Prevalence of coagulation factor II G20210A and factor V G1691ALeiden polymorphisms in Chechans, a genetically isolatedpopulation in Jordan

Rana Dajani • Raja Fatahallah • Abdelrahman Dajani •

Mohammad Al-Shboul • Yousef Khader

Received: 18 November 2011 / Accepted: 9 June 2012 / Published online: 29 June 2012

� Springer Science+Business Media B.V. 2012

Abstract Background Coagulation factor II G20210A

and coagulation factor V (Leiden) G1691A single nucleo-

tide polymorphisms (SNPs) are major inherited risk factors

of venous thromboembolism. In view of the heterogeneity

in their world distribution and lack of sufficient informa-

tion about their distribution among Chechans, we addres-

sed the prevalence of these SNPs in the Chechan

population in Jordan, a genetically isolated population.

Methods and Results factor II G20210A and factor V

Leiden SNPs were analysed by polymerase chain reaction

and restriction fragment length polymorphism (PCR–

RFLP) method and Amplification refractory mutation

detection system (ARMS) respectively in 120 random

unrelated subjects from the Chechan population in Jordan.

Among the subjects studied for factor II G20210A muta-

tion there were three individuals carrying this mutation as

heterozygous (one female and two male), giving a

prevalence of 2.5 % and an allele frequency of 1.25 %. No

homozygous factor II allele was found. Factor V Leiden

G1691A mutation was detected as heterozygous in 22 of

120 of individuals (17 female and five male) indicating a

prevalence of 18.3 % and allele frequency of 9.2 %. No

homozygous allele was found. Conclusion Our results

indicated that prevalence of factor II G20210A mutation in

the Chechan population is similar to prevalence in Jordan

and Caucasian populations (1–6 %) while the prevalence

of factor V Leiden was higher in the Chechan population

compared to Jordan and Caucasian populations (2–15 %).

Keywords Genetic polymorphisms � Ethnicity �Thrombosis

Introduction

Thrombosis represents one of the most common causes of

morbidity and mortality in societies [1]. Haemostatic dis-

equilibrium is the key mechanism for all types of thrombo-

ses. Venous thrombosis (VTE) is a common multifactorial

disease involving the interaction of environmental factors

such as aging, obesity, surgery, pregnancy and post-partum,

and oral contraceptives or cancer, with genetic predisposing

risk factors [1]. The most common genetic risk factors

implicated in VTE, are the polymorphisms of the pro-

thrombin G20210A and factor V Leiden [2–4]. The study of

their frequencies in various populations provides perspec-

tives for both clinical medicine, population genetics and

biological anthropology [1].

Dahlback et al. [5]. reported activated protein C resis-

tance (APCR) which until now has constituted the most

frequently encountered genetic abnormality in patients

with VTE (20–30 % of cases). This condition is due in

R. Dajani (&)

Department of Biology and Biotechnology, Hashemite

University, Zarqa, Jordan

e-mail: rdajani@hu.edu.jo

R. Fatahallah

National Center for Diabetes, Endocrinology and Genetics,

Amman, Jordan

A. Dajani

Faculty of Medicine, Hashemite University, Zarqa, Jordan

M. Al-Shboul

Laboratory of Human Embryology, Institute of Medical Biology,

A*STAR, Singapore, Singapore

Y. Khader

Department of Community Medicine, Public Health and Family

Medicine, Faculty of Medicine, Jordan University for Science

and Technology, Irbid, Jordan

123

Mol Biol Rep (2012) 39:9133–9138

DOI 10.1007/s11033-012-1785-7

more than 90 % of cases to a single mutation (G1691A) in

the factor V gene [2]. In 1996, Poort et al. [4] described

factor II G20210A which is present in 8–10 % of throm-

bosis patients. The coinheritance of these relatively com-

mon genetic conditions, which is not a rare event, further

increases the relative risk of thrombosis, i.e., factor V

Leiden plus factor II G20210A [6, 7]. Similarly, the com-

bination with an environmental risk factor is associated

with a substantial increased risk of venous thromboembo-

lism [8].

