J Thromb Thrombolysis (2008) 26:229–233
DOI 10.1007/s11239-007-0163-0
Prevalence of thrombotic risk factors among b-thalassemia
patients from Western Iran
Zohreh Rahimi Æ Mandana Ghaderi Æ
Ronald L. Nagel Æ Adriana Muniz
Published online: 4 November 2007
Ó Springer Science+Business Media, LLC 2007
Abstract Background There is evidence for increased risk
of thrombosis in patients with b-thalassemia intermedia and
b-thalassemia major. The present study investigated the
prevalence of thromboembolic risk factors of prothrombin
G20210A, factor V Leiden G1691A and methylentetrahy-
drofolate reductase (MTHFR) C677T, as well as the
hematological and clinical profiles in b-thalassemia major and
intermedia patients from Western Iran. Methods Patients
consisted of 158 b-thalassemia patients, 151 b-thalassemia
major and 7 b-thalassemia intermedia patients, including 82
males and 76 females aged 13.6 ± 6.3 years. The control
group were 180 healthy blood donors and school students,
consisting of 103 males and 77 females aged 16.8 ± 2.1.
Genotyping was done by PCR-RFLP using Mnl I, Hind III and
Hinf I for factor V Leiden and prothrombin G20210A and
MTHFR, respectively. Results The prevalence of prothrombin
G20210A variant in patients and healthy individuals were 1.3
Z. Rahimi (&)
Medical Biology Research Center, Medical School,
Kermanshah University of Medical Sciences, Daneshgah
Avenue, P.O. Box 67148-69914, Kermanshah, Iran
e-mail: zrahimi@kums.ac.ir
Z. Rahimi
Department of Biochemistry, Medical School,
Kermanshah University of Medical Sciences,
Kermanshah, Iran
M. Ghaderi
Department of Pediatrics, Children Hospital of Shahid
Fahmideh, Kermanshah University of Medical Sciences,
Kermanshah, Iran
R. L. Nagel
A. Muniz
Department of Medicine, Division of Hematology,
Department of Physiology and Biophysics, Albert Einstein
College of Medicine, Bronx, NY, USA
and 3.3%, respectively. Factor V Leiden G1691A was insig-
nificantly higher in b-thalassemia patients (prevalence 5.7%
and allele frequency 3.2%) compared to healthy individuals
(2.8%). This mutation was found in eight b-thalassemia major
(5.3%) and one b-thalassemia intermedia (14.3%) patients.
The prevalence of MTHFR C677T polymorphism was slightly
higher in patients (50%) compared to healthy individuals
(48.3%). Around 71% of b-thalassemia intermedia and 38.4%
of b-thalassemia major patients had undergone splenectomy.
In b-thalassemia major patients, 5.3% had insulin dependent
diabetes mellitus (IDDM) and 6.6% had HCV antibodies. All
patients with IDDM were splenectomized and in one of them
the prothrombin G20210A variant was found. Two patients, a
7-year-old boy with b-thalassemia intermedia receiving regu-
larly blood transfusion and a b-thalassemia major patient (a 22-
year-old splenectomized female), were found to be homozy-
gous for MTHFR 677TT and heterozygous for factor V Leiden
G1691A. Double heterozygosity for factor V Leiden G1691A
and MTHFR C677T and also homozygous factor V Leiden
1691AA were found in two b-thalassemia major patients. No
thromboembolic event has been recorded in the files of
patients. Conclusions The results of present study establish the
prevalence of biological risk factors of thrombosis in b-thal-
assemia patients from Western Iran. It seems that
thrombophilic mutations may not be associated with throm-
botic events in thalassemic patients, which needs to be
confirmed by the study of larger sample sizes.
Keywords Factor V Leiden
Prothrombin G20210A

MTHFR
b-Thalassemia
Iran
Introduction
The increased risk of thrombosis in patients with
b-thalassemia intermedia (TI) and b-thalassemia major
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(TM) has been reported [1]. The study of Eldor et al. [2]
demonstrated the existence of a chronic hypercoagulable
state in thalassemia, and that platelet activation and
enhanced thrombin generation already existed in early
childhood.
There are several reports related to the frequency of
thromboembolic events (TE) among thalassemic patients.
In three from Italy, an overall prevalence of TE was
reported to be 1.65 and 5.2% in thalassemic patients, with a
prevalence of 0.9, 1.1 and 3.3% for TM and 4 and 16.2%
for TI [3–5].
