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 4
Selectin haplotypes and the risk of
venous thrombosis; influence of linkage
disequilibrium with the Factor V Leiden mutation
Shirley Uitte de Willige, Hans L. Vos, Marieke C.H. de Visser, Jeanine J.
Houwing-Duistermaat, Frits R. Rosendaal and Rogier M. Bertina Submitted for publication
Abstract
Background Selectins (E-, L- and P-selectin) and their most important counter- receptor P-selectin glycoprotein ligand (PSGL-1) facilitate the interaction of platelets, leukocytes and endothelial cells at inflammatory sites. Selectin polymorphisms/haplotypes have been associated with cardiovascular disease. We investigated the association between haplotypes (H) of these four genes and venous thrombosis (VT) risk. We additionally explored the effect of linkage disequilibrium (LD) with the nearby Factor V Leiden mutation (FVL). Furthermore, interactions between PSGL-1 polymorphisms and selectin polymorphisms, and the association between P-selectin haplotypes and P-selectin levels were investigated.
Methods and Results In the Leiden Thrombophilia Study (LETS), subjects were genotyped for 24 polymorphisms by TaqMan or PCR-RFLP, detecting all common haplotypes in four blocks. P-selectin was analysed in two blocks, upstream (SELPup) and downstream (SELPdown) of the recombination hotspot. In E-selectin and L- selectin, none of the haplotypes was associated with VT risk. In SELPup, H2-carriers had a 1.3-fold increased risk (95% confidence interval (CI): 1.0-1.7), whereas H4- carriers had a 1.4-fold decreased risk (95%CI: 0.5-1.0). In SELPdown, H2-carriers had a 1.3-fold increased risk (95%CI: 1.0-1.7). Because of LD with FVL we subsequently excluded all FVL-carriers and all risks disappeared (all odds ratios to 1.0). Mutual adjustment within a logistic regression model resulted in disappearance of the risks for the SELP haplotypes, whereas FVL risk remained.
Conclusions After adjustment for LD with FVL, none of the selectin haplotypes was associated with VT risk, showing that the increased risks of the selectin haplotypes were a reflection of the effect of FVL on thrombosis risk.
Introduction
The initial attachment and subsequent movement of leukocytes and platelets to vascular surfaces is mediated by the selectins, a family of three vascular cell adhesion molecules (CD62).1,2 This family of type I membrane proteins includes E- selectin, L-selectin and P-selectin, encoded by SELE, SELL and SELP, respectively.
Recent data support a role of selectins and their ligands in hemostasis and thrombosis.3,4 In mouse models, P-selectin and E-selectin seem to be critical for the recruitment of leukocytes to venous thrombi5 and overexpression of P-selectin in mice induces a pro-coagulant state.6 Celi at al. reported that P-selectin can induce tissue factor (TF) expression in human monocytes.7 Furthermore, there seems to be a major role for P-selectin and PSGL-1 in the accumulation of TF in the thrombus and the subsequent generation of fibrin via the recruitment of TF bearing microparticles and leukocytes.4 In addition, elevated plasma levels of soluble P- selectin have been shown to be associated with an increase in venous thrombosis risk.8-10
P-selectin is stored in platelet alpha granules, as well as in Weibel-Palade bodies of endothelial cells and is translocated to the surface when these cells are activated.11 P-selectin and the endothelial E-selectin are essential for leukocyte attachment to, and movement on, the monolayer of active vascular endothelial cells,12 and both play a role in the accumulation of inflammatory cells and fibrin generation after venous thrombosis.5,13 L-selectin, which is constitutively expressed at the leukocyte cell surface, is critical for the interaction of lymphocyte homing to the lymphatic organs. In addition, it triggers the adhesion of circulating leukocytes on activated endothelial cells, thereby amplifying inflammatory reactions.14 In these processes the most well-defined and biologically important counter receptor on leukocytes and platelets is the P-selectin glycoprotein ligand-1 (PSGL-1).15,16
The genes encoding the three selectins are localized in a cluster of 160 kb on the long arm of chromosome 1, 1q24-q25, closely linked to the gene for coagulation factor V.17,18 SELE contains 14 exons and spans over 11 kb of DNA (610 amino acids), while SELL is larger, extending over 20 kb, but consisting of just 9 exons (372 amino acids).18 SELP (830 amino acids) spans >50 kb and contains 17 exons, most of which encode structurally distinct domains.17 SELP is located just upstream of the gene for coagulation Factor V. All genes are transcribed in the same direction.
