Thrombosis Research 100 (2000) 127–131

ORIGINAL ARTICLE
Prevalence of Three Prothrombotic
Polymorphisms: Factor V G1691A, Factor II
G20210A and Methylenetetrahydrofolate
Reductase (MTHFR) C 677T in Argentina
Valeria Genoud1, Mercedes Castañon1, Joyce Annichino-Bizzacchi2, Jorge Korin1
and Lucı́a Kordich1 on behalf of the Grupo Cooperativo Argentino de Hemostasia y Trombosis
1
Facultad de Ciencias Exactas y Naturales. Dpto. Quı́mica Biológica,
Laboratorio de Hemostasia y Trombosis, Universidad de Buenos Aires,
and 2Hematology-Hemotherapy Center, State University of Campinas, Campinas SP, Brazil.

(Received 13 October 1999 by Editor J. Aznar; revised/accepted 30 May 2000)

Key Words: Thrombophilia; Factor V Leiden; Prothrom- the presence of antiphospholipid antibodies, preg-
bin 20210; MTHFR; Prevalence nancy or oral contraceptives (OC) may induce
APCR [3]; folic acid deficiency or renal insuffi-
ciency can increase homocysteine levels; and OC,

E
ach year, venous thromboembolism (VTE)
affects 50–150/100,000 individuals in the
global population [1]. Acquired (surgery,
pregnancy, contraceptives, cancer, antiphospho-
lipid antibodies, immobilization, etc.), as well as
hereditary, factors interact to promote the develop-
ment of deep vein thrombosis and pulmonary em-
bolism. Hereditary factors that induce VTE devel-
opment can be classified as classical (e.g., ATIII,
Protein C and Protein S deficiencies or dysfunc-
tions) and polymorphisms. In recent years, Factor
V Leiden, MTHFR CT 677, and Prothrombin
20,210 mutations [2] were found to be responsible
for producing Activated Protein C Resistance
(APC R), hyperhomocysteinemia and increasing
levels of Factor II, respectively. Some acquired
conditions may have similar effects. For instance,
Abbreviations: MTHFR, methylenetetrahydrofolate reductase;
VTE, venous thromboembolism; AT III, Antithrombine III; APC
R, Activated Protein C Resistance; OC, oral contraceptives.
Corresponding author: Lucı́a Kordich, Facultad de Ciencias Ex-
actas y Naturales, UBA, Ciudad Universitaria, Pabellón II, 4⬚
Piso, Dpto. Quı́mica Biológica, Laboratorio de Hemostasia y
Trombosis. Tel: ⫹54 (11) 4576 3300 ext. 209; Fax: ⫹54 (11) 4576
3342; E-mail: ⬍lht@qb.fcen.uba.ar⬎.
and presumably other yet unknown mutations,
might increase plasmatic levels of prothrombin.
Thus, genetic analyses are the better tool to detect
hereditary thrombophilic cases in general popula-
tion studies.
Factor V Leiden, which consists of a point muta-
tion in factor V gene (nucleotide 1691 G to A
substitution), had so far emerged as the most fre-
quent genetic condition associated with VTE, with
a frequency of 18% in clinical series of VTE (11.5–
37.0%) [4–8]. Heterozygous individuals have a
5–10 times higher risk of VTE during their lives
compared to wild type population; this risk in-
creases 10 times in homozygous subjects compared
to heterozygous.
A G→A variant in Factor II (Prothrombin
20,210) has been associated with high plasma levels
of this coagulation factor, and with a higher risk of
venous and arterial thrombosis. This characteristic
has been reported in 18% of VTE patients with a
positive familial clinical history of the disease and
in 6.2% of consecutive patients with a first deep
vein thrombosis. The risk of VTE attributed to the
mutation is 2.8 times higher when compared to the
wild type [9–11].

