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Keywords:

  • ABO blood group;
  • factor VIII;
  • quantitative trait loci;
  • von Willebrand factor

Abstract

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Summary.  Elevated factor (F)VIII levels contribute to venous thrombotic risk. FVIII levels are determined to a large extent by levels of von Willebrand factor (VWF), its carrier protein which protects FVIII against proteolysis. VWF levels are largely dependent on ABO blood group. Subjects with blood group non-O have higher VWF and FVIII levels than individuals with blood group O. Apart from ABO blood group no genetic determinants of high FVIII levels have been identified, whereas clustering of FVIII levels has been reported within families even after adjustment for ABO blood group and VWF levels. We investigated the FVIII and VWF loci as possible quantitative trait loci (QTL) influencing FVIII and VWF levels. Two sequence repeats in the FVIII gene and three repeats in the VWF gene were typed in 52 FV Leiden families. Multipoint sib-pair linkage analysis was performed with the MAPMAKER/SIBS program. FVIII levels adjusted for VWF levels and age, and VWF levels adjusted for ABO blood group and age, were used for this linkage analysis. No linkage of FVIII levels to the FVIII locus was found, whereas we found evidence that the VWF locus contains a QTL for VWF levels [maximum likelihood no dominance variance lod score = 0.70 (P = 0.04) and non-parametric Z-score = 1.92 (P = 0.03)]. About 20% of the total variation in VWF levels may be attributed to this VWF locus.

Elevated levels of coagulation factor (F)VIII have been reported to be associated with the risk of venous thrombosis [1], recurrent venous thrombosis [2], coronary heart disease [3] and stroke [4]. Individuals with levels of FVIII above 150 IU dL−1, which are found in over 10% of the general population, have a 6-fold increased risk of thrombosis compared with individuals with levels below 100 IU dL−1[1]. In a population-based case-control study on venous thrombosis (Leiden Thrombophilia Study, LETS) blood group non-O, elevated von Willebrand factor (VWF) levels and elevated FVIII levels were all positively associated with thrombotic risk in univariate analysis [1]. In multivariate analysis, only FVIII levels remained a strong risk factor, suggesting that VWF and blood group exert their thrombotic risk via final effector FVIII. The relationship between ABO blood group type and plasma levels of FVIII and VWF has been recognized for a long time [5,6]. Numerous studies have shown that subjects with blood group non-O have increased FVIII and VWF levels compared with subjects with blood group O. Ørstavik et al. reported that 30% of the genetic variance of VWF levels was attributable to the effect of ABO blood group type [5]. Significant linkage between the ABO locus and VWF antigen level was found in the families of the Genetic Analysis of Idiopathic Thrombosis (GAIT) Study [7]. Blood group A, B and H(O) oligosaccharide structures have been found on VWF, that may affect the clearance of VWF and of the FVIII/VWF complex [8]. The effect of ABO blood group on FVIII levels is mediated by VWF. VWF serves as the carrier protein of FVIII and protects FVIII against proteolysis, so there is a strong correlation between VWF levels and FVIII levels (for a review see [9]). Unbound FVIII is unstable with a half-life of 1 h [10].

Apart from ABO blood group no genetic determinants of high FVIII levels have been identified. However, clustering of FVIII levels within families has been reported even after adjustment for ABO blood group and VWF levels [11–13], suggesting that there are additional genetic determinants of FVIII levels. Recently, de Lange et al. assessed heritabilities of FVIII and VWF in a classic twin study [14]. They reported that genetic factors contributed 61% and 75% to the variation in FVIII and VWF levels, respectively [14]. These values are similar to the heritabilities (57% for FVIII and 66% for VWF) found earlier in the twin study of Ørstavik et al. [5]. In families of the GAIT study Souto et al. found heritabilities of 40% for FVIII and 32% for VWF [15]. In a large pedigree of French Canadian descent with protein C deficiency heritabilities of 59% for FVIII levels and 44% for VWF levels were found [16].

Elevated FVIII levels seem to persist over time [17] and are in general not attributable to an acute-phase reaction [17,18]. Furthermore O'Donnell et al. reported that only about 50% of patients with high FVIII levels also had high VWF levels [17]. This again suggests that there are causes of high FVIII levels that are independent of VWF levels and blood group. To date no sequence variations in the FVIII gene have been found that could account for the variation in FVIII level [19]. In the promoter region of the VWF gene, four polymorphisms have been identified, but their association with VWF levels has not been convincingly demonstrated [20–23].