Each of the selectins contains an N-terminal C-type lectin domain, followed by an epidermal growth factor (EGF)-like motif, a series of short consensus repeats, a transmembrane domain and a cytoplasmic tail.19 The PSGL-1 gene maps on chromosome 12q24 and contains two exons and one intron with the complete coding sequence residing in exon 2 (412 amino acids), in total spanning approximately 13 kb.16,20
All four genes contain many single nucleotide polymorphisms (SNPs) and there are recombination hotspots present within the SELP and PSGL-1 genes.21,22 The majority of studies on polymorphisms or haplotypes of the selectin genes focused on arterial disease and did not include the entire gene cluster.23-29 The present study was aimed at investigating the association between haplotypes of the three genes of the selectin cluster and their main counter-receptor and the risk of deep venous thrombosis (DVT). Since the gene for coagulation Factor V is located just downstream of SELP, and the former gene harbors the most important genetic risk factor for DVT, the Factor V Leiden mutation (FVL),30 we additionally explored the degree of linkage between polymorphisms in the selectin genes and the FVL polymorphism and adjusted risk estimates for this linkage.
Methods Study population
The design of the Leiden Thrombophilia Study (LETS) has been described in detail previously.31,32 In short, 474 consecutive patients with an objectively confirmed first
episode of DVT and 474 controls, frequency matched for sex and age, were included. The patients were all younger than 70 years and individuals with malignancies were excluded. Acquaintances and partners of patients with no cancer history served as controls. The mean age for patients and controls was 45 years (range 15-69 for patients, 15-72 for controls). Both groups consist of 202 (42.6%) men and 272 (57.4%) women. Venous blood was collected into 0.1 volume of 0.106 mol L-1 trisodium citrate. Plasma was prepared by centrifugation for 10 minutes at 2000 g at room temperature and stored at -70oC. High molecular weight DNA was isolated from leukocytes by standard methods and stored at -20oC. DNA samples were available from 471 patients and 471 controls. Informed consent was obtained from all participants and the study was approved by the local ethics committee.
Genetic analysis
The group of SeattleSNPs re-sequenced the three genes of the selectin cluster as well as of PSGL-1 in 23 individuals of European-American descent.21 For each gene, haplotypes for the 46 chromosomes were reconstructed from the unphased SNP genotype data, using the software PHASE 2.0.33 We used these data to select 24 single nucleotide polymorphisms (SNPs) (Table 1) to cover the 23 common haplotype groups of the genes. Because of the presence of a recombination hotspot in intron 8 of SELP, we analyzed the regions upstream (SELPup) and downstream (SELPdown) from the recombination hotspot separately. Genotyping for SELE SNP 135A>G was performed by polymerase chain reaction and restriction fragment length polymorphism analysis. All other SNPs were genotyped using the 5' nuclease/TaqMan assay. The polymerase chain reactions with fluorescent allele- specific oligonucleotide probes (Assay-by-Design/Assay-on-Demand, Applied Biosystems) were performed on a PTC-225 thermal cycler (Biozym) and fluorescence endpoint reading for allelic discrimination was done on an ABI 7900 HT instrument (Applied Biosystems). Information on primer and probe sequences and restriction enzyme used is available upon request. The Factor V Leiden mutation (1691G>A [rs6025] 30) was genotyped previously.34
Soluble P-selectin measurement
Soluble P-selectin levels were measured previously in a subgroup of 89 patients and 126 control subjects.9
Statistical analysis
In the healthy controls, Hardy-Weinberg equilibrium for each SNP was tested by χ2- analysis. The software program TagSNPs (Version 2),35 which minimizes the uncertainty in predicting common haplotypes for individuals with unphased genotype data, was used to calculate the number of haplotypes for each gene, the frequency of the haplotypes and the statistic R2h, the squared correlation between true haplotype dosage (0, 1 or 2 copies of a haplotype) and the haplotype dosage
predicted by TagSNPs.36 A high R2h indicates that a haplotype could be assigned with high certainty. We assigned haplotypes individually to the patient and control subjects. An R2h>0.95 and overall haplotype frequency ≥1% were used as criteria for assigning haplotypes of SELE, SELL and SELP (Figure 1A). Because a recombination hotspot covers part of the PSGL-1 gene, haplotype construction for this gene was less accurate. Only one of the 11 set PSGL-1 haplotypes had an R2h>0.95 and frequency ≥1%. Consequently, no haplotypes were constructed for PSGL-1, and the SNPs were analyzed separately. To investigate whether SNPs and haplotypes of the selectin genes were associated with DVT risk, odds ratios (ORs) and 95% confidence intervals (95%CI) were calculated. The degree of linkage disequilibrium (D’) in our study population between the SNPs of the selectin genes and FVL was estimated using the software program Haploview.37 This analysis indicated that significant linkage existed between some selectin SNPs and FVL. To adjust for the effect on risk due to this linkage, risks were calculated after exclusion of all FVL carriers. Furthermore, FVL and the selectin haplotypes were adjusted for each other in one logistic regression model. Interactions between PSGL-1 SNPs and SNPs of SELE, SELL and SELP were tested under a dominant model within the logistic regression model. The association between SELP haplotypes and soluble SELP levels was tested in control subjects by ANOVA. The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
Figure 1A Haplotype groups and typed SNPs (bold and circled) of SELE, SELL and SELP (upstream and downstream of the recombination hotspot) upstream of the Factor V gene. SNP numbering according to SeattleSNPs.21 F: haplotype allele frequency in LETS population (TagSNPs results). R2h: squared correlation between true and predicted haplotype dosage.