Flanking SNPs and microsatellites demonstrate a single

origin for both factor II G20210A and factor V G1691A

and indicate that the polymorphisms arose 1,050–1,200

generations ago (21,000–36,000 years ago for 20–30 year

generations) [9, 10]. Based on the frequencies of the two

polymorphisms, it has been suggested that they originated

in the Middle East. Several studies argued that factor II

G20210A and factor V G1691A were maintained at poly-

morphic frequencies among Caucasoids, because they

conferred an evolutionary advantage of reduced bleeding

[11]. The various prevalences of thrombophilia in different

ethnic populations have been established. A high preva-

lence of the factor V Leiden mutation in Middle Eastern

subjects and a virtual absence of this mutation in Asian

population has been suggested [9]. The ethnic and geo-

graphic distribution prevalence of factor II G20210A and

factor V Leiden among general population ranges from 1 to

4 and 3 to 15 %, respectively [1].

These genetic prothrombotic factors demonstrate

peculiar patterns of geographical distribution and there-

fore represent valuable tools for population genetics. We

are interested in studying the prevalence of these

thrombophilic mutations in the Chechan population in

Jordan. The Chechans, who call themselves Noxchii and

their land Noxchiin moxk, are the largest indigenous

nationality of the North Caucasus. Most of Chechans in

Jordan belong to the Naqshbandi tariqat [12]. The

Chechans immigrated to Jordan about 140 years ago and

are genetically isolated because of cultural reasons.

Chechans in Jordan have managed to keep their separate

sense of identity and ethnicity during the last one hundred

years, even after large waves of Bedouin and Palestinian

immigration into Jordan over the course of the twentieth

century. The Chechan population is around 10,000 [13].

Jaradat has confirmed that the mitochondrial DNA vari-

ation in the HV1 region is significantly different between

the Chechan and Arab populations in Jordan (S. Jaradat,

personal communication).

This study aimed to assess the prevalence of factor II

G20210A and factor V Leiden in 120 unrelated random

samples from the Chechan population, living in Jordan. We

hypothesize that the prevalences of these thrombophilic

mutations in this population are different from other

populations because of their separate ethnicity. Since the

prevalences of these thrombophilic mutations are studied in

the Chechan population in Jordan we also wanted to

compare the prevalences of these factors in the general

Jordanian population and to other countries and world

regions. This is the first report on the prevalence of these

factors in the Chechan population.

Materials and methods

This study has been approved by the IRB committee at the

Hashemite University.

A random sample of unrelated individuals (N = 120) of

both sexes, mean age of 45 (range 13–81 years) from the

Chechan population in Jordan were recruited after signing

a consent form indicating their acceptance to participate in

the study. The sample size was calculated based on the

assumption that the prevalence of factor II mutation in the

Caucasian population is 2 %. The sample size needed to

estimate the prevalence of factor II mutation with a pre-

cision of 3 % at a level of confidence of 95 % is 88 sub-

jects. The sample size was calculated using Epicalc 2000.

The samples were taken during the period between August

2008 and March 2009. Each participant in the study filled

out a survey that included pedigree information. The names

and ethnicity of parents, grandparents, great grand parents

both maternal and paternal and any individual with non

Chechan heritage for even one person in his/her pedigree

was excluded.

Sample collection

Nine millilitre of whole blood was drawn in EDTA tubes

from 120 peoples by Vacutainer system. Genomic DNA

was isolated from whole blood sample using the phenol–

chloroform protocol [14].

PCR-reactions

The PCR-reactions [4] were performed in a final volume of

25 lL containing 5 lL from 5X Go Taq buffer (Promega)

for factor II, and 109 buffer (Applied Biosystem) for factor

V, 2.5 lL from 2 mM dNTPs (invitrogen) 15 lL sterile

distilled water, 10 pmol forward and 10 pmol reverse

primers (Alpha DNA, Canada), 1 U (5 U/lL) Taq poly-

merase (Go Taq� DNA polymerase, Promega) and 1.5 lL

target DNA (50–100 lg/mL).