In order to find genetic determinants of FVIII levels we investigated by a sib-pair linkage analysis the FVIII and VWF loci as possible quantitative trait loci (QTL) influencing FVIII and VWF levels.

Subjects and methods

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Study population

For our analysis we studied 59 families (47 families derived from a case-control study and 12 thrombophilic families) with a proband with venous thrombosis and the FV Leiden mutation. The recruitment of the families has been described elsewhere [24,25].

Briefly, forty-seven probands were derived from a population-based case-control study on venous thrombosis, the Leiden Thrombophilia Study (LETS) [24,26]. Cases were 474 consecutive patients younger than 70 years with a first episode of deep vein thrombosis. The presence of FV Leiden was detected in 92 cases (84 heterozygotes and eight homozygotes). Of these 92 cases, all homozygous patients (n = 8) and the heterozygous patients who resided in the area of the Leiden University Medical Center were invited (n = 40). One homozygous proband was not willing to participate in the study. All siblings, parents and children of the remaining 47 probands were invited for the study. These 47 families are referred to as ‘LETS families’.

Twelve families were thrombophilic families of symptomatic FV Leiden carriers who were referred to our center for diagnostic work-up for venous thrombophilia [25]. These probands had a positive family history of venous thrombosis (in addition to the proband at least two symptomatic relatives) and did not have deficiencies of protein C, protein S or antithrombin. Relatives (first, second or third degree) of these 12 probands were invited. These 12 families are referred to as ‘thrombophilic families’.

The probands and family members were not selected on the basis of their FVIII or VWF levels, so there was no need to correct for ascertainment bias.

Biochemical measurements

Blood was collected into tubes containing 0.1 volume 0.106 mol L−1 trisodium citrate. Plasma was prepared by centrifugation for 10 min at 2000 × g at room temperature and stored at −70 °C.

ABO blood group phenotypes were determined in plasma by routine serum blood grouping procedures. FVIII antigen was measured by a sandwich type enzyme linked immunosorbent assay (ELISA) using two monoclonal antibodies directed against the light chain of FVIII [23]. VWF antigen was measured by an in-house developed ELISA with use of polyclonal antibodies. Plasma levels were expressed in units per deciliter (U dL−1). By definition 1 mL pooled normal plasma contains 1 unit.

Genotyping

High molecular weight DNA was isolated from leukocytes and stored at 4 °C. DNA samples were genotyped for five polymorphic markers by polymerase chain reaction (PCR). Three markers in the VWF gene, located on chromosome 12p13, were typed: A (GT)n repeat in the promoter of VWF (GDB Accession ID 63847, M1) [27] and two tetranucleotide repeats in intron 40 (GDB accession ID 61879 [M2] and GDB accession ID 56220 [M3]) [28,29]. Two markers were typed in the FVIII gene, located on chromosome Xq28: A (CA)n repeat in intron 13 (GDB accession ID 155465, M4) [30] and a (GT)n(AG)n repeat in intron 22 (M5) [31]. Primer sequences were the same as described before [27–31] with two modifications: For each marker one primer was labeled at the 5′-end using fluorescent dyes and the other primer was extended at the 5′-end with the sequence-GTGTCTT. Many DNA polymerases can catalyze the addition of a single nucleotide (predominantly adenosine) to the 3′ ends of double-stranded PCR products [32]. Extension of a primer with the above mentioned sequence-GTGTCTT-encourages this adenosine addition leading to double-stranded PCR products that will all have the same size. So, each allele is represented by a single peak and not by two peaks one base pair apart. The PCR mixtures consisted of 25 pmol of both oligonucleotides, 200 µmol L−1 of each dNTP, a variable amount of MgCl2 (1.5–2.5 mmol L−1), 1 × PCR buffer II (Perkin-Elmer) and 0.75 units AmpliTaq Gold DNA polymerase (Perkin-Elmer) in a total volume of 25 µL. The reactions were performed in a T3 Thermocycler (Biometra, Göttingen, Germany). The PCR conditions were as follows: 10 min initial denaturation at 95 °C, followed by 33 cycles of 1 min at 94 °C, 1 min at 50 °C (M4)/55 °C (M5)/59 °C (M1, M2, M3) and 1 min at 72 °C. A final extension was performed at 60 °C for 45 min. Diluted amplification products of one individual were pooled and analyzed by capillary electrophoresis using the ABI Prism 310 Genetic Analyzer (Applied Biosystems). Fragment sizing was performed by use of GeneScan 2.1 software. The heterozygosity for the VWF markers was 0.73 for M1, 0.77 for M2 and 0.78 for M3. The heterozygosity for the FVIII markers was 0.63 (M4) and 0.57 (M5). Incompatibilities between the haplotypes of parents and children were searched for with the PedCheck 1.1 program [33] and inconsistencies were resolved.

