Thrombosis Research 129 (2012) 629–634

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Thrombosis Research

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Regular Article

Lack of association between polymorphisms in the interleukin-1 gene cluster andfamilial thrombophilia

Luke Marsden a, Angela Cox b, Mike Makris a, Martina E. Daly a,⁎a Department of Cardiovascular Science, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, UKb Department of Oncology, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, UK

Abbreviations: IL-1, Interleukin-1; SNP, single nucleovein thrombosis; TF, tissue factor; TNF, tumour necrosisreaction.⁎ Corresponding author at: Department of Cardiov

Sheffield Medical School, Beech Hill Road, Sheffield,2713213; fax: +44 114 2711863.

E-mail address: m.daly@sheffield.ac.uk (M.E. Daly).

0049-3848/$ – see front matter © 2011 Elsevier Ltd. Aldoi:10.1016/j.thromres.2011.07.002

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 18 March 2011Received in revised form 31 May 2011Accepted 5 July 2011Available online 11 August 2011

Keywords:InflammationthrombosisIL-1 gene clustercase control study

Introduction: Inflammation and venous thrombosis are intimately linked, and there is evidence that levels ofinflammatory cytokines influence risk of venous thrombosis. We investigated the hypothesis that allelicvariation within the IL-1 gene cluster, which encompasses the genes encoding the inflammatory cytokinesIL-1α and IL-1β and the competitive IL-1 receptor antagonist, is associated with venous thrombosis amongpatients with heritable thrombophilia.Subjects and Methods: Genomic DNA samples from 181 index cases with heritable thrombophilia and 323control subjects were genotyped for four SNPs, and four microsatellite markers located within the IL-1 genecluster. The distributions of SNP genotypes and of microsatellite marker alleles were then compared betweenthe patient and control groups.Results: There was no significant difference in the distribution of alleles between the patients and control

subjects for any of the four microsatellite loci studied. Likewise, the distribution of genotypes for each of thefour SNPs investigated was similar among the cases and control subjects. Haplotype analysis showed nodifference in the estimated frequencies of any of the IL-1 gene cluster haplotypes between the patients andcontrol subjects.Conclusions: Our findings in this study suggest that inherited variation within the IL-1 gene cluster is notassociated with thrombosis among patients with heritable thrombophilia and that alterations ininflammatory cytokines encoded by loci in the IL-1 gene cluster are more likely to occur as a result, ratherthan a cause, of venous thrombosis.

© 2011 Elsevier Ltd. All rights reserved.

Introduction

Venous thromboembolism, as manifested by deep vein thrombosis(DVT) and pulmonary embolism, is a major cause of morbidity andmortality affecting 0.1 to 0.3% of the population annually [1,2]. It is amultifactorial disorder, the risk of disease being influenced by bothgenetic and acquired risk factors, with overall thrombotic risk beingdetermined by the combination of risk factors present. Five genetic riskfactors are well established; deficiencies of antithrombin, protein C andprotein S and the gain of function mutations factor V Leiden and theprothrombin 20210A allele. Elevated circulating levels of factor VIII,

tide polymorphism; DVT, deepfactor; PCR, polymerase chain

ascular Science, University ofS10 2RX, UK. Tel.: +44 114

l rights reserved.

fibrinogen and other coagulation factors are also recognised toindependently increase thrombotic risk, though apart from the knownassociationbetweenABOblood group and factorVIII levels, the extent towhich coagulation factor levels are genetically determined remains tobe elucidated [3]. It is possible, for some coagulation factors at least, thatelevated levels reflect the presence of an underlying inflammatorycondition that may itself predispose to thrombosis.

