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Background: Leukotrienes (LTs) have been identified as central mediators in asthma and allergy. Pharmacological inhibition of cysteinyl-LT activity improves asthma symptoms and control. Accumulating evidence suggests a role for the dihydroxy leukotriene LTB4 in airway disease. LTA4 hydrolase and 5-lipoxygenase activating protein have key roles in LTB4 production. Single nucleotide polymorphism (SNPs) and haplotypes spanning the LTA4H and ALOX5AP genes have been associated with LTB4 production and myocardial infarction (MI).
Objective: To assess the contribution of LTA4H and ALOX5AP polymorphism to asthma and allergy susceptibility.
Methods: Three hundred and forty-one Caucasian families (two asthmatic siblings) were genotyped for eight SNPs spanning ALOX5AP and five SNPs spanning LTA4H. Association analyses of asthma and related phenotypes (total IgE, atopy, bronchial hyper-responsiveness, FEV1) were undertaken using the Family Based Association Test.
Results: Single point analyses identified association (P < 0.05) between SNPs SG13S114, SG13S89, SG13S41 (ALOX5AP), rs1978331 (LTA4H) and asthma and/or related phenotypes. Haplotype analyses using all LTA4H SNPs identified a single key risk haplotype for the development of asthma (P = 0.006) and related phenotypes (P = 0.042–0.005). Haplotype analyses using all ALOX5AP SNPs identified several asthma and atopy risk and protective haplotypes. There was limited correlation with previously identified MI risk haplotypes in both genes. Carriers of both ALOX5AP SG13S41 and LTA4H rs1978331 alleles had an increased risk of developing asthma (OR 2.17, CI 1.41–3.32).
Conclusions: These data provide evidence for the role of SNPs spanning the ALOX5AP and LTA4H genes in asthma and atopy susceptibility in the Caucasian population and support a role for LTB4 in disease pathogenesis.
Asthma is a complex multifactorial disease, involving genetic and environmental components leading to disease expression (1). Leukotrienes (LTs) are lipid mediators generated from the metabolism of arachadonic acid via a series of enzymes that constitute the 5-lipoxygenase pathway. In particular, 5-lipoxygenase activating protein (FLAP) is involved in the conversion of arachidonate to the unstable intermediate LTA4 and metabolism of LTA4 by the enzyme LTA4 hydrolase (LTA4H) results in the formation of the dihydroxy acid leukotriene B4 (LTB4). Alternatively, conjugation of LTA4 with glutathione by LTC4 synthase forms LTC4, the first member of the family of cysteinyl-leukotrienes (cys-LTs) LTC4, LTD4 and LTE4 (2). It has been established that the cys-LTs play a significant role in bronchoconstriction and airway inflammation in asthma (3). However, more recently, it has become apparent that LTB4 and its high affinity receptor LTB4R1 also play a significant role in the pathogenesis of asthma (4, 5). Concentrations of LTB4 are increased in the blood (6, 7), and bronchoalveolar lavage (8, 9) of asthma subjects. LTB4 is the major LT product of neutrophils, monocytes and alveolar macrophages and has been shown to be involved in the recruitment of various cell types to the airways including neutrophils and eosinophils (10, 11). In mild/moderate and severe asthmatic patients, where neutrophils are a feature of airways inflammation (12, 13), levels of LTB4 have been shown to be increased in exhaled breath condensate in comparison to mild asthmatics (14). A role for the LTB4-LTB4R1 interaction in T-cell recruitment to the lung has also been established (15).
Recently, studies have shown that genetic variation in enzymes in the LT biosynthetic pathway may influence LTB4 levels. Helgadottier et al. (16) identified the gene encoding 5-lipoxygenase activating protein (ALOX5AP) as underlying a susceptibility locus for myocardial infarction (MI) in the Icelandic population. They identified a specific haplotype (HapA) of ALOX5AP that was associated with increased susceptibility to MI and increased LTB4 production by neutrophils stimulated with ionomycin. In a second study, Helagdottir et al. (17) showed that haplotypes of the gene encoding leukotriene A4 hydrolase (LTA4H) were also associated with risk of MI and the ‘at-risk’ haplotype (HapK) was correlated with increased LTB4 production by ionomycin stimulated neutrophils. Subsequently, single SNPs defining these ALOX5AP haplotypes have been shown to be significant risk factors for the development of MI however replication of haplotype association has been limited (18).