Statistical analysis

Allele frequencies were estimated via gene counting using founder genotypes (n = 76). A founder is a person whose parents are not in the pedigree. Founders are assumed to be unrelated.

Prior to the linkage analysis multiple linear regression analysis was performed to adjust for the influence of age and ABO blood group on VWF levels. ABO blood group was entered into the regression model as a dichotomous variable (blood group O vs. blood group non-O) and age as a continuous variable. FVIII levels were adjusted by multiple linear regression for VWF levels and age. To investigate the genetic influence on FVIII and VWF levels these adjusted values were used.

Fifty-two (12 Thrombophilic families and 40 LETS families) of the 59 families contained sibships with ≥two sibs with available plasma and DNA material. These 52 families yielded 95 nuclear (father, mother and children) families with ≥2 sibs with available genotype and phenotype information. In view of calculation times, children with phenotype values around the family mean were omitted (n = 11) in families with more than eight children. For three children without phenotypic information genotypes were included in the analysis enabling a better reconstruction of genotypes of missing parents.

QTL multipoint linkage analysis was performed in the 95 nuclear families. In general, QTL linkage analysis approaches focus on identity by descent (IBD) sharing at a locus between relatives. Two alleles at a single locus are IBD if they are copies of a common ancestral allele. At every position on the genome a pair of relatives may share 0, 1 or 2 alleles IBD. The underlying idea of QTL analysis is that relatives who share more alleles IBD at a marker locus linked to the QTL are expected to have more similar trait values than relatives who share fewer alleles. We performed QTL analyses in sib-pairs with the MAPMAKER/SIBS 2.0 program [34]. Analysis of VWF was performed by the maximum likelihood (ML) method assuming no dominance variance and by the non-parametric method (see [34,35] for more information on the statistics). For FVIII analysis the ML method and the non-parametric method were used [34,35]. Analysis of X-linked data (in case of FVIII) in MAPMAKER/SIBS assumes that the difference in phenotype between two sibs is a function of whether 0 or 1 alleles are inherited IBD from the mother. When more than two sibs occurred in a sibship, a weighting (two/number of sibs) was applied. To calculate P-values lod scores (log10 of the likelihood ratio) were converted to a χ2 statistic by multiplying by 2ln10, which follows a χ2 distribution with one degree of freedom.

Testing for differences in effect of the VWF locus between the thrombophilic families and LETS families was performed with a likelihood ratio test statistic. In this test, a model with separate data set dependent variances in sib differences was compared with a model with identical variances in both data sets.

The additive genetic variance of the VWF QTL was calculated according to Sham [35]. The population variance was calculated in founders (n = 72) in the extended families.

Results

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the 52 extended families (12 thrombophilic and 40 LETS families), the individual mean FVIII level was 142 U dL−1 (range, 48–355) and the mean VWF level was 123 U dL−1 (range, 41–298). Plasma FVIII levels were not different between FV Leiden carriers (mean = 141 U dL−1) and non-carriers (mean = 142 U dL−1). Also VWF levels did not differ between FV Leiden carriers (mean VWF = 124 U dL−1) and non-carriers (mean VWF = 121 U dL−1). FVIII levels and VWF levels were higher in subjects with blood group non-O {mean FVIII = 155 U dL−1[95% confidence interval (CI) 149–161], mean VWF = 136 U dL−1 (95% CI 131–141)} compared with subjects with blood group O [mean FVIII = 117 U dL−1 (95% CI 110–124), mean VWF = 100 U dL−1 (95% CI 94–106)]. In this data set of 52 families multiple linear regression analysis was performed to adjust VWF levels for the influence of ABO blood group and age and to adjust FVIII levels for VWF levels and age. About 30% of the variation in VWF levels was explained by ABO blood group and age (R2 = 0.27) and about 70% of the variation in FVIII levels was explained by VWF levels and age (R2 = 0.72). The adjusted FVIII and VWF levels were used for linkage analysis. The 52 extended families yielded 95 nuclear families that were used for linkage analysis. The total number of sibling pairs was 583, but when a weighting factor was applied to reduce the information coming from sibships with more than two sibs, the total information content of the data set was equivalent to 242 sibling pairs.

