Genetic modulation of the FVLeiden/normal FV ratio and risk of venous thrombosis in factor V Leiden heterozygotes

Authors

  • O. SEGERS,

    1. Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, the Netherlands
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  • P. SIMIONI,

    1. Department of Cardiologic, Thoracic and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy
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  • D. TORMENE,

    1. Department of Cardiologic, Thoracic and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy
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  • C. BULATO,

    1. Department of Cardiologic, Thoracic and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy
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  • S. GAVASSO,

    1. Department of Cardiologic, Thoracic and Vascular Sciences, 2nd Chair of Internal Medicine, University of Padua Medical School, Padua, Italy
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  • J. ROSING,

    1. Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, the Netherlands
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  • E. CASTOLDI

    1. Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, the Netherlands
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Elisabetta Castoldi, Department of Biochemistry, Maastricht University, P.O. Box 616, 6200 MD Maastricht, the Netherlands.
Tel.: +31 43 3884160; fax: +31 43 3884159.
E-mail: e.castoldi@maastrichtuniversity.nl

Abstract

Summary. Background and Objectives: The factor (F) V Leiden mutation causes activated protein C (APC) resistance by decreasing the susceptibility of FVa to APC-mediated inactivation and by impairing the APC-cofactor activity of FV in FVIIIa inactivation. However, APC resistance and the risk of venous thromboembolism (VTE) vary widely among FV Leiden heterozygotes. Common F5 genetic variation probably contributes to this variability. Patients/methods: APC resistance was determined in 250 FV Leiden heterozygotes and 133 normal relatives using the prothrombinase-based assay, which specifically measures the susceptibility of plasma FVa to APC. The effects of 12 F5 single-nucleotide polymorphisms (SNPs) on the normalized APC sensitivity ratio (nAPCsr) and on FV levels were determined by multiple regression analysis. Results: In FV Leiden heterozygotes, VTE risk increased with increasing nAPCsr, reaching an odds ratio (OR) of 9.9 (95% confidence interval [CI] 1.2–80.5) in the highest nAPCsr quartile. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G (K830R, T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, R2 haplotype), increased the nAPCsr in FV Leiden heterozygotes, but not in their normal relatives. Most of these effects could be attributed to a shift in the FVLeiden/normal FV ratio. Four FV Leiden heterozygotes with extremely high nAPCsr turned out to be pseudo-homozygotes, i.e. they carried a deleterious mutation on the non-Leiden allele. Conclusions: In FV Leiden heterozygotes, the prothrombinase-based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FVLeiden/normal FV ratio in plasma.

Introduction

Coagulation factor (F) V is a large glycoprotein that is produced in the liver and circulates in plasma as a single-chain inactive precursor. After limited proteolysis by thrombin or FXa, FV is converted to its active form (FVa), which acts as an essential non-enzymatic cofactor of FXa in prothrombin activation. FVa is inactivated by activated protein C (APC) via proteolytic cleavage at Arg306, Arg506 and Arg679 [1,2]. Although Arg506 is the kinetically preferred APC-cleavage site, cleavage at Arg306 is required for complete loss of cofactor activity. Therefore, FVa is mainly inactivated via initial cleavage at Arg506, which yields an intermediate with ∼40% activity, followed by slow cleavage at Arg306 [1,2]. However, FVa can also be inactivated by a single slow cleavage at Arg306 [2]. Cleavage at Arg306 is greatly stimulated by the APC-cofactor protein S [3]. APC also cleaves non-activated FV [1] and Arg506-cleaved FV acts as a cofactor of APC in the inactivation of FVIIIa [4].