Given the evidence for the role of LTB4 in allergic inflammation in the lung, we hypothesized that genetic variation in the ALOX5AP and LTA4H genes would increase susceptibility and severity of asthma. To examine this, we have genotyped single nucleotide polymorphisms (SNPs) in the ALOX5AP and LTA4H genes in an asthma cohort of 341 Caucasian families with two affected siblings and tested for single SNP and haplotype association with asthma and related phenotypes.
In addition to the primary analyses, we investigated gene–gene interactions between associated SNPs within the ALOX5AP and LTA4H genes and with other polymorphisms within genes involved in LT production/activity i.e. 5-lipoxygenase (ALOX5), leukotriene C4 synthease (LTC4S), cysteinyl leukotriene receptor 1 (CYSLTR1) and multidrug resistance associated protein 1 (MRP1).
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We present the first report demonstrating that polymorphism spanning the ALOX5AP and LTA4H genes that have been implicated as determinants of LTB4 production, are also risk factors for the development of asthma and related traits. Using a well-characterized cohort of 341 asthma families, we have identified key polymorphisms and haplotypes and identified an additive affect in carriers of risk alleles in both the ALOX5AP and LTA4H genes for the development of asthma. Interestingly, in contrast to the MI risk haplotypes reported previously, alternative haplotypes based on the same series of SNPs were identified as asthma susceptibility risk factors. These findings have broader implications and suggest that LTB4 production and activity are important determinants in asthma and allergy pathogenesis.
The importance of the LTs in asthma has been highlighted by the fact that CysLT receptors and biosynthesizing enzymes (such as 5-LO) have been used as pharmacological targets in asthma (27). Recently, a more prominent role for LTB4 in asthma has been proposed. LTB4 production is elevated in subjects with asthma (28) and LTB4 levels in the airways have been shown to correlate with asthma severity (13).
We and others previously investigated the hypothesis that genetic variation controlling the expression of LT biosynthetic enzymes and receptors predispose individuals to develop asthma (19, 23, 24, 29) or can determine the therapeutic response to pharmacological targeting of this pathway (30). There has now been an extensive body of work completed; however, these studies have generally involved the examination of single polymorphisms in single genes. In the current analyses, we specifically addressed these issues by evaluating the contribution of single SNPs, haplotypes and combinations of SNPs in the ALOX5AP and LTA4H genes to asthma susceptibility.
ALOX5AP single SNP analyses identified SG13S114/Intron1 (A allele) and SG13S41/Intron4(G allele) as risk factors for the development of asthma and related phenotypes. Interestingly, both lung function (BHR, FEV1) and atopy scores showed association indicating a potential role of LTB4 in mediating inflammatory cell influx and BHR. Similarly, there was association with atopy and asthma severity scores complementing previous data identifying a correlation between disease severity and LTB4 levels (13). These SNPs have not been investigated in asthma; however, in contrast, the SNP SG13S114/Intron1 (T) not (A) was identified as a risk factor for MI in studies of two Caucasian populations (16, 18) and is a key determinant of both previously identified MI risk haplotypes HapA and HapB (16). The SG13S114/Intron1 and SG13S41/Intron4 SNPs show limited LD (D′ 0.66) and so potentially identify independent risk factors located in or close to intron 1 and intron 4 of ALOX5AP. Haplotype analyses of the ALOX5AP SNPs identified multiple risk and protective haplotypes; however, there was no dominant haplotype. Overall, there was a limited correlation with MI related Haplotypes and alternative haplotypes based on these SNPs demonstrated association with asthma phenotypes; however, these effects may be driven by the presence or absence of asthma risk alleles at the SG13S114/Intron1 and SG13S41/Intron4 loci, e.g. GAGA (frequency 0.24, HapA SNPs) was identified as a risk haplotype and contains the SG13S114 risk allele (A). Interestingly, a recent study questioned the functional significance of the MI susceptibility Haplotypes HapA and HapB in the Caucasian population (31). Stimulated LTB4 production from neutrophils from a healthy UK population failed to identify any haplotype specific effects on the level of LTB4 generated (31). Using the same data from Maznyczka et al. (31) we explored the functional significance of ALOX5AP SNPs and haplotypes identified in this study. We did not identify any genotype or haplotype effect on LTB4 production (data not shown). These subjects were recruited on the basis of no history of cardiovascular disease or inflammatory conditions and were selected based on Hap A and B haplotypes and so we cannot exclude the possibility of a selection bias. Similarly, it is not unreasonable to predict that the functional effect of ALOX5AP genotypes identified in this study is only of relevance under the appropriate inflammatory stimuli e.g. in asthma. Overall, these data confirm the role of ALOX5AP polymorphism as asthma susceptibility markers, but highlight that the specific molecular basis underlying these observations remains to be resolved.