To examine whether the region encoding the FVIII gene contains a QTL for FVIII levels, we undertook a linkage analysis of this region. All subjects were genotyped for two polymorphic sites within the FVIII gene. Results of the multipoint linkage analysis are shown in Table 1. There is no significant evidence for a QTL at the FVIII locus. When the analysis was performed for the thrombophilic and LETS families separately also no evidence for linkage was obtained.

Table 1.  Results of multipoint sib-pair linkage analysis for FVIII locus and FVIII level
 Maximum likelihood (ML) lod scoreNon-parametric Z-score
  1. FVIII levels adjusted for VWF levels and age were used for analysis. A weighting of two/(number of sibs) was applied.

All sibships (n = 95)00.883 (P = 0.189)
Thrombophilic sibships (n = 46)0.118 (P = 0.461)0.295 (P = 0.384)
LETS sibships (n = 49)01.019 (P = 0.154)

Three polymorphic sites in the VWF gene were typed to test the VWF locus as QTL for VWF levels. Results are shown in Table 2. Multipoint linkage analysis in all nuclear families resulted in a lod score of 0.70 (P = 0.036) with the ML no dominance variance method and a Z-score of 1.92 (P = 0.027) with the non-parametric method. Separate analyses of thrombophilic and LETS sibships resulted in lod scores of 0.77 and 0.15, respectively. We tested whether there was a significant difference in the contribution of this locus in the two data sets but this was not the case.

Table 2.  Results of multipoint sib-pair linkage analysis for VWF locus and VWF level
 ML no dominance variance lod scoreNon-parametric Z-score
  1. VWF levels adjusted for ABO blood group (O vs. non-O) and age were used for analysis. A weighting of two/(number of sibs) was applied.

All sibships (n = 95)0.70 (P = 0.036)1.92 (P = 0.027)
Thrombophilic sibships (n = 46)0.77 (P = 0.030)1.37 (P = 0.085)
LETS sibships (n = 49)0.15 (P = 0.200)1.29 (P = 0.099)

The population variance, as calculated in the founders, of VWF levels adjusted for ABO blood group (O vs. non-O) and age was 1747. The additive genetic variance of the VWF QTL was 469. This means that the VWF locus is responsible for 27% of the variation in adjusted VWF levels. Regression analysis already showed that ABO blood group and age explain 27% of the total variation in VWF levels. Of the remaining 73% of the total variation that is not explained by ABO blood group and age, 27% can be attributed to the VWF locus. So, the VWF locus explains 20% of the total variation in VWF levels in this population.

Discussion

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

High FVIII levels are a common risk factor for venous thrombosis. The mechanisms that lead to high FVIII levels are not clear. An important determinant of FVIII level is its carrier protein VWF, a polymeric glycoprotein possessing ABO blood group antigens. FVIII levels are highly correlated with VWF levels and VWF levels are correlated with ABO blood group. After adjustment for these known influences we examined whether FVIII and VWF levels are linked to the FVIII and VWF loci. Our data suggest that the VWF locus is a QTL for VWF levels (lods = 0.70), but we did not find evidence for a QTL effect of the FVIII locus on FVIII levels.

It may be debated whether a lod score of 0.70 represents evidence for linkage. In a genome-wide search for linkage, when no a priori candidate regions have been identified, stronger statistical evidence may be required. The present study, however, specifically targeted two candidate regions, namely the FVIII locus as QTL for FVIII levels and the VWF locus as QTL for VWF levels. Therefore the stringent correction for multiple testing that is common to a genome-wide search is not required here.

We performed a linkage analysis in which we studied allele sharing in related individuals (sibs), based on the idea that relatives with similar trait values are expected to share more alleles IBD at a marker locus linked to the QTL. For this approach it is necessary to select highly polymorphic markers to enable a good estimation of the IBD sharing. To estimate the IBD sharing as good as possible we included more than one marker per locus. A linkage analysis, like the one we performed, should not be confused with an association analysis in which the association between a specific functional polymorphism and a trait value is determined. By association analysis genetically determined differences in plasma levels for some components of the blood coagulation system have been found before. Polymorphisms in the promoter region of the genes for protein C [36], PAI-1 [37], β-fibrinogen [38], FXII [39], FVII [40] and thrombin activatable fibrinolysis inhibitor (TAFI) [41], the HR2 haplotype of FV [42] and a variation in the 3′-untranslated region of the prothrombin gene [43] have been reported to be associated with protein levels. In contrast to these association studies, we did not look at association between marker and levels.