Detection of the G20210A polymorphism in factor II

gene was performed by means of polymerase chain reac-

tion followed by restriction enzyme analysis. The forward

9134 Mol Biol Rep (2012) 39:9133–9138

123

primer for factor II (G20210A) is 50-TCTAGAAA-

CAGTTGCCTGGC-30 and the reverse primer is 50-ATA-

GCACTGGGAGCATTGAAGC-30 [4]. The thermal

cycling conditions for factor II consisting of 5 min dena-

turation at 95� followed by 35 cycles of denaturation at

95 �C for 25 s, annealing at 55 �C for 25 s, extension at

72 �C for 40 s, then the final extension step at 72 �C for

5 min and kept at 4 �C until use. After overnight incuba-

tion with 5 U of HindIII restriction enzyme for the

G20210A at 37 �C, PCR reactions were run on 3 % aga-

rose gels for 1.30 h at 90 V stained with ethidium bromide.

The factor II wild type had only one band: 345 bp,

G20210A genotype had three bands: 345, 322 and 23 bp,

and AA homozygote had two bands: 322 and 23 bp.

For factor V Leiden (G1691A) the PCR reaction were

carried out by Amplification refractory mutation detection

system (ARMS), the wild-type primer is 50-GGA-

CAAAATACCTGTATTCCTC-30, the mutant primer is 50-GGACAAAATACCTGTATTCCTT-30, and the common

primer is 50-CTTTCAGGCAGGAACAACACC-30 [15].

The thermal cycling conditions consisting of 5 min dena-

turation at 95� followed by 35 cycles of denaturation at

95 �C for 25 s, annealing at 61 �C for 25 s, extension at

72 �C for 40 s, then the final extension step at 72 �C for

5 min, kept at 4 �C until use. PCR reaction was run on 2 %

agarose gel for 45 min at 150 V and stained with ethidium

bromide, the product size was 233 bp.

Statistical analysis

Statistical analysis was performed using the Statistical

Package for Social Sciences (SPSS), version 15. Frequen-

cies, percentages, and means were used to describe data.

Data were expressed as percentages of the mean or as

frequency of the allele. Percentages were compared using

v2-test or Fisher exact test wherever appropriate. A p value

of less than 0.05 was considered statistically significant.

Results

The prevalences and allele frequencies of factor II

G20210A and factor V Leiden G1691A were determined

for 120 random unrelated Chechan subjects. The subjects

were of both sexes 43 males (36 %) and 77 females (64 %)

with a mean age of 45 (range 13–81 years).

Among the 120 individuals studied for factor II

G20210A mutation there were three individuals carrying

this mutation as heterozygous (one female and two male),

giving a prevalence of 2.5 % (95 % CI: 0–5.3) and an

allele frequency of 1.25 %. No homozygous factor II allele

was found (Fig. 1). There was no significant difference in

factor II G20210A frequency with respect to gender (4.7 %

in males vs. 1.3 % in females) (Table 1). Furthermore, the

observed homozygote to heterozygote ratio was consistent

Fig. 1 Detection of factor II

gene mutation G20210A by

PCR–RFLP analysis. HindIII

digested fragment were

separated by 3 % agarose gel

electrophoresis and visualized

by ethidium bromide staining.

Lanes 1, 3–9, 11–15 represent

an individuals with normal

factor II genotype, 345 bp.

