Hersberger, Martin

Madi, Zoltan Laszlo

Marti-Jaun, Jacqueline

Waechter, Vanessa

Weber, Angelika

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Clin Chem Lab Med 2012;50(1):177–179 © 2011 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/CCLM.2011.738 Letter to the Editor  No evidence for the involvement of the lipoxin A4 receptor ( FPR2/ALX ) gene in the susceptibility to coronary artery disease  Vanessa  Waechter,  Jacqueline  Marti-Jaun,  Angelika  Weber,  Zoltan  Laszlo Madi  and  Martin  Hersberger *  Division of Clinical Chemistry and Biochemistry ,  University Children ’ s Hospital Zurich and Center for Integrative Human Physiology, University of Zurich, Zurich ,  Switzerland  Keywords:  atherosclerosis;  FPRL1;  FPR2;  FPR2/ALX;  lipoxin A4 receptor;  polymorphism.  The lipoxin A4 receptor (FPR2/ALX) belongs to the class A rhodopsin G protein-coupled receptor superfamily and plays a role in chemotaxis and activation of phagocytes  (1) . Several structurally diverse agonistic ligands, including peptides and lipid mediators have been shown to bind to the FPR2/ALX  (2) . Since the discovery of serum amyloid A (SAA) as a pro-infl ammatory ligand for FPR2/ALX, more peptides were identifi ed, which all mediate a pro-infl ammatory activation of leukocytes through FPR2/ALX  (1) . In contrast, FPR2/ALX can also bind anti-infl ammatory ligands, such as lipoxin A4 (LXA4) and annexin-1  (2) . Indeed, LXA4 was shown to inhibit neutrophil migration, to induce chemotaxis of mono-cytes and to promote the non-phlogistic phagocytosis of apoptotic neutrophils by macrophages  (2) . Thus, FPR2/ALX might induce anti-infl ammatory effects when pro-resolving mediators are present.  Expression of the FPR2/ALX has been observed in sev-eral human chronic infl ammatory diseases, such as der-matitis, rheumatoid arthritis, and severe asthma, however, the role of the FPR2/ALX in atherosclerosis has not been investigated  (3) .  Atherosclerosis is a multifactorial disease that is char-acterized by chronic infl ammation at every stage with leu-kocytes infi ltrating the arterial intima  (4) . To investigate the role of FPR2/ALX in atherosclerosis we screened the  FPR2/ALX gene for polymorphisms and analyzed whether the detected polymorphisms are associated with coronary artery disease (CAD) in a case-control study involving 497 Caucasians  (5, 6) . We initially screened the promoter, the coding region and the 3 ′ region of the  FPR2/ALX gene of 98 Caucasians for polymorphisms by DHPLC and detected eight variations, fi ve of which were located in the promoter region, one in the coding region, and two in the 3 ′ region (Table  1 ). Seven of these variations proved to be polymor-phisms with minor allele frequencies between 5 % and 32 % , while variant c.410G > A was a private mutation. The geno-type frequency for all polymorphisms was in agreement with those predicted by the Hardy-Weinberg equilibrium in the case-control sample.  This case-control sample for CAD included 259 patients with  > 50 % stenosis in at least one coronary artery and 238 subjects without a history of CAD, stroke, or peripheral vas-cular disease  (5, 6) . Written informed consent was obtained from all participants and the Local Ethics Committee approved the study. The case-control study has a power of 25 % – 80 % to detect an OR of 1.7 for minor allele frequencies of 5 % – 30 % .  Logistic regression analysis revealed no association between fi ve polymorphisms of the  FPR2/ALX gene and CAD (Table  1 ), while the T allele of the c.-7893C > T poly-morphism was associated with an increased risk for CAD (OR = 2.9; confi dence interval 1.2 – 7.1; p-value: 0.018). This c.-7893C > T polymorphism is located in the core promoter of the  FPR2/ALX gene and could therefore modify the binding site for a transcription factor and hence alter transcription of the gene  (8) .  