Approximately 5% of the Caucasian population carries a FV gene (F5) mutation causing the substitution of Arg506 by a Gln (FV Leiden) [5], which originated from a single and relatively recent mutational event [6]. Owing to the absence of the APC-cleavage site at Arg506, FV(a)Leiden is less susceptible to APC-mediated inactivation [2] and cannot be converted into a functional APC-cofactor for FVIIIa inactivation [4]. As a consequence, plasma from FV Leiden carriers shows a decreased anticoagulant response to the addition of APC in the activated partial thromboplastin time (APTT) test, a condition known as APC resistance [7]. This plasma phenotype (reviewed in [8]) is a prevalent risk factor for venous thromboembolism (VTE), VTE risk increasing progressively at increasing APC resistance [9,10]. However, APC resistance and VTE risk vary widely among FV Leiden carriers, suggesting the existence of common phenotype modulators.

In previous studies, we have shown that FV Leiden heterozygotes in whom the normal (non-Leiden) FV allele is not expressed because of a null mutation (FV Leiden pseudo-homozygotes [11]) have considerably higher APC resistance [12] and VTE risk [13] than the average FV Leiden heterozygote. Although FV Leiden pseudo-homozygosity is a rare condition, common F5 genetic variation with small effects on gene expression may also modulate the APC resistance of FV Leiden heterozygotes by shifting the ratio between FVLeiden and normal FV in plasma. In addition, missense single-nucleotide polymorphisms (SNPs) could modify the APC-susceptibility of FVa or the APC-cofactor activity of FV. Although a few F5 SNPs, including F5 R2 [14–17], F5 T2 [16], F5 409 G/C [17] and F5 6533 T/C [18], have been reported to modulate APC resistance in FV Leiden carriers, most F5 genetic variation has remained unexplored. Moreover, in previous studies APC resistance was measured with the classic APTT-based assay [7], which has several determinants besides FV [19] and is therefore not optimally sensitive to FV-related effects.

In the present study, we have genotyped a large group of FV Leiden heterozygotes (and their normal relatives) for several SNPs tagging all common and a few uncommon F5 haplotypes, and analyzed the effects of F5 genetic variation on APC resistance determined with a test that exclusively probes the susceptibility of FVa to APC.

Patients and methods

Study population

Ninety-seven families segregating the FV Leiden mutation were identified at Padua University Hospital (Italy) via a proband that underwent thrombophilia screening because of a personal or family history of thrombosis. Probands with FV Leiden and all available relatives including spouses were invited to donate blood for genetic and functional studies. In total, 461 individuals (204 males and 257 females), aged between 4 and 82 years, agreed to participate: 153 did not carry the FV Leiden mutation, 282 were heterozygous carriers and 26 were homozygous carriers. Sixteen female participants were on oral contraceptives (OC) and four on hormone-replacement therapy (HRT) at the time of blood collection. A history of VTE (defined as deep vein thrombosis, pulmonary embolism or venous thrombosis at unusual sites) was present in 49 participants and 20 of these were still on oral anticoagulant treatment (OAT) at the time of blood collection. The present study was conducted in accordance with the Declaration of Helsinki and informed consent was obtained from all participants. Parents provided informed consent for their children younger than 18 years.

Only FV Leiden heterozygotes and their normal (i.e. not carrying FV Leiden) relatives were included in the present study. The specific characteristics of these genotype subgroups are shown in Table 1.

Table 1.   Characteristics of the study population
FV Leiden genotypenGender (M/F)OC/HRT use (n)VTE (n)OAT (n)FV (%)
  1. n, number of subjects; M/F, male/female ratio; OC, oral contraceptives; HRT, hormonal replacement therapy; VTE, venous thromboembolism (deep vein thrombosis, pulmonary embolism or thrombosis at unusual sites); OAT, oral anticoagulant therapy. *One of the VTE patients turned out to be pseudo-homozygous for FV Leiden.

Normal13365/6834196.9 ± 22.6
Heterozygous250106/1441527*1198.6 ± 23.5
All383171/21218311298.0 ± 23.2

Blood collection and plasma preparation

Blood was drawn by venipuncture in 3.8% sodium citrate (1:9 vol/vol). Platelet-poor plasma was obtained by centrifugation at 2000 g for 15 min. Aliquots were snap-frozen and stored at −80 °C until use. Buffy coats were stored at −20 °C for later DNA isolation.