0049-3848/00 $–see front matter  2000 Elsevier Science Ltd. All rights reserved.
PII S0049-3848(00)00314-5
128 V. Genoud et al./Thrombosis Research 100 (2000) 127–131
Since mutation of methylenetetrahydrofolate re-
ductase at nucleotide 677 (C to T) results in a
thermolabile variant with reduced enzymatic activ-
ity, homocysteine levels may increase, particularly
in conjunction with low plasma folate levels (⬍15.4
nmol/L). Although generally considered a mild risk
(2.5 times) for VTE, there are still some controver-
sial data about its contribution as an isolated risk
factor or as an element that enhances other throm-
bogenic conditions [12–15].
A previous study carried out in Argentina
showed 1.6% prevalence of Factor V Leiden [16],
but this survey included data mainly from Buenos
Aires, the capital city. Thus, it does not actually
represent the whole country, which might show
many ethnic differences. We are not aware of any
previous studies carried out in our country on the
prevalence of Prothrombin 20,210 and MTHFR.
1. Materials and Methods
1.1. Patients
Samples from 418 healthy unrelated blood donors
(21–65 years old) from all the different regions of
our country, and devoid of previous hemorrhagic
or thrombotic disorders, were analyzed. The num-
ber of samples from each place was calculated pro-
portionally to the population of the region, with
slight overrepresentation of the Patagonian and
northern provinces in order to study a relative large
proportion of Amerindians.
1.2. DNA Extraction and Mutations Detection
DNA extraction was performed as originally de-
scribed [17] from an entire blood sample kept at
–20⬚C. To identify Factor V Leiden, a 267-bp DNA
fragment of factor V gene that included nucleotide
1691 was amplified by PCR technique and digested
with Mnl 1 as previously described [18]. To identify
G→A mutation of the prothrombin gene, a 345-
bp fragment was obtained and then digested using
Hind III endonuclease as reported [9]. Screening
for the MTHFR C→T677 substitution was per-
formed by amplification of a 198-bp DNA fragment
and followed by Hinf I digestion, as originally de-
scribed [19].
1.3. Statistical Analysis
Differences in prevalence between provinces were
tested for heterogeneity by the chi-square test.
2. Results
From a total of 418 samples taken throughout the
country, heterozygosity for Factor V Leiden was
detected in 12 (2.9%), and heterozygosity for Pro-
thrombin 20,210 in 11 (2.6%). No homozygous in-
dividuals were found for these two mutations. In
the case of MTHFR, on the other hand, 65 subjects
were mutant homozygous (15.8%) and 180 were
heterozygous (42.8%) (Table 1). No distribution
differences were observed in the provinces sam-
pled, perhaps due to the low prevalence found for
Factor V Leiden and Prothrombin 20,210 (p⫽0.54
for both).
Volunteers were asked about the nationality of
their ancestors, especially their grandparents, in
order to establish the main source of migration to
the country. However, no relationship between this
data and Factor V Leiden, Prothrombin 20,210 mu-
tation or MTHFR T 677 was found (data not shown).
This study also gave us the opportunity to detect
asymptomatic combined mutations in the general
population. Thus, three cases of Factor V Leiden
with MTHFR T 677, and two cases of Prothrombin
20,210 with MTHFR T 677 were observed. These
results represented 1.2% of the general population.
No double heterozygosity for Factor V Leiden and
Prothrombin 20,210 was discovered.
3. Discussion
Considerable variations of the prevalence of Factor
V Leiden have been found in different surveys
carried out in many countries throughout the
world. In Argentina, there is virtually no black
population, with a Caucasian migration that came
mainly from Spain and Italy and secondarily from
Northern and Eastern Europe. A small number of
Asian immigrants arrived during this century. As
a result of this distribution, we expected to find
similar values of Factor V prevalence to those re-
ported for Southern Europe [20–21] (Italy: 2.5%
 V. Genoud et al./Thrombosis Research 100 (2000) 127–131 129