Results SNPs
The re-sequencing data from SeattleSNPs were used to select 24 SNPs to cover all common haplotype groups of the four selected genes (Figure 1A). Table 1 shows the minor allele frequencies of the 24 genotyped SNPs in 471 patients and 471 controls.
For almost all SNPs the distribution of the genotypes among control subjects was in Hardy-Weinberg equilibrium. Only for SELP SNPs 20732G>A and 28972G>A there
was a small deviation. DVT risks for the separate SNPs of SELE, SELL and SELP are shown in supplemental Tables I, II and III.
Table 1 SELE, SELL, SELP and PSGL-1 polymorphisms, rs numbers and minor allele frequencies in patients and controls
Gene (GenBank Patients Controls
Accession number) SNP* rs number
n=471 n=471 SELE (AF540378) 135A>G rs3917389 0.416 0.401
3689A>C rs5361 0.130 0.123 4930C>T rs3917419 0.409 0.391 5924C>A rs1534904 0.327 0.349 12228A>G rs5359 0.109 0.101 SELL (AY233976) 4318C>T rs4987284 0.130 0.122
8813C>T rs4987310 0.110 0.102 17100C>A rs4987358 0.269 0.240 19572C>A rs2298902 0.081 0.099 24467T>C rs4987391 0.472 0.495 SELP (AF542391)
upstream 5300A>T rs2236866 0.401 0.436 10703A>G rs3917690 0.261 0.256 14668A>G rs2244526 0.091 0.119 20732G>A rs6131 0.221 0.175 downstream 23627C>T rs3917744 0.349 0.305
28972G>A rs2205895 0.355 0.374 34080G>T rs3917793 0.092 0.100 36060C>A rs2205896 0.444 0.409 36279G>T rs6133 0.108 0.115 37674A>C rs6136 0.089 0.102 PSGL-1 (AY331789) 7436C>T rs4964269 0.462 0.442
7768C>T rs7138370 0.329 0.346 10546A>G rs8179137 0.174 0.172 11819T>A rs7137098 0.372 0.408
*SNP numbering according to SeattleSNPs; minor alleles shown in bold and underlined
Linkage disequilibrium
SELE, SELL and SELP are located in a large cluster on chromosome 1q. We estimated the degree of LD between the SNPs of these genes in our study population (Figure 1B). Haploview analysis showed that SELE and SELL are present in a single haplotype block with a high degree of LD (D’ ranges from 0.42 to 1.00), indicating that recombination in this region is rare. In SELP a clear recombination hotspot is present, dividing the gene into two parts. Within each part, the degree of LD is high. In SELPup, D’ ranges from 0.83 to 0.94, in SELPdown, D’ ranges from 0.65 to 1.00. In addition, SELPup forms a haplotype block together with SELE and SELL, although the degree of LD is somewhat lower (D’ ranges from 0.00 to 0.90).
B Haploview LD plot of E-selectin, L-selectin and P-selectin (upstream and downstream of the recombination hotspot), together with the Factor V Leiden mutation. LD (number shown in squares) ranges from 0 (D’=0) to 100 (D’=1). The color of the squares indicates the LOD- score, with white color for LOD<2 and gray color for LOD≥2 (the higher the LOD-score, the darker the gray color).