Inflammation and venous thrombosis are linked intimately. Thus,increased activation of the coagulation network as a result of theinflammatory response to stimuli such as sepsis and acute or chronicinflammatory conditions is well recognised and DVT is a feature ofinflammatory conditions such as inflammatory bowel disease andatherosclerosis [4–6]. The mechanisms underlying thrombus forma-tion in the absence of obvious damage to the vascular endotheliumhave been the subject of much investigation. Evidence that tissuefactor (TF)-bearing monocyte-derived microvesicles circulate innormal plasma, and can participate in thrombus formation bybinding, and transferring TF, to the surface of platelets at sites ofvascular injury has led to the suggestion that TF-bearing micro-vesicles could interact similarly with P-selectin-expressing activatedendothelial cells to initiate thrombus formation [7–10]. There is

630 L. Marsden et al. / Thrombosis Research 129 (2012) 629–634

evidence supporting this proposal, including the observation thatmany of the disorders in which DVT is a feature, are characterised byelevated levels of inflammatory mediators such as tumour necrosisfactor-α (TNF-α) which trigger the generation of TF-bearingmonocyte-derived microvesicles [11,12]. Other mechanisms linkinginflammation and thrombosis include the inhibitory effects of TNF-αand interleukin-1 (IL-1) on thrombomodulin expression [13,14], andthe potential for both cytokines to reduce endothelial fibrinolyticactivity [15–17]. Data from case-control studies also support a rolefor inflammatory mediators in thrombosis. Thus, elevated levels ofthe inflammatory cytokines IL-6 and IL-8 have been shown to beassociated with a doubling in the risk for recurrent thromboembolicevents [18], while data from the Leiden Thrombophilia Study (LETS)have shown an approximate 2-fold increased risk of a first DVT in thepresence of detectable levels of IL-6, IL-8 and TNF-α [19].

Given the recognised relationship between the inflammatory andcoagulation responses, it is conceivable that polymorphic variation inthe genes encoding pro- and anti-inflammatory cytokines couldcontribute to the expression of a thrombotic tendency. Indeed, weakevidence for a thrombosis susceptibility locus in the region of theInterleukin-1 (IL-1) locus has been reported in a genetic study ofthrombophilic families [20]. The genes encoding the two inflamma-tory IL-1 cytokines IL-1α (IL1A) and IL-1β (IL1B), and theircompetitive antagonist, the IL-1 receptor antagonist (IL1RN) arelocated, together with six other similar genes encoding members ofthe IL-1 family, in a region of chromosome 2 (2q13-14) referred to asthe IL-1 gene cluster, which spans approximately 400 kb of genomicDNA from the IL1A gene at the centromeric end to the IL1RN gene atthe other end [21]. Several polymorphisms have been describedwithin the IL-1 gene cluster and been associated with susceptibility toinflammatory, autoimmune and infectious diseases [22,23].

In the present study, we have adopted a case control approach toexamine whether specific alleles of polymorphic markers locatedwithin the IL-1 gene cluster are associated with venous thrombosisby genotyping thrombophilic patients and control subjects for fourmicrosatellite markers and four single nucleotide polymorphisms(SNPs) at loci in the IL-1 gene cluster (Fig. 1). The selection ofpolymorphic markers for genotyping was based on their use inprevious studies to determine the degree of linkage disequilibriumin the IL-1 gene cluster [24]. Moreover, genotyping of these markershas previously allowed the identification of a common IL-1 genecluster haplotype and provided evidence supporting an association

113.

55M

113.

6M

113.

7M

113.

65M

IL1A IL1F7 IL1B

IL1A

222

/223

IL1A

rs1

7561

IL1A

gz5

/gz6

IL1B

rs1

6944

IL1B

rs1

1436

34

Centromere

Fig. 1. The IL-1 Gene Cluster. Schematic representation of the IL-1 gene cluster showing therelative positions of the genes on chromosome 2 are shown according to the scale which is ishown. The arrows below the gene names indicate the direction of transcription. The direc

between the IL-1 gene cluster and erosive rheumatoid arthritis[24,25].

