Novel genetic risk factors for venous thrombosis; a haplotype-
based candidate gene approach
Uitte de Willige, S.
Citation
Uitte de Willige, S. (2007, May 23). Novel genetic risk factors for venous
thrombosis; a haplotype-based candidate gene approach. Hemostasis and
Thrombosis Research Center, Department of Hematology, Faculty of Medicine,
Leiden University. Retrieved from https://hdl.handle.net/1887/11970
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis
in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/11970
Note: To cite this publication please use the final published version (if
applicable).
Chapter 1
General Introduction
Thrombosis
The development of thrombotic disorders in humans is one of the most common causes of morbidity and mortality in the Western world. There are two distinct forms of thrombosis; venous thrombosis, which occurs in the venous system of low flow and pressure and arterial thrombosis, which occurs in the high flow and pressure arterial system. The most important differences between venous and arterial thrombosis are the composition of the blood clot or thrombus (fibrin rich in venous and platelet rich in arterial) and the presence of vascular damage (atheroma) in arterial thrombosis.
Venous thrombosis
Venous thrombosis is a common disorder with an annual incidence of 1-3 per 1000 individuals.1-4 The key event in venous thrombosis is the formation of a thrombus, most often in the deep veins of the legs. Rarely, the thrombus occurs in other veins (upper extremities, liver, cerebral sinus, retina, mesentery). Once formed, a thrombus may either develop into an obstructive clot, impeding the blood flow, or become unstable and form smaller emboli, obstructing the small arterioles of the lungs (pulmonary embolism), occasionally with fatal outcome. Other complications are recurrent events and morbidity due to the development of leg ulcers (postthrombotic syndrome). Venous thrombosis may develop spontaneously, but may also occur in reaction to an acute and short-lasting risk. Treatment with anticoagulants is generally effective but is associated with an increased risk of bleeding (hemorrhage).
Arterial thrombosis
Arterial thrombosis (atherothrombosis) is characterized by a sudden disruption (rupture or erosion/fissure) of an atherosclerotic plaque, which leads to platelet activation and thrombus formation. Plaque formation results from the progressive accumulation of lipids and fibrous elements in the large arteries (atherosclerosis), a diffuse process that starts early in life and progresses largely asymptomatically through adult life.5 Atherosclerosis is the underlying condition that later in life results in events leading to coronary artery disease (myocardial infarction, stable or unstable angina pectoris, ischemic stroke, transient ischemic attack) or peripheral arterial disease. Arterial thrombosis can be seen as a chronic disorder related to the slowly increasing severity of atherosclerosis with acute symptoms due to the blocking of the blood flow to a vital organ (heart, brain).
Risk factors for venous thrombosis
In 1856, Rudolph Virchow famously postulated that thrombosis was the result of at least one of three underlying etiologic factors: stasis of the blood flow, changes in the composition of the blood (hypercoagulability), and vascular endothelial
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damage.6 Whereas the known risk factors for arterial thrombosis fall mainly in the third group (vascular endothelial damage), most of the known risk factors for venous thrombosis fall in the first (stasis) and second group (changes in blood coagulability). Currently an additional classification is made, which includes genetic and non-genetic (acquired or environmental) risk factors. Non-genetic risk factors are those risk factors that a person can be exposed to during lifetime. These factors often relate to stasis and include advanced age, immobilization, surgery, trauma, plaster cast, or to hypercoagulability, as with lupus anticoagulans, malignancy, use of female hormones, pregnancy and puerperium. Additionally, a first thrombotic event is a strong risk factor for recurrent events.7,8 In contrast to most acquired risk factors, genetic risk factors are present lifelong and are almost exclusively associated with hypercoagulability. Current disease models assume that an individual will develop venous thrombosis when the cumulative effect of genetic and environmental risk factors (and their interactions) exceeds a certain threshold value, which makes venous thrombosis a multicausal disease.9-12
Genetic risk factors
During the past 40 years, several genetic risk factors for venous thrombosis have been identified. Some of these risk factors are rare and genetically very heterogeneous and include deficiencies of antithrombin,13,14 protein C15,16 and protein S.17 These deficiencies all lead to ‘loss of function’ of the protein, i.e.