Haplotypes
Based on our genotyping data in 942 subjects, TagSNPs analysis resulted in 9 SELE haplotypes, 13 SELL haplotypes, 12 haplotypes for SELPup, 18 haplotypes for SELPdown and 11 haplotypes for PSGL-1. Implementing the criteria used for individually assigning haplotypes (R2h>0.95 and frequency ≥1%) resulted in assignment of 5 SELE haplotypes, 6 SELL haplotypes, 5 haplotypes for SELPup and 6 haplotypes for SELPdown (Figure 1A). Since SELE haplotype 1 (H1) had a frequency <1%, it was excluded from further analysis. Because of the criteria used, we did not assign haplotypes to 7 patients and 15 controls for SELE, to 19 patients and 19 controls for SELL, to 22 patients and 22 controls for SELPup and to 50 patients and 58 controls for SELPdown. These individuals were excluded from further haplotype analysis for these genes or regions.
In Tables 2a and 2b, crude ORs and 95%CIs for the haplotypes of SELE, SELL and SELPup and SELPdown are shown. In SELE and SELL none of the haplotypes was associated with VT risk. In SELPup, H2-carriers had a slight increase in risk (OR=1.3, 95%CI: 1.0-1.7), whereas H4-carriers had a slight decrease in risk (OR=0.7, 95%CI: 0.5-1.0). These effects already had been detected in the single SNP analyses (Supplemental Table III, SNPs 20732G>A and 14668A>G). In SELPdown, H2-carriers had a slight increase in risk (OR=1.3, 95%CI: 1.0-1.7),
whereas H6-carriers had a slight decrease in risk (OR=0.7, 95%CI: 0.5-1.1), although this protection was not significant.
Table 2a Thrombosis risk for the haplotype groups of SELE and SELL
SELE SELL
Patients
(%) Controls
(%) Patients
(%) Controls Haplotype (%)
n=464 n=456
OR 95% CI
n=452 n=452
OR 95% CI
Haplotype 1
H1Hx NA NA NA 65 (14.4) 77 (17.0) 0.8 0.6-1.2
H1H1 NA NA NA 4 (0.9) 6 (1.3) 0.6 0.2-2.3
H1Hx/H1H1 NA NA NA 69 (15.3) 83 (18.4) 0.8 0.6-1.1
Frequency H1 0.081 0.098
Haplotype 2
H2Hx 196 (42.2) 220 (48.2) 0.9 0.6-1.1 231 (51.1) 229 (50.7) 0.9 0.7-1.3 H2H2 92 (19.8) 68 (14.9) 1.3 0.9-1.9 69 (15.3) 81 (17.9) 0.8 0.5-1.2 H2Hx/H2H2 288 (62.1) 288 (63.2) 1.0 0.7-1.3 300 (66.4) 310 (68.6) 0.9 0.7-1.2
Frequency H2 0.409 0.390 0.408 0.433
Haplotype 3
H3Hx 196 (42.2) 214 (46.9) 0.8 0.6-1.1 175 (38.7) 158 (35.0) 1.2 0.9-1.6 H3H3 56 (12.1) 55 (12.1) 0.9 0.6-1.4 35 (7.7) 30 (6.6) 1.3 0.8-2.1 H3Hx/H3H3 252 (54.3) 269 (59.0) 0.8 0.6-1.1 210 (46.5) 188 (41.6) 1.2 0.9-1.6
Frequency H3 0.332 0.355 0.271 0.241
Haplotype 4
H4Hx 84 (18.1) 81 (17.8) 1.0 0.7-1.4 25 (5.5) 24 (5.3) 1.0 0.6-1.9 H4H4 9 (1.9) 6 (1.3) 1.5 0.5-4.2 2 (0.4) 2 (0.4) 1.0 0.1-7.1 H4Hx/H4H4 93 (20.0) 87 (19.1) 1.1 0.8-1.5 27 (6.0) 26 (5.8) 1.0 0.6-1.8
Frequency H4 0.110 0.102 0.032 0.031
Haplotype 5
H5Hx 108 (23.3) 95 (20.8) 1.1 0.8-1.6 104 (23.0) 92 (20.4) 1.2 0.8-1.6 H5H5 7 (1.5) 10 (2.2) 0.7 0.3-1.9 7 (1.5) 10 (2.2) 0.7 0.3-1.9 H5Hx/H5H5 115 (24.8) 105 (23.0) 1.1 0.8-1.5 111 (24.6) 102 (22.6) 1.1 0.8-1.5
Frequency H5 0.131 0.126 0.131 0.124
Haplotype 6
H6Hx 16 (3.4) 20 (4.4) 0.8 0.4-1.5 64 (14.2) 62 (13.7) 1.0 0.7-1.5 H6H6 0 (0.0) 2 (0.4) - - 3 (0.7) 2 (0.4) 1.5 0.3-9.1 H6Hx/H6H6 16 (3.4) 22 (4.8) 0.7 0.4-1.4 67 (14.8) 64 (14.2) 1.1 0.7-1.5 Frequency H6 0.017 0.026 0.077 0.073 All odds ratios were calculated with HxHx as the reference category (OR=1);
Hx indicates all haplotypes but the one given; NA: Not Applicable
Linkage disequilibrium with FVL
Since the gene for coagulation Factor V is located only 2.4 kb downstream of SELP, and this gene contains the most important risk factor for DVT, FVL, we additionally explored the degree of linkage between SNPs in the selectin genes and FVL.
