Journal of Thrombosis and Thrombolysis 19(3), 189–196, 2005. C  2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands. A Case Control Study on the Contribution of Factor V-Leiden, Prothrombin G20210A, and MTHFR C677T Mutations to the Genetic Susceptibility of Deep Venous Thrombosis Wassim Y. Almawi, 1 Hala Tamim, 2 Raghid Kreidy, 3 Georgina Timson, 1 Elias Rahal, 3 Malak Nabulsi, 4 Ramzi R. Finan, 3 Noha Irani-Hakime 3 1 Al-Jawhara Center for Molecular Medicine, Genetics, and Inherited Diseases, Arabian Gulf University, Manama, Bahrain; 2 Faculty of Health Sciences, American University of Beirut; 3 St. Georges University Hospital, Beirut; 4 Islamic Hospital, Tripoli, Lebanon Abstract. Background: Insofar as the inherited pro- thrombotic single nucleotide polymorphisms (SNPs) factor V G1691A (FV-Leiden), prothrombin (PRT) G20210A, and methylenetetrahydrofolate reductase (MTHFR), C677T are inherited risk factors of venous thromboembolism (VTE), the aim of this study was to determine the prevalence of single and combined SNPs in 198 patients with documented deep venous thrombo- sis (DVT), and 697 control subjects, and to estimate the associated risks. Methods: Factor V-Leiden, PRT G20210A, and MTHFR C677T were analyzed by PCR and restriction fragment length polymorphism (RFLP). Results: The prevalence of the heterozygote and ho- mozygous variants for FV-Leiden (52.02 vs. 14.78%, RR 6.28), PRT G20210A (19.2 vs. 3.6%; RR 6.38), and to a lesser extent the T/T genotype of MTHFR C677T (20.71 vs. 11.0%; RR 1.49) were higher among DVT patients vs. controls, respectively. Two or more SNPs were de- tected in 90 of 198 patients (45.5%) and in 60 of 697 controls (8.6%), with odds ratios of 16.754 for joint oc- currence of FV-Leiden and PRT G20210A, 10.471 for FV- Leiden and MTHFR C677T, and 6.283 for PRT G20210A SNPs and MTHFR 677T/T. Logistic regression analy- sis showed a further increased odds for FV-Leiden in combination with PRT G20210A (85.198) or homozy- gous MTHFR C677T (81.133), and to a lesser extent for PRT G20210A in combination with homozygous MTHFR C677T (20.812). Conclusions: This indicates that FV-Leiden and PRT G20210A, more than MTHFR C677T, are important risk factors for DVT, and that the presence of more than one prothrombotic SNPs was associated with a significant risk of DVT. Key Words. Venous Thrombosis; Factor V-Leiden, Prothrombin G20210A; MTHFR C677T; PCR Introduction Venous thromboembolism (VTE) is a multi-factorial disease, resulting from the interaction of genetic and environmental factors [1]. Among the inherited risk factors are single nucleotide polymorphisms (SNPs) in the genes coding for blood coagulation factors which induce either the synthesis of a defective pro- tein, or the enhanced production of a procoagulant protein, and hence precipitate VTE events. The for- mer mechanism is exemplified by the factor V gene G1691A SNP (FV-Leiden) which renders factor V re- sistant to activated protein C (APC) degradation [2]. The latter is exemplified by the prothrombin/factor II (PRT) G20210A, a SNP in the 3 ′ -untranslated region of the PRT gene, which alters PRT mRNA stability, resulting in higher PRT levels. Both FV-Leiden and PRT G20210A SNPs are associated with heightened risk of VTE [3,4], and are relatively frequent among white/Caucasian populations [5], with a founder ef- fect being suggested [6]. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrah- ydrofolate to 5-methyltetrahydrofolate, and plays a critical role in the homocysteine-to-methionine methylation. A reduction in MTHFR level or activity leads to hyperhomocysteinemia, characterized by in- creased plasma total homocysteine (Hcy) levels, and is often seen in patients with vascular diseases [7]. The C677T SNP in the MTHFR gene (A223V) results in a thermolabile enzyme and reduced enzymatic ac- tivity, and homozygotes (677 T/T) for this SNP were associated with a 50% reduction in the MTHFR ac- tivity. Whereas FV-Leiden or PRT G20210A poly- morphism were considered risk factors for deep ve- nous thrombosis (DVT), similar association between MTHFR C677T SNP and heightened risk of DVT was Address for correspondence: Wassim Y. Almawi, PhD, Al-Jawhara Center for Molecular Medicine, Genetics and In- herited Diseases, Arabian Gulf University, P.O. Box 22979, Manama, Bahrain. Tel.: +973-39 71 7118; Fax. +973- 17271090; E-mail: wyalmawi@yahoo.co.uk 189