absence or impaired function of the protein. Another mutation, ABO blood group, also influences the risk of venous thrombosis. Compared to blood group O carriers, which lack the enzymes glycosyltransferase (blood group A) and galactosyltransferase (blood group B), non-O carriers have an increased risk for venous thrombosis.18 These individuals also have higher levels of Factor VIII and higher levels of von Willebrand factor (the carrier protein of Factor VIII), which is probably the cause of the thrombotic risk associated with non-O blood group.19 Other risk factors are single nucleotide polymorphisms (SNPs) that are relatively common in the population and lead to ‘gain of function’ of the protein, i.e.
improvement of the function of the protein or higher plasma concentrations of the protein. Examples of such risk factors are the Factor V Leiden mutation (FVL),20,21 a G>A transition on position 1691 of the Factor V gene, and the Prothrombin 20210A mutation.22 There are also some plasma phenotypes, which may be either acquired or genetic, that are associated with thrombotic risk. These include elevated levels of Factors II,22 VIII,19 IX23 and XI,24 fibrinogen,25,26 homocysteine,27 thrombin activatable fibrinolysis inhibitor (TAFI),28 and a reduced sensitivity for APC in the absence of FVL.29 Another, at most very mild risk factor is the 677C>T polymorphism in the gene encoding 5, 10-Methylenetetrahydrofolate reductase (MTHFR).30 The prevalence and relative risk of these risk factors are shown in Table 1 (reviewed in refs 31 and 32).
Table 1 Prevalence and relative risk of risk factors for venous thrombosis
Risk Factor Prevalence* Relative Risk
Deficiencies of
Antithrombin < 1% >8
Protein S < 1% >8
Protein C < 1% 7
Mutations
Factor V Leiden 5%-8% 3 to 8 in heterozygotes
50 to 80 in homozygotes
Prothrombin 20210A 2%-3% 3
Non-O blood group 53%-55% 2 to 4
MTHFR -677TT 10%-12% 1.2
Elevated levels of
Fibrinogen (>5g/L) 1% 4
Factor II (>115 U/ml) 31% 2
Factor VIII (≥150IU/dL) 25% 5
Factor IX (>P90) 10% 3
Factor XI (>P90) 10% 2
Homocysteine (>18 mmol/L) 5%-10% 2
TAFI (>P90) 10% 2
Other
Reduced APC sensitivity 10% 4
without FV Leiden (< P10)
*Prevalence in the Caucasian (Northern Europe) population; the relative risks for antithrombin, protein S and protein C are rough estimations; P90: 90th percentile as measured in the control population; P10: 10th percentile as measured in the control population.
Missing genetic risk factors
About 20-30% of consecutive patients with a first event of venous thrombosis report at least one first-degree relative with venous thrombosis.33,34 In addition, there are many families that stand out by the clustering of venous thrombotic events. A heritability of 50%-60% was estimated for venous thrombosis, suggesting that genes play an important role in the development of venous thrombosis.35-37
Familial thrombophilia is considered to be an oligogenetic disease, in which at least two genetic defects segregate in the family.12,38 At least two of the known genetic risk factors (FVL, Prothrombin 20210A and deficiencies of antithrombin, protein C and protein S) were found in 13% of thrombophilia families, whereas only one of these genetic risk factors was found in 60% of the families. In 27% of the families none of the genetic risk factors was found, although these families are thought to carry multiple genetic defects.39 Furthermore it has been reported that elevated plasma levels of fibrinogen,25 factor VIII,19 factor IX,23 and factor XI24 are associated with an increased thrombosis risk and show a high degree of heritability.35,40-43 However, variations in the genes coding for these proteins that influence these
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levels are still unknown. Therefore, we hypothesized that there are genetic determinants of venous thrombosis that have not yet been identified.