LTA4H single SNP analyses identified a significant association (protective) with the rs1978331(C) allele for asthma and suggestive evidence for related traits. The rs1978331 SNP is located within intron 11 of the LTA4H gene and was found to be common in our population (frequency 0.42). Haplotype analyses identified a risk haplotype (TTAAC, frequency 0.40) for asthma and all of the phenotypes examined. No association was observed for the MI susceptibility haplotype HapK (16). The associated TTAAC haplotype includes the rs1978331/Intron11(T) protective allele and this may be the basis for the association; however, the magnitude of the association is potentially more significant suggesting additional loci have a role. The SNPs that determine the TTAAC haplotype span the LTA4H gene including the 5′ region and so it is tempting to speculate that expression levels may be influenced via enhancer/transcriptional mechanisms.
Finally, we hypothesized that combinations of SNPs in multiple LT synthesizing enzymes and receptors may determine the overall expression/activity of this pathway and therefore the relative risk of developing asthma. We combined data for ALOX5AP and LTA4H SNPs showing significant association with previous data for polymorphisms present in the ALOX5, LTC4S, MRP1 and CYSLTR1 genes (19, 23, 24). The combined odds ratio for individuals carrying both the risk alleles of ALOX5AP and LTA4H (G allele in SG13S41/Intron4 and a T allele in rs1978331/Intron11) was 2.17 demonstrating an additive, but not a synergistic effect. From a biological stance, these data can be explained by the potentially increased activity at multiple locations in the 5-LO pathway leading to an overall increase in LTB4 production that is additive; however, this requires validation using isolated cells of known genotype. Little is known about the functional significance of the ALOX5AP and LTA4H intronic SNPs showing the most robust association with asthma susceptibility in the current analyses i.e. SG13S114/Intron1, SG13S41/Intron4 and rs1978331/Intron11 respectively and this requires investigation. Recent analyses of an intronic SNP (BC+1) located in the ADAM33 gene identified a significant effect of this polymorphism on gene transcription (32). A similar mechanism may be of relevance to the ALOX5AP and LTA4H genes.
It is important to note that for the main three SNPs within ALOX5AP and LTA4H showing association with asthma i.e. SG13S114, SG13S41 and rs1978331, there is good concordance with clinically relevant phenotypes e.g. if the allele is over transmitted to the asthma subject, it is also over transmitted to subjects with elevated IgE. However, this was not the case with the FEV1 (per cent predicted) analyses, which indicated the same direction of association as asthma for all SNPs i.e. clinically counter intuitive. This finding was also present in the haplotype analyses. The basis of this observation remains to be resolved; however, these analyses are correlated outcomes (asthma + FEV1) and cannot be interpreted as independent associations.
In conclusion, we have identified multiple ALOX5AP and LTA4H SNPs and haplotypes that constitute risk factors for the development of asthma and allergy and demonstrate an additive effect between polymorphism in these genes with an increased risk of asthma.
While these data require validation in additional large scale cohorts, they strongly suggest a prominent role for LTB4 production in the pathogenesis of asthma and indicate a potential clinical benefit for therapies based on LT inhibition over and above those provided by cysteinyl -LT receptor antagonism. A greater understanding of the molecular mechanisms underlying these observations may lead to new therapeutic opportunities for asthma treatment and also facilitate the targeting of therapies based on LT inhibition to those patients most likely to gain benefit.