Prothrombinase-based APC resistance assay

APC resistance was determined using a prothrombinase-based assay [20], essentially as described previously [12]. Briefly, plasma was diluted 1:1000 in HNBSA/Ca2+ buffer (25 mm Hepes, 175 mm NaCl, 3 mm CaCl2, 5 mg mL−1 bovine serum albumin [BSA], pH 7.7 at room temperature) and FV was activated by adding 3 nm thrombin and 25 μm phospholipid vesicles (DOPS/DOPC 10/90 mol/mol). After 10-min incubation in the absence or presence of 0.45 nm APC, the (residual) activity of FVa was quantified via a prothrombinase-based assay. The outcome of the assay was expressed as the ratio of the FVa cofactor activities determined in the presence and absence of APC (FVa+APC/FVa−APC). The FVa ratio of each sample was normalized to the FVa ratio of a FV Leiden heterozygous plasma pool (prepared by pooling plasma from three FV Leiden heterozygotes) measured in parallel, yielding a normalized APC sensitivity ratio (nAPCsr). All samples were measured at least in duplicate.

In contrast to the more commonly used APTT-based and thrombin generation-based APC resistance assays, this assay exclusively measures the susceptibility of plasma FVa to APC-mediated inactivation. As a consequence of the high dilution of plasma, the prothrombinase-based nAPCsr is not influenced by variations in plasma FX, prothrombin or protein S levels [20]. The inter-assay variation of the nAPCsr, determined by measuring the FV Leiden heterozygous plasma pool in triplicate on 38 separate plates, was 5.5%.

As FV(a) is the limiting factor in the assay, the rate of prothrombin activation in the absence of APC is a measure of the plasma FV concentration. This was expressed as a percentage of normal pooled plasma measured in parallel.

Genetic analysis

Genomic DNA was isolated from buffy coats using the QiaAmp Blood Mini Kit (QIAGEN, Venlo, the Netherlands). FV Leiden genotypes were determined using a 5′ nuclease (TaqMan®) assay (Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands). Carriership of FV Cambridge (R306T) [21] and FV Liverpool (I359T) [22] was ascertained by PCR-mediated amplification of F5 exons 7 and 8 followed by restriction analysis with BstNI and BsrI, respectively. F5 sequencing in the FV Leiden pseudo-homozygotes was performed as previously described [23].

To identify a minimal set of SNPs representing all common genetic variation in the F5 gene, we searched the HapMap (http://www.hapmap.org) and SeattleSNPs (http://pga.mbt.washington.edu) databases for F5 SNPs with minor allele frequencies (MAF) ≥ 0.05 in the Caucasian population. SNPs with r2 ≥ 0.6 were considered to belong to the same haplotype, and only one (preferably exonic missense) SNP per haplotype was included in the analysis. Besides these common SNPs, a few missense SNPs with MAF < 0.05 were also included, two of which had been previously associated with APC resistance or thrombosis (i.e. 6533 T/C [18] and 1628 G/A [24–26]). These criteria led to the selection of 12 non-redundant SNPs (Table 2). SNP genotyping was performed by PCR-mediated amplification followed by restriction analysis, except for F5 6755 A/G (rs6027), for which a TaqMan® assay (Applied Biosystems) was used (Table 2).

Table 2. F5 SNP genotypes in the normal and FV Leiden heterozygous individuals
 Normal individuals (n = 133)FV Leiden heterozygotes (n = 246*)
SNP IDGene locationNucleotide positionAmino acid changeRestriction enzymeMAF HapMapMAF Seattle SNPsFV Leiden haplotypeNormal (n)Hetero (n)Homo (n)Normal (n)Hetero (n)Homo (n)
  1. SNP, single-nucleotide polymorphism; EX, exon; IVS, intron; MAF, (reported) minor allele frequency. cDNA numbering according to Jenny et al. 1987 [38]. *The four FV Leiden pseudo-homozygotes were excluded. The allele distributions of the SNPs located upstream of intron 6 were determined by genotyping 23 unrelated FV Leiden homozygotes from the same geographical region as the study population. Mutagenic primer used to introduce the restriction site. §Genotyped using TaqMan® assay.