Although the Factor V gene is located downstream from SELP and separated from SELP by a recombination hotspot in the Factor V gene, we observed significant linkage between FVL and SNPs in all three selectin genes (Figure 1B). Because of this high degree of linkage with FVL, a risk association of a selectin haplotype may reflect the effect of FVL on thrombosis risk (OR=8.0, 95%CI: 4.5-14.2 in LETS).
Table 2b Thrombosis risk for the haplotype groups the upstream and downstream part of SELP
SELP upstream SELP downstream
Patients
(%) Controls
(%) Patients
(%) Controls (%)
Haplotype n=449 n=449 OR 95% CI n=421 n=413 OR 95% CI Haplotype 1
H1Hx 159 (35.4) 166 (37.0) 1.0 0.7-1.3 88 (20.9) 94 (22.8) 0.9 0.7-1.3 H1H1 34 (7.6) 25 (5.6) 1.4 0.8-2.4 9 (2.1) 5 (1.2) 1.7 0.6-5.3 H1Hx/H1H1 193 (43.0) 191 (42.5) 1.0 0.8-1.3 97 (23.0) 99 (24.0) 1.0 0.7-1.3
Frequency H1 0.253 0.241 0.126 0.126
Haplotype 2
H2Hx 140 (31.2) 117 (26.1) 1.3 1.0-1.8 188 (44.6) 168 (40.7) 1.2 0.9-1.7 H2H2 25 (5.6) 21 (4.7) 1.3 0.7-2.4 50 (11.9) 41 (9.9) 1.4 0.9-2.2 H2Hx/H2H2 165 (36.7) 138 (30.7) 1.3 1.0-1.7 238 (56.5) 209 (50.6) 1.3 1.0-1.7
Frequency H2 0.212 0.177 0.342 0.303
Haplotype 3
H3Hx 203 (45.2) 214 (47.7) 0.8 0.6-1.1 69 (16.4) 62 (15.0) 1.1 0.8-1.6 H3H3 74 (16.5) 83 (18.5) 0.8 0.5-1.2 2 (0.5) 4 (1.0) 0.5 0.1-2.7 H3Hx/H3H3 277 (61.7) 297 (66.1) 0.8 0.6-1.1 71 (16.9) 66 (16.0) 1.1 0.7-1.5
Frequency H3 0.391 0.423 0.087 0.085
Haplotype 4
H4Hx 78 (17.4) 99 (22.0) 0.7 0.5-1.0 149 (35.4) 151 (36.6) 0.9 0.7-1.3 H4H4 2 (0.4) 4 (0.9) 0.5 0.1-2.6 28 (6.7) 30 (7.3) 0.9 0.5-1.5 H4Hx/H4H4 80 (17.8) 103 (22.9) 0.7 0.5-1.0 177 (42.0) 181 (43.8) 0.9 0.7-1.2
Frequency H4 0.091 0.119 0.243 0.255
Haplotype 5
H5Hx 44 (9.8) 32 (7.1) 1.4 0.9-2.3 80 (19.0) 85 (20.6) 0.9 0.6-1.3 H5H5 2 (0.4) 2 (0.4) 1.0 0.1-7.4 6 (1.4) 10 (2.4) 0.6 0.2-1.6 H5Hx/H5H5 46 (10.2) 34 (7.6) 1.4 0.9-2.2 86 (20.4) 95 (23.0) 0.9 0.6-1.2
Frequency H5 0.053 0.040 0.109 0.127
Haplotype 6
H6Hx NA NA NA 43 (10.2) 55 (13.3) 0.7 0.5-1.1
H6H6 NA NA NA 3 (0.7) 6 (1.5) 0.5 0.1-1.9
H6Hx/H6H6 NA NA NA 46 (10.9) 61 (14.8) 0.7 0.5-1.1
Frequency H6 0.058 0.081
All odds ratios were calculated with HxHx as the reference category (OR=1);
Hx indicates all haplotypes but the one given; NA: Not Applicable
To account for this LD effect, we first excluded all FVL carriers from the analyses.