Materials and methods

Subjects

All patients were investigated at the Sheffield Haemophilia andThrombosis Centre as part of a prospective study of thrombophilia. Atotal of 181 index cases (132 female, 49 male; mean age 50.3 years,range 23–86 (ages calculated as that attained on 1st March 2007 forboth patients and control subjects)) diagnosedwith at least one of fiveestablished heritable thrombophilic defects (factor V Leiden (n=83),prothrombin 20210A allele (n=36), deficiency of antithrombin(n=24), protein C (n=16) or protein S (n=22)) were studied. Adetailed thrombotic history, recording site of thrombosis, whether ornot it was confirmed, and whether it occurred spontaneously or inassociation with acquired risk factors was documented. Otherpotential risk factors such as body mass index, varicose veins,smoking and the use of anti-thrombotic prophylaxis were alsorecorded. All patients had experienced at least one confirmed venousthromboembolic event. Confirmation of thrombotic events was withDoppler scanning or venography in the case of limb thrombosis andventilation-perfusion scanning or pulmonary angiography in the caseof pulmonary emboli. The 323 control subjects (215 female, 108male;mean age 53.5 years, range 24–93) were healthy individuals recruitedby random sampling from the registers of two local collaboratinggeneral practices and were age (± 10 years) and sex matched to theindex cases. Subjects were excluded as controls when known to havemalignancy. Each control subject completed a questionnaire forknown risk factors for thrombosis and a previous history of venousthromboembolism. The project was approved by the South SheffieldResearch Ethics Committee and all patients and control subjects gavetheir signed consent before participating in the study.

Fibrinogen levels

Plasma fibrinogen levels were measured using a prothrombin-timebased assay. Samples from patients were collected during routineoutpatient visits at least 3 months after the thrombotic episode and thepatients didnot have any acute thrombosis, inflammation or infection atthe time of sampling.

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IL1F10 IL1F5

IL1F8 IL1F6 IL1F9 IL1RN

IL1F

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3333

0

IL1F

8 Y

31

IL1R

N r

s419

598

genes that form the cluster as solid black rectangular boxes. The approximate sizes andn megabases. The locations of the polymorphic markers genotyped in the study are alsotion of the centromere is indicated.

631L. Marsden et al. / Thrombosis Research 129 (2012) 629–634

Genotyping

Genomic DNA was extracted from peripheral blood samples fromall subjects and genotyped for four SNPs (rs17561 in IL1A; rs1143634and rs16944 in IL1B; rs419598 in IL1RN) and four microsatellitemarkers (222/223, gaat.p3330, Y31, Gz5/6) located within the IL-1gene cluster. The characteristics of the markers used, and theirlocationswithin the IL-1 gene cluster are indicated in Table 1. To avoidconfusion, we have used the same names to identify themicrosatellitemarkers as in previous studies [21,24]. To provide a reference pointexternal to the IL-1 gene cluster, a fifth microsatellite marker, located400 kb centromeric of the IL1A gene, D2S160 (Genome Data Baseaccession no. 133520) was also genotyped.

The SNPs rs17561, rs1143634, rs16944 and rs419598 weregenotyped as described previously on an ABI Prism™ 7200 sequencedetector by 5’ nuclease PCR using assays designed for the TaqMan™allelic discrimination system, and probes and primers designedwith thePrimer Express v 1.0 software [24,25]. Microsatellite markers 222/223,gaat.p3330 (hereafter referred to as gaat), Y31, gz5/gz6 andD2S160wereamplified using fluorescently labelled forward primers as describedpreviously [24,25]. Aliquots of the PCR products were examined byelectrophoresis on 2% agarose gels. The remainder of the products werepooled according to the intensity of ethidium bromide staining beforeanalysis on an ABI 377 automated sequencer. Allele sizes weredetermined by comparison with the GeneScan™ 500™ ROX SizeStandard (ABI) or GenoTYPE-ROX 50–500 DNA ladder (Life Technolo-gies), using the Genescan v3.0 and Genotyper v2.5 software from ABI.

Data analysis

The χ2 test was used to compare the distribution of SNP genotypesand allele frequencies between the patient and control groups. Oddsratios were calculated as estimates of the relative risk for venousthrombosis and thrombophilia associated with carriage of the rarer“2” allele of each SNP, with homozygosity for the common “1” allelebeing the reference category, and 95% confidence intervals weredetermined according to the method of Woolf [26].

The distribution of microsatellite marker alleles was comparedbetween patients and control subjects using CLUMP [27]. Thisprogramme uses the Monte Carlo method to assess the significance

Table 1SNP and Microsatellite Markers in the IL-1 Gene Cluster.