rs2269648Promoter−426 G/ABstN I0.087G (89.1%)/A (10.9%)90403170697
rs3753305IVS 1+1409 G/CMfe I0.4750.457G (76.1%)/C (23.9%)3467327313142
rs6028EX 2327 A/GQ51QHpyCH4 III0.3000.396A (91.3%)/G (8.7%)863413158817
rs6019EX 3409 G/CD79HMbo I0.0500.022G (100%)123100226200
rs6020EX 101628 G/AR485KBsmA I0.009G (100%)1294024240
rs4524 (T2)EX 132663 A/GK830RHpy188 I0.2580.214A (100%)75544176700
rs9332695EX 132833 A/TT887SPle I0.0080.068A (100%)1330024240
rs1046712EX 133943 C/AL1257IBsrD I0.167C (100%)108232184620
rs9332608EX 134300 C/TP1376SHinf I0.0490.023C (100%)1285023880
rs6007EX 134679 A/CQ1502AEar I0.017A (100%)1312024510
rs9332701EX 246533 T/CM2120THpyCH4 IV0.0350.045T (100%)12112024060
rs6027 (R2)EX 256755 A/GD2194G§0.0400.045A (100%)114190232140

Statistical analysis

Data are reported as mean ± standard deviation. Correlations are expressed as Pearson’s coefficients (r). Means were compared with Student’s t-test, carrier frequencies with the χ2 test. The risk of VTE in the different quartiles of nAPCsr was assessed by logistic regression analysis and expressed as odds ratio (OR) and 95% confidence intervals (95% CI). The effects of F5 SNPs on FV levels and nAPCsr were analyzed by multiple linear regression analysis. Effects are reported as unstandardized regression coefficients (B), representing the absolute change in FV level or nAPCsr per mutated allele. Statistical analysis was performed using SPSS 15.0 (SPSS Inc., Chicago, IL, USA).

Results

Population characteristics

Twenty normal individuals were excluded from the study because plasma or DNA was not available for analysis. Of the 282 FV Leiden heterozygotes, 32 were excluded for various reasons, including a previous diagnosis of pseudo-homozygous APC resistance (n = 7), no plasma or DNA available (n = 23) and plasma/DNA mismatch as a result of liver transplantation (n = 1) or homonymy (n = 1). The demographic and clinical characteristics of eligible participants, as well as their plasma FV levels, are reported in Table 1. VTE cases were more numerous among FV Leiden carriers than in normal relatives (P = 0.006). FV levels were similar in the two groups.

Prothrombinase-based APC resistance and VTE risk

The distributions of the prothrombinase-based nAPCsr in the FV Leiden heterozygotes and their normal relatives are shown in Fig. 1. The nAPCsr (normalized against a FV Leiden heterozygous plasma pool) was 0.22 ± 0.03 (% CV = 15.9%) in normal individuals and 0.99 ± 0.11 (% CV = 11.5%) in FV Leiden heterozygotes. Interestingly, four FV Leiden heterozygotes had nAPCsr values typical of homozygotes (Fig. 1). As they also had reduced FV levels, pseudo-homozygous APC resistance was suspected; F5 gene sequencing indeed revealed a deleterious mutation in each of them. Two siblings, a 28-year-old female (nAPCsr 1.72, FV level 65%) and a 23-year-old male (nAPCsr 1.69, FV level 69%), carried a previously described [27] insertion of an extra adenine (A) in a poly-A tract in exon 15 (F5 5123–5127 insA), predicting a frame-shift and premature termination of translation at codon 1659. The third subject, a 72-year-old female (nAPCsr 1.71, FV level 65%), showed a previously reported [28] nonsense mutation in exon 13 (F5 C2308T; Arg712Stop), whereas the last one, a 47-year-old male (nAPCsr 1.64, FV level 80%), carried a novel mutation in exon 15 (F5 C5237T) predicting the substitution of a serine by a leucine at position 1688. These individuals were excluded from further analysis because their high nAPCsr could be attributed to non-expression of the normal F5 allele. After the exclusion of these four subjects, the nAPCsr in FV Leiden heterozygotes was 0.98 ± 0.07 (% CV = 7.3%).

