for the FV:Q506carriers, their results are difficult to assess.

We report here different and intriguing data showing a highly prevalent coinheritance of the prothrombin variant 20210 GA as an additional prothrombotic risk allele among young symptomatic FV:Q506carriers.

After obtaining informed consent, FII genotyping was performed in 200 apparently healthy controls and unpreferentially in 200 carriers of FV:Q506, including 150 unrelated patients who had had an objectively confirmed VTE before 45 years of age. The FV genotype at nucleotide 1691 was determined by polymerase chain reaction (PCR) andMnl I restriction analysis of PCR-amplified genomic FV DNA fragments.2,3 Screening of the prothrombin variant due to a G to A transition at nucleotide 20210 of the FII gene was performed byHindIII cleavage of a 345-bp fragment amplified by PCR using a mutagenic primer as described previously.13 The 20210 A allele was found in 4 of 200 healthy subjects with a normal FV genotype (100 men and 100 women; age range, 18 to 47 years; median age, 26 years), corresponding to a prevalence of 2%, whereas among 50 asymptomatic heterozygous FV:Q506 carriers (22 men and 28 women; median age, 31 years; range, 24 to 64 years), the prothrombin variant was detected in 2 subjects (4%). Among 115 symptomatic subjects affected by the heterozygous FV:R506Q mutation (69 women and 46 men; median age at onset of VTE, 28 years; range, 18 to 45 years), 14 (12.2%) also had the FII 20210 A allele. In the presence of the 20210 A allele, the relative risk of juvenile VTE was additionally threefold increased in patients carrying the FV:R506Q mutation in a heterozygous form (95% confidence interval, 0.8 to 11.7), which itself was found to increase the risk of VTE approximately fourfold.18 Patients affected by double heterozygous defects presented with thrombosis at a slightly younger age (median age at onset of VTE, 27 years) as compared with patients suffering from either FII 20210 A (33 years) or FV:Q506 in a heterozygous (29 years) form. In the group of 35 symptomatic patients affected by homozygous FV:R506Q mutation (21 women and 14 men; median age at onset of VTE, 27 years; range, 18 to 33 years), a coexistence of the prothrombin 20210 AG genotype was detected in 5 subjects, corresponding to a prevalence of 14%.

Persons homozygous for the 20210 A allele were not found.

With respect to the coexistence of the prothrombin variant 20210 GA in carriers of the FV:R506Q mutation, the rate observed in the presented study of relatively young thrombophilic patients was clearly higher compared with the rare association published for other populations.14-17,19 However, when assuming the theory that a high proportion of combined inherited hemostatic abnormalities predispose for thrombophilia already at a young age, the significance of the uncommon coinheritance of both FV:Q506and prothrombin variant observed in previous studies is difficult to assess; either the age was not mentioned at all17 or the majority of patients investigated were over the age of 60 years,15 much older than our patient population.14,16 By contrast, Poort et al13reported that the prothrombin variant was identified in 18% of selected patients, segregated in 40% with the FV:R506Q mutation.13 Furthermore, the 20210 A allele possibly has a similar distinctive racial and/or geographical distribution, as has been described for the FV mutant.20 These observations need to be kept in mind for prediction of the risk of VTE emanating in different populations from either FV:R506Q or FII 20210 GA or their coinheritance.

In summary, the high frequency of additional carriership for FII 20210 GA found in young thrombophilic patients with the FV:R506 mutation indicates that the prothrombin 20210 A allele is an important additional risk factor for VTE and might contribute to the thromboembolic manifestations. A careful search for the prothrombin 20210 G to A variant should therefore be included in thrombophilia screening programs, particularly in young patients carrying other genetic defects predisposing for thrombosis. However, whether the coinheritance of FV:Q506 and FII 20210 GA is also associated with a higher recurrence rate of thrombotic events is one issue in an ongoing prospective study.

