Association of 5-lipoxygenase gene polymorphisms with bronchial asthma

Leukotrienes are important pro-inflammatory mediators in bronchial asthma (BA) and are derived from arachidonic acid by the action of 5-lipoxygenase (5-LO). We investigated the association of 5-LO gene polymorphisms with BA. Thirty-six single-nucleotide polymorphisms (SNPs) of the 5-LO gene, as referenced in the dbSNP gene bank, were analyzed with sequencing and allele-specific PCR (AS-PCR) in genomic DNA from individuals with BA and controls. Of these 36 SNPs, 4 were identified in our study. The c.760 G>A (E254K) (rs2228065) was detected in 15 out of 215 BA patients and 6 out of 212 controls (P<0.05). There were no differences in the frequencies of the other three silent polymorphisms, rs2228064 (c.270 G>A), rs116961353 (c.780G>A) and rs2229136 (c.1728 A>G) between individuals with BA and controls (P>0.05). With our designed primers for AS-PCR, the detection of the 5-LO gene E254K polymorphism was clear and accurate, and the genotype was directly distinguished. Our findings contribute to the evaluation of one of the genetic risk factors for BA and we report an accurate and simple method to rapidly detect the 5-LO E254K polymorphism. It is important to further study the correlation between drug response in BA patients using 5-LO inhibitors with the E254K polymorphism in the clinic.


Introduction
The incidence of asthma in numerous countries is increasing and the cause of the disease is complex (1,2). Leukotrienes (LTs) play an important pro-inflammatory role in both earlyand late-phase asthmatic responses (3). LTs constitute a class of potent biological mediators of inflammation and anaphylaxis. 5-Lipoxygenase (5-LO) is an essential enzyme which catalyzes the first committed steps in the biosynthetic pathway leading to the production of LTs (4)(5)(6). The 5-LO gene is located on chromosome 10q11.2 (7). In several studies, the addition of an Sp-1 binding motif (-GGGCGG-) or the deletion of one or two Sp-1 binding motifs in the 5-LO core promoter have been shown to be associated with reduced gene expression (8,9). Our previous study revealed that the E254K polymorphism in the 5-LO gene may be associated with bronchial asthma (BA) in Japanese children (10). However, few studies have investigated the role of polymorphisms of the 5-LO gene in a Chinese population. Therefore, it is necessary to identify such single-nucleotide polymorphisms (SNPs) in the 5-LO gene and to research the correlation between SNPs in the 5-LO gene and the incidence of BA in the Chinese population.  (11). The controls were healthy and did not have a history of allergic diseases. All subjects were randomly selected among patients in our hospital or our college. Informed consent was obtained from all individuals or their parents.

Patients and methods
Peripheral blood collection and separation of cells. Peripheral blood (2 ml) was obtained from each patient and treated with 0.2% NaCl to collect leukocytes. Genomic DNA was extracted from the leukocytes with a DNA Extract kit (Bioteke, Beijing, China) (12).
Allele-specifc (AS)-PCR primer design. AS-PCR is selective PCR amplification of one allele and is used to detect SNPs. Selective amplification is usually achieved by designing a primer such that it will match/mismatch one of the alleles at the 3' end and the penultimate base is designed to incorporate the SNP in order to improve the specificity of the primer. We designed the following primers: P1, 5'-cgc tgc aca gag ctg cct g (254Glu); P2, 5'-cgc tgc aca gag ctg cct a (254Lys); P3, 5'-cgc aat tcc tcc tct gat gt (co-reverse primer); PCR product, 301-bp. We also designed a pair of reference primers: P4, 5'-aga ggc gaa gtt ctc caa ca; P5, 5'-aac agg gac gga gag tga tg; PCR product, 600-bp. The primer for c.780 G>A (c.780G) was 5'-aga agc tcc cgg tga cca tg-3' and that for c.780 G>A (c.780A) was 5'-aga agc tcc cgg tga cca ta-3'. AS-PCR system and PCR conditions. P3, P4 and P5 primers were added to both A and B tubes (P1,P3,P4 and P5 primers in A tube; P2,P3,P4 and P5 primers in B tube). A and B tubes were prepared and added to 2 µl (10 µM) wild-type P1 and mutant P2 primers, respectively. Two tubes were added per 1 µl (10 µM) a pair of reference primers, P4 and P5, as well as 2 µl (10 µM) common reverse primer P3 were also used. The reaction mixture consisted of 25 µl 2X PCR Mastermix (Bioteke, Beijing, China), 1 µl genomic DNA (100-150 ng/µl) and 18 µl sterilized water in a total volume of 50 µl. The PCR conditions were as follows: 95˚C for 5 min, then 95˚C for 30 sec, 56˚C for 30 sec and 72˚C for 1 min for 35 cycles, the last cycle after 72˚C was extended for 10 min. The products were stored at 4˚C. were detected in 65 individuals with BA and 56 controls. The primer details for the PCR used in the detection of these polymorphisms are shown in Table I.
Statistical analyses. Allele and genotype frequencies were calculated for each locus and tested for Hardy-Weinberg equilibrium. The distribution of the genotype of E254K in the 5-LO gene was analyzed by Fisher's exact test. P<0.05 was considered to indicate a statistically significant result. The significance of the differences in clinical characteristics was analyzed using the two-sample t-test.

