Cardiovascular disease and stroke remain one of the

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1 Genetic Variation in the Leukotriene Pathway and Carotid Intima-Media Thickness A 2-Stage Replication Study Steve Bevan, BSc, PhD; Matthias W Lorenz, MD; Matthias Sitzer, MD; Hugh S. Markus, FRCP Background and Purpose The recent finding that genetic variation in the leukotriene biosynthesis pathway may confer an increased risk of ischemic stroke and atherosclerosis has implicated the leukotriene family as potential mediators of cardiovascular disease. Using a 2-stage replication design, we have examined whether polymorphisms in 8 genes related to this pathway are associated with early atherosclerosis and remodeling as measured by carotid artery intima-media thickness. Methods We assessed 969 individuals from the Carotid Atherosclerosis Progression Study (CAPS), a community based study of normal subjects, for 39 variants in the leukotriene pathway. Significant associations and gene environment interactions were found for 21 variants in the initial cohort and were examined in the next 1905 consecutive cases from the same CAPS population. Results No replicable association between any individual polymorphism and carotid intima-media thickness itself was present after correction for multiple testing. A single gene environment interaction was replicated between rs on bifurcation intima-media thickness and alcohol consumption exceeding 30 grams per day. Conclusion The genetic variants we examined in the leukotriene biosynthesis pathway have little effect on early atherosclerosis and remodeling risk as determined by carotid intima-media thickness. Our study cannot exclude them as being risk factors for more advanced stages in the atherosclerotic process. (Stroke. 2009;40: ) Key Words: atherosclerosis genetics Cardiovascular disease and stroke remain one of the leading causes of death in the developed world, 1 although the genetic basis of the condition is largely uncharacterized. Considerable evidence suggests inflammation may play an important role in cardiovascular disease, and recently genetic variants in inflammatory response genes have been shown to interact with both conventional proinflammatory risk factors such as smoking, as well as with chronic infection. 2,3 One such pathway is the leukotriene biosynthesis pathway; initially, variants in 5-lipoxygenase activating protein (FLAP) and leukotriene A4 hydrolase (LTA4H) were associated with increased stroke risk. 4 6 More recently variants associated with the leukotriene B4 receptors BLT1 and BLT2 receptor region were also associated with ischemic stroke risk, implicating the full leukotriene synthesis pathway. 7 The leukotriene pathway has also been implicated in atherosclerosis, with leukotriene B4 (LTB4) shown to be expressed in carotid plaques 8 and coronary artery smooth muscle cells. 9 It is unclear at what stages of the atherosclerotic process genetic alterations in leukotriene activity act to increase disease. The enhanced inflammatory processes could promote a number of stages, including early endothelial dysfunction, established plaque progression, and plaque instability. Carotid intima-media thickness (IMT) is widely used as a measure of arterial remodeling and early atherosclerosis, and has been extensively used to examine the contribution of genetic variants to early atherosclerosis. It is an independent predictor of both myocardial infarction and stroke. 10 We performed a 2-stage replication study in a large community population to determine whether genetic variation in the leukotriene pathway was associated with early atherosclerosis and remodeling as indicated by increased IMT. IMT is a continuous variable and can be measured in all individuals. Its use therefore offers considerably more power than case control studies of symptomatic disease. This, and our large sample size, allowed us to also evaluate gene environment interactions with proinflammatory risk factors in the same 2-stage replication design. Methods Subjects We studied subjects from the Carotid Atherosclerosis Progression Study. 