Lanes 2, 10 represent an

individuals with heterozygous

for FII-G20210A digested by

HindIII, 345 and 322 fragments

Table 1 Genotype and allele frequency of factor II G20210A mutation

Group N Genotype freq

of G/G

N (%)

Genotype freq

of G/A

N (%)

95 % confidence

interval

p value for G/A

(males vs. females)

Genotype freq

of A/A %

Allele freq

of A %

Total 120 117 (97.5) 3 (2.5) 0–5.3 0.292 0.00 1.25

Male 43 41 (95.3) 2 (4.7) 0–10.9 0.00 2.3

Female 77 76 (98.7) 1 (1.3) 0–3.8 0.00 0.65

Mol Biol Rep (2012) 39:9133–9138 9135

123

with the Hardy–Weinberg equilibrium (p = 0.975,

pq = 0.025, and q = 0) v2 = 0.02, p [ 0.05.

Factor V G1691A mutation was detected as heterozy-

gous in 22 of 120 of individuals (17 female and five male)

indicating a prevalence of 18.3 % (95 % CI 11.4–25.2) and

allele frequency of 9.2 %. No homozygous allele was

found, Fig. 2. There was no significant difference in factor

V Leiden frequency with respect to gender (11.6 % in

males vs. 22.1 % in females) (Table 2). Furthermore, the

observed homozygote to heterozygote ratio was consistent

with the Hardy–Weinberg equilibrium (p = 0.817,

pq = 0.183, and q = 0.00) v2 = 0.99, p [ 0.05.

There are no subjects with both mutations in factor II

and factor V Leiden.

Discussion

The prevalence of factor II mutation in the Caucasian

population varies between 1 and 6 %, with an overall

prevalence of about 2 % [1]. In Jordan the prevalence of

factor II G20210A mutation was 2 % [16]. Our studies

have found that the prevalence of factor II G20210A is

2.5 % in the Chechan population comparable to the prev-

alence in Jordan. The prevalence of factor II mutation is

rarely seen in Asian populations [17]. Among Arab popu-

lations the allele frequency is 1.36 % in Lebanon, 1.28 %

in Tunis, 0.52 % in Bahrain and 0.0 % in Saudi Arabia

[18]. In a separate study on Middle Eastern Arab popula-

tions the prevalence has been reported to be 1.7 % [19].

Fig. 2 Detection of factor V

Leiden gene mutation G1691A

by polymerase chain reaction–

Amplification refractory

mutation system (PCR–ARMS)

analysis. Bands were separated

by 2 % agarose gel

electrophoresis and visualized

by ethidium bromide staining.

Lane M represents a 100-bp

molecular weight marker. Every

number represents two lanes;

numbers 1, 3, 4–11, 13, 15–20represents normal FV genotype

with one band in first lane for

every numbers (241 bp).

Numbers 2, 12, and 14represents heterozygous factor

V Leiden mutation (R506Q)

with two bands in both lanes for

every number. Number 21represents positive control for

factor V Leiden mutation.

Number 22 represents negative

control for factor V Leiden

mutation. Number 23 represents

blank

Table 2 Genotype and allele frequency of factor V Leiden mutation

Group N Genotype freq

of G/G

N (%)

Genotype freq

of G/A

N (%)

95 % confidence

interval

p value for G/A

(males vs. females)

Genotype freq

of A/A %

Allele freq

of A %

Total 120 98 (81.7) 22 (18.3) 11.4–25.2 0.156 0.0 9.2

Male 43 38 (88.4) 5 (11.6) 2.0–21.2 0.0 5.8

Female 77 60 (77.9) 17 (22.1) 12.8–31.3 0.0 11.0

9136 Mol Biol Rep (2012) 39:9133–9138

123

The allele frequency of factor II in the Turkish population

and Turkish Cypriotes is 1.37 and 4.0 % respectively [20].

Among southern Europeans a rate of 3 % for factor II

polymorphism has been reported which is higher than the

1.7 % reported for northern European populations [21]

(Table 3).

The prevalence of factor V Leiden varies between 2 and

15 % in the healthy Caucasian population [1]. The preva-

lence factor V Leiden in the Chechan population is 18.3 %.

