Hum Genet (2011) 129:617���627 DOI 10.1007/s00439-011-0963-3 123 ORIGINAL INVESTIGATION Genetic contribution of the leukotriene pathway to coronary artery disease Jaana Hartiala �� Dalin Li �� David V. Conti �� Susanna Vikman �� Yesha Patel �� W. H. Wilson Tang �� Marie-Louise Brennan �� John W. Newman �� Charles B. Stephensen �� Patrice Armstrong �� Stanley L. Hazen �� Hooman Allayee Received: 22 October 2010 / Accepted: 27 January 2011 / Published online: 4 February 2011 �� The Author(s) 2011. This article is published with open access at Springerlink.com Abstract We evaluated the genetic contribution of the leukotriene (LT) pathway to risk of coronary artery disease (CAD) in 4,512 Caucasian and African American subjects ascertained through elective cardiac evaluation. Of the three previously associated variants, the shorter ���3��� and ���4��� alleles of a promoter repeat polymorphism in ALOX5 increased risk of CAD in African Americans (OR = 1.4, 95% CI 1.0���1.9 p = 0.04), whereas a haplotype of LTA4H (HapK) was associated with CAD in Caucasians (OR = 1.2, 95% CI 1.01���1.4 p = 0.03). In Caucasians, Wrst-stage analysis of 254 haplotype-tagging SNPs in 15 LT pathway genes with follow-up of 19 variants in stage 2 revealed an LTA4H SNP (rs2540477) that increased risk of CAD (OR = 1.2, 95% CI 1.1���1.5 p = 0.003) and a PLA2G4A SNP (rs12746200) that decreased risk of CAD (OR = 0.7, 95% CI 0.6���0.9 p = 0.0007). The PLA2G4A rs12746200 variant also decreased risk of experiencing a major adverse cardiac event (MACE = myocardial infarction, stroke, or death) over 3 years of follow-up (HR = 0.7, 95% CI 0.5���0.9 p = 0.01), consistent with its cardioprotective eVect. Func- tional experiments demonstrated that stimulated monocytes from carriers of LTA4H variants HapK or rs2540477 had 50% (p = 0.002) and 33% (p = 0.03) higher LTB4 produc- tion, respectively, compared to non-carriers. These ex vivo results are consistent with LTB4 being the direct product of the reaction catalyzed by LTA4H and its role in promoting monocyte chemotaxis to sites of inXammation, including the artery wall of atherosclerotic lesions. Taken together, this study provides additional evidence that functional genetic variation of the LT pathway can mediate athero- genic processes and the risk of CAD in humans. Introduction Class four leukotrienes (LTs) are potent pro-inXammatory mediators synthesized from arachidonic acid, an omega-6 polyunsaturated fatty acids (PUFAs) (Peters-Golden and Henderson 2007). The rate-limiting step in this pathway is catalyzed by the enzyme arachidonic acid 5-lipoxygenase (ALOX5). The biologically active LTs are synthesized by Electronic supplementary material The online version of this article (doi:10.1007/s00439-011-0963-3) contains supplementary material, which is available to authorized users. J. Hartiala �� D. Li �� D. V. Conti �� S. Vikman �� Y. Patel �� H. Allayee Department of Preventive Medicine, USC Keck School of Medicine, Los Angeles, CA 90033, USA J. Hartiala �� S. Vikman �� Y. Patel �� H. Allayee (&) Institute for Genetic Medicine, USC Keck School of Medicine, 2250 Alcazar Street, CSC 206, Los Angeles, CA 90033, USA e-mail: hallayee@usc.edu W. H. Wilson Tang �� M.-L. Brennan �� S. L. Hazen Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH 44195, USA M.-L. Brennan �� S. L. Hazen Department of Cell Biology, Cleveland Clinic, Cleveland, OH 44195, USA M.-L. Brennan �� S. L. Hazen Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, Cleveland, OH 44195, USA J. W. Newman �� C. B. Stephensen �� P. Armstrong USDA Western Human Nutrition Research Center, University of California Davis, Davis, CA 95616, USA C. B. Stephensen �� P. Armstrong Program in International and Community Nutrition, Department of Nutrition, University of California Davis, Davis, CA 95616, USA

618 Hum Genet (2011) 129:617���627 123 subsequent conversion to LTB4 and the cysteinyl LTs (LTC4, LTD4, and LTE4) via enzymatic reactions by LTA4 hydrolase (LTA4H) and LTC4 synthase (LTC4S), respec- tively (Peters-Golden and Henderson 2007). LTs then aVect the function of target cells, including monocytes and other pro-inXammatory leukocytes, through receptor-mediated signal transduction. While LTs have long been known to be involved in chronic allergic inXammatory conditions, such as asthma, the LT pathway has also recently garnered attention for its potential role in coronary artery disease (CAD)-related traits. This stems from a series of biochemical, genetic, and pharmacological studies over the last few years that have provided evidence for the pro-atherogenic role of LTs (Back and Hansson 2006 Mehrabian and Allayee 2003 Tymchuk