11 All members of a German primary health care service Received May 13, 2008; final revision received July 28, 2008; accepted July 30, From Clinical Neuroscience (S.B., H.S.M.), St. George s University of London, London, UK; Department of Neurology (M.W.L., M.S.), Johann Wolfgang Goethe-University, Frankfurt am Main, Germany. Correspondence to Steve Bevan, Clinical Neuroscience, St. Georges University of London, Cranmer Terrace, Tooting, London SW17 0RE. sbevan@sgul.ac.uk 2009 American Heart Association, Inc. Stroke is available at DOI: /STROKEAHA

2 Bevan et al Genetic Variation in the Leukotriene Pathway 697 population (n ) who lived within a radius of 50 kilometers from 5 study sites in Western Germany were invited to participate. Within a predefined time limit, 6962 (21.3%) agreed to participate. In this study we included the first 969 consecutively recruited subjects as the initial study population, and the next 1905 consecutively recruited subjects as the replication population. Vascular risk factors were assessed using a standardized computer-assisted interview performed by a physician experienced in vascular medicine. Risk factors determined included pack-years of cigarette smoking and smoking category (never/ex/current smoker), history of arterial hypertension, history of diabetes mellitus, and body mass index. The mean value of 3 supine blood pressure measurements was taken as the actual arterial blood pressure. Fasting blood samples were taken for estimation of serum cholesterol and glycosylated hemoglobin A1 (HbA1c). Total serum cholesterol was determined enzymatically using a commercial kit (Boehringer). Baseline high-sensitivity C-reactive protein circulating levels were measured using an IMMAGE automatic immunoassay system (Beckmann-Coulter). Informed written consent was obtained from all participants, and the study protocol was approved by the ethical review committee of the Hospital of J.W. Goethe-University Frankfurt am Main. Ultrasound Imaging For ultrasonic examinations, a 7.5- to 10.0-MHz linear array transducer was used (P700SE; Phillips Medical System). Preprocessing configurations (log gain compensation [60 db], and image persistence) were held constant during all examinations. The gain was adjusted so that the least dense arterial wall interface was just visible. With the use of antero-oblique insonation, far-wall carotid IMT was visualized within the common carotid artery (CCA) 20 to 60 mm proximally from the flow divider, the carotid bifurcation (bifurcation IMT) 0 to 20 mm proximally from the flow divider, and the interior carotid artery (ICA) bulb (ICA bulb IMT) 0 to 20 mm distally from the flow divider, bilaterally. The images were digitally captured during the systole of a single heartbeat for off-line measurements. Vertical and horizontal calibration measurements were performed every 100 th measurement using an ultrasound assurance phantom. Automated image analysis software (Matlab; The Mathworks, Inc) was used to determine both the blood/intimal and the medial/ adventitial borderline automatically with a gray value-based edge detection algorithm combined with higher-degree polynomial fitting along these borderlines. The mean distance between the blood/ intimal and the medial/adventitial interfaces was calculated and defined as the IMT of the corresponding arterial segment. The mean length of the arterial segment in which IMT was determined was mm for the left CCA IMT, mm for the right CCA IMT, 5.75 mm for the left bifurcation IMT, 5.8 mm for the right bifurcation IMT, and 3.45 mm for the ICA bulb IMT on both sides. Choice of Genetic Polymorphisms and Overall Study Design In the initial association study in the first 969 consecutive subjects, we studied 39 variants in the 8 genes regulating the leukotriene synthesis pathway (Table 1, Figure). The genes were: FLAP, 5-LO, LTA4H, BLT1/BLT2, leukotriene C4 synthase (LTC4S), and cysteinyl leukotriene receptors 1 and 2 (CYSLTR1/CYSLTR2). In addition, variants in ALOX15 were also examined on the basis of familial similarity and previous findings in a stroke case control analysis. 