SHORT REPORT
Factor V Arg
306
→
Thr (factor V Cambridge) and factor V
Arg
306
→
Gly mutations in venous thrombotic disease
R. F. FR A N C O,1 , 2F. H. MA F F E I,3D. LO U R E N C¸ O,4V. MO R E L L I,4I. A. TH O M A Z I N I,3C. E. PI C C I NAT O,5
M. H. TAV E L L A1A N D M. A. ZAG O1 1Department of Clinical Medicine and5Department of Vascular Surgery,
School of Medicine of Ribeira˜o Preto, USP,2Blood Centre of Ribeira˜o Preto, FUNDHERP, Ribeira˜o Preto, 3
Department of Vascular Surgery, School of Medicine of Botucatu, UNESP, and 4
Department of Clinical Medicine, UNIFESP, Brazil
Received 7 April 1998; accepted for publication 24 August 1998
Summary.We investigated the prevalence of two reported mutations of the factor V gene (factor V Arg306
→Thr, or factor V Cambridge, and factor V Arg306→Gly) in 104 relatively young patients with verified venous thrombosis and in 208 age-, sex- and race-matched controls, in order to establish whether the two mutations are associated with increased predisposition for venous thrombosis. PCR ampli-fication followed byBstNI andMspI digestion was employed to determine the genotypes, and each mutation was confirmed by DNA sequencing. Among the controls, one
individual was found to be heterozygous for the factor V Arg306
→Thr mutation and one heterozygous for the factor V Arg306→Gly mutation; none of the patients carried either mutation. Our findings do not support factor V Cambridge and factor V Arg306→Gly as risk factors for venous thrombosis.
Keywords: factor V Arg306→Thr, factor V Arg306→Gly, venous thrombosis, thrombophilia, risk factor.
Resistance to activated protein C (APC) is presently considered the commonest known genetic cause of venous thrombosis, found in 20–60% of patients with venous thromboembolic disease (Da¨hlback et al, 1993; Da¨hlback, 1997). In most cases inherited resistance to APC is caused by a single point mutation in the factor V gene: a G to A transition at nucleotide 1691 in exon 10, which results in an Arg to Gln substitution at amino acid position 506 (Bertina
et al, 1994). The mutant factor V (Arg506→Gln), known as factor V Leiden, exhibits resistance to APC-mediated neutralization, is associated with a hypercoagulable state, and increases risk for venous thrombosis (Bertina et al, 1994; Da¨hlback, 1997).
Recently, two mutations affecting the Arg306 APC cleavage site in the factor V gene have been described (Williamson et al, 1998; Chan et al, 1998). The first mutation, referred to as factor V Cambridge (Williamsonet al, 1998), is a G to C transition at nucleotide position 1091 and predicts replacement of arginine with a threonine at amino acid position 306 (Arg306→Thr). This mutant factor V was identified in a British patient with venous thrombosis
and was found to be associated with APC resistance in the absence of the factor V Leiden mutation, but it was absent in a large sample of patients with venous thromboembolism and among healthy blood donors (Williamsonet al, 1998). The second mutation (a A to G transition at nucleotide position 1090 in the exon 7 of the factor V gene, Arg306→Gly) was found in 2/43 patients with venous thrombosis and in one out of 40 nonthrombotic patients from Hong Kong; a plasma sample was available for only one carrier and a normal APC resistance assay was obtained (Chanet al, 1998).
In the present study we investigated the prevalence of the Arg306→Thr and Arg306→Gly mutations in the factor V gene in 104 relatively young patients with objectively diagnosed deep vein thrombosis and in 208 age-, sex- and race-matched healthy controls, in order to test whether these mutations are associated with increased predisposition for venous thrombophilia.
MATERIALS AND METHODS
Subjects. A hundred and four consecutive subjects aged
<45 years (49 men and 55 women; mean age 31 years,
range 1–45 years) who had an episode of deep venous thrombosis objectively confirmed by phlebography British Journal of Haematology, 1998,103, 888–890
888
q1998 Blackwell Science Ltdor ultrasonography (colour duplex scan) composed the patient group. None of the patients had evidence of malignant disease. Patients were admitted to the University Hospital between 15 October 1996 and 16 February 1998. Based on phenotype criteria, 82 patients were identified as Whites, 13 as Blacks and nine as Mulattos. For each patient, two age-, sex- and race-matched healthy unrelated indivi-duals (selected from donors in the Blood Centre of Ribeira˜o Preto, Sa˜o Paulo, Brazil) without a personal or family history of venous thrombosis were invited to participate in the study as controls. Final response rate among patients and controls was 94·5% and 86·6%, respectively. Informed consent was obtained from all individuals and the study was approved by the local Ethics Committee.
Methods. Genomic DNA was extracted from peripheral blood leucocytes by employing standard methods. A 228 bp DNA fragment containing exon 7 of the factor V gene was amplified by PCR using the primers and amplification conditions reported by Williamson et al (1998), and was submitted to restriction digestion with BstNI. Both the 1091G→C and the 1090A→G mutations in the factor V gene abolish the recognition site forBstNI (Fig 1). Hence,
BstNI restriction-digestion does not differentiate the two mutations. For the samples in which absence of digestion was observed afterBstNI digestion, the PCR product of factor V exon 7 was submitted toMspI restriction-digestion. The 1090A→G (but not the 1091G→C) mutation creates a recognition site for this enzyme, therefore allowing the identification of the mutations (Fig 1). The presence of both mutations was also confirmed by dideoxynucleotide chain termination DNA sequencing (Sangeret al, 1977) (Fig 2). Odds ratios (OR) and 95% confidence intervals (CI95) were calculated employing standard methods.