ReferenceID

Type Alternatename§

Location* Variation Position onchromosome2

IL1A222/223

Microsatellite 222/223 069703 [GT]n 2:113535810

IL1Ars17561

SNP IL1A +4845 340 GNT 2:113537223

IL1A gz5/gz6 Microsatellite gz5/gz6 072247 [AAT]n 2:113538316IL1Brs1143634

SNP IL1B +3954 315 CNT 2:113590390

IL1Brs16944

SNP IL1B −511 −511 CNT 2:113594867

IL1F9gaat.p33330

Microsatellite gaat.p33330 271521 [GAAT]n 2:113737416

IL1F8 Y31 Microsatellite Y31 335956 [GT]n 2:113801838IL1RNrs419598

SNP IL1RN +2018

117 TNC 2:113887207

* SNPs are numbered according to their position in the cDNA for the correspondinggene, where +1 is the A of the initiator codon. Microsatellite markers are numberedaccording to their position within a sequence based map of the IL-1 gene cluster(GenBank Acc No. BN000002). The position of each marker on chromosome 2, asdefined by the GRCh37 assembly, is also indicated. §Alternate names used to identifythese polymorphic markers in previous studies. To avoid confusion, microsatelliteshave been identified by the names used in previous studies [21,24].

of any differences in allele frequencies between cases and controlsubjects when multiallelic markers are used.

Haplotype analysis was performed using the Haploview® software[28]. Microsatellite marker alleles were grouped into pseudo-biallelicmarkers using a grouping method known as ‘δ’, which uses theobserved allelic associations between pairs of markers to determinethe grouping [24].

All analyses were performed using Microsoft Excel 2003 and theGraphpad Instat™, and Prism™ statistical analysis programs.

Results

A total of 504 subjects, 181 patients with heritable thrombophilicdefects and 323 control individuals, were studied. DNA samples weregenotyped for four microsatellite markers and four SNPs located in theIL-1 gene cluster. A fifth microsatellite marker, D2S160, located 400 kbcentromeric of the IL1A locuswas alsogenotyped. The precise numberofalleles genotyped at each locus varied according to the marker tested.Microsatellite marker alleles were assigned the same identifyingnumbers as in previous studies, and were numbered according totheir size, with allele 1 corresponding to the allele having the lowestnumber of repeat units [24].

The distributions and frequencies of the alleles of each of the fivemicrosatellite markers genotyped, among both the patients and controlsubjects, are shown inTable 2. Allele1 of the IL1A222/223marker, alleles1 and 2 of the IL1A gz5/gz6marker and allele 2 of the IL1F8 Y31marker,were not represented among the subjects genotyped here. This was notsurprising as a previous study by Cox and colleagues reported that eachof these alleles occurred at a frequency of 0.008 or less when the samemarkers were genotyped in 212 healthy blood donors from Sheffieldand Manchester [24]. Similarly, alleles 13 and 14 of the IL1F8 Y31marker, which occurred with frequencies of 0.007 and 0.003 among thepatients respectively, were novel alleles which were not representedamong the blood donors genotyped by Cox et al. [24]. There was nosignificant difference in the distribution of alleles between the patientsand controls at any of the five microsatellite loci studied (D2S160χ2=2.68, p=0.76; 222/223 χ2=7.60, p=0.09; gaat χ2=3.71,p=0.14; Y31χ2=7.97, p=0.31; gz5gz/6χ2=0.33, p=0.56) (Table 2).

Table 3 shows the distribution of genotypes, and correspondingallele frequencies, for each of the four SNPs genotyped, among thepatients and the control subjects. The distribution of genotypes foreach SNP did not differ significantly from that expected for Hardy-Weinberg equilibrium among the control subjects and there was nosignificant difference in the genotype distributions, between patientsand control subjects, at any of the loci tested (Table 3). Compared topatients who were homozygous for the common allele (genotype1/1), and the relevant control subgroups, there was no increasedassociation with thrombosis in thrombophilia patients who wereeither homozygous (genotype 2/2) for, or carried (genotypes 1/2 and2/2), the rare allele of any of the four SNPs investigated (Table 3).