7 Eighteen of the variants were in FLAP and 21 were in the remaining 7 genes. This was followed by a replication study in the next 1905 consecutive subjects of variants for which associations with either carotid IMT itself, or via gene environment interactions, had been found. On this basis we tested for replication of 21 variants. Statistical Analysis We tested for associations between gene variants and IMT at 3 sites in the carotid artery, namely the CCA ICA and the bifurcation of the carotid artery (BIF). All analyses were conducted using SPSS v15. Table 1. Variants Investigated in the Study and Frequency of Successful Genotyping Initial Cohort Replication Cohort rs N Gene Genotyped % Genotyped % rs FLAP 967/969 99% 1866/ % rs FLAP 947/969 98% 1875/ % rs FLAP 945/969 98% 1849/ % rs FLAP 910/969 94% rs FLAP 923/969 95% rs FLAP 919/969 95% rs FLAP 935/969 96% rs FLAP 955/969 98% 1905/ % rs FLAP 914/969 94% rs FLAP 893/969 92% rs FLAP 931/969 96% rs FLAP 965/969 99% rs FLAP 918/969 95% 1898/ % rs FLAP 945/969 98% 1761/ % rs FLAP 922/969 95% 1556/ %* rs FLAP 963/969 99% 1832/ % rs FLAP 944/969 97% rs FLAP 959/969 99% 1005/ %* rs LO 943/969 97% rs LO 965/969 99% 1552/ %* rs LTC4S 969/ % 1811/ % rs LTA4H 956/969 99% 1865/ % rs LTA4H 969/ % rs LTA4H 958/969 99% 1881/ % rs LTA4H 957/969 99% 1903/ % rs CYSLTR1 954/969 98% rs CYSLTR1 954/969 98% 1869/ % rs CYSLTR1 927/969 96% 1533/ %* rs CYSLTR1 939/969 97% 1817/ % rs CYSLTR2 938/969 97% rs CYSLTR2 952/969 98% 1816/ % rs LTB4R1/2 960/969 99% rs LTB4R1/2 960/969 99% rs LTB4R1/2 964/969 99% rs LTB4R1/2 926/969 96% 1761/ % rs LTB4R1/2 965/969 99% rs ALOX15 959/969 99% 1899/ % rs ALOX15 962/969 99% rs ALOX15 965/969 99% 1770/ % *Reduced genotyping because of limited DNA. DNA plates and variants were genotyped randomly and no systematic bias was identified in those 4 variants whose genotyping was less than complete. 5-LO indicates 5 lipoxygenase; CYSLTR, cysteinyl leukotriene receptor; FLAP, 5-lipoxygenase activating protein; LTA4, leukotriene A4; LTB4, leukotriene B4; LTB4R, leukotriene B4 receptor; LTC4, leukotriene C4; LTD4, leukotriene D4; LTE4, leukotriene E4.

3 698 Stroke March 2009 LTA4 Hydrolase Arachidonic Acid LTA 4 LTB 4 LTC 4 LTB4R / LTB4R2 5-LO FLAP LTC4 Synthase LTD 4 LTE 4 CYSLT1R / CYSLT2R Figure. Leukotriene biosynthesis pathway. Solid arrows indicate the pathway, dashed arrows indicate the binding of components to their respective receptors. Block arrows indicate the action of an enzyme to generate the next step in the pathway. 5-LO indicates 5 lipoxygenase; CYSLTR, cysteinyl leukotriene receptor; FLAP, 5-lipoxygenase activating protein; LTA4, leukotriene A4; LTB4, leukotriene B4; LTB4R, leukotriene B4 receptor; LTC4, leukotriene C4; LTD4, leukotriene D4; LTE4, leukotriene E4. After univariate analysis, adjustment was made for age and sex, and then for common cardiovascular risk factors. The following risk factors were included: body mass index, hypertension, smoking (pack-years), low-density lipoprotein cholesterol, alcohol intake 30 grams/day, and a measure of impaired glucose intolerance (defined as diabetes mellitus or HbA1C 6%). Our prespecified hypothesis was that interactions would exist with proinflammatory environmental stimuli; therefore, we determined interactions with the following risk factors: smoking, body mass index, alcohol, and impaired glucose tolerance. Correction for multiple testing in the regression model was performed based on the number of genes examined, with a nominal threshold of P 0.01 for the initial screen. A Bonferroni correction based on the number of genes would represent a threshold of P However, to ensure associations in the initial cohort were not missed, a less conservative threshold of P 0.01 was chosen. A full Bonferroni correction based on the number of genes was made for the replication cohort, setting a significance threshold as P because variants in 8 