Figure 1.

 Distribution of the prothrombinase-based normalized activated protein C sensitivity ratio (nAPCsr) in normal individuals and in factor (F) V Leiden heterozygotes. The prothrombinase-based APC resistance assay was performed as described under Patients and methods. The APCsr of each individual was normalized against the APCsr of a FV Leiden heterozygous plasma pool (nAPCsr). Open circles represent patients with a history of venous thromboembolism (VTE). The four FV Leiden heterozygotes with an extremely high nAPCsr (> 1.6) turned out to be pseudo-homozygotes.

Factor V Leiden heterozygotes with a history of VTE (n = 26, open symbols in Fig. 1) had higher nAPCsr than healthy FV Leiden heterozygotes (1.02 ± 0.07 vs. 0.97 ± 0.07, P = 0.004). Moreover, when FV Leiden heterozygotes were stratified according to their nAPCsr, VTE risk increased progressively from the lowest to the highest quartile (Fig. 2). Compared with the lowest nAPCsr quartile, which was taken as a reference, the OR for VTE was 1.4 (95% CI 0.5–3.6) in the second quartile, 2.8 (95% CI 0.8–9.4) in the third quartile and 12.9 (95% CI 1.6–102.9) in the fourth quartile. After adjustment for age and gender, this trend persisted and the OR for VTE in the highest nAPCsr quartile was 9.9 (95%CI 1.2–80.5).

Figure 2.

 The effect of the prothrombinase-based normalized activated protein C sensitivity ratio (nAPCsr) on venous thromboembolism (VTE) risk in factor (F) V Leiden heterozygotes. FV Leiden heterozygotes were stratified according to their prothrombinase-based nAPCsr. The odds ratio (OR) for VTE in each nAPCsr quartile was calculated by logistic regression analysis. The lowest nAPCsr quartile was taken as a reference (OR = 1.0 by definition).

Determinants of prothrombinase-based APC resistance

To investigate the effects of F5 genetic variation on the prothrombinase-based nAPCsr, all eligible normal and FV Leiden heterozygous individuals were genotyped for the FV Cambridge (R306T) and Liverpool (I359T) mutations as well as for the 12 selected F5 SNPs (Table 2). No carriers of FV Cambridge or FV Liverpool were observed. The genotype distributions for the F5 SNPs in the normal and FV Leiden heterozygous individuals are reported in Table 2. For most SNPs, all FV Leiden heterozygotes were either homozygous for the more common allele or heterozygous, but never homozygous for the minor allele (Table 2), strongly suggesting that the minor alleles of these SNPs always reside on the non-Leiden allele. The reason for this is that the FV Leiden mutation originally occurred on a F5 haplotype containing the common alleles of all investigated SNPs [6] (Table 2). However, for the SNPs located at the 5′ end of the gene (rs2269648, rs3753305 and rs6028) all three genotypes were represented among FV Leiden heterozygotes (Table 2), indicating that the minor alleles of these SNPs can also reside on the FV Leiden allele. This is explained by the presence of a recombination hot-spot in intron 6 [29], which allows relatively frequent recombination between the FV Leiden mutation (located in exon 10) and the 5′ portion of the gene (Fig. 3). For all SNPs located upstream of the recombination hot-spot, the proportion of FV Leiden alleles bearing the minor allele of that SNP, estimated by genotyping 23 unrelated FV Leiden homozygotes from the same geographical region as the study population, is reported in Table 2.

Figure 3.