Biologic risk factors for thrombosis include splenec-
tomy, red cell phosphatidylserine exposure and platelet and
plasma coagulation factor abnormalities [5]. TE has fre-
quently reported in b-thalassemic patients in association
with known risk factors such as diabetes, complex cardio-
pulmonary abnormalities, hypothyroidism, liver function
anomalies, and postsplenectomy thrombocytosis [6].
In a few studies, no increased prevalence of thrombo-
philic mutations in TM and TI patients has been found [2,
7]. However, recurrent thromboembolism in a b-thalasse-
mia major patient double heterozygote for prothrombin
G20210A and factor V Leiden G1691A [8] was demon-
strated. Also, Brankovic-Sreckovic et al. [9] reported a boy
with thalassemia minor who presented arterial ischemic
stroke due to homozygosity for MTHFR C677T mutation.
b-thalassemia is the most common single gene disorder
in Iran, and more than 25,000 affected individuals have
been reported. High prevalence of b-thalassemia (around
10%) occurs in the north of Iran, close to the Caspian Sea,
and in the south close to the Persian Gulf. The prevalence
of b-thalassemia alleles in other parts of the country has
been estimated to be 4–8% [10]. Recently, in the Kurdish
population of Kermanshah, we established a prevalence of
1.6% for prothrombin G20210A mutation and 2.97% for
factor V G1691A mutation [11].
In order to find the prevalence of thromboembolic risk
factors and the occurrence of thrombotic events among
b-thalassemia major and intermedia patients, the presence
of prothrombin G20210A, factor V Leiden G1691A and
MTHFR C677T as well as hematological and clinical
profiles was studied in 151 b-thalassemia major and 7
b-thalassemia intermedia patients from Western Iran.
Materials and methods
Studied individuals consisted of 158 b-thalassemia
patients, 151 b-thalassemia major and 7 b-thalassemia
intermedia patients, including 82 males and 76 females
aged 13.6 ± 6.3 years, who referred to the Children Hos-
pital of Shahid Fahmideh in Kermanshah for blood
transfusion. The median age of adult patients was
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Z. Rahimi et al.
21.5 ± 3.4 years (range 18–34 years) and of children was
10.8 ± 4.4 years (range 1–17 years). All b-thalassemia
major patients received blood transfusion every 4 weeks,
while 57.1% of b-thalassemia intermedia patients received
blood transfusion regularly every 4 weeks, and three
patients (42.9%) received blood transfusion sporadically. A
total of 5 out of 7 b-thalassemia intermedia patients
(71.4%) and 58 out of 151 (38.4%) of the b-thalassemia
major patients were splenectomized. Most of the patients
had received desferrioxamine. The last blood transfusion
was undertaken at least 4 weeks prior to blood sampling
for the present study. The mean ± SD levels of hemoglo-
bin in b-thalassemia major and intermedia patients were
8.9 ± 1.1 and 9.3 ± 1.6 g/dl, respectively. The control
group were 180 healthy blood donors and school students,
consisting of 103 males and 77 females aged 16.8 ± 2.1.
DNA was extracted from the leukocyte fraction of the
EDTA-treated whole blood by using the phenol-chloroform
method [12]. Identification of factor V Leiden G1691A and
prothrombin G20210A was according to the standard
procedure as previously described [13].
For detection of MTHFR C677T, a 198-bp region in exon
4 was amplified using the forward primer of 50 TGA AGG
AGA AGG TGT CTG CGG GA 30 and the reverse primer of
50 AGG ACG GTG CGG TGA GAG TG 30 as described by
Frosst et al. [14]. Amplification was carried for 40 cycles at
94°C for 0.5 min, 62°C for 0.5 min, 72°C for 0.5 min, with a
final extension period of 5 min at 72°C. The 198-bp PCR
product (10–15 ll) was digested with 5 units of the restric-
tion enzyme Hinf I at 37°C overnight. The C to T substitution
in the 198 bp fragment creates a Hinf I recognition sequence
which digests the 198 bp fragment into 175 and 23 frag-
ments. The Hinf I treated PCR fragments were analyzed by
3% agarose gel electrophoresis.
Statistics
The allelic frequencies were calculated by the gene
counting method. The distribution of the genotype fre-
quencies in both groups did not deviate from the Hardy–
Weinberg expectation. The significance of the difference of
observed alleles and genotypes between the groups was
tested using the v2 analysis. Data on quantitative charac-
teristics are expressed as means ± standard deviations.
Levene’s test was used to compare the variances based on
the assumption that the variances of the two groups are not
different. According to the results obtained by the Levene’s
test, this test was followed by an independent t-test to
compare the mean value of platelet counts between patients
with or without splenectomy. Statistical significance was
assumed at the P \ 0.05 level. The SPSS software package
version 11.5 was used for the statistical analysis.
