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Keywords:

  • allergy;
  • asthma;
  • leukotriene;
  • polymorphism;
  • susceptibility

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

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).

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Subjects and clinical assessments

Caucasian families (n = 341) from the Southampton area were recruited with at least two biological siblings with a current physician diagnosis of asthma and taking asthma medication on a regular basis (Table 1). Serum total IgE levels and specific IgE levels for grass, house dust mite, cat, dog, Alternaria and tree allergens were determined by RAST. Skin prick testing was also completed for the same common allergens. Baseline lung function tests [Forced Expiratory volume in 1 s (FEV1) best of three values within 5%] were performed using Vitalograph® dry wedge bellows spirometer (Vitalograph, Maids Moreton, UK). Bronchial hyper-responsiveness (BHR) was measured as the provocation concentration of inhaled methacholine required to reduce FEV1 by 20% (PC20 FEV1). Methacholine dilutions (0–16 mg/ml) were given at 5 min intervals by DeVilbiss nebuliser with KoKo dosimeter (PDS Instruments Inc. Louisville, CA, USA). This population and the generation of phenotypic scores has been described previously (19). Phenotypes examined in the association analyses included; asthma, FEV1 (percent predicted), BHR (methacholine), total IgE, atopy severity and asthma severity. Ethical approval was obtained from the Southampton & SouthWest Hampshire Joint Ethics Committee.

Table 1.   Clinical characteristics of the Southampton asthma family cohort
 Pedigrees (n = 1508)Parents (n = 681)Non asthmatic parents (n = 492)Asthmatic parents (n = 189)Sibling 1 (n = 341)Sibling 2 (n = 328)
  1. *Age corrected log IgE levels represent the mean log of the standard deviation from the median for each of the following age groups (≥5 and ≤10, ≥10 and ≤15, ≥15 and ≤18, ≥18).

Age (years)24.640.540.740.213.09.9
Gender (% male)51.849.951.047.156.953.6
Asthma (%, doctor)60.127.80100100100
Eczema (%, questionnaire)45.632.725.850.857.862.4
Hayfever (%, questionnaire)48.946.838.069.864.247.0
FEV1 (% predicted)98.05100.81103.3994.1294.7495.62
BHR (methacholine) (1/LSlope+30) × 100019.0324.3326.7917.2214.5512.00
Log IgE (*Age corrected) 1.250.640.491.011.841.93

Selection of haplotype tagging SNPs and haploview analyses

ALOX5AP SNPs were selected for their ability to tag haplotypes that were previously shown to be associated with MI and LTB4 production, SNPs SG13S25(5′UTR), SG13S114(Intron1), SG13S89(Intron3), SG13S32(Intron4), SG13S377(5′UTR), SG13S41(Intron4), SG13S35(3′UTR) (16). In addition, two SNPs that define another ALOX5AP haplotype (Hap1) that has previously shown an association with asthma susceptibility were genotyped, rs3803277(Intron2) and rs4468448(Intron4) [US Patent 6 531 279 B1 (2003)]. LTA4H SNPs were selected for their ability to tag the HapK haplotype previously shown to be associated with risk if MI and LTB4 production, SNPs rs1978331(Intron11), rs17677715(Intron6), rs2540482(5′UTR), rs2660845(5′UTR) and rs2540475(5′UTR) (17). Details of SNP sequence variation were obtained from NCBI. SNPs were genotyped by Kbioscience using KASPar or TaqMan methodology (Kbiosciences, Hitchin, UK). The linkage disequilibrium (LD) pattern across the genes was established using haploview software (20).

Association analyses

The Family Based Association Test (FBAT, version 1.5.1) was used to test for association with the series of phenotypic scores described (21, 22). Single SNP and SNP Haplotype analyses were completed using FBAT or HBAT under the additive and bi-allelic model (21, 22). A P < 0.05 was considered significant. We did not correct for multiple testing as we did not consider the tests to be independent because of LD spanning the two genes. However, simulation minimal P values for the haplotype analyses were generated using the FBAT –P option (100 000 simulations) in order to facilitate interpretation.