Aim of this thesis
The aim of this thesis was to identify novel genetic risk factors for deep venous thrombosis. Key objectives were to identify genes that contribute to the genetic susceptibility to venous thrombosis and to identify the functional (i.e. disease causing) mutations in these genes. More detailed knowledge of risk factors for venous thrombosis and understanding their interactions leads to better insight in the mechanisms that lead to venous thrombosis. In addition, it would improve clinical management of thrombosis patients (and prevention of thrombosis in predisposed individuals) through individualized risk profiling.
Strategy
One way to find new candidate genes is to perform a genome scan in affected individuals, e.g. in families or sib pairs. A genome scan encompasses the unbiased approach of linkage analysis and subsequent fine mapping. The basic idea is that within a family a genetic marker will be linked to the disease-causing variant, due to its proximity. In this method, the genome is covered with a grid of markers to search for a high degree of allele sharing between the affected family members.
After that, the original region is narrowed, and the disease gene is identified.
The research described in this thesis comprises a different approach, namely the haplotype-based analyses of candidate genes in a large association (case-control) study. The basic idea of such association studies is that the marker studied is either the disease causing variant itself, or is in close proximity and linked to the disease causing variant in the population.
The strategy followed in this thesis is shown in Figure 1. Candidate genes were selected on the basis of theoretical knowledge of the proteins encoded by the genes and their putative role in blood coagulation. Subsequently, the common haplotypes of the candidate gene were identified. A haplotype (Haploid Genotype) is a set of closely linked genetic markers present on one chromosome which tend to be inherited together. Haplotypes may have particular significance with regard to functionality or as markers for unknown functional variants, and in order to better characterize the role of a candidate gene, it is best to explore the full haplotypic information, in stead of single nucleotide polymorphisms (SNPs) only.44
As marker alleles haplotype-tagging SNPs (htSNPs) were used. These are SNPs that uniquely identify or ‘tag’ the common haplotypes. By using these htSNPs, only a small set of SNPs was needed to capture the full haplotypic information. Information on the haplotypes and their htSNPs was obtained from the database of
SeattleSNPs.45 SeattleSNPs focuses on identifying, genotyping, and modelling the associations between SNPs in candidate genes and pathways that underlie inflammatory responses in humans. To identify these SNPs, SeattleSNPs resequenced two series of DNA samples; 23 samples of European American descent and 24 samples of African American descent.
Figure 1 Followed approach of the research described in this thesis.
After selection of the htSNPs, a large case-control study, the Leiden Thrombophilia Study (LETS),46,47 was genotyped for these htSNPs. Subsequently, haplotypes were assigned to the subjects and the association was calculated between the haplotypes and venous thrombosis risk and potential intermediate phenotypes. The main hypothesis in our candidate gene approach was that relatively common functional variants exist which influence the risk of venous thrombosis and are the product of unique mutational events in a founder haplotype, and that the frequency of such haplotypes will be increased in a patient population. The magnitude of this increase will dependent on the risk associated with the causal polymorphism and its frequency in the risk haplotype and on the frequency of the risk haplotype itself.
To identify the causal polymorphism of a risk haplotype, the relevant gene(s) were
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resequenced in homozygous carriers of this haplotype to identify additional SNPs that are specific to this haplotype or that contribute to a subhaplotype. By selecting homozygous patients, the chance of finding such SNPs is maximized. When a functional variant was found, the biological mechanism behind this variant was investigated. It had been demonstrated previously that an earlier discovered common genetic risk factor for venous thrombosis (Factor V Leiden) would have been found with this strategy when the Factor V gene was treated as a candidate gene in a population-based association study with a case-control design.48
Candidate genes
The candidate genes investigated in this thesis were selected on the basis of theoretical knowledge of the proteins encoded by the genes. In normal hemostasis there is a dynamic balance between the pro- and anticoagulant and pro- and antifibrinolytic systems. Since the biochemistry of blood coagulation has been well defined,49-51 there are many potential candidate genes known that might confer a risk for deep venous thrombosis, although several of these potentials have already been investigated in detail (reviewed in refs 52 and 53).