Haplotype analysis was carried out to investigate the potentialassociationof oneormore IL-1 geneclusterhaplotypeswith thrombosis.Using the ‘δ’ method described earlier, microsatellite marker alleleswere grouped as pseudo-biallelic markers, and assigned as either “A” or“T” as indicated in Table 4 [24]. Haplotypes of the biallelic SNP andpseudo-biallelic microsatellite markers were then predicted, and theirfrequencies estimated using the Haploview® software. The IL1F8 Y31marker was excluded from the haplotype analysis as the distribution ofgenotypes was not in Hardy-Weinberg equilibrium among the controlsubjects following the grouping of alleles for this marker. There was nosignificantdifference in theestimated frequencies of anyof the IL-1 genecluster haplotypes between the patients and control subjects (Table 5).

Using plasma fibrinogen level as a marker of inflammation, weexamined the possible association of IL-1 gene cluster SNPmarkerswitha pro-inflammatory phenotype. However, therewasnodifference in the

Table 2Distribution of microsatellite marker alleles (and allele frequencies) among thrombophilic patients and control subjects.

AlleleNo.

Marker allele frequency

D2S160 222/223 gz5/gz6 gaat Y31

ControlsN=460

PatientsN=316

ControlsN=480

PatientsN=316

ControlsN=478

PatientsN=314

ControlsN=478

PatientsN=320

ControlsN=426

PatientsN=286

1 47 (.102) 34 (.107) 0 (.000) 0 (.000) 0 (.000) 0 (.000) 305 (.638) 218 (.681) 30 (.070) 24 (.084)2 10 (.022) 9 (.028) 3 (.006) 5 (.016) 0 (.000) 0 (.000) 0 (.000) 2 (.006) 0 (.000) 0 (.000)3 125 (.272) 82 (.259) 155 (.323) 114 (.360) 332 (.695) 212 (.675) 148 (.309) 79 (.247) 186 (.437) 129 (.451)4 92 (.200) 77 (.244) 138 (.288) 92 (.290) 146 (.305) 102 (.325) 25 (.052) 21 (.066) 19 (.045) 10 (.035)5 142 (.309) 93 (.294) 8 (.017) 9 (.028) …. …. …. …. 6 (.014) 3 (.010)6 32 (.069) 18 (.057) 134 (.279) 65 (.205) …. …. …. …. 48 (.113) 44 (.154)7 5 (.011) 1 (.003) 34 (.071) 31 (.098) …. …. …. …. 17 (.039) 6 (.021)8 6 (.013) 1 (.003) 5 (.010) 0 (.000) …. …. …. …. 25 (.059) 13 (.045)9 1 (.002) 1 (.003) 3 (.006) 1 (.003) …. …. …. …. 26 (.061) 20 (.069)10 …. …. …. …. …. …. …. …. 56 (.131) 26 (.091)11 …. …. …. …. …. …. …. …. 14 (.033) 6 (.021)12 …. …. …. …. …. …. …. …. 0 (.000) 2 (.007)13 …. …. …. …. …. …. …. …. 0 (.000) 2 (.007)14 …. …. …. …. …. …. …. …. 0 (.000) 1 (.003)

N=Number of chromosomes analysed; alleles were numbered according to their size, with allele 1 having the lowest number of repeat units.

Table 3Distribution of SNP genotypes among thrombophilia patients and control subjects, and odds ratios for thrombosis in thrombophilia patients.

SNP Patients Control Subjects P-value†

OR (95% CI)*

1/1 1/2 2/2 1/1 1/2 2/2 1/1‡ vs 1/2+2/2 1/1 vs 2/2

IL1A rs17561 89 65 20 160 130 29 0.44 0.96 (0.66-1.39) 1.24 (0.66-2.32)(51.15) (37.36) (11.49) (50.16) (40.75) (9.09)

IL1B rs1143634 100 66 9 190 114 19 0.36 1.07 (0.74-1.55) 0.90 (0.39-2.06)(57.14) (37.71) (5.14) (58.82) (35.29) (5.88)

IL1B rs16944 70 85 18 143 151 27 0.44 1.18 (0.81-1.72) 1.36 (0.70-2.64)(40.46) (49.13) (10.45) (44.55) (47.04) (8.41)

IL1RN rs419598 101 60 11 163 139 21 0.66 0.72 (0.49-1.04) 0.85 (0.39-1.83)(58.72) (34.88) (6.40) (50.46) (43.03) (6.51)

Genotypes are represented as 1/1, 1/2 and 2/2 where “1” and “2” refer to the common and rare alleles of each SNP marker respectively. Numbers in parentheses are percent ofpatients or control subjects.† p value derived from χ2 test comparing the distribution of genotypes between patients and control subjects.* Odds ratios for thrombosis (with 95% confidence interval) in thrombophilia patients associated with carriage or homozygosity for the rare allele of SNP markers.‡ Reference group.