We found that the odds ratios for SELPup H4 and SELPdown H6 slightly increased (OR=0.8, 95%CI: 0.6-1.1 and OR=0.8, 95%CI: 0.5-1.3, respectively) as a result of linkage of the common allele of this haplotype to FVL. The risks for SELPup H2 and SELPdown H2 completely disappeared (OR=1.0, 95%CI: 0.8-1.4 and OR=1.0, 95%CI: 0.7-1.3, respectively), showing that the earlier observed increased risks were indeed a reflection of the effect of FVL on thrombosis risk. Mutual adjustment of FVL and either SELPup or SELPdown haplotypes in one logistic regression model gave the same results for the SELP haplotypes as after exclusion of all FVL carriers, whereas the risk of FVL remained (data not shown).
SNPs in PSGL-1
Since haplotype assignment for PSGL-1 was not accurate, the SNPs were analyzed
separately. Table 3 shows the DVT risk for PSGL-1 SNPs. None of the PSGL-1 SNPs was significantly associated with thrombosis risk.
Interactions
Since PSGL-1 is the main counter-receptor for each of the three selectin genes, we investigated possible interactions between SNPs of PSGL-1 and SELE, SELL and SELP, but no interactions between SNPs of the different genes were found, since none of the ORs exceeded the risk of the SNPs themselves (data not shown).
Table 3 Thrombosis risk for PSGL-1 SNPs
Patients (%) Controls (%) SNP Genotype
n=471 n=471 OR 95% CI
7436C>T CT 251 (53.3) 234 (49.7) 1.2 0.9-1.6 TT 92 (19.5) 91 (19.3) 1.2 0.8-1.7 CT+TT 343 (72.8) 325 (69.0) 1.2 0.9-1.5 7768C>T CT 206 (43.7) 196 (41.6) 1.0 0.8-1.4 TT 52 (11.0) 65 (13.8) 0.8 0.5-1.2 CT+TT 258 (54.8) 261 (55.4) 1.0 0.8-1.3 10546A>G AG 136 (28.9) 128 (27.2) 1.1 0.8-1.4 GG 14 (3.0) 17 (3.6) 0.8 0.4-1.7 AG+GG 150 (31.8) 145 (30.8) 1.1 0.8-1.4 11819T>A TA 230 (48.8) 238 (50.5) 0.9 0.6-1.1 AA 60 (12.7) 73 (15.5) 0.7 0.5-1.1 TA+AA 290 (61.6) 311 (66.0) 0.8 0.6-1.1 All odds ratios were calculated with homozygous wild-type as the reference category (OR=1)
Soluble P-selectin levels
In the control subjects, none of the haplotypes was associated with soluble P- selectin levels (data not shown).
Discussion
In this study, we investigated the effect of haplotypes of SELE, SELL, SELP upstream and downstream of the recombination hotspot and SNPs of the P-selectin ligand PSGL-1 on DVT risk. In SELE and SELL, none of the haplotypes was associated with risk. In SELPup, H2-carriers had a 1.3-fold increased risk, whereas H4-carriers had a 1.4-fold decreased risk. In SELPdown, H2-carriers had a 1.3-fold increased risk, whereas H6-carriers had a 1.4-fold decreased risk. After adjustment for linkage with FVL, the crude risk associations of the selectin haplotypes disappeared, whereas the effect of FVL on DVT risk remained. This shows that the risk associations found for the selectin haplotypes were caused by linkage to FVL.
In the risk analyses of the haplotypes for each gene, we used the subjects with genotype HxHx (Hx: all other haplotypes but the one being analyzed) as the
reference group. Although in this way each haplotype has a different reference group, the analysis is in fact the same as the risk analysis of single SNPs, where the genotype of carriers of the rare allele (heterozygous and/or homozygous) is compared to that of non-carriers. We additionally analyzed the haplotype risks with the subjects homozygous for the most common haplotype of the haplotype block as the reference group, thereby avoiding different reference groups. This did not result in strikingly different odds ratios, but gave wider confidence intervals, since the reference groups were smaller (data not shown).
Several polymorphisms and haplotypes of the selectin genes have been studied in cardiovascular disease, but these studies mainly involved coronary arterial disease and myocardial infarction and were often directed at a single gene of the selectin family. SELPup H2, in this study associated with a 1.3-fold increased DVT risk, is tagged by SNP 20732G>A. This polymorphism is located in exon 7 and confers a serine to asparagine change in position 290. The haplotype carrying the rare allele of this polymorphism was found to increase the risk of myocardial infarction 2- to 3- fold in populations of Belfast and France.26 Although FVL only moderately increases the risk of myocardial infarction,38 it may be interesting to see what happens to this risk after adjustment for FVL, as in our study the risk associated with this SNP disappeared after adjustment for FVL.