Thrombotic risk factors in b-thalassemia patients 231

Results a prevalence of 5.7% (allele frequency 3.2%). Among 8

b-thalassemia major patients with factor V Leiden
Eight patients (5.3%) with b-thalassemia major, including G1691A, there were seven carrier patients and one patient
five males and three females, had insulin dependent dia- homozygous for this mutation (prevalence 5.3% and allele
betes mellitus (IDDM). Ten males and three females with frequency 3%). Only one out of seven patients with
b-thalassemia major had HCV antibodies. Two males with b-thalassemia intermedia (14.3%) was a carrier of factor V
IDDM were HCV positive. All patients with IDDM were Leiden G1691A. In healthy individuals, 5 out of 180
splenectomized and in one of them the prothrombin individuals (2.8%) were heterozygous for factor V Leiden
G20210A variant existed. G1691A. Although the prevalence of factor V Leiden
The platelet counts in splenectomized thalassemia mutation was twice as high in patients than in healthy
major and intermedia patients were 674.9 ± 175.8 9 103/ subjects the difference between the two groups was not
ll and 897.4 ± 220.1 9 103/ll, respectively that were statistically significant. MTHFR C677T polymorphism was
significantly (P = 0.0) higher compared to non-splenec- detected as heterozygous in 68 patients (66 b-thalassemia
tomized thalassemic patients (292.1 ± 128.2 9 103/ll major and 2 b-thalassemia intermedia) and as homozygous
and 271.5 ± 77.1 9 103/ll, respectively). in 11 patients (10 b-thalassemia major and 1 b-thalassemia
The prevalence and allele frequency of three thrombo- intermedia). The overall prevalence of MTHFR C677T was
philic mutations are demonstrated in Table 1. The 50% (the heterozygous state 43% and homozygous state
prothrombin G20210A variant was found in two b-thalas- 7%). The frequency of alleles C and T of MTHFR in
semia patients as heterozygous, giving a prevalence of patients was detected to be 71.5 and 28.5%, respectively. In
1.3% (allele frequency 0.65%). The two patients with healthy individuals, 74 heterozygous (41.1%) and 13
prothrombin G20210A mutation were b-thalassemia major homozygous (7.2%) individuals were found for MTHFR
and this mutation was not found among b-thalassemia C677T polymorphism giving a prevalence of 48.3%. The C
intermedia patients, while 6 out of 180 healthy individuals and T allele frequencies in healthy individuals were 72.2
were carriers of this mutation (3.3%). No homozygous and 27.8%. No significant difference was found between
prothrombin 20210AA was found among patients and patients and healthy individuals related to the prevalence of
healthy individuals. Factor V Leiden G1691A was found in MTHFR C677T polymorphism. Two patients, one b-thal-
9 patients with b-thalassemia major and intermedia giving assemia intermedia, a 7-year-old boy regularly transfused,
and one b-thalassemia major patient, a 22-years-old sple-
nectomized female receiving blood transfusion regularly,
Table 1 The prevalence and allele frequencies of factor V Leiden, were found to be homozygous for MTHFR 677TT and
prothrombin G20210A and MTHFR C677T among BM and BI heterozygous for factor V Leiden G1691A. Further, a girl
patients compared to healthy subjects
(13-years-old) with b-thalassemia major was found to be
Genotype BM BI Healthy double heterozygous for MTHFR C677T and factor V
patients patients subjects
Leiden G1691A. Factor V Leiden G1691A as homozygous
Factor V Leiden G1691A (n) 151 7 180 was found in an 18-years-old splenectomized male with
AA 1 – – b-thalassemia major. There were no records related to the
GA 7 1 5 clinical signs or symptoms of thrombosis in their files.
GG 143 6 175
Prevalence (%) 5.3 14.3 2.8
Frequency of A allele (%) 3.0 7.15 1.4 Discussion
Prothrombin G20210A (n) 151 7 180
AA – – – Thalassemic patients have a higher than normal incidence
GA 2 – 6 of thromboembolic events [2]. A prevalence of 1.65% for
GG 149 7 174 TE has been reported among 8,860 studied thalassemia
Prevalence (%) 1.3 0 3.3 patients from the Mediterranean and Iran. It was found that
Frequency of A allele 0.65 0 1.65
thromboembolism occurred 4.38 times more frequently in
MTHFR C677T (n) 151 7 180
thalassemia intermedia than in thalassemia major patients
[15]. In a multicenter study performed in Italy, the inci-
CC 75 4 93
dence of thromboembolic events was 3.95% among TM
CT 66 2 74
and 9.61% among TI [6]. In the study of Moratelli et al. [4]
TT 10 1 13
in Italy, a prevalence of 5.2% was found for thrombotic
Prevalence (%) 50.3 42.9 48.3
events among thalassemia patients (3.3% in TM and 16.2%
Frequency of T allele 28.5 28.6 27.8
in TI patients). A retrospective study by Cappellini et al.