genes were replicated. For gene environment interactions, only a single gene was tested at a time. Bonferroni correction was therefore applied to the number of environmental risk factors tested for, namely 4. A significance level of P was therefore considered as significant for gene environment interactions. Power calculations suggest that assuming a significance level of allows the detection of a genetic variant contributing between 1% and 1.5% variation of IMT thickness with 80% power in a sample size of 1000 individuals (Quanto; gxe/). Thus our initial population is adequate in size to identify changes mm in IMT, a figure previously observed with single polymorphisms in previous genetic association studies in this population. 3 The use of a larger replication population ensures that such a finding should be replicated if it is a true association in the initial population of 969 individuals. Results Demographic characteristics of the study population are given in Table 2. The replication cohort was younger than the Table 2. Population Characteristics for the Initial and Replication Cohorts Initial 969 Individuals 1905 Replication Individuals Age, (mean SD) 57.7 (4.7) 48.9 (12.5) Sex, % male Hypertension, % Smokers, % Alcohol 30 grams/day, % IGT, % BMI 30, % LDL 5.2, % BMI indicates body mass index; IGT, impaired glucose tolerance, defined as presence of diabetes or HbA1c 6%; LDL, low-density lipoprotein. primary cohort and had a lower frequency of individuals with hypertension. All other variables were not significantly different between the 2 populations. Genotypes were assessed for Hardy-Weinberg equilibrium and a full listing of variants investigated are available in Table 1, whereas the leukotriene pathway is shown in the Figure. Data in the manuscript refer only to positive associations after correction for multiple testing because of the large number of variants investigated. Initial Study in 969 Individuals Demographic details including prevalence of risk factors and mean IMT values are shown in Table 2. Significant findings for age-/sex-adjusted and multivariable models after correction for multiple testing are shown in Table 3. There was no significant association between CCA IMT and any variant. A single variant in FLAP (rs ) showed a significant association with ICA IMT after correction in a multivariable model. Associations with BIF IMT were shown for 3 variants in 3 genes (rs in FLAP; rs in LTA4H; and rs in ALOX15) after similar correction. No individual variant was associated with IMT at 1 carotid site. Gene environment interactions were determined with the potentially proinflammatory stimuli: body mass index, alcohol, smoking, and impaired glucose tolerance After correction for multiple testing, numerous interactions in CCA, ICA, and BIF IMT were identified in the initial cohort (Table 4). Interactions were shown with smoking (rs in LTA4H) and impaired glucose tolerance (rs in CYS- LTR1 and rs in ALOX15) on ICA IMT alone, in addition to multiple interactions with alcohol in excess of 30 grams/day in CCA and BIF IMT independently. Of these, 2 variants in FLAP showed an interaction with alcohol excess at 1 carotid site (rs and rs in CCA and BIF). Both variants showed a similar direction of effect in the 2 vessels, with the rare allele being associated with higher IMT at both sites for rs and lower IMT at both sites for rs (Table 4). Replication Study in 1905 Subjects Any variant that showed an association with IMT on multivariable analysis, or that showed gene environment interactions in the initial sample of 969 individuals, was then replicated in a further sample of 1905 individuals from the