REFERENCES

1
Dahlbäck
B
Carlsson
M
Svensson
PJ
Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C.
Proc Natl Acad Sci USA
90
1993
1004
2
Bertina
RM
Koeleman
BPC
Koster
T
Rosendaal
FR
Dirven
RJ
de Ronde
H
van der Velden
PA
Reitsma
PH
Mutation in blood coagulation factor V associated with resistance to activated protein C.
Nature
369
1994
64
3
Greengard
JS
Sun
X
Xu
X
Fernandez
JA
Griffin
JH
Evatt
B
Activated protein C resistance caused by Arg506Gln mutation in factor Va.
Lancet
343
1994
1361
4
Lane
DA
Mannucci
PM
Bauer
KA
Bertina
RM
Bochkov
NP
Boulyjenkov
V
Chandy
M
Dahlbäck
B
Ginter
EK
Miletich
JP
Rosendaal
FR
Seligsohn
U
Inherited thrombophilia: Part 1.
Thromb Haemost
76
1996
651
5
Miletich
JP
Prescott
SM
White
R
Majerus
PW
Bovill
EG
Inherited predisposition to thrombosis.
Cell
72
1993
477
6
Koster
T
Rosendaal
FR
de Ronde
H
Briet
E
Vandenbroucke
JP
Bertina
RM
Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study.
Lancet
342
1993
1503
7
Seligsohn U, Zivelin A: Thrombophilia as a multigenic disorder: Thromb Haemost 78:297, 1997
8
van Boven
HA
Reitsma
PH
Rosendaal
FR
Bayston
TA
Chowdhury
V
Bauer
KA
Scharrer
I
Conard
J
Lane
DA
Factor V Leiden (FVR506Q) in families with inherited antithrombin deficiency.
Thromb Haemost
75
1996
417
9
Koelemann
BPC
Reitsma
PH
Allaart
CF
Bertina
RM
Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families.
Blood
84
1994
1031
10
Gandrille
SG
Greengrad
JS
Alhenc-Gelas
M
Juhan-Vague
I
Abgrall
JF
Jude
B
Griffin
JH
Aiach
M
The French Network on behalf of INSERM
Incidence of activated protein C resistance caused by the ARG 506 GLN mutation in factor V in 113 unrelated symptomatic protein C-deficient patients.
Blood
86
1995
219
11
Zöller
B
Berntsdotter
A
de Frutos
PG
Dahlbäck
B
Resistance to activated protein C as an additional genetic risk factor in hereditary deficiency of protein S.
Blood
85
1995
3518
12
Koelemann
BPC
van Rumpt
D
Hamulyak
K
Reitsma
PH
Bertina
RM
Factor V Leiden: An additional risk factor for thrombosis in protein S deficient families?
Thromb Haemost
74
1995
580
13
Poort
SR
Rosendaal
FR
Reitsma
PH
Bertina
RM
A common genetic variation in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis.
Blood
88
1996
3698
14
Cumming
AM
Keeney
S
Salden
A
Bhavnani
M
Shwe
KH
Hay
CRM
The prothrombin gene G20210A variant: Prevalence in a U.K. anticoagulant clinic population.
Br J Haematol
98
1997
353
15
Hillarp
A
Zöller
B
Svensson
PJ
Dahlbäck
B
The 20210 A allele of the prothrombin gene is a common risk factor among Swedish outpatients with verified deep venous thrombosis.
Thromb Haemost
78
1997
990
16
Brown
K
Luddington
R
Williamson
D
Baker
P
Baglin
T
Risk of venous thromboembolism associated with a G to A transition at position 20210 in the 3′-untranslated region of the prothrombin gene.
Br J Haematol
98
1997
907
17
Alhenc-Gelas
M
Le Cam-Duchez
V
Emmerich
J
Frebourg
T
Fiessinger
JN
Borg
JY
Aiach
M
The A20210 allele of the prothrombin gene is not frequently associated with the factor V Arg 506 to Gln mutation in thrombophilic patients.
Blood
90
1997
1711
18
Ehrenforth
S
Zwinge
B
Scharrer
I
High prevalence of factor V R506Q mutation in German thrombophilic and normal population.
Thromb Haemost
79
1998
384
19
Rosendaal
FR
Siscovic
DS
Schwartz
SM
Psaty
BM
Raghunathan
TE
Vos
HL
A common prothrombin variant (20210 G to A) increases the risk of myocardial infarction in young women.
Blood
90
1997
1747
20
Rees
DC
Cox
M
Clegg
JB
World distribution of factor V Leiden.
Lancet
346
1995
1133