 Linkage disequilibrium map of the F5 gene. The map was generated with the Genome Variation Server of the SeattleSNPs database (http://pga.mbt.washington.edu) using the genotype data from the 60 unrelated individuals of Caucasian descent belonging to the HapMap-CEU panel. Top: schematic representation of the F5 gene (oriented from right to left). Middle: Chart showing the genotypes of each individual for each SNP. Each row in the chart represents an individual and each column represents a single-nucleotide polymorphism (SNP). SNPs are identified by their rs numbers and by their positions on the gene (according to SeattleSNPs numbering). Genotypes are colour-coded: blue indicates homozygosity for the common allele, red heterozygosity and yellow homozygosity for the rare allele. Gray means that the genotype could not be determined. Bottom: linkage disequilibrium between SNPs. Each dot represents the correlation coefficient (r2) between the two SNPs it connects. The degree of linkage disequilibrium is colour-coded: red indicates complete linkage disequilibrium (r2 = 1); orange, yellow, green and blue indicate progressively decreasing degrees of linkage disequilibrium; and white indicates no linkage at all (r2 = 0). This graphical representation reveals the existence of two distinct haploblocks in the F5 gene, separated by a recombination hot-spot which can be roughly mapped to intron 7 (previously assigned to intron 6 [29]).

Multiple regression analysis including age, gender, the F5 SNPs and FV levels as independent variables indicated that common F5 genetic variation is a major determinant of the prothrombinase-based nAPCsr in FV Leiden heterozygotes but not in normal individuals (Table 3). The minor alleles of several F5 SNPs increased the nAPCsr in FV Leiden heterozygotes, including 327G (B = 0.023/allele, P = 0.032), 409C (B = 0.035/allele, P = 0.034), 2663G (B = 0.025/allele, P = 0.014), 6533C (B = 0.099/allele, P < 0.001) and 6755G (B = 0.089/allele, P < 0.001), whereas 3943A showed a trend to decrease the nAPCsr (B = −0.019/allele, P = 0.065). In contrast, none of the F5 SNPs had an effect on the nAPCsr in normal individuals, except for F5 3943 C/A whose minor allele showed a trend to increase the nAPCsr (B = 0.013/allele, P = 0.093).

Table 3.   Determinants of the prothrombinase-based normalized activated protein C sensitivity ratio (nAPCsr) in normal individuals and in factor (F) V Leiden heterozygotes Thumbnail image of

Among demographic variables, age and gender did not affect the nAPCsr. FV levels modestly but significantly increased the nAPCsr in FV Leiden carriers (B = 0.001/1% increase in FV, P < 0.001), but not in normal individuals.

Effects of F5 SNPs on FV levels

Since the effects of F5 genetic variation on the nAPCsr of FV Leiden heterozygotes might be mediated by effects on gene expression that modify the FVLeiden/normal FV ratio in plasma, we also investigated the relationship between F5 SNPs and FV levels (Table 4). In a regression model including age, gender, FV Leiden and the F5 SNPs as independent variables, F5 327G (B = −7.2%/allele, P = 0.023), 2663G (B = −7.9%/allele, P = 0.004) and 6755G (B = −10.3%/allele, P = 0.020) all significantly decreased FV levels. F5 409C (B = −8.4%/allele, P = 0.084) and 6533C (B = −8.6%/allele, P = 0.147) also decreased FV levels, but their effects did not reach statistical significance. A good correlation between the effects (B) of these SNPs on FV levels and on the nAPCsr of FV Leiden heterozygotes (r = −0.746, P = 0.148) was observed. Whether the effect of F5 409 G/C on FV levels is due to this missense SNP or to the genetically linked F5−426 G/A promoter SNP, whose minor allele also showed a trend to decrease FV levels (B = −6.7%/allele, P = 0.063), is still a matter of debate [30].

Table 4.   Determinants of factor (F) V levels Thumbnail image of

In line with previous reports [31–33], FV levels increased with age (B = 0.28%/year, P < 0.001) and were not affected by gender or by the FV Leiden mutation.