4 Bevan et al Genetic Variation in the Leukotriene Pathway 699 Table 3. Significant Results From Age-/Sex-Adjusted and Multivariable Analysis on Carotid IMT Vessel Variant Gene Genotype Age/Sex Significance Multivariable Significance N Age/Sex Mean IMT Replication Multivariable Significance ICA rs FLAP AG/GG P P AA/GG mm P AG/GG mm BIF rs FLAP AG/GG P P AA mm P AG/GG mm BIF rs LTA4H CT/CC P P CC mm P CT/TT mm BIF rs ALOX15 CG/GG P P CC mm P CG/GG mm A nominal correction of P 0.01 has been applied, being less conservative than a full Bonferroni correction but accounting for the multiple genes examined. Replication results refer to a multivariable analysis in the replication cohort for the same variant as was significantly associated in the initial cohort. No variant remained significant on replication. same Carotid Atherosclerosis Progression Study population. A total of 21 variants were replicated. After correction for multiple testing, no variant was identified as significant in the replication sample (Table 3). When examining gene environment interactions, only a single association was replicated in the second sample set, namely rs in FLAP and alcohol consumption on BIF IMT. A second variant was identified in a different IMT segment, rs in LTA4H, with BIF IMT in the replication cohort rather than ICA in the initial cohort (Table 5). Under the initial replication hypothesis, after correction for multiple testing, no variant studied was shown to be significantly associated with carotid IMT. When considering gene environment interactions under the same model and after correction for multiple testing, only a single gene environment interaction has been identified and confirmed in the same carotid segment. Discussion In this primary replication study, in a total of 3000 subjects we found no evidence of a major role of variants in genes encoding components of the leukotriene pathway in increasing carotid IMT risk. Previous studies have implicated a number of these variants in ischemic stroke risk, but the stage of the pathogenic process at which this occurs is unclear. Our study suggest that these associations are not accounted for by accelerated early atherosclerosis or vessel remodeling, as evidenced by increased IMT. Given the large number of variants investigated, we chose a 2-stage replication design of sufficient size to allow associations to be detected in the first sample, with positive associations being confirmed in a larger replication sample from the same population. This methodology represented a balance between cost and power, an issue of increasing importance with the advent of large-scale genetic association studies. Although not part of our initial study design, for completeness we did perform a global analysis of all data combined (ie, combining the initial and replication samples and analyzing jointly), but did not identify any significant finding for the 21 variants when this was possible (data not shown). The initial analysis in 969 individuals revealed few associations with IMT, with no single variant associated with increased IMT at 1 carotid site. In contrast, multiple gene environment interactions, most notably with excess alcohol consumption, were identified. However, despite the initial large sample size, the chance of detecting false-positive gene environment interactions is much higher than that for primary association with IMT itself. This is a consequence of the requirement to split a sample by both a genetic and an environmental component, particularly if the genetic variant is rare. Therefore, it is particularly important that these interactions are replicated. In the replication study of a further 1905 individuals from the same Carotid Atherosclerosis Progression Study, no association with IMT itself was replicated. Only 1 of the gene environment interactions was replicated, and this was 1 out of many and may therefore be a chance finding. Consequently, we uncovered little evidence for these genetic variants playing a significant role in IMT risk. Whereas a difference in the 2 populations cannot be excluded given the age and sex demographics, both cohorts were consecutively collected series of patients without selection bias. The younger age and consequently slightly lower frequency of hypertension in the replication cohort could be masking a replication effect, although analysis before correction for risk factors also revealed no replication