Gene–gene interactions

To test for interaction between polymorphisms showing positive association with asthma phenotypes in the current analyses and previously implicated polymorphism in related genes [i.e. ALOX5 Sp1 repeat in promoter region (23), LTC4S -444 A/C(promoter/rs730012) (19) and CYSLTR1 927 T/C(coding region/F309F/ rs320995) (24)], a dataset of cases and pseudo controls was created using David Clayton’s pseudocc program in Stata (25). The ‘one’ option was used to create only one pseudo control per case, with the untransmitted alleles assigned to the pseudo control. Each of the above SNPs (apart from CYSLTR1) was investigated individually for their influence on case (asthma) pseudo control status using conditional logistic regression. Robust estimates of the standard deviations were used (clustered according to family) as more than one affected sibling from each family was included. ALOX5, ALOX5AP, LTA4H and MRP1(Intron1/rs119774) (26) were then put in the same model to investigate the effect of other genes as covariates. These were removed one at a time. ALOX5, ALOX5AP and LTC4S were put into a different model to investigate covariates. Again these were removed one at a time.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Genotyping

Genotyping completion rates were >95% for SNPs; however, SG13S377 (ALOX5AP/5UTR) failed quality control using the two different genotyping chemistries and was omitted from the analyses. The genotyping data for the 13 SNPs analysed did not show deviation from Hardy–Weinberg Equilibrium, P > 0.05. FBAT inheritance check identified five errors (0.02%) and these families were excluded from the analyses of the relevant SNP. The minor allele frequencies for the ALOX5AP SNPs were similar to allele frequencies observed in other Caucasian populations [Table 2, (16)]. Similarly, the LTA4H SNP allele frequencies were comparable to previous data generated in the Caucasian population (17). Figure 1 illustrates the location of SNPs genotyped.

Table 2.   Family based association test results between ALOX5AP SNPs and asthma and allergy phenotypes
dbSNP referenceGene locationAllelesMAFAsthma (n)Z-score (P-value)+BHR (n)Z-score (P-value)+FEV1 (n)Z-score (P-value)+IgE (n)Z-score (P-value)+atopy (n)Z-score (P-value)+severity (n)Z-score (P-value)
  1. SNP, single nucleotide polymorphism; UTR, untranslated region; MAF, minor allele frequency; BHR, bronchial hyper-responsiveness; FEV1, forced expiratory volume in 1 s; IgE, immunoglobulin E; atopy, atopy severity; severity, asthma severity (19). No. represents number of families analysed. *P < 0.05. Where positive associations occurred we have included the Z score which indicates the direction of the association (+ means the minor allele was over transmitted = risk and − means under transmitted = protection with respect to asthma affection, for continual traits a negative sign indicates that the presence of the allele confers lower trait values; a positive sign indicates that the allele confers higher values). The Z score is a measure of transmission equilibrium i.e. the deviation from the number of times an allele was transmitted to affected offspring (statistic S) and the number of times it should be transmitted (expected statistic E) under the null hypothesis (no association, no linkage).

SG13S255′UTRG/A0.1061040.6391000.5091050.5581050.432960.402960.566
SG13S114Intron 1T/A0.331216+2.349  0.018*2210.236222+2.127 0.033*2220.155205+1.974 0.048*2150.188
rs3803277Intron 2C/A0.4512460.1442470.3132500.1582500.2442240.2162410.187
SG13S89Intron 3G/A0.043470.157480.701480.177480.21645+2.064 0.039*480.156
rs4468448Intron 4C/T0.2472130.1842150.3892160.2902160.5051940.2312100.684
SG13S32Intron 4C/A0.4792380.2012390.1892430.2222430.3412220.4592330.459
SG13S41Intron 4A/G0.06769+2.681 0.007*720.16073+2.541 0.011*72+2.220 0.026*69+2.578 0.010*72+2.311 0.021*
SG13S353′UTRG/A0.079830.466850.545860.568860.366800.201820.593
image