Table 1. Distribution of three gene alterations in the Argentine population

Factor V Prothrombin MTHFR
G1691A G20210A C677T
Province/City G/G G/A A/A G/G G/A A/A C/C C/T T/T

Buenos Aires (n⫽47) 45 2 – 45 2 – 19 26 2

Capital Federal (n⫽72) 70 2 – 72 – – 24 36 12
Córdoba (n⫽40) 39 1 – 38 2 – 21 14 5
Corrientes (n⫽21) 20 1 – 21 – – 11 4 6
Chaco (n⫽20) 19 1 – 20 – – 5 13 2
Chubut (n⫽34) 33 1 – 32 2 – 13 11 10
Formosa (n⫽19) 18 1 – 19 – – 11 6 2
Mendoza (n⫽47) 45 2 – 47 – – 16 24 7
Misiones (n⫽20) 20 – – 20 – – 9 9 2
San Juan (n⫽20) 20 – – 19 1 – 9 7 4
Santa Fé (n⫽38) 38 – – 35 3 – 14 15 9
Tierra del Fuego (n⫽20) 19 1 – 20 – – 7 10 3
Tucumán (n⫽20) 20 – – 19 1 – 14 5 1
Totals (n⫽418) 406 12 – 407 11 – 173 180 65

of 4676 and Spain 3.7% of 132), but with some 4. Summary

possible variations between provinces. However,
this has not been observed in our survey.
In Spain, Prothrombin 20,210 is reported to be
the most prevalent genetic risk factor for VTE,
with 6.5% of carriers in the general population.
Factor V Leiden prevalence in Argentina (2.9%)
is close to the value expected, considering that our
main sources of immigration were Caucasians from
Southern Europe. Prothrombin 20,210 prevalence
(2.6%) is quite comparable to Factor V Leiden.
Homozygous MTHFR T 677 was found in a rela-
tively high percentage of the Argentine population
(15.8%). Even higher values have been reported
in Colombia, another South American country
(25.3%) [22]. Regional variations of the genetic
background should be considered in order to ob-
tain useful information about the relative weight
of hereditary and environmental factors for VTE
in each country. This could be useful for making
public health decisions such as folic acid intake
recommendations.
The detection of a joint concurrence of two pro-
thrombotic risk factors in 1.2% of the asymptom-
atic population agrees with reports on control
groups of cohorts of patients with VTE (0.9%)
[23], emphasizing the importance of acquired as
well as other yet unknown genetic factors for the
development of VTE.
Screening in selected but still wide populations
such as women with recurrent fetal loss, placental
insufficiency, pre-eclampsia, or needing oral con-
traception, or programmed high-risk surgery in
both sexes, is still controversial. Knowledge of the
prevalence of these polymorphisms in the general
population is useful in order to take decisions that
could considerably increase the burden to health
resources, even in developed countries.
The authors are very grateful to Labs Rhone Poulenc Argentina
and Otsuka Argentina for their assistance with sample handling.
We are indebted to Mrs. Ch. Fukuoka for her cooperation with our
Group, and to Eliane Baldinelli and Marisa Farber for their advice.
We wish to thank the Members of the Grupo Cooperativo de
Hemostasia y Thrombosis who have been involved in the sending
of the samples: Capital Federal: Dr Alicia Rovó; Buenos Aires: Dr
Osvaldo Gioseffi; Córdoba: Dr Susana Villafañe and Dr Luis Del
Val; Santa Fe: Dr Silvia Mori and Dr Daniel Bar; Mendoza: Dr
Diana Marcipar; Corrientes: Dr Emilio Lanari Zubiaur; Chaco:
Dr Nancy Pujal; Chubut: Dr Marı́a del Carmen Rı́os Part and Dr
Oscar Gómez; Formosa: Dr Victoria Welch; Misiones: Dr Sandra
De Haro; San Juan: Dr Mercedes Gómez de Herrera; Tierra del
Fuego: Dr Alejandro Dates; Tucumán: Dr Hugo Medici.
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