et al. 2006). For example, genetic deWciency for ALOX5 in mice protects against aortic lesion formation and leads to other metabolic disturbances (Mehrabian et al. 2002, 2005, 2008). Other mouse studies have reported the involvement of LT pathway genes in atherosclerosis- related traits as well, including the LT receptors and ALOX5 activating protein (ALOX5AP) (Ahluwalia et al. 2007 Aiello et al. 2002 Heller et al. 2005 Jawien et al. 2006, 2008 Subbarao et al. 2004). Studies in humans have also provided evidence supporting the notion that LTs participate in atherosclerotic processes. Immunohistochemical studies have shown that ALOX5, ALOX5AP, and LTA4H are abundantly expressed in arterial walls of CAD patients, with ALOX5 having markedly increased expression in advanced lesions and localizing to macrophages, dendritic cells, and neutrophilic granulocytes (Qiu et al. 2006 Spanbroek et al. 2003). In addition, indi- viduals carrying the shorter alleles of a functional ALOX5 promoter polymorphism, consisting of tandem Sp1 binding sites, have signiWcantly increased carotid atherosclerosis and risk of myocardial infarction (MI), particularly in the context of high dietary arachidonic acid levels (Allayee et al. 2008 Dwyer et al. 2004). This is supported by studies that have reported associations between other ALOX5 and ALOX5AP variants with CAD-related phenotypes (Burdon et al. 2010 Carlson et al. 2007 Crosslin et al. 2009 Helga- dottir et al. 2004). More recently, a 10-SNP haplotype of LTA4H, designated HapK, has been associated with MI in Caucasians and African Americans, with a more pro- nounced eVect in the latter group (Helgadottir et al. 2006), and LTC4S variants have been associated with surrogate measures of CAD, including coronary artery calciWcation and carotid atherosclerosis (Iovannisci et al. 2007). Impor- tantly, these genetic studies are bolstered by functional data showing that the associated variants/haplotypes lead to increased gene expression or LT production (Allayee et al. 2008 Helgadottir et al. 2004, 2006 Sanak et al. 2000 Vikman et al. 2009). Despite these reports, evidence for association of LT pathway genes with CAD traits has not been consistently observed across all studies (Assimes et al. 2008 Koch et al. 2007 Zee et al. 2006 Zintzaras et al. 2009). Thus, the aim of the present study was to compre- hensively evaluate the genetic contribution of the LT path- way to CAD in a large cohort of subjects undergoing elective cardiac evaluation. Materials and methods Study subjects GeneBank is a single site (Cleveland Clinic) sample repository generated from patients undergoing elective diagnostic coronary angiography or elective cardiac computed tomo- graphic angiography with extensive clinical and laboratory characterization and longitudinal observation (Bhatta- charyya et al. 2008 Nicholls et al. 2010). Ethnicity was self-reported and information regarding demographics, medical history, and medication use was obtained by patient interviews and conWrmed by chart reviews. All clinical outcome data were veriWed by source documentation. CAD was deWned as adjudicated diagnoses of stable or unstable angina, MI (adjudicated deWnition based on deW- ned electrocardiographic changes or elevated cardiac enzymes), angiographic evidence of ��50% stenosis of one or more major epicardial vessel, and/or a history of known CAD (documented MI, CAD, or history of revasculariza- tion). Prospective cardiovascular risk was assessed by the incidence of major adverse cardiac events (MACE) during 3 years of follow-up from the time of enrollment, which included nonfatal MI, nonfatal stroke, and all-cause mortal- ity. Nonfatal events were deWned as MI or stroke in patients who survived at least 48 h following the onset of symp- toms. Adjudicated outcomes ascertained over the ensuing 3 years for all subjects following enrollment were conWrmed using source documentation. All patients pro- vided written informed consent prior to being enrolled in GeneBank and the study was approved by the Institutional Review Board of the Cleveland Clinic. The present genetics study was approved by the Institutional Review Boards of the Cleveland Clinic and USC Keck School of Medicine. Clinical laboratory measurements Samples were collected from overnight fasted subjects on the day of elective cardiac catheterization. Plasma aliquots were isolated from whole blood collected into EDTA tubes, maintained at 0���4��C immediately following phlebotomy, processed within 4 h of blood draw, and stored at ��80��C until analysis. Plasma levels of total cholesterol, low-den- sity lipoproteins (LDL), high-density lipoproteins (HDL),