632 L. Marsden et al. / Thrombosis Research 129 (2012) 629–634

mean fibrinogen levels among the control subjects according togenotype, for any of the four SNPs genotyped (data not shown).

Discussion

Given the considerable evidence for cross talk between theinflammatory and coagulation cascades, we hypothesised that variationin inflammation related genes contributes to the thrombotic risk inpatients with heritable thrombophilia. In particular, we investigatedwhether inherited variation within the IL-1 gene cluster, which haspreviously beenassociatedwith susceptibility to inflammatory diseases,was associatedwith the occurrence of venous thrombosis by comparingthe frequencies of eight polymorphic markers at loci in this regionamong 181 patients with thrombophilia and 323 age and sex-matchedcontrol subjects. Therewereno significantdifferences in thedistribution

Table 4Grouping of microsatellite markers as pseudo-biallelic markers using the ‘δ’ method.

MicrosatelliteMarker

Allele grouping

Allele “A” Allele “T”

IL1A 222/223 4, 5 1, 2, 3, 6, 7, 8, 9, 10IL1A gz5/gz6 1, 2, 3 4IL1 F9 gaat 1, 2, 4 3IL1 F8 Y31 1, 5, 6 2, 3, 4, 7, 8, 9, 10, 11-14

of alleles between the cases and control subjects for any of the 4microsatellite loci genotyped. Similarly, thedistributionof genotypes foreach of the 4 SNPs investigated did not differ significantly between thecases and control subjects. Further analysis to identify one or more IL-1gene cluster haplotypes which may be associated with thrombosis alsofailed to demonstrate a difference in the frequencies of any of thepredicted IL-1 gene cluster haplotypes between the cases and controlsubjects. Our findings would therefore suggest that there is nocontribution from genes in the IL-1 gene cluster to risk of venousthrombosis among patients with familial thrombophilia.

Weperformed a post hoc analysis to estimate the power of our study.Thus, accepting that the frequency of the least common SNP allele,which was the T allele of rs1143634 in IL1B, was 0.235, with an alphavalue of 0.05, the power of this study to detect a 1.5-fold change in therisk of venous thrombosis was 78%. This value assumes a multiplicativemodel in apopulation of 175 cases and323 control subjects. Byusing thelowest allele frequency in the calculation, the lowest power is obtained.Thus, the power to detect a change in risk of venous thrombosis usingany of the other SNP markers would exceed 78% under the sameconditions. Thus, we are able to exclude effect sizes greater than 1.5, butwe do not have sufficient power to exclude smaller effects.

There is a well recognised tendency for heritable risk factors forvenous thrombosis to co-segregate in affected individuals [29]. For thisreason, we chose to investigate cases with previously identifiedthrombophilic defects in this study and we studied more controlsubjects than cases (ratio of control subjects to cases, 1.8:1) in order to

Table 5Frequencies of IL-1 gene cluster haplotypes among cases and control subjects.

Haplotype† Patients Controls P-value* Haplotype† Patients Controls P-value*

TGACCAC 0.202 0.193 0.7396 AGTCTTC 0.028 0.018 0.3403TTATCAC 0.121 0.112 0.6932 TTACTTT 0.019 0.023 0.6599AGTCTTT 0.069 0.088 0.2970 TGTCCAC 0.021 0.019 0.8075AGTCCAC 0.067 0.062 0.7822 TTATTAC 0.025 0.015 0.2359AGTCTAC 0.062 0.044 0.2180 TGACTTC 0.015 0.018 0.7385TGACCAT 0.047 0.048 0.9250 TTACTAC 0.018 0.012 0.4657TTACCAC 0.043 0.038 0.6868 TTATCTC 0.007 0.016 0.2001TGACCTC 0.022 0.046 0.0591 AGTCCAT 0.010 0.014 0.5326TGACTAC 0.033 0.024 0.4125 TGACCTT 0.008 0.012 0.4898TGATCAC 0.030 0.021 0.4008 TGTCTAC 0.013 0.009 0.5932