SELP polymorphism 37674A>C confers a threonine to proline change in position 715 in exon 13, and the rare allele of this polymorphism has been found to be protective against myocardial infarction,23,26,39 and to be associated with low levels of soluble P-selectin.24,25,40 In our study this polymorphism tagged SELPdown H6 (Figure 1A), which was associated with a slightly decreased risk, but had no effect on soluble P- selectin levels. Actually, in our study none of the SELP haplotypes was significantly associated with P-selectin levels, but this may be due to the low number (n=126) of control subjects in whom P-selectin levels were measured.
Another polymorphism of interest is the Ser128Arg in SELE, corresponding to polymorphism 3689A>C in our study. This polymorphism has been found to be functional, since it alters ligand affinity,41 enhances tethering of myeloid cells,42 and regulates leukocyte endothelial interaction in vitro.43 Additionally, it is associated with enhanced endotoxin-triggered, tissue factor-mediated coagulation in humans.44 Clinically, the Arg-allele has been associated with atherosclerosis,45-47 myocardial infarction,43 and restenosis after angioplasty.48 Recently, homozygosity for the Arg- allele was found to be associated with an increased risk of recurrent venous thromboembolism.49 In contrast, in our study of first events of DVT, homozygous carriers of SELE H5, tagged by the rare allele of this polymorphism, had a non- significant reduction in risk (OR=0.7, 95%CI:0.3-1.9), indicating that SELE H5 might be protective against a first event of DVT. There is no clear explanation for
the observed difference in risk between a first and a recurrent episode of DVT for homozygous carriers of this polymorphism, but such differences have been seen before, since carriers of FVL only have an increased risk for a first event of DVT, not for a recurrent event.49,50
In conclusion, after adjustment for LD with FVL, none of the selectin haplotypes was associated with VT risk, showing that the increased risks of the selectin haplotypes were a reflection of the effect of FVL on thrombosis risk.
Acknowledgements
This study was financially supported by grant 912-02-036 from the Netherlands Organization for Scientific Research (NWO). The LETS study was supported by grant 89-063 from the Netherlands Heart Foundation.
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Supplemental data
Thrombosis risk for SNPs and haplotypes of E-selectin, L-selectin and P-selectin
Table I Thrombosis risk for E-selectin SNPs
Patients (%) Controls (%) SNP Genotype
n=471 n=471 OR 95% CI
135A>G AA 176 (37.4) 168 (35.7) 1*
AG 198 (42.0) 228 (48.4) 0.8 0.6-1.1 GG 97 (20.6) 75 (15.9) 1.2 0.9-1.8 AG+GG 295 (62.6) 303 (64.3) 0.9 0.7-1.2 3689A>C AA 356 (75.6) 365 (77.5) 1*
AC 108 (22.9) 96 (20.4) 1.2 0.8-1.6 CC 7 (1.5) 10 (2.1) 0.7 0.3-1.9 AC+CC 115 (24.4) 106 (22.5) 1.1 0.8-1.5 4930C>T CC 178 (37.8) 173 (36.7) 1*