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232
[3] of 83 patients with thalassemia intermedia and 65
patients with thalassemia major demonstrated a prevalence
of venous thromboembolic events of 29 and 2%,
respectively.
Splenectomy, profound anemia, enhanced aggregability
of RBC and abnormality of coagulation factors are known
risk factors of TE in thalassemia patients [2, 5, 15]. The
study of Taher et al. [15] revealed that thrombosis in
thalassemia was also more common in females, splenec-
tomized patients and those with profound anemia
(hemoglobin \ 9 g/dl). Although splenectomy does not
appear to increase procoagulant activity in splenectomized
healthy individuals due to trauma, in thalassemic patients
under regular transfusion, the abnormal red cells with
procoagulant activity still persisted postsplenectomy [16].
The mean level of Hb in TM patients analyzed in the
present study was 8.9 ± 1.1. Also, 71% of b-thalassemia
intermedia and 38.4% of b-thalassemia major patients
underwent splenectomy and the platelet counts in sple-
nectomized thalassemia major and intermedia patients
were more than 600,000/ll. Low-dose daily aspirin has
been recommended for all splenectomized thalassemic
patients and also for splenectomized patients with thalas-
semia major who have platelet counts in excess of 600,000/
ll [1].
Heterozygous carriers of factor V Leiden have a seven-
fold increased risk of venous thrombosis and homozygous
individuals have a risk that is increased up to 100-fold
compared to those without the mutation [17]. Few studies
have identified the three known inherited mutations of
factor V Leiden, prothrombin G20210A and MTHFR
C677T in thalassemic patients. Zalloua et al. [7] in small
subset samples of Lebanese TI patients (50) indicated a
prevalence of 14% for heterozygous factor V Leiden
compared to 12.4% in healthy individuals. However, only
0.08% (4/50) of TI patients developed TE. Also, in the
study of Eldor et al. [2], no increased prevalence of
thrombophilic mutations was observed in TM patients. In
contrast, Kahn et al. [8] indicated the presence of double
heterozygosity for prothrombin G20210A, factor V Leiden
G1691A in a b-thalassemia patient with recurrent throm-
boses and recommended that patients with TM and
congenital thrombophilic mutations need intensive and
long-lasting anticoagulant treatment. Further, the presence
of arterial ischemic stroke due to homozygosity for
methylenetetrahydrofolate reductase C677T mutation has
been reported in a thalassemia minor patient [9]. We
found a higher prevalence of factor V Leiden among TM
(5.3%) and TI (14.3%) patients compared to healthy
individuals. Also, in the present study, we established a
prevalence of 48.3% for MTHFR C677T among healthy
individuals from Western Iran that was slightly lower than
that in thalassemia patients (50.3%). Further, the presence
123
Z. Rahimi et al.
of two thrombophilic mutations in four cases (2.5%), 1 TI
patient (14.3%) and 3 TM patients (2%), homozygous for
factor V Leiden or double heterozygous for factor V
Leiden and MTHFR C677T along with the presence
of additional risk factors of splenectomy and anemia
(Hb \ 9 g/dl), could indicate the need for antithrombotic
therapy to prevent thrombotic events in these patients.
Although the predisposing factors to thrombosis existed in
a relatively high prevalence among TM (heterozygous FV
Leiden: 5.3%, MTHFR 677TT: 6.6%, splenectomy: 38.4%
and diabetes: 5.3%) and TI patients (heterozygous FV
Leiden: 14.3%, MTHFR 677TT: 14.3%, splenectomy:
71.4%) no occurrence of thromboembolism was found in
their files.
Although our findings indicate that thrombophilic
mutations may not be associated with thrombotic events in
our thalassemic patients, due to the presence of a life-long
hypercoagulable state in thalassemia patients and the
higher than normal prevalence of thrombotic risk factors, it
has been suggested that those patients who are at higher
risk of developing TE events should be closely monitored.
Studying the prevalence of thrombophilic mutations and
their association with thromboembolic events in larger
sample sizes could elucidate the role of these factors in TE.
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