Discussion

Although FV Leiden is a well-established risk factor for VTE, most carriers of this mutation never develop thrombosis [34], whereas others experience severe and recurrent thrombotic events. Presently, we cannot predict which FV Leiden carriers will develop VTE and risk assessment is largely based on personal or a family history of thrombosis, in combination with precipitating conditions. On the other hand, APC resistance varies widely among different FV Leiden carriers and may be a good indicator of VTE risk, as a dose-response relationship between APTT-based APC resistance and VTE risk has been previously demonstrated [9,10]. Among all potential modulators of APC resistance, FV represents an obvious candidate as it is both a substrate and a cofactor of APC [8].

F5 genetic variation may modulate the APC resistance phenotype of FV Leiden heterozygotes by modifying the FVLeiden/normal FV ratio in plasma and/or by affecting the APC-susceptibility of FVa or the APC-cofactor activity of FV in FVIIIa inactivation. In particular, all F5 variants that decrease the expression of the non-Leiden allele shift the FVLeiden/normal FV ratio in favour of FVLeiden and are therefore expected to increase APC resistance in FV Leiden heterozygotes. This is well illustrated by the four FV Leiden heterozygotes with extremely high nAPCsr (Fig. 1), who all carried mutations preventing the expression of the non-Leiden allele and leading to the exclusive presence of FVLeiden in plasma. However, this ‘pseudo-homozygosity’ for FV Leiden is a rare condition [35].

Although the role of common F5 genetic variation as a modulator of APC resistance in FV Leiden heterozygotes has been addressed before by ourselves and others [14–18], the present study offers several advantages over earlier studies, namely: (a) a larger population of FV Leiden heterozygotes (n = 250), which affords greater statistical power to detect infrequent/weak modulators; (b) the systematic coverage of F5 genetic variation (all non-redundant common SNPs and a few uncommon missense SNPs), including the often neglected 5′ portion of the gene; and (c) the use of an APC resistance assay which is highly specific for FV. This prothrombinase-based assay measures exclusively the susceptibility of plasma FVa to APC-mediated inactivation and is not affected by other coagulation factors or inhibitors nor by acquired factors such as OC/HRT use or OAT [20].

In the present study, we show for the first time that the risk of VTE in FV Leiden heterozygotes increases with increasing prothrombinase-based nAPCsr, with an age- and gender-adjusted OR of ∼10 for the highest vs. the lowest nAPCsr quartile. This indicates that the overall susceptibility of plasma FVa to APC-catalyzed inactivation is a determinant of VTE risk in FV Leiden heterozygotes. Whether this is also the case in the absence of FV Leiden remains unclear, because only four normal individuals in our population had experienced VTE. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G (K830R, tagging the T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, tagging the R2 haplotype), increased the prothrombinase-based nAPCsr of FV Leiden heterozygotes (Table 3). Interestingly, these same alleles were also associated with reduced FV levels (Table 4), suggesting that their effects on the nAPCsr are mediated by a shift in the FVLeiden/normal FV ratio rather than by a qualitative change in the APC-susceptibility of the FVa encoded by the non-Leiden allele. In fact, a strong correlation was observed between the effects of these SNPs on FV levels and their effects on the nAPCsr of FV Leiden heterozygotes. In addition, none of these SNPs affected the nAPCsr in normal individuals, in line with the expectation for SNPs that only affect gene expression without modifying the functional properties of FVa.

