Resistance to activated protein C due to Factor V R506Q (Factor V Leiden)

David Keeling BSc MD MRCP MRCPath
Consultant Haematologist
Oxford Haemophilia Centre, Churchill Hospital, Oxford.

The Discovery of Resistance to Activated Protein C and Identification of the Mechanism.

The phenomenon of resistance to activated protein C (APC) was discovered only three years ago. Dahlback and colleagues identified a middle aged man with a personal and a family history of thrombosis whose APTT did not show the expected prolongation when exogenous APC was added to his plasma [1]. The same phenomenon was found in several of the patient's relatives. The mechanism was unknown but inheritance of a deficiency of a cofactor for APC was hypothesised. This proved not to be the case and one year later the molecular defect was identified as a point mutation in factor V (FV) [2]. The mutation was a G to A substitution at nucleotide position 1,691. This results in the arginine at position 506 (coding triplet CGA) being replaced by a glutamine (coding triplet CAA). Using the single letter amino-acid code the mutant FV can therefore be written as FV R506Q but is more often referred to as FV Leiden (Figure 1).

Figure 1. The G to A point mutation results in the arginine at the protein C cleavage site being replaced by glutamine.


Factor V Leiden (R506Q)
505 506 507
Arg Arg Gly
AGG C G A GGA
  |  
AGG C A A GGA
Arg Gln Gly

APC is a serine protease which acts as a natural anticoagulant by inactivating FVa and FVllla. Normally FVa is inactivated by an initial cleavage of the peptide bond on the carboxyl side of arginine 506 followed by a second cleavage at arginine 306. The mutant FV is inactivated by cleavage at arginine 306, but this cleavage is ten fold slower without prior cleavage at position 506 which is prevented by the R506Q mutation [3]. Thus the FV Leiden mutation leads to the phenomenon of resistance to the anticoagulant activity of APC.

THE PREVALENCE OF FACTOR V LEIDEN IN THE GENERAL POPULATION.

When different groups and populations were investigated for FV Leiden its prevalence was scarcely believable. In white Europeans and North Americans this mutation is present in approximately 5% of the general population. The C to A point mutation occurs at a CpG dinucleotide (Figure 1), these are mutation hot-spots, and the question arose as to whether the FV Leiden mutation was a common new event or whether a founder effect had spread the disorder in the population. Analysis of a FV polymorphism in the original Nature paper suggested the latter [2]. This is supported by the finding that the mutation is rare in the rest of the world [4].

Why should a mutation which is associated with venous thromboembolism (VTE) have spread so successfully? It is first important to note that even in subjects with the mutation fatal pulmonary embolism is more likely to occur after rather than before the individual has reproduced. Nevertheless it is tempting to speculate that the gene could have offered a selective advantage at some point in the past. Our modern way of life may have increased the risk of thrombosis and in the past the risk of fatal bleeding from post- partum haemorrhage or traumatic injury was higher than today.

THE RISK OF THROMBOSIS ASSOCIATED WITH FACTOR V LEIDEN.

FV Leiden is present in 5% of the population but is found in 20% of all cases of deep vein thrombosis (DVT). In cases of DVT where there is a family history it is found in 50% and it is found in 60% of pregnancy associated thrombosis. The increased risk of thrombosis that it confers has been estimated from The Leiden Thrombophilia Study. The odds ratio for thrombosis was 7 for heterozygotes and 80 for homozygotes [5] (if FV Leiden is found in 5% of a population then approximately 1 in 1,600 of the population will be homozygotes).

Of major importance is the interaction of FV Leiden with other risk factors for thrombosis. It had previously been noted that many kindreds with inherited defects in the natural anticoagulants (protein C, protein S, antithrombin) had variable thrombotic tendencies. Those with a high incidence of thrombosis may well also have had the common FV Leiden mutation in the family. FV Leiden has been shown to present an additional risk factor in hereditary protein S deficiency [6], protein C deficiency [7] and antithrombin deficiency [8]. In the latter case both defective genes can be on the same chromosome (chromosome 1) and the two risk factors can be inherited together in successive generations.

FACTOR V LEIDEN, THE CONTRACEPTIVE PILL AND SCREENING.

Of major importance is the interaction between FV Leiden and the thrombotic risk imposed by the combined oral contraceptive pill (COC). This issue was also addressed by The Leiden Thrombophilia Study. In this analysis the COC had an odds ratio for thrombosis of 4, the FV Leiden mutation an odds ratio of 8 and for both together the odds ratio was 35 [9]. The four fold increase in risk due to the COC in this study, in which a mixture of pills were used, is in keeping with the three fold and six fold increases in risk estimated for pills without and with gestodene or desogestrel (CMO's Update 8). Assuming the risk of VTE in women of this age distribution is 5 per 100,000 (CMO's Update 8) then using these odds ratios (and assuming 5% of the population have FV Leiden) we can construct Table 1. We thus have an estimate of the excess cases of VTE due to use of the COC.