Results
Polymorphisms in the 5-LO gene. We identified 4 SNPs in the 5-LO gene in individuals with BA ( Fig. 1). Three SNPs were silent polymorphisms [c.270 G>A (exon 2), c.780 G>A (exon 6) and c.1728 A>G (exon 13)]. There were no differences in the frequencies of these three SNPs between individuals with BA and controls (Table Ⅱ).
One SNP was a missense polymorphism and the amino acid at 254 was changed from Glu (E) to Lys (K) (rs2228065). We determined the prevalence of c.760 G>A (E254K) in the 5-LO genes of individuals with BA and controls. This SNP was found in 15 (8 males and 7 females; mean age, 2.19±4.81 years; 0.070) out of the 215 individuals with BA and 6 (0.028) of the 212 controls. The mutant allele frequency was 0.035 in 215 individuals with BA and 0.014 in the 212 controls (Table III). There was a significant difference in the E254K frequency between individuals with BA and controls (Fisher's exact test, P<0.05). We detected the E254K polymorphism in  50 Chinese Mongolian BA patients by AS-PCR and it was present in 3 patients.
Agarose gel electrophoresis. DNA marker I (600, 500, 400, 300, 200 and 100 bp; 5 µl) and 10 µl AS-PCR products were analyzed by 2% agarose gel electrophoresis. With our designed primers for AS-PCR, it was possible to accurately and clearly detect the 5-LO gene rs2228065 (E254K) and rs116961353 (c.780G>A) polymorphisms, and the genotype was directly distinguished (Fig. 2). Compared with the sequencing and restriction enzyme method, this method is economical, quick and simple.
Associations of E254K with clinical characteristics. We compared the clinical characteristics with the results of a routine blood test by automatic blood analyser (Sysmex 1800i;. Sysmex Corp., Kobe, Japan) among the controls and patients with BA with and without E254K (Table IV). The percentages of lymphocytes and monocytes and the levels of platelets were significantly higher in individuals with BA (without or with E254K) than in controls (P<0.05). However, the percentage and number of neutrophils were significantly lower in individuals with BA (without or with E254K) than in the controls (P<0.05). No significant difference was identified between the clinical features in BA patients with E254K compared with those without E254K. The percentage of eosinophils (EO%) was significantly lower in individuals with BA and E254K than in controls (P=0.04). There was no significant difference in age or gender between BA patients with and without E254K (P>0.05).

Discussion
Allergic diseases, including asthma and atopic dermatitis, are complex genetic disorders that do not conform to a simple Mendelian pattern of inheritance (13). The etiology is extremely complex and the incidence of asthma is correlated with environmental and genetic factors. Clinically similar asthma symptoms may be caused by different mechanisms (14).     In this study, we studied polymorphisms of the 5-LO gene and attempted to investigate the association of 5-LO gene polymorphisms with BA in a Chinese Han population. We compared the difference in the 5-LO gene polymorphism frequency between asthma patients and normal controls and evaluated the correlation of the SNPs with asthma onset, age, gender, severity of asthma and other clinical characteristics of BA. We found a missense SNP and three silent SNPs in the 5-LO gene in BA patients.
To examine the functional effects of c.760 G>A (E254K) in the 5-LO gene, we compared the clinical characteristics with results from routine blood test reports performed in the last three years in our hospital among the controls, BA with E254K and BA without E254K. However, the clinical features (including the age and gender) in our study were not signifi-cantly different between individuals with BA with and without E254K (P>0.05). This result may be due to the number of individuals with BA and E254K being too low to obtain accurate statistical results.
The percentages of lymphocytes, monocytes and platelets (10 9 /l) levels were significantly higher in individuals with BA (without or with E254K) than in controls (P<0.05). Monocytes are the largest cells in normal blood. They act as phagocytes in certain inflammatory diseases and are the body's second line of defense against infection (16). Lymphocytes are the primary components of the body's immune system and increase in number in numerous viral infections and with tuberculosis (17). Inflammatory disorders may cause a high platelet count in a similar way to infections. Our date support the theory that inflammation is a cause of asthma attack.
The percentage and number of neutrophils were significantly lower in individuals with BA (without or with E254K) than in controls (P<0.05). A decrease in neutrophil levels is known as neutropenia. Although most bacterial infections stimulate an increase in neutrophils, certain bacterial infections, including typhoid fever and brucellosis, and numerous viral diseases, including hepatitis, influenza, rubella, rubeola and mumps, decrease the neutrophil count. Our data support the hypothesis that viral infection is a cause of asthma attack.
The mean level of eosinophils showed a tendency to increase in individuals with BA without E254K more than in controls. However, the percent eosinophil levels were significantly lower in individuals with BA and E254K than in controls (P=0.04). Eosinophils are associated with antigenantibody reactions (18). The most common reasons for an increase in the eosinophil count are allergic reactions such as hay fever, asthma or drug hypersensitivity. Decreases in the eosinophil count may be observed when a patient is receiving corticosteroid drugs.  BA without E254K --------------------------------------------------characteristics Normal range mean ± SD (n=14) mean ± SD (n=114) mean ± SD P1 P2 P3 Pharmacogenetics is the study of how genetic differences influence the variability of patients' responses to therapy (19). Therefore, we intend to study the correlation between these four SNPs and patients' responses to therapy. With our designed primers for AS-PCR, the detection of the 5-LO gene E254K and c.780G>A polymorphisms is accurate and clear and the genotype may be directly distinguished. Compared with the sequencing and restriction enzyme method, our method was economic, quick and simple.
In summary, our findings contribute to the evaluation of one of the genetic risk factors for BA and we report an accurate and simple method to quickly detect the 5-LO E254K polymorphism. It is important to further study the correlation between the response to 5-LO inhibitors with the E254K polymorphism in the clinic. These results are important for clarifying the different mechanisms of BA.