Endothelial cell Protein C Receptor (EPCR)
In chapter 2, we assessed whether haplotypes of the endothelial cell protein C receptor (EPCR) gene or soluble plasma EPCR (sEPCR) levels were associated with the risk of deep venous thrombosis. EPCR functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation of protein C by the thrombin-thrombomodulin complex.54 EPCR binds to both protein C and activated protein C (APC) with high affinity.55 sEPCR circulates in plasma and inhibits APC anticoagulant activity.54,55 EPCR is an attractive candidate gene, since it is a small gene of only four exons with a clear haplotype structure and without recombination within the gene. In addition, other abnormalities in the protein C pathway have been found that increase the risk of venous thrombosis, such as deficiencies of protein C15,16 and protein S,17 and low levels of circulating APC.56
Fibrinogen
The candidate genes explored in chapter 3 were the three genes of the fibrinogen cluster (fibrinogen alpha (FGA), beta (FGB) and gamma (FGG)). Fibrinogen is an essential component of the haemostatic system, being the precursor of fibrin, the end-product of blood coagulation.57 Of all the components of the coagulation system, elevated fibrinogen levels have been most consistently associated with vascular disorders.58 In addition, genetic variants of fibrinogen (dysfibrinogenemias) have been found in patients with thrombosis and a prolonged thrombin time.59,60 The analysis of the fibrinogen genes was more complicated, since the three genes are clustered on the long arm of chromosome 4 with a high degree of linkage disequilibrium between the genes.
In chapter 3.1 the association between haplotypes of the three genes, total fibrinogen levels, fibrinogen γ' levels, resulting from alternative processing of the FGG pre-mRNA, and the risk of deep venous thrombosis was investigated. We found that haplotype 2 of FGG (FGG-H2) was associated with an increased risk for venous thrombosis, with decreased fibrinogen γ' levels and decreased fibrinogen γ'/total fibrinogen ratios and proposed that FGG-H2 tagging SNP 10034C>T is the causal variation in this haplotype. This finding was further investigated in chapter 3.2.
In chapter 3.3 we aimed to assess whether FGG 3’-end SNPs were associated with the risk of venous thromboembolism in the African-American population and to replicate the association of SNP 10034C>T (FGG-H2) with the risk of venous thrombosis in the Caucasian population.
Elevated fibrinogen levels have also been associated with arterial disease.61 The association of the four common haplotypes of the FGG gene with the risk of myocardial infarction and with total fibrinogen levels was studied in chapter 3.4. In chapter 3.5 the role of fibrinogen γ' and FGG haplotypes in ischemic stroke was determined.
Selectins
In chapter 4, members of the selectin family (P-selectin, E-selectin, L-selectin and the P-selectin ligand) have been studied. Deep venous thrombosis is associated with a significant inflammatory response in the vein wall and the thrombus.62 At sites of vascular injury or inflammation, leukocytes form initial attachments to vascular endothelial cells, then roll on the vessel wall and finally arrest, spread and migrate from endothelial cells into the surrounding tissues. The rolling, adhesion and binding of leukocytes from the bloodstream to the blood vessel wall is mediated by the selectin family.63 Recent data support the role of the selectins in hemostasis and thrombosis, It was demonstrated that overexpression of P-selectin can induce a pro- coagulant state,64 that circulating microparticles bearing the P-selectin ligand deliver tissue factor to the growing platelet thrombus,65 and that procoagulant microparticles can partially correct the hemostatic defect in hemophilic mice.66 In addition, elevated levels of soluble P-selectin have been shown to be associated with an increase in venous thrombosis risk.67-69
Like the three genes of the fibrinogen cluster, the genes for E-selectin (SELE), L- selectin (SELL) and P-selectin (SELP) are clustered. They are located next to each other on the long arm of chromosome 1, 2.4 kb upstream of the gene for coagulation Factor V. The analyses of these genes were more complicated, since there is a recombination hotspot present within SELP and the P-selectin ligand (PSGL-1). Therefore, SELP was analysed in two parts, upstream (SELPup) and downstream (SELPdown) from the recombination hotspot, whereas for PSGL-1 the
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SNPs were analyzed separately. We additionally adjusted for linkage between the selectin genes and the factor V Leiden mutation. Furthermore, interactions between PSGL-1 SNPs and selectin SNPs, and the association between SELP haplotypes and soluble P-selectin levels were investigated.