Table 1. Cases of VTE per 100,000 women years

Without COC With COC Excess cases
All women 5 20 15
Normals 4 15 11
Factor V Leiden 30 130 100

VTE = venous thromboembolism, COC = combined oral contraceptive pill


This has raised the question of screening for FV Leiden before prescribing the pill. How the data is presented can have a great effect on interpretation [10], so in Table 2 the risk of thrombosis in women who use the pill for 10 years is presented in three different ways.


Table 2. Risk of VTE for women taking the COC for 10 years

All Normals Factor V Leiden
Absolute increase in risk 0.15% 0.11% 1.00%
Probability of no VTE reduced from 99.95% 99.96% 99.70%
to 99.80% 99.85% 98.70%
NNT for 10 years for one extra event 667 909 100

VTE = venous thromboembolism, COC = combined oral contraceptive pill, NNT=number needed to treat


A women who is heterozygous for the FV Leiden mutation and uses the pill for 10 years has an absolute increase in risk of 1%, her chance of being free of thrombosis being reduced from 99.7% to 98.7%, and 100 women with the mutation would need to take the pill for 10 years each for one extra thrombotic event. These figures are very helpful when considering the individual. In terms of the population, three million women are on the pill in the UK and we can estimate that the pill results in 450 extra episodes of VTE and nine deaths per year (assuming a mortality of 2%). Denying the estimated 150,000 women with FV Leiden the pill would prevent 150 episodes of VTE and three deaths per year. These figures do, however, have one important assumption which is that other methods of contraception do not increase the risk of VTE by having a higher failure rate. If denying women with FV Leiden the pill results in an increase in pregnancies then the overall number of thromboses may increase as pregnancy is a greater thrombotic risk than being on the pill. It is instructive to note that the recent pill scare in the UK resulted in a significant increase in the number of abortions (British Pregnancy Advisory Service).

On balance I would not offer screening to women without a personal or family history of thrombosis. Women with a personal history of thrombosis should not be prescribed the pill. Women with a family history of thrombosis should not be given the pill without screening for all the inherited causes of thrombophilia.

DETECTION OF FACTOR V LEIDEN IN THE LABORATORY.

The initial test for resistance to APC was based on the APTT. Most commonly the APTT is performed with and without the addition of exogenous APC and the two clotting times expressed as a ratio. Resistance to APC can then be defined as a ratio more than 1.96 standard deviations below the mean of the log transformed data. Ideally patients with FV Leiden should be excluded in the subjects used to define the normal range. This test costs approximately [[sterling]]2 and in expert hands it has been reported to have a sensitivity of 90% and a specificity of 95% for the FV Leiden mutation [11]. Such a test would have a negative predictive value of 99% when the prevalence in the population tested is 10% and on this basis it was suggested that DNA testing is unnecessary if the APC resistance test is negative [11]. However, I think there are two problems with this. Firstly, the prevalence in a population of patients selected for investigation may be as high as 50% and if this is the case the negative predictive value would fall to 90%. Secondly, in the routine service laboratory I think the sensitivity and specificity are rather less than those quoted above. I do not think the standard APC resistance test enables us to forgo DNA analysis and recent reports support this [12]. A modification of the APC resistance test is to dilute the patient's plasma in FV deficient plasma. This increases the cost to approximately [[sterling]]4 per test but the sensitivity and specificity for the FV Leiden mutation approach 100% [12, 13].

DNA analysis is simple but relatively expensive at approximately [[sterling]]25 per test. A common method is to amplify the DNA around the mutation site using PCR and then to perform a restriction digest with the enzyme Mnl 1. This recognises the sequence GAGG which is present in normal FV but is lost in FV Leiden (see Figure 1). DNA testing clearly distinguishes normals, heterozygotes and homozygotes.

At present I think a reasonable strategy is to use the modified APC resistance test with FV deficient plasma and to assume a normal result excludes FV Leiden but to confirm the positive results with DNA analysis. This will of course also distinguish heterozygotes and homozygotes. Professor Dahlback does this but also performs the standard APC resistance test in order to identify those subjects with APC resistance not due to an abnormality in FV [13]. This is of considerable interest for research purposes, however, I feel such patients are uncommon and do not think this is necessary in a service laboratory. As to which patients with thrombosis should be tested I think the standard criteria for thrombophilia screening apply [14], the main indications being age less than 45 years, a family history, thrombosis at an unusual site or recurrent thrombosis.

SUMMARY

APC resistance due to FV Leiden is the most important medical discovery of the last decade. It is extremely common, occurring in approximately 5% of the population, and is a significant risk factor for thrombosis. Thrombosis is a common and often serious clinical problem and FV Leiden plays an important contributory role in a large proportion of cases, including those related to the contraceptive pill and pregnancy. All doctors must have access to reliable diagnostic testing which can be achieved by a combination of coagulation and genetic tests.

REFERENCES

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14. Standard Haematology Practice, Blackwell Scientific Publications, Oxford, 1991. Ed. Roberts B. Chapter 11 The investigation and management of thrombophilia p112-127.