Although the minor alleles of most SNPs included in the present study always reside on the non-Leiden allele, the minor alleles of the SNPs located upstream of the recombination hot-spot may also reside on the FV Leiden allele at low (but not negligible) frequencies (Table 2). This introduces a complication for SNPs, such as F5 327 A/G and 409 G/C, which affect FV expression levels. In fact, the minor alleles of these SNPs will increase the FVLeiden/normal FV ratio (and hence the nAPCsr) when they are present on the non-Leiden allele, but they will decrease the FVLeiden/normal FV ratio (and the nAPCsr) when they are present on the FV Leiden allele. As the phase between these SNPs and the FV Leiden mutation could only be determined in few individual families in our population, we could not correct for these opposite effects in the statistical analysis. As a consequence, the effects of the 327G and 409C alleles on the nAPCsr of FV Leiden heterozygotes presented in Table 3 may be under-estimated. In addition, it should be realized that the effect of a particular SNP on the nAPCsr may be modulated by other SNPs residing on the same F5 allele and that different combinations of SNPs located upstream and downstream of the recombination hot-spot may be present/prevalent in different populations.

F5 SNPs that shift the FVLeiden/normal FV ratio in favour of FV Leiden are expected to decrease not only the susceptibility of FVa to APC, but also the APC-cofactor activity of FV in FVIIIa inactivation, because FVLeiden expresses hardly any APC-cofactor activity [4,36]. If this is the case, these SNPs should increase the APC resistance of FV Leiden heterozygotes in other APC resistance assays as well. Accordingly, three of the genetic modulators identified in the present study (i.e. the F5 R2 haplotype [14–17], 409C [17] and 6533C [18]) have previously been associated with increased APTT-based APC resistance in FV Leiden heterozygotes. In contrast, the F5 T2 haplotype has been reported to decrease the APTT-based APC resistance in FV Leiden heterozygotes [16]. The reason for this apparent discrepancy is that the F5 T2 haplotype (also known as the ‘G-allele’) encodes several amino acid changes in the B domain of FV, which make FVT2 a more efficient cofactor of APC in FVIIIa inactivation [37]. This gain in APC-cofactor activity tends to mitigate APC resistance determined with the APTT-based assay and is apparently large enough to compensate for the increased FVLeiden/normal FV ratio. Finally, the F5 327 A/G SNP is a novel and very common (reported allele frequency 0.300) modulator of APC resistance in FV Leiden heterozygotes. As this SNP had the smallest effects on FV levels and nAPCsr, it might have escaped detection in previous studies.

The minor allele of another common F5 SNP (3943 C/A) showed a strong trend to decrease the prothrombinase-based nAPCsr, but no association with FV levels. Although it is a missense SNP, the predicted amino acid substitution (Leu1257Ile) resides in the B domain, which is removed upon FV activation, making it unlikely that this amino acid change directly affects the APC-susceptibility of FVa. Therefore, its effect on APC resistance may be mediated by another genetically linked variant. Unfortunately, no information is available on the effect of this common SNP on the nAPCsr determined with the APTT-based assay.

Given the correlation between the prothrombinase-based nAPCsr and VTE risk in FV Leiden heterozygotes, we wondered whether the F5 SNPs that affect the nAPCsr are also associated with the occurrence of VTE. In spite of the small number of VTE patients in our population, the minor alleles of all F5 SNPs that increased the prothrombinase-based nAPCsr in FV Leiden heterozygotes (except for 6533 T/C) also showed a tendency to be enriched in VTE patients as compared with non-patients (data not shown), but differences were not statistically significant. In particular, F5 409C carriers were almost three times more represented (19.2% vs. 6.8%, P = 0.049) and F5 6755G (R2 haplotype) carriers two times more represented (11.5% vs. 5.0%, P = 0.139) among FV Leiden heterozygotes with VTE than among those without VTE.

In conclusion, we have shown that in FV Leiden heterozygotes the overall APC-susceptibility of plasma FVa is a marker of VTE risk and is modulated by several common F5 SNPs that shift the FVLeiden/normal FV ratio in plasma. Given their high prevalence in the general population, these SNPs are likely to contribute substantially to the large inter-individual variability in APC resistance (and VTE risk) observed among FV Leiden carriers.

Acknowledgements

This work was supported by a VIDI grant (nr. 917-76-312, to E. Castoldi) from the Dutch Organisation for Scientific Research (NWO).

Disclosure of Conflict of Interest

The authors state that they have no conflict of interest.

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