Figure 1.  Schematic representation of ALOX5AP and LTA4H genes including linkage disequilibrium data. (A) The ALOX5AP gene on chromosome 13q12 showing the location of SNPs genotyped in the current study. Polymorphisms SG13S25, SG13S114, SG13S89, SG13S32 define HapA, SG13S114, SG13S41, SG13S35 partially define HapB previously implicated in MI susceptibility [(16), see text] and rs3803277, rs4468448 define Hap1. Exons are represented by black boxes, introns are depicted by lines between these boxes. (B) Linkage disequilibrium pattern of genotyped ALOX5AP SNPs. The chromosomal location of each SNP is illustrated at the top of the diagram. The pairwise linkage disequilibrium values were generated using haploview software (20). The intensity of shading represents D′ and numerical values are provided. (C) The LTA4H gene (reverse complement) on 12q22 showing the location of the five SNPs that define HapK that has been associated with MI susceptibility (17). (D) Linkage disequilibrium pattern of genotyped LTA4H SNPs.

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Family based association analyses for ALOX5AP and LTA4H SNPs

Family based association analyses of the eight ALOX5AP SNPs using 341 Caucasian families with two asthma siblings [Table 1, (19)] identified a significant association between increased risk of asthma and SNPs; SG13S114(ALOX5AP/Intron1) and SG13S41(ALOX5AP/Intron4) (P = 0.018 and P = 0.007 respectively) (Table 2). Similarly, SG13S114 showed association with BHR (P = 0.033), FEV1 (P = 0.033) and atopy severity (P = 0.048) confirming the significance of this SNP. The SG13S41 SNP also showed association with FEV1 (P = 0.011), atopy severity (P = 0.026) and asthma severity (P = 0.021). The SG13S89(ALOX5AP/Intron3) SNP also showed an association (risk) with atopy severity (P = 0.039). Family based association analyses of the five LTA4H SNPs identified a significant association (protection) between rs1978331(LTA4H/Intron11) and asthma (P = 0.036) and atopy severity (P = 0.05), but there was also suggestive evidence of association for the other phenotypes examined (Table 3).

Table 3.   Family based association test results between LTA4H SNPs and asthma and allergy phenotypes
dbSNP referenceGene locationAllelesMAFAsthma (n)Z-score (P-value)+BHR (n)Z-score (P-value)+FEV1 (n)Z-score (P-value)+IgE (n)Z-score (P-value)+atopy (n)Z-score (P-value)+severity (n)Z-score (P-value)
  1. SNP, single nucleotide polymorphism; UTR, untranslated region; MAF, minor allele frequency; BHR, bronchial hyper-responsiveness; FEV1, forced expiratory volume in 1 s; IgE, immunoglobulin E; atopy, atopy severity; severity, asthma severity (19). No. represents number of families analysed. *P < 0.05. Where positive associations occurred we have included the Z score which indicates the direction of the association (+ means the minor allele was over transmitted = risk and − means under transmitted = protection with respect to asthma affection, for continual traits a negative sign indicates that the presence of the allele confers lower trait values; a positive sign indicates that the allele confers higher values). The Z score is a measure of transmission equilibrium i.e. the deviation from the number of times an allele was transmitted to affected offspring (statistic S) and the number of times it should be transmitted (expected statistic E) under the null hypothesis (no association, no linkage).

rs1978331Intron 11T/C0.417227−2.095   0.036*2330.462235−1.863  0.063235−1.727  0.084213−1.960   0.050*227−1.731  0.083
rs17677715Intron 6T/C0.1951630.2161660.9991680.3801670.3171560.4261620.257
rs25404825′UTRA/G0.2241810.2481840.3401880.2521880.1281720.6501800.150
rs26608455′UTRA/G0.2601990.8532040.8052060.8502050.4731880.8602010.551
rs25404755′UTRC/T0.2161810.3031820.6461840.5471840.1671690.1951770.495