We found no evidence for linkage of FVIII levels to the FVIII locus. However, the presence of rare functional variations, which are not sufficiently represented in our study population, cannot be excluded. Up to now, no sequence variations have been found in the promoter and the 3′-untranslated region of the FVIII gene that might lead to high FVIII levels [19]. Although the FVIII gene is large and sequence variations may be present in the gene itself, these data support our finding that the FVIII locus is not linked to FVIII levels. In the same set of 12 thrombophilic families that was used in the present study, Kamphuisen et al. investigated correlations of FVIII levels between first degree relatives [12]. FVIII levels were found to be correlated in siblings and in mother–son pairs, but not in mother–daughter, father–daughter and father–son pairs [12]. Especially the absence of a correlation in father–daughter pairs, in which the paternal X chromosome is always passed to the daughter, argued against an X-linked determinant of FVIII levels.

In theory, a gain of function mutation in the FVIII-binding domain of VWF may lead to high FVIII levels. However, sequence analysis in 13 thrombosis patients with high FVIII levels did not show any novel mutations in this binding domain [44]. Also no association of FVIII levels with two polymorphisms in the region coding for the FVIII-binding domain of VWF was found [23].

So, sequence variations that are associated with high FVIII levels have not been reported up to now. On the other hand, numerous deleterious mutations in the FVIII gene have been identified that cause the severe reduction or complete absence of FVIII levels in patients with the X-linked bleeding disorder hemophilia A (for a review see [45]). Similarly several mutations in the VWF gene have been reported which cause reduced VWF and concomitant FVIII levels in von Willebrand disease (VWD) (for a review see [46]).

Controlling for ABO blood group we found evidence that VWF levels are linked to the VWF locus. This VWF locus would explain 20% of the total variation in VWF levels. Most likely this QTL is the VWF gene itself, but the causal genetic variation could also be in the neighborhood of the VWF gene. Four common polymorphisms in the promoter of the VWF gene (−1793C/G, −1234C/T, −1185 A/G and −1051G/A) have been described, which are in strong linkage disequilibrium with each other [20]. In 261 blood donors with blood group O an association between VWF levels and these promoter polymorphisms was reported [21,22]. After stratification for age this association remained in subjects ≤40 years of age but the association was no longer present in subjects above 40 years of age [21,22]. Kamphuisen et al. investigated the same promoter polymorphisms in 301 male thrombosis patients and 301 age-matched controls and did not find an association of genotype with VWF levels [23]. So, the presence of an association of these promoter polymorphisms with VWF levels has not been convincingly demonstrated. Apart from mutations that cause VWD, no other sequence variations in the VWF gene or the 5′- and 3′-untranslated region have been described so far that influence VWF levels. Our results suggest that there is a QTL for VWF levels in the VWF region. Additional investigations are needed to find the causal genetic variations either in the VWF gene itself or in its neighborhood.

Further research may also focus on other genes that could affect FVIII and VWF levels, e.g. possible modifiers that are involved in the expression of FVIII and VWF levels. Such a modifier locus involving lineage-specific expression of a glycosyltransferase that affects VWF levels was recently found in a mouse model [47]. Also genes involved in the secretion and clearance of the proteins may be investigated. For instance, mutations in the ER-golgi intermediate compartment protein ERGIC-53, involved in the transport from ER to Golgi of a specific subset of secreted proteins, were found to be associated with combined FV and FVIII deficiency [48].

In conclusion, we did not find linkage of FVIII levels to the FVIII locus. However, we did find evidence that the VWF locus contains a QTL for VWF levels. This locus would explain 20% of the total variation in VWF levels, which is comparable with the effect of ABO blood group on VWF levels. Causal genetic variations remain to be identified.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by grant no. 95.001 from the Netherlands Thrombosis Foundation. We thank Ted Koster, who interviewed and collected blood samples of the LETS patients, Ank Schreijer who performed data management, and Hans de Ronde and Thea Visser who performed several of the assays described in this paper. We also wish to thank all patients and their family members who kindly participated in our study.

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  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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