Finally, in chapter 5 the findings presented in this thesis are summarized and discussed.
Study populations LETS
The analyses described in chapters 2, 3.1 and 4 of this thesis were performed in the Leiden Thrombophilia Study (LETS); a large population-based case-control study on venous thrombosis. The design of this study has been described in detail elsewhere.46,47 In short, patients participating in LETS were identifiedfrom the files at the anticoagulation clinics in Leiden, Amsterdam,and Rotterdam between January 1988 and December 1992. In total, 474 consecutive patients younger than 70 years with an objectively confirmed first episode of deep venous thrombosis (453 in the leg, 21 in the arm) and 474 age- and sex-matched controls were included. Patients with known malignant disorders were excluded. The median time between a thrombotic event and venipuncturewas 19 months (range, 6-68 months). Controls were acquaintances of patients or partners of other patients, without a history of venous thrombosis, no use of coumarin-derivatives for at least three months and no known malignant disorder.
GATE
In chapter 3.3 the Genetic Attributes and Thrombosis Epidemiology (GATE) study was used, a case-control study on risk factors for venous thromboembolism (VTE) in an American population, including subjects of the Caucasian population and subjects of the African-American population.70 In short, patients, aged 18-70 years, were diagnosed between March 1998 and December 2002 with a recent first or recurrent episode of deep venous thrombosis and/or pulmonary embolism. They were hospitalized at two University hospitals in Atlanta, Georgia, USA. Patients with severe illness or with cognitive deficits were excluded. Control subjects were selected from a list of patients who visited the office of one of 10 physicians at a university-affiliated primary care clinic between January 1997 and December 2000.
Controls were frequency matched to patients in age, sex and race. Racial assignment was self-reported. Individuals with a history of VTE, currently taking anticoagulant medication, or with a mental or physical problem were excluded.
Venipuncture of patients and controls was performed at the time of enrolment. From case subjects a second blood sample was obtained during a follow-up appointment, at least one month after completion of anticoagulation therapy and at least three months after the index thrombotic event.
SMILE
In chapter 3.4 the Study of Myocardial Infarctions Leiden (SMILE), another large population-based case-control study was used. Full details of the SMILE study have been described elsewhere.71 In short, patients consisted of 560 Dutch men below the age of 70 years with a first myocardial infarction who were hospitalized in a university orgeneral hospital in Leiden, the Netherlands, between January1990 and January 1996. Control subjects were 646 Dutch men, frequency matched for 10- year age groups to the patients, who had undergone an orthopedic intervention between January 1990 and May 1996 andhad received prophylactic anticoagulants for a short periodafter the intervention. The control subjects were identified in the records of the LeidenAnticoagulant Clinic, but did not have a history of myocardial infarction. For patients, the median time between myocardial infarction and blood collection was 2.6 years (range 0.2-6.0 years). For control subjects, the median time between orthopaedic intervention and blood collection was 2.9 years (range 0.6-6.3 years).
COCOS
In chapter 3.5 the COntrolled study of genetically determined COagulation disorders in patients with transient ischemic attack or ischemic Stroke (COCOS), a case- control study with prospective inclusion of the participants was used. The design and full details of this study have been published elsewhere.72 In short, patients were consecutively recruited with a first ischemic stroke, admitted to the department of Neurology of the Erasmus Medical Center, Rotterdam, the Netherlands. Ischemic stroke was defined as the acute onset of local cerebral dysfunction because of cerebral ischemia with symptoms lasting more than 24 hours. Patients with a definite non-atherosclerotic cause of the stroke, such as mechanical heart valve, endocarditis or dissection, as well as patients above 75 years or using anticoagulants were excluded. Venipunctures were performed in the acute phase of the stroke (7-14 days after the event) and in the convalescent phase of the stroke (three months after the event). Control subjects were neighbours or friends of the patients. They were age- and sex-matched to the patients, but did not have a history of stroke and were not related to the patient. Patients, controls and their parents were born in northern Europe and were of Caucasian race.
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