for any variant investigated (data not shown). Thus, although a replication cannot be totally excluded, it remains extremely unlikely. Correction for multiple testing can be approached in a number of ways, from the most conservative full Bonferroni on all variants, genes, and phenotypes examined, through evidence-based correction on linkage disequilibrium between single nucleotide polymorphisms such as the spectral decomposition found by Nyholt, 12 to no correction but cautious interpretation. We have adopted a modified Bonferroni correction based on the number of genes investigated or a full Bonferroni correction based on the number of gene environment interactions investigated as appropriate. Such a process reduces the risk of false-positive associations, particularly in gene environment interactions in which stratification by both a genetic factor and an environmental factor is likely to lead to groups with small sample sizes. Genetic variation in the 5-lipoxygenase promoter has previously been associated with increased atherosclerosis. 13

5 700 Stroke March 2009 Table 4. Positive Gene Environment Interactions From the First Sample of 969 People Vessel Variant Gene Genotype Interaction Split Significance N a/s mean IMT CCA rs FLAP AG/GG Alcohol 30 g/day P AA mm AG/GG mm CCA rs FLAP AC/CC Alcohol 30 g/day P AA mm AC/CC mm CCA rs FLAP CC Alcohol 30 g/day P AA/AC mm CC mm CCA rs FLAP CT/TT Alcohol 30 g/day P CC mm CT/TT mm CCA rs FLAP TT Alcohol 30 g/day P CC/CT mm TT mm CCA rs FLAP AG/GG Alcohol 30 g/day P AA mm AG/GG mm CCA rs FLAP CC Alcohol 30 g/day P AA/AC mm CC mm CCA rs FLAP AG/GG Alcohol 30 g/day P AA mm AG/GG mm ICA rs LTA4H GG Smoking Never P AA/AG mm GG mm ICA rs CYSLTR1 CT/TT IGT Yes P CC mm CT/TT mm ICA rs CYSLTR2 GG Alcohol 30 g/day P AA/AG mm GG mm ICA rs ALOX15 GG IGT No P CC/CG mm GG mm BIF rs FLAP TT Alcohol 30 g/day P GG/GT mm TT mm BIF rs FLAP GG Alcohol 30 g/day P AA/AG mm GG mm BIF rs FLAP AG/GG Alcohol 30 g/day P AA mm AG/GG mm BIF rs FLAP TT Alcohol 30 g/day P CC/CT mm TT mm BIF rs FLAP AG/GG Alcohol 30 g/day P AA mm AG/GG mm Only results significant after Bonferroni correction for the number of gene environment interactions (n 4) are shown. We have failed to replicate this finding in our cohort, although we have looked at single nucleotide polymorphisms rather than the tandem Sp1 motif previously studied. The effects observed by Dwyer et al 13 were also related to dietary intake, particularly levels of n-6 polyunsaturated fatty acid and marine n-3 fatty acids, which were found to promote and inhibit, respectively, leukotriene-mediated inflammation leading to atherosclerosis. We do not have such dietary data for our cohort, and it may be that this gene environment interaction is more significant than the risk factors examined as part of this study. Similarly, the Carotid Atherosclerosis Progression Study population represents a community population with predominantly early atherosclerosis, and there were insufficient Table 5. Positive Gene Environment Interactions From the Replication Sample of 1905 People Vessel Variant Gene Genotype Interaction Split Significance N Age/Sex Mean IMT BIF rs FLAP AG/GG Alcohol 30 g/day P AA mm AG/GG mm BIF rs LTA4H AG/GG IGT Has IGT P AA mm AG/GG mm Only results significant after Bonferroni correction for the number of gene environment interactions (n 4) are shown.

6 Bevan et al Genetic Variation in the Leukotriene Pathway 701 advanced plaque and stenoses to exclude associations with advanced atherosclerosis. As such, we cannot exclude the possibility that the risk previously identified with ischemic stroke is manifest through later pathogenic mechanisms such as plaque instability and plaque rupture rather than the early atherosclerotic mechanisms of vessel remodeling or changes in IMT. Further specific studies will be required to assess the role of leukotrienes in such processes. In summary, no variant was identified that associated with carotid IMT in both samples. A single gene environment interaction was detectable in both samples