ALOX5AP linkage disequilibrium and haplotype specific association analyses

Figure 1B shows the LD plot for the eight ALOX5AP SNPs spanning ∼40 kbp genotyped in the families. These data show that there are regions of high LD (measured using D′) between SNPs e.g. SG13S41(Intron4) and SG13S35(3′UTR); however, there are also regions of limited LD e.g. SG13S114(Intron1)-rs3803277(Intron2). The two most distal SNPs show relatively high LD across the region (SG13S25(5′UTR)-SG13S35(3′UTR), D′ = 89) (Fig. 1B). Haplotype association analyses based on all SNP data identified multiple protective and risk haplotypes most notable a potentially common protective haplotype (GTCCGCCAC, 0.411 frequency) for asthma (P = 0.069) and FEV1 (P = 0.069) (Table 4). Investigation of the MI associated haplotype, HapA did not identify a significant association (GTGA, frequency 0.14), but did identify suggestive evidence for an alternative risk haplotype (GAGA, frequency 0.63) for asthma (P = 0.062), BHR (P = 0.062) and FEV1 (P = 0.070) which was supported by simulation (Table 4). Analyses of the partially tagged MI susceptibility Haplotype, HapB (AAG, frequency 0.217) did identify a suggestive association with BHR (P = 0.060), but this was not supported by simulation (Table 4). The partial HapB haplotype examined in the current analyses had a frequency of 0.21 in contrast to 0.075 for the completely tagged MI susceptibility haplotype HapB (including SG13S377) observed in another British population (16). The common TAG haplotype (frequency 0.639) was identified as a potentially protective haplotype for asthma (P = 0.022), FEV1 (P = 0.034) and atopy severity (P = 0.054). The TGG haplotype (0.015 frequency) also showed an association (P < 0.05) with asthma and related phenotypes (Table 4). Analyses based on Hap1 identified a potentially protective haplotype (CT, 0.012 frequency) for asthma severity (P = 0.065) in contrast to previous findings for Hap1 (AT, frequency 0.235).

Table 4.   Family based association test results between ALOX5AP haplotypes and asthma and allergy phenotypes
Haplotype analysesNo. familiesFreq. Phenotype (risk/protection)HBAT (P < 0.1) P-valueSimulation minimal P-value
  1. BHR, bronchial hyper-responsiveness; FEV, forced expiratory volume in 1 s; IgE, immunoglobulin E. ‘All’ defined by SG13S25, SG13S114, rs3803277, SG13S89, rs4468448, SG13S32, SG13S41, SG13S35. HapA defined by SG13S25, SG13S114, SG13S89, SG13S32. ^HapB defined by SG13S114, SG13S41, SG13S35 [SG13S377omitted in contrast to (16)]. Hap1 defined by rs3803277 and rs4468448. Haplotypes showing a P-value <0.1 are shown. Permutation (100 000 simulations) was used to derive minimal simulation P-values allowing interpretation of the observed values.

All SNPs
GTCGCCAC2010.411Asthma (protection)0.0690.274
GTCGCCAC2090.411Asthma + FEV1 (protection)0.0690.252
GACGCAAG430.044Asthma + BHR (risk)0.0050.299
GACGCAAG430.044Asthma + FEV1 (risk)0.0590.252
GACACCGG420.041Atopy severity (risk)0.0550.584
GTAGTAAG150.016Atopy severity (protection)0.0430.584
GTAGTAAG200.016Asthma + IgE (protection)0.0250.299
GTAGTCGG150.013Asthma (risk)0.0250.274
GTAGTCGG150.013Asthma + BHR (risk)0.0080.299
GTAGTCGG150.013Asthma + FEV1 (risk)0.0180.252
GTAGTCGG150.013Asthma severity (risk)0.0820.507
GTAGTCGG150.013Asthma + IgE (risk)0.0420.299
GACGTAAG130.012Asthma severity (protection)0.0800.507
MI HapA (GTGA)
  GAGA1840.241Asthma (risk)0.0620.339
  GAGA1850.241Asthma + BHR (risk)0.0620.343
  GAGA1860.241Asthma + FEV1 (risk)0.0700.401
  GAAC450.041Atopy severity (risk)0.0260.171
MI ^HapB (AAG)
  TAG2190.639Asthma (protection)0.0220.081
  TAG2250.639Asthma + IgE (protection)0.0880.158
  TAG2250.639Asthma + FEV1 (protection)0.0340.082
  TAG2080.639Atopy severity (protection)0.0540.221
  AAG*1680.217Asthma + BHR (risk)0.0600.035
  AGG530.051Atopy severity (risk)0.0420.221
  TGG170.015Asthma (risk)0.0160.081
  TGG190.015Asthma + IgE (risk)0.0320.158
  TGG180.015Asthma + BHR (risk)0.0040.035
  TGG190.015Asthma + FEV1 (risk)0.0150.082
  TGG180.015Asthma severity (risk)0.0490.232
Hap1(AT)
  CT280.012Asthma severity (protection)0.0650.178

