ABO blood group genotypes and the risk of venous thrombosis: effect of factor V Leiden

Abstract

ABO blood group and more recently high von Willebrand factor (VWF) and factor (F)VIII levels have been associated with thrombotic disease. An excess of non-O blood group has long been recognized in patients with ischemic heart disease [1] and venous thrombosis [2]. In 1995, we demonstrated that nonO blood group, high VWF levels and high FVIII levels all increased the risk of deep vein thrombosis [3]. In multivariate analysis only FVIII remained a risk factor, whereas the thrombosis risk associated with VWF and ABO blood group largely disappeared. Since then, several other studies have identified high FVIII levels as a risk factor for venous thrombosis [4–7]. Usually blood group phenotypes are used to study the associationbetweenbloodgroupandvenous thrombosis.Blood group genotypes may be more informative since genotypes can distinguish between heterozygous and homozygous carriers of A, B and O alleles and between A and A alleles. Therefore we studied the effect ofABOgenotype on thrombosis risk in a large population-based case–control study of venous thrombosis (Leiden Thrombophilia Study, LETS). This study, which included 474 patients and 474 control subjects, has been previously described [3]. For the present study DNA was available for 471 patients and 471 control subjects. Blood was collected into 0.1 volume 0.106 mol L trisodium citrate. Plasma was prepared by centrifugation for 10 min at 2000 · g at room temperature and stored at )70 C. FVIII coagulant activity (FVIII:C), FVIII:Ag, VWF:Ag and blood group phenotype were measured as previously reported [3–5]. High-molecular-weight DNA was isolated from leukocytes and stored at 4 C. Polymerase chain reaction (PCR) was designed to amplify exons 6 and 7 of the ABO blood group gene in two separate reactions. The sequences of the primers have been described previously [8]. The amplified DNA fragments corresponding to exons 6 and 7 were digested with Acc65I (MBI Fermentas) or MspI (MBI Fermentas), respectively, in two separate reactions and separated by electrophoresis on 3.5% agarose gels. With this method we discriminated A, A, B, O and O alleles. There was 99% agreement between ABO blood group phenotype and genotypes in all patients and controls. Table 1 (upper part) shows the frequency of the ABO blood group genotypes in patients and controls. Odds ratios (OR) were calculated as estimates of the relative risk by anunmatched method. Ninety-five percent confidence intervals were assessed according to Woolf [9]. All non-OO genotypes except A homozygotes or A–O combinations, i.e. AO/AO/AA, were associated with an increased thrombosis risk when compared with OO genotypes. This reinforces the concept that blood group exerts its thrombotic risk largely via FVIII levels, since AO/AA genotypes correspond to the lowest FVIII levels among non-OO genotypes (data not shown). Adjustment for age and sex did not alter the risk estimates. Because blood group is known to affect plasma levels of VWF and FVIII and because VWF and FVIII levels influence thrombosis risk, we adjusted the thrombosis risk associated