Mol Biol Rep (2012) 39:91339138 DOI 10.

1007/s11033-012-1785-7

Prevalence of coagulation factor II G20210A and factor V G1691A Leiden polymorphisms in Chechans, a genetically isolated population 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 nucleotide polymorphisms (SNPs) are major inherited risk factors of venous thromboembolism. In view of the heterogeneity in their world distribution and lack of sufcient information about their distribution among Chechans, we addressed 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 Amplication 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 mutation 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 ve 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 (16 %) while the prevalence of factor V Leiden was higher in the Chechan population compared to Jordan and Caucasian populations (215 %). Keywords Genetic polymorphisms Ethnicity Thrombosis

Introduction Thrombosis represents one of the most common causes of morbidity and mortality in societies [1]. Haemostatic disequilibrium is the key mechanism for all types of thromboses. 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 prothrombin G20210A and factor V Leiden [24]. The study of their frequencies in various populations provides perspectives for both clinical medicine, population genetics and biological anthropology [1]. Dahlback et al. [5]. reported activated protein C resistance (APCR) which until now has constituted the most frequently encountered genetic abnormality in patients with VTE (2030 % 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

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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 810 % of thrombosis patients. The coinheritance of these relatively common 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 combination with an environmental risk factor is associated with a substantial increased risk of venous thromboembolism [8]. Flanking SNPs and microsatellites demonstrate a single origin for both factor II G20210A and factor V G1691A and indicate that the polymorphisms arose 1,0501,200 generations ago (21,00036,000 years ago for 2030 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 polymorphic 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 prevalence 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 geographic 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 therefore 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 conrmed that the mitochondrial DNA variation in the HV1 region is signicantly 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 rst 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 1381 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 precision of 3 % at a level of condence of 95 % is 88 subjects. 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 lled 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 nal 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 polymerase (Go Taq DNA polymerase, Promega) and 1.5 lL target DNA (50100 lg/mL). Detection of the G20210A polymorphism in factor II gene was performed by means of polymerase chain reaction followed by restriction enzyme analysis. The forward

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primer for factor II (G20210A) is 50 -TCTAGAAACAGTTGCCTGGC-30 and the reverse primer is 50 -ATAGCACTGGGAGCATTGAAGC-30 [4]. The thermal cycling conditions for factor II consisting of 5 min denaturation 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 nal extension step at 72 C for 5 min and kept at 4 C until use. After overnight incubation with 5 U of HindIII restriction enzyme for the G20210A at 37 C, PCR reactions were run on 3 % agarose 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 Amplication refractory mutation detection system (ARMS), the wild-type primer is 50 -GGACAAAATACCTGTATTCCTC-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 denaturation 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 nal 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.
Fig. 1 Detection of factor II gene mutation G20210A by PCRRFLP analysis. HindIII digested fragment were separated by 3 % agarose gel electrophoresis and visualized by ethidium bromide staining. Lanes 1, 39, 1115 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

Statistical analysis Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS), version 15. Frequencies, 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 signicant.

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 1381 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: 05.3) and an allele frequency of 1.25 %. No homozygous factor II allele was found (Fig. 1). There was no signicant 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

Table 1 Genotype and allele frequency of factor II G20210A mutation Group N Genotype freq of G/G N (%) 117 (97.5) 41 (95.3) 76 (98.7) Genotype freq of G/A N (%) 3 (2.5) 2 (4.7) 1 (1.3) 95 % condence interval 05.3 010.9 03.8 p value for G/A (males vs. females) 0.292 Genotype freq of A/A % 0.00 0.00 0.00 Allele freq of A % 1.25 2.3 0.65

Total Male Female

120 43 77

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with the HardyWeinberg equilibrium (p = 0.975, pq = 0.025, and q = 0) v2 = 0.02, p [ 0.05. Factor V G1691A mutation was detected as heterozygous in 22 of 120 of individuals (17 female and ve male) indicating a prevalence of 18.3 % (95 % CI 11.425.2) and allele frequency of 9.2 %. No homozygous allele was found, Fig. 2. There was no signicant 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 HardyWeinberg 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 prevalence in Jordan. The prevalence of factor II mutation is rarely seen in Asian populations [17]. Among Arab populations 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 populations the prevalence has been reported to be 1.7 % [19].

Fig. 2 Detection of factor V Leiden gene mutation G1691A by polymerase chain reaction Amplication refractory mutation system (PCRARMS) 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, 411, 13, 1520 represents normal FV genotype with one band in rst lane for every numbers (241 bp). Numbers 2, 12, and 14 represents heterozygous factor V Leiden mutation (R506Q) with two bands in both lanes for every number. Number 21 represents 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 (%) 98 (81.7) 38 (88.4) 60 (77.9) Genotype freq of G/A N (%) 22 (18.3) 5 (11.6) 17 (22.1) 95 % condence interval 11.425.2 2.021.2 12.831.3 p value for G/A (males vs. females) 0.156 Genotype freq of A/A % 0.0 0.0 0.0 Allele freq of A % 9.2 5.8 11.0

Total Male Female

120 43 77

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Mol Biol Rep (2012) 39:91339138 Table 3 Genotype frequency of factor II G20210A mutation in different populations Population Chechans Jordan Lebanon Tunis Bahrain Saudi Arabia Turkish Turk cypriotes Southern Europeans Northern Europeans Asians Prevalence (%) 2.5 2 1.36 1.28 0.52 0.0 1.37 4.0 3 1.7 Rare [16] [18] [18] [18] [18] [20] [20] [21] [21] [17] Reference

9137 Table 4 Allele frequency of factor V Leiden mutation in different populations Population Chechans Jordan Cyprus Lebanon Tunis Bahrain Saudi Arabia Turkey North India Western Iran Tehran Sweden Allele frequency (%) 9.2 8.5 12 7.88 3.5 1.5 1 4.54.9 1.9 2.97 5.5 15 [16] [23] [18] [18] [18] [18] [23] [24] [25] [25] [23] Reference

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 prevalence factor V Leiden in the Chechan population is 18.3 %. In comparison with percentage of prevalence for Jordanians 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 nonCaucasians [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 Lebanon 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 4.54.9 % [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 different 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 posterior to the separation of the Caucasoids and Mongoloids (21,00034,000 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 Europe 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 multifactorial [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 Diabetes, Endocrinology and Genetics. We would also like to thank the Chechan community for their cooperation in this study.

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9138 Conict of interest None.

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