LTA4H linkage disequilibrium and haplotype specific association analyses

Figure 1D shows the LD plot for the five LTA4H SNPs genotyped. These data show limited LD between individual SNPs overall; however, there is high LD between selected SNPs e.g. rs1978331(Intron11)-rs17677715(Intron6) and potentially there are two blocks of LD at the 5′ and 3′ regions respectively.

Haplotype association analyses did not identify an association between the MI associated haplotype HapK (TTGGC, frequency 0.139) and asthma related phenotypes (data not shown). A common risk haplotype was identified (TTAAC, frequency 0.404) for the development of asthma (P = 0.006) and all other phenotypes analysed i.e. IgE levels (P = 0.005), FEV1 (P = 0.012), atopy severity (P = 0.042) and asthma severity (P = 0.014), which was supported by simulation (Table 5).

Table 5.   Family based association test results between LTA4H haplotypes and asthma and allergy phenotypes
Haplotype analysesNo. familiesFreq. Phenotype (risk/protection)HBAT (P < 0.1) P-valueSimulation minimal P-value
  1. BHR = bronchial hyper-responsiveness; FEV1, forced expiratory volume in 1 s; IgE, immunoglobulin E. Hap-K defined by rs1978331 rs17677715 rs2540482 rs2660845 rs2540475 (17). Haplotypes showing P value <0.1 are shown. Permutation (100 000 simulations) was used to derive minimal simulation P-values allowing interpretation of the observed values.

MI HapK (TTGGC)
 TTAAC2050.404Asthma (risk)0.0060.065
 TTAAC2210.404Asthma + IgE (risk)0.0050.049
 TTAAC2220.404Asthma + FEV1 (risk)0.0120.130
 TTAAC2010.404Atopy severity (risk)0.0420.376
 TTAAC2130.404Asthma severity (risk)0.0140.173
 CTAAC980.103Asthma + BHR (protection)0.0660.668
 CTGGC160.008Atopy severity (protection)0.0320.376

Gene–gene interaction analysis

To test for potential synergistic interaction between polymorphisms in different components of the LT-biosynthetic pathway on affection status, polymorphisms showing the strongest association in ALOX5AP (SG13S41/Intron4), LTA4H (rs1978331/Intron11) and MRP1 (26), (rs119774/Intron1, FBAT(risk) BHR (P = 0.003), data not shown) together with previously reported data generated using this cohort ALOX5 (Sp1 promoter repeat) (23), LTC4S (promoter/A-444C/rs730012) (19), CYSLTR1 (T927C/coding region/F309F/rs320995) (24) were analysed using conditional logistic regression. No evidence on synergistic interaction was observed (data not shown). However, the combined odds ratio for asthma susceptibility individuals carrying both the risk alleles of ALOX5AP and LTA4H (G allele in SG13S41/Intron4 and a T allele in rs1978331/Intron11 respectively) was 2.17 (CI 1.41–3.32). Individual odds ratios for risk alleles were: SG13S41(G) 1.78 (CI 1.21–2.60) and rs1978331(T) 1.22 (1.02–1.48).

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

I. Sayers is supported by the Medical Research Council (New Investigator Award). This study was also supported by The Allergy, Asthma & Inflammation Research Trust. The Southampton Asthma Family Cohort was originally recruited in collaboration with Genome Therapeutics Corporation and Schering-Plough. We thank Professor N. J. Samani and A. Maznyczka (University of Leicester) for providing the LTB4 data from stimulated neutrophils from genotyped healthy individuals.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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