independently at the same carotid site and remains significant after correction for multiple testing. The lack of association at 1 single carotid site, however, suggests this finding is likely to be of limited importance. As such, we would conclude that the genetic variants investigated in this study have little influence on early atherosclerosis and remodeling as measured by changes in IMT. Further, these results also emphasize that when examining gene environment interactions, sample sizes should be as large as possible to ensure false-positive associations are not detected as a consequence of population substratification. None. Disclosures References 1. Yusuf S, Reddy S, Ounpuu S, Anand S. Global burden of cardiovascular diseases: part I: general considerations, the epidemiologic transition, risk factors and impact of urbanisation. Circulation. 2001;104: Jerrard-Dunne P, Sitzer M, Risley P, Steckel DA, Buehler A, Kegler S, Markus HS. Inetrleukin-6 promoter polymorphism modulates the effects of heavy alcohol consumption on early carotid artery atherosclerosis. Stroke. 2003;34: Jerrard-Dunne P, Sitzer M, Risley P, Buehler A, Kegler S, Markus HS. Inflammatory gene load is associated with enhanced inflammation and early carotid atherosclerosis in smokers. Stroke. 2004;35: Helgadottir A, Manolescu A, Thorleifsson G, Gretarsdottir S, Jonsdottir H, Thorsteinsdottir U, Samani N, Gudmundsson G, Grant SFA, Thorgeirsson G, Sveinbjornsdottir S, Valdimarrson E, Matthiasson SE, Johannsson H, Gudmundsdottir O, Gurney ME, Sainz J, Thorhallsdottir M, Andresdottir M, Frigge ML, Topol EJ, Kong A, Gudnason V, Hakonaeson H, Gulcher JR, Stefansson K. The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke. Nat Gen. 2004;36: Helgadottir A, Manolescu A, Helgason A, Thorleifsson G, Thorsteinsdottir U, Gudbjartsson D, Gretarsdottir S, Magnusson KP, Gudmundsson G, Hicks A, Jonsson T, Grant SFA, Sainz J, O Brien SJ, Sveinbjornsdottir S, Valdimarrson E, Matthiasson SE, Levey AI, Abramson JL, Reilly MP, Vaccarino V, Wolfe ML, Gudnason V, Quyyumi AA, Topol E, Rader DJ, Thorgeirsson G, Gulcher J, Hakonaeson H, Kong A, Stefansson K. A variant of the gene encoding leukotriene A4 hydrolase confers ethnicity-specific risk of myocardial infarction. Nat Gen. 2006;38: Helgadottir A, Gretarsdottir S, St.Clair D, Manolescu A, Cheung J, Thorleifsson G, Pasdar A, Grant SFA, Whalley LJ, Hakonarson H, Thorsteinsdottir U, Kong A, Gulcher J, Stefansson K, MacLeod MJ. Association between the gene encoding 5-lipoxygenase activating protein and stroke replicated in a Scottish population. Am J Hum Genet. 2005;76: Bevan S, Dichgans M, Wiechmann E, Gschwendtner A, Meitinger T, Markus HS. Genetic variation in members of the leukotriene biosynthesis pathway confer an increased risk of ischaemic stroke a replication study in two independent populations. Stroke. 2008;39: Zhou YJ, Wang JH, Li L, Yang HW, Wen DL, He QC. Expanding expression of the 5-lipoxygenase/leukotriene B4 pathway in atherosclerotic lesions of diabetic patients promotes plaque instability. Biochem Biophys Res Comm. 2007;363: Back M, Bu D, Branstrom R, Sheikine Y, Yan Z-Q, Hansson GK. Leukotriene B4 signalling through NF-KB-dependent BLT1 receptors on vascular smooth muscle cells in atherosclerosis and intimal hyperplasa. Proc Natl Acad Sci U S A. 2005;102: Lorenz MW, Markus HS, Bots ML, Rosvall M, Sitzer M. Prediction of clinical cardiovascular events with carotid intima-media thickness: a systematic review and meta-analysis. Circulation. 2007;115: Sitzer M, Markus HS, Mendall MA, Liehr R, Knorr U, Steinmetz H. C-reactive protein and carotid intimal medial thickness in a community population. J Cardiovasc Risk. 2002;9(2): Nyholt DR. A simple correction for multiple testing for single nucleotide polymorphisms in linkage disequilibrium with each other. Am J Hum Genet. 2004;74: Dwyer JH, Allayee H, Dwyer KM, Fan J, Wu H, Mar R, Lusis AJ, Mehrabian M. Arachidonate 5-lipoxygenase promoter genotype, dietary arachidonic acid and atherosclerosis. N Engl J Med. 2004;350:29 37.