To test whether the C > T transition affects the transcrip-tional activity of the promoter, we subcloned the  FPR2/ALX core promoter upstream of a luciferase reporter gene and introduced all the variations of the minor alleles of the three promoter polymorphisms into individual constructs. These constructs were then transiently transfected into THP-1 mac-rophages and the transcriptional activity of the different pro-moter alleles was judged from the intensity of the luciferase reporter enzyme (Figure  1 , dual-luciferase reporter assay). None of the three polymorphisms c.-7978A > G, c.-7986A > G and c.-7893C > T altered the transcriptional activity of the  FPR2/ALX promoter in macrophages, suggesting that the observed association of the SNP c.-7893C > T with CAD was rather a chance fi nding than a real association. This is  *Corresponding author: Martin Hersberger, Division of Clinical Chemistry and Biochemistry, University Children ’ s Hospital Zurich, Steinwiesstr. 75, 8032 Zurich, Switzerland Phone:  + 41-44-266-75-41, Fax:  + 41-44-266-71-69 , E-mail:  martin.hersberger@kispi.uzh.ch Received June 14, 2011; accepted September 9, 2011; previously published online October 6, 2011 178  Waechter et al.: FPR2/ALX and coronary artery diseasesupported by the skewed distribution of the homozygous TT genotypes between cases and controls in the study sample. While the TT genotype was observed to a low frequency in cases, this genotype was absent in controls (1.95 % vs. 0 % , respectively). The  FPR2/ALX haplotypes derived from the seven polymorphisms also showed no association with CAD.  Table 1  Logistic regression model predicting CAD status for cases. NM01005738 Allele frequency,  % Genotype controls,  % Genotype cases,  % OR 95 % CI p-Valuec.-8751A > G A: 68.6   G: 31.4 47.3/43.9/8.9 47.5/41.2/11.3 1.72 0.75 – 3.94 0.20rs11666254c.-8461T > G T: 68.3    G: 31.7 46.4/43.9/9.7 47.5/41.6/10.9 0.60 0.26 – 1.36 0.22rs11670660c.-7986A > G A: 73      G: 27 49.8/46/4.2 52.1/42/5.8 0.98 0.69 – 1.40 0.92rs35215887c.-7978A > G A:95        G: 5 92.4/7.6/0 89.1/9.7/1.2 1.42 0.75 – 2.68 0.29rs35225016c.-7893C > T C: 88.2      T: 11.8 77.2/22.8/0 77.4/20.6/1.9 2.93 1.20 – 7.13 0.02rs56238033c.410G > A Private mutationc.1315A > G A: 89.1      G: 10.9 76.8/23.2/0 80.5/18.3/1.2 0.66 0.19 – 2.28 0.51rs17695052c.1346C > T C: 89.4       T: 10.6 77.2/22.4/0.4 81.3/17.9/0.8 0.51 0.17 – 1.58 0.24rs17695064 DNA extracted from EDTA-supplemented blood was genotyped by tetra-primer analysis for each variation  (7) . Association with CAD was analyzed by logistic regression using an additive genetic model adjusted for age and sex. p < 0.05. 2.01.51.00.5Fold induction0.0P1-414c.-7986A>Gc.-7978A>Gc.-7893C>T Figure 1  Basal transcription of the different  FPR2/ALX core pro-moter alleles observed.  Luciferase expression of the four  FPR2/ALX core promoter alleles was determined (c.-7986A > G: A-to-G base exchange at position -7986; c.-7978A > G: A-to-G base exchange at position -7978; c.7893-C > T: C-to-T base exchange at position -7893). Human THP-1 mono-cytes were transfected with the constructs mentioned and a renilla control construct, differentiated into macrophages and the luciferase activity was measured and normalized against the activity of the renilla luciferase 48 h later. Results were calculated as fold induction over the  FPR2/ALX wild-type core promoter. All experiments were done in triplicate and bars indicate the means of three independent experiments with three plasmid preparations. Values represent fold induction compared to the P1-414 vector and the error bars represent the standard deviation ( ± SD). We observed six haplotypes with a frequency  > 1 % which summed up to 94 % of all haplotypes in the sample (data not shown).  