RESULTS
One out of the 208 control subjects was found to be heterozygous for the factor V Arg306→Thr mutation; this
mutation was not observed among patients with venous thrombosis. The factor V Arg306→Gly mutation was detected in heterozygous form, in one control subject and was absent in the patient group. Both carriers, found in the control group, were Whites.
Considering each mutation separately, a prevalence of zero among 104 cases and 1 among 208 controls yielded an OR for venous thrombosis of zero (CI95 0–78). The OR was still zero when both mutations were analysed together (i.e. a prevalence of zero among 104 patients and 2 among 208 controls), with a CI95 of 0–10·6.
DISCUSSION
The recent description of two mutations affecting the Arg306 APC-cleavage site of coagulation factor V stimulated investigation of these genetic variations as candidate risk factors for venous thrombosis. In the present study we determined the prevalence of factor V Arg306
→Thr and factor VArg306→Thr mutations in relatively young patients with venous thrombosis and in healthy controls. These two groups have been previously examined for the presence of factor V Leiden and of the factor II G20210A mutation. Factor V Leiden was found in 2% of the controls and in 13·5% of the patients, whereas the frequency of the factor II G20210A mutation was 1·6% in controls and 8·5% in patients (unpublished observation). These data indicated that both control and patient groups here investigated were representative samples.
The Arg306→Thr mutation, or factor V Cambridge, was originally described in a patient suffering from venous thrombosis with resistance to APC, but was not found among 585 patients with venous thromboembolism or among 226 blood donors (Williamsonet al, 1998). In the present study the mutation was not found in a selected population of relatively young patients who had suffered from deep venous thrombosis, whereas it was observed in one out of 208 healthy controls. These data are in agreement with the original report by Williamsonet al(1998), in so far as they suggest that the Arg306→Thr mutation in the coagulation factor V is not a risk factor for venous thrombophilia. The original publication reports factor V Cambridge as a cause of APC resistance, but this aspect was not assessed in the present investigation since a plasma
889
Short Report
q1998 Blackwell Science Ltd,British Journal of Haematology103: 888–890
Fig 1. Silver-stained polyacrylamide gel electrophoresis following
BstNI (lanes 1–3) andMspI (lanes 4–6) restriction digestion of a 228 bp DNA fragment of exon 7 of factor V gene. M, molecular weight marker; ND, fragment not digested. Lanes 1 and 4: normal genotype; lanes 2 and 5: heterozygosity for the factor VArg306→Gly
mutation; lanes 3 and 6: heterozygosity for the factor V Arg306
→Thr mutation (factor V Cambridge).
Fig 2.DNA sequencing of a fragment of exon 7 of factor V gene. Factor V Arg306→Thr (1091 G→C) and Arg306→Gly (1090
sample from the carrier control was not available to perform the assay.
A second recently reported mutation affecting the APC-cleavage site of factor V (Arg306→Gly) was found in two patients with venous thrombosis and in one individual without venous thrombosis (Chan et al, 1998). The only plasma sample from a carrier of this mutation did not exhibit an APC resistance phenotype. However, since the mutation was observed in 2/43 patients with venous thrombosis, its exact role as a putative risk factor for venous thrombo-embolism had not been properly investigated, so far. In the present study the factor V Arg306
→Gly mutation was not detected among relatively young thrombotic patients but was found in 1/208 healthy controls. Hence, our findings provide evidence against factor V Arg306→Gly mutation being a risk factor for venous thrombophilia.
As discussed above, the findings from the present study diminish the likelihood that factor V Cambridge and factor V Arg306
→Gly are risk factors for venous thrombophilia. However, it must be mentioned that although our results do not suggest a risk associated with these mutations, this possibility cannot be completely ruled out, given the statistical uncertainty of the data, which yield a relative risk of zero for venous thrombosis, but with a 95% confidence interval of 0–10·6 (considering both mutations together) and of 0–78 (considering each mutation sepa-rately). For the same reason, an analysis of a large number of healthy individuals is recommended before we conclude that both mutations are indeed rare polymorphisms.
Venous thromboembolism is a prevalent clinical condition currently understood as an entity in which genetic factors play an important role (Da¨hlback, 1997; Seligsohn & Zivelin, 1997). In fact, during this decade several genetic risk factors for venous thrombosis have been discovered and included in the diagnostic investigation of prothrombotic states (Poortet al, 1996; Da¨hlback, 1997; Seligsohn & Zivelin, 1997). To establish which genetic abnormalities are of clinical sig-nificance and should be screened in thrombotic patients is therefore an important step for the management of venous thromboembolism. Our present data do not support factor V
Cambridge or factor V Arg306
→Gly as risk factors for thrombophilia, suggesting that screening for these muta-tions is probably not recommended for patients suffering from venous thromboembolic disease.
ACKNOWLEDGMENTS
We are grateful to Amelia G. Araujo and Walter J. Cassinelli for excellent technical assistance. This research was partly supported CNPq, FUNDHERP and FAPESP.
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