CT 201 (42.7) 228 (48.4) 0.9 0.6-1.1 TT 92 (19.5) 70 (14.9) 1.3 0.9-1.9 CT+TT 293 (62.2) 298 (63.3) 1.0 0.7-1.2
5924C>A CC 219 (46.5) 198 (42.0) 1*
CA 196 (41.6) 217 (46.1) 0.8 0.6-1.1 AA 56 (11.9) 56 (11.9) 0.9 0.6-1.4 CA+AA 252 (53.5) 273 (58.0) 0.8 0.6-1.1 12228A>G AA 377 (80.0) 382 (81.1) 1*
AG 85 (18.0) 83 (17.6) 1.0 0.7-1.5 GG 9 (1.9) 6 (1.3) 1.5 0.5-4.3 AG+GG 94 (20.0) 89 (18.9) 1.1 0.8-1.5
*Reference Category
Table II Thrombosis risk for L-selectin SNPs
Patients (%) Controls (%) SNP Genotype
n=471 n=471 OR 95% CI
4318C>T CC 356 (75.6) 366 (77.7) 1*
CT 108 (22.9) 95 (20.2) 1.2 0.9-1.6 TT 7 (1.5) 10 (2.1) 0.7 0.3-1.9 CT+TT 115 (24.4) 105 (22.3) 1.1 0.8-1.5 8813C>T CC 376 (79.8) 382 (81.1) 1*
CT 86 (18.3) 82 (17.4) 1.1 0.8-1.5 TT 9 (1.9) 7 (1.5) 1.3 0.5-3.5 CT+TT 95 (20.2) 89 (18.9) 1.1 0.8-1.5 17100C>A CC 253 (53.7) 275 (58.4) 1*
CA 183 (38.9) 166 (35.2) 1.2 0.9-1.6 AA 35 (7.4) 30 (6.4) 1.3 0.8-2.1 CA+AA 218 (46.3) 196 (41.6) 1.2 0.9-1.6
19572C>A CC 399 (84.7) 384 (81.5) 1*
CA 68 (14.4) 81 (17.2) 0.8 0.6-1.1 AA 4 (0.8) 6 (1.3) 0.6 0.2-2.3 CA+AA 72 (15.2) 87 (18.5) 0.8 0.6-1.1 24467T>C TT 122 (25.9) 120 (25.5) 1*
TC 253 (53.7) 236 (50.1) 1.1 0.8-1.4 CC 96 (20.4) 115 (24.4) 0.8 0.6-1.2 TC+CC 349 (74.1) 351 (74.5) 1.0 0.7-1.3
*Reference Category
Table III Thrombosis risk for P-selectin SNPs
Patients (%) Controls (%) SNP Genotype
n=471 n=471 OR 95%
CI 5300A>T AA 177 (37.6) 154 (32.7) 1*
AT 210 (44.6) 223 (47.3) 0.8 0.6-1.1 TT 84 (17.8) 94 (20.0) 0.8 0.5-1.1 AT+TT 294 (62.4) 317 (67.3) 0.8 0.6-1.1 10703A>G AA 265 (56.3) 261 (55.4) 1*
AG 166 (35.2) 179 (38.0) 0.9 0.7-1.2 GG 40 (8.5) 31 (6.6) 1.3 0.8-2.1 AG+GG 206 (43.7) 210 (44.6) 1.0 0.7-1.3 14668A>G AA 388 (82.4) 364 (77.3) 1*
AG 80 (17.0) 102 (21.7) 0.7 0.5-1.0 GG 3 (0.6) 5 (1.1) 0.6 0.1-2.4 AG+GG 83 (17.6) 107 (22.7) 0.7 0.5-1.0 20732G>A GG 294 (62.4) 327 (69.4) 1*
GA 146 (31.0) 123 (26.1) 1.3 1.0-1.8 AA 31 (6.6) 21 (4.5) 1.6 0.9-2.9 GA+AA 177 (37.6) 144 (30.6) 1.4 1.0-1.8
23627C>T CC 199 (42.3) 229 (48.6) 1*
CT 215 (45.6) 197 (41.8) 1.3 1.0-1.6 TT 57 (12.1) 45 (9.6) 1.5 0.9-2.3 CT+TT 272 (57.7) 242 (51.4) 1.3 1.0-1.7 28972G>A GG 201 (42.7) 196 (41.6) 1*
GA 206 (43.7) 198 (42.0) 1.0 0.8-1.3 AA 64 (13.6) 77 (16.3) 0.8 0.6-1.2 GA+AA 270 (57.3) 275 (58.4) 1.0 0.7-1.2 34080G>T GG 386 (82.0) 383 (81.3) 1*
GT 83 (17.6) 82 (17.4) 1.0 0.7-1.4
TT 2 (0.4) 6 (1.3) 0.3 0.1-1.6
GT+TT 85 (18.0) 88 (18.7) 1.0 0.7-1.3 36060C>A CC 151 (32.1) 165 (35.0) 1*
CA 222 (47.1) 227 (48.2) 1.1 0.8-1.4 AA 98 (20.8) 79 (16.8) 1.4 0.9-2.0 CA+AA 320 (67.9) 306 (65.0) 1.1 0.9-1.5 36279G>T GG 375 (79.6) 373 (79.2) 1*
GT 90 (19.1) 88 (18.7) 1.0 0.7-1.4 TT 6 (1.3) 10 (2.1) 0.6 0.2-1.7 GT+TT 96 (20.4) 98 (20.8) 1.0 0.7-1.3
37674A>C AA 390 (82.8) 381 (80.9) 1*
AC 78 (16.6) 84 (17.8) 0.9 0.6-1.3
CC 3 (0.6) 6 (1.3) 0.5 0.1-2.0
AC+CC 81 (17.2) 90 (19.1) 0.9 0.6-1.2
*Reference Category