In comparison with percentage of prevalence for Jordani-

ans 15 % [16] and more recently 21.8 % in a study done by

Nusier et al. [22], the Chechans have a higher percentage

than other populations. A high prevalence of factor V

Leiden has been reported in Caucasians but not in non-

Caucasians [1]. For example the allele frequency of factor

V Leiden polymorphism among Caucasoid subpopulations

ranges from 1 to 8.5 %. This polymorphism is not found

among African blacks Chinese, Japanese and native North

and South Americans or Greenland Inuits [9].

The mean allele frequency among European populations

is 2.7 %. In Cyprus the incidence is 12 % and in Sweden is

15 % [23]. Among Arabs the highest frequency is in Leba-

non 7.88 % followed by Tunis 3.5 %, Bahrain 1.5 % and

Saudi Arabia 1.0 % [18]. The allele frequency in north India

is 1.9 % [24], in western Iran 2.97 % and in Tehran 5.5 %

[25]. The frequency in Turkey is 4.5–4.9 % [23]. The allele

frequency in the Chechan population is 9.2 % which is high

compared to other populations (Table 4). Thus suggesting a

single origin of the mutation. The data obtained from dif-

ferent studies support and suggest that this mutation arose in

the Eastern Mediterranean and migrated to European regions

with the migrants [9]. This supports the history that states the

Chechans to be more ancient as a race having a higher

prevalence of factor V Leiden.

Thus, factor II G20210A and factor V Leiden are

restricted to European populations, which tends to argue

for their monocentric origin. Their founding effect is pos-

terior to the separation of the Caucasoids and Mongoloids

(21,000–34,000 years ago according to haplotype analysis)

[9, 10] and their dispersion was probably associated with

the Neolithic migrations into Europe [11]. In their review

of about 6,000 individuals from 26 populations from Eur-

ope and neighbouring countries, Lucotte and Mercier [23]

concluded that factor V Leiden may have expanded from

the Anatolian region, close by the zone where the process

of agriculture is thought to have begun.

Factor V Leiden is the largest inherited risk factor of

VTE [26]. However, the pathogenesis of VTE is multi-

factorial [1]. Although our results do not support random

screening for factor V Leiden, its high prevalence among

apparently healthy individuals in the Chechan population

recommends screening for factor V Leiden in relatives of

factor V Leiden carriers, in individuals with family history

of VTE, and in high-risk situations, including pregnancy,

use of oral contraceptives, and surgery.

Thus, these two inherited prothrombotic polymorphisms

represent interesting tools for population genetics studies.

The knowledge of these frequencies in the Middle East

region through population-based studies will contribute to

a better understanding of the interaction between genetic

and environmental risk factors underlying thrombosis. The

relationship between venous thrombophilia and these

mutations have to be further studied in the Chechan

population.

Acknowledgments This study has been supported by the Hashemite

University. We would like to thank Dr. M El Khateeb for giving us

the opportunity to use the facilities at the National Center for Dia-

betes, Endocrinology and Genetics. We would also like to thank the

Chechan community for their cooperation in this study.

Table 3 Genotype frequency of factor II G20210A mutation in dif-

ferent populations

Population Prevalence (%) Reference

Chechans 2.5

Jordan 2 [16]

Lebanon 1.36 [18]

Tunis 1.28 [18]

Bahrain 0.52 [18]

Saudi Arabia 0.0 [18]

Turkish 1.37 [20]

Turk cypriotes 4.0 [20]

Southern Europeans 3 [21]

Northern Europeans 1.7 [21]

Asians Rare [17]

Table 4 Allele frequency of factor V Leiden mutation in different

populations

Population Allele frequency (%) Reference

Chechans 9.2

Jordan 8.5 [16]

Cyprus 12 [23]

Lebanon 7.88 [18]

Tunis 3.5 [18]

Bahrain 1.5 [18]

Saudi Arabia 1 [18]

Turkey 4.5–4.9 [23]

North India 1.9 [24]

Western Iran 2.97 [25]

Tehran 5.5 [25]

Sweden 15 [23]

Mol Biol Rep (2012) 39:9133–9138 9137

123

Conflict of interest None.

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