In this study, we identifi ed seven polymorphisms in the promoter and in the 3 ′ region of the  FPR2/ALX gene, but no polymorphism was detected in the coding region of the gene. Such a lack of polymorphisms in the coding region of the  FPR2/ALX gene has previously been observed in a small study comparing the number of polymorphisms in the coding region between the  FPR2/ALX and the closely related  FPR1 gene  (9) . This study found that the two genes originated from a common gene but that they had undergone markedly different evolutionary events since then. While seven poly-morphisms have been detected in the coding region of the  FPR1 gene in this small Caucasian, Black and Asian sample, no polymorphism was detected in the  FPR2/ALX coding region in the same racial groups  (9) . Our study supports and expands these fi ndings, because we screened an additional 196 Caucasian alleles and did not fi nd a polymorphism in the coding region of the  FPR2/ALX . Such an absence of func-tional polymorphisms in the coding region and the lack of functional polymorphisms in the core promoter region sug-gest that the  FPR2/ALX gene is highly conserved in humans and that it may be important for the correct function of leuko-cytes in innate immunity.  In conclusion, the  FPR2/ALX gene was not associated with CAD in our small case-control study, however, our study suggests that the  FPR2/ALX gene is conserved in humans, since no polymorphism in the coding region and no func-tional polymorphisms in the core promoter region have been identifi ed.  Acknowledgments  This work was supported by grants from the Swiss National Science Foundation and the Hermann Klaus-Stiftung. Waechter et al.: FPR2/ALX and coronary artery disease  179Confl ict of interest statement  Authors ’ confl ict of interest disclosure: The authors stated that there are no confl icts of interest regarding the publication of this article. Research support played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.  Research funding: None declared.  Employment or leadership: None declared.  Honorarium: None declared.  References  1.  Ye RD, Boulay F, Wang JM, Dahlgren C, Gerard C, Parmentier M, et al. International union of basic and clinical pharmacology. Lxxiii. Nomenclature for the formyl peptide receptor (fpr) family. Pharmacol Rev 2009;61:119 – 61.  2.  Chiang N, Serhan CN, Dahlen SE, Drazen JM, Hay DW, Rovati GE, et al. The lipoxin receptor alx: potent ligand-specifi c and ste-reoselective actions in vivo. Pharmacol Rev 2006;58:463 – 87.  3.  Hersberger M. Potential role of the lipoxygenase derived lipid mediators in atherosclerosis: leukotrienes, lipoxins and resolvins. Clin Chem Lab Med 2010;48:1063 – 73.  4.  Paoletti R, Gotto AM Jr, Hajjar DP. Infl ammation in athero-sclerosis and implications for therapy. Circulation 2004;109:III20 – 6.  5.  Hersberger M, Muntwyler J, Funke H, Marti-Jaun J, Schulte H, Assmann G, et al. The cag repeat polymorphism in the andro-gen receptor gene is associated with hdl-cholesterol but not with coronary atherosclerosis or myocardial infarction. Clin Chem 2005;51:1110 – 5.  6.  Wittwer J, Bayer M, Mosandl A, Muntwyler J, Hersberger M. The c.-292c > t promoter polymorphism increases reticulocyte-type 15-lipoxygenase-1 activity and could be atheroprotective. Clin Chem Lab Med 2007;45:487 – 92.  7.  Hersberger M, Marti-Jaun J, Rentsch K, Hanseler E. Rapid detec-tion of the cyp2d6*3, cyp2d6*4, and cyp2d6*6 alleles by tetra-primer pcr and of the cyp2d6*5 allele by multiplex long pcr. Clin Chem 2000;46:1072 – 7.  8.  Wittwer J, Marti-Jaun J, Hersberger M. Functional polymorphism in alox15 results in increased allele-specifi c transcription in mac-rophages through binding of the transcription factor spi1. Hum Mutat 2006;27:78 – 87.  9.  Sahagun-Ruiz A, Colla JS, Juhn J, Gao JL, Murphy PM, McDermott DH. Contrasting evolution of the human leukocyte n-formylpeptide receptor subtypes fpr and fprl1r. Genes Immun 2001;2:335 – 42. 

No evidence for the involvement of the lipoxin A4 receptor (FPR2/ALX) gene in the susceptibility to coronary artery disease

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