†Haplotypes are presented in the following order: IL1A 222/223, IL1A rs17561, IL1A gz5/gz6, IL1B rs1143634, IL1B rs16944, IL1F9 gaat, IL1RN rs419598; Microsatellite alleles weredivided into two groups, denoted “A” or “T” as indicated in Table 5.*P-value derived by comparing the frequency of each haplotype between the patient and control subjects using aχ2 test.

633L. Marsden et al. / Thrombosis Research 129 (2012) 629–634

increase the likelihood of detecting differences in allele frequenciesbetween the groups.While wewould predict prothrombotic risk allelesto be enriched among our cases, it is also possible that smallcontributions to the thrombotic risk thatmay be associatedwith geneticvariation in the IL-1 gene cluster might not have been detected usingthis approach due to the presence of the relatively stronger prothrom-botic defects. Thus, we cannot exclude the possibility that geneticvariation in the IL-1 gene cluster contributes to thrombotic risk inunselected thrombosis patients. It is therefore interesting that there isconflicting evidence from prospective studies which have investigatedthe potential contribution of IL-1 gene locus variation to risk of venousthrombosis. The study by van Minkelen et al. examined whetherhaplotypes of IL1B and IL1RN influenced risk of venous thrombosis inpatients and control subjects recruited to the Leiden ThrombophiliaStudy (LETS) [30]. While there was no significant difference in allelefrequency between patients and controls for any of the SNPs tested inthese two genes, homozygous carriers of the rare allele of a SNP in IL1RN(rs2232354),were found tohave an increased riskof venous thrombosis(OR=2.8; 95%CI:1.3 to 6.1; P=0.007), though no effect on risk wasfound for heterozygous carriers of this SNP [30]. Similarly, thromboticrisk was also increased for homozygous carriers of an IL1RN haplotypethat was tagged by rs2232354 (OR=3.9, 95% CI: 1.6-9.7: P=0.002)[30]. However, the authors of this study advised caution wheninterpreting these results due to the low numbers of homozygouscarriers of the rare allele identified among the case and control groups[30].More recently, thepotential associations of 51 candidate SNPs in32inflammation-related genes, including the IL1B SNPs rs16944 andrs1143634 genotyped in this study, to risk of venous thromboembolismwere evaluated in a prospective cohort of 22,413 women recruitedthrough the Women's Genome Health Study [31]. Interestingly, whilethere was no significant association of IL1B rs16944 with risk of venousthrombosis, the rare T allele of IL1B rs1143634 was associated with areduced risk of idiopathic venous thromboembolism (Hazard Ratio,0.59; 95% CI 0.44 to 0.80; P=0.0007) [31]. This finding contrasts withour study, and with other studies which have reported an associationbetween the T allele of rs1143634 and elevated levels of C-reactiveprotein among patients referred for coronary angiography [32], and inpatients with coronary heart disease [33].

It was not possible to determine plasma levels of IL-1α or β in ourstudypopulationdue to the lowsample volumes available.We thereforeassessed plasmafibrinogen as a biomarker thatwould be expected to beincreased in subjects with a pro-inflammatory phenotype. Fibrinogenlevels were not associated with any of the four IL-1 gene cluster SNPsgenotyped, similar to the findings of van Minkelen et al. who failed todemonstrate an association between fibrinogen or C reactive proteinlevels and haplotypes of IL1B and IL1RN [30].

We conclude that inherited variation within the IL-1 gene clusterdoes not appear to be associated with thrombosis among patients withheritable thrombophilia. Our findings would suggest that alterations ininflammatory cytokines encoded by loci in the IL-1 gene cluster are

more likely to occur as a result, rather than a cause, of venousthrombosis.

Conflict of interest statement

No conflicts of interest have been disclosed by the authors.

Acknowledgements

This study was supported by grants from the British HeartFoundation and the Sheffield Hospitals Charitable Trust.

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