A functional Sp1/Egr1-tandem repeat polymorphism in the 5-lipoxygenase gene is not associated with myocardial infarction

Arachidonate 5-lipoxygenase is an enzyme encoded by the ALOX5 gene, and plays an important role in the synthesis of leukotrienes. These are inflammatory mediators, and have been involved in atherosclerosis and other pathological processes that require proinflammatory activities. Human and animal studies have suggested a role for the ALOX5 gene in atherosclerosis, including a significant association between a promoter polymorphism and a carotid intimal-medial thickness in response to dietary fat. This polymorphism was three- to six-tandem repeats of a Sp1/Egr1 binding motif (GGGCGG)(n), and the number of repeats has been linked with the amount of gene expression. We hypothesized that this ALOX5 polymorphism could influence the risk for myocardial infarction (MI). First, we analysed the effect of the four alleles on gene expression by transfecting the HEK-293 cell line with luciferase reporter-constructs. We found that luciferase activities are dependent on the number of the Sp1/Egr1 repeats, with the three and six repeats having the lowest and highest values. We genotyped 312 male MI survivors, aged < 55 years, and 376 healthy controls matched with patients for sex, age, and ethnicity. Ninety-six per cent of the patients were smokers, compared to only 42% among the controls (P < 0.001; OR = 31.84). The 55 + 56 repeat genotypes were less frequent in patients (55 = 56%, 56 = 0.6%) compared to controls (55 = 60%, 56 = 3%). However, these were non-significantly different frequencies. In addition, no difference in MI-onset age and biochemical values was found between the allele and genotypes. In conclusion, we confirmed the effect of the ALOX5-promoter polymorphism on gene expression, but our data did not support a significant effect of this functional variation on MI risk.     

ALOX5AP基因SG13S114A/T多态性与颈动脉粥样硬化斑块稳定性的相关性

目的 探讨花生四烯酸5-脂氧合酶激活蛋白(arachidonate 5-lipoxygenase acttvating protein,ALOX5AP)基因SG13S114 A/T的多态性与颈动脉粥样硬化斑块稳定程度的关系.方法 应用聚合酶链反应-限制性片段长度多态性分析法检测132例颈动脉易损斑块及152例稳定斑块的急性脑梗死者ALOX5AP基因SG13S114 A/T基因型,比较两组间该位点多态性的差异.结果 易损斑块组ALOX5AP基因SG13S114 AA基因型和A等位基因频率高于稳定斑块组,两组差异有统计学意义(P＜0.01),其中男性和女性上述基因型和等位基因频率的比较,易损斑块组均高于稳定斑块组,两组差异有统计学意义(P＜0.01).结论 ALOX5AP基因SG13S114 A/T多态性可能与动脉粥样斑块的稳定性有关,其中SG13S114 A等位基因可能是易损斑块的风险因素.     

Polymorphisms in the 5-lipoxygenase activating protein (ALOX5AP) gene are not associated with asthma in an Australian population

BACKGROUND: The cysteinyl-leukotrienes (cys-LTs) are important pro-inflammatory mediators in asthma, and have been shown to have a role in specific disease subtypes, including asthma severity. Few studies have investigated the role of polymorphisms in the ALOX5AP gene, encoding 5-lipoxygenase activating protein (FLAP), and asthma. We hypothesized that polymorphisms in this gene are associated with asthma and in particular, with asthma severity, in an Australian population. OBJECTIVE: To screen the coding region of the ALOX5AP gene for polymorphisms and to determine the association between previously described polymorphisms and asthma and asthma severity in an Australian population. METHODS: We used PCR-SSCP and PCR-RFLP analysis to examine a previously described promoter polyA variable repeat polymorphism and two intronic polymorphisms (IVS2+12C>A, IVS2+105T>C), and to screen all five exons of the gene for new polymorphisms, in a large Australian population of randomly selected, non-asthmatic controls (n=457), mild asthmatics (n=274), moderate asthmatics (n=231) and severe asthmatics (n=79). RESULTS: We confirmed the presence of two polymorphisms in intron 2 and found no association between these polymorphisms and asthma or asthma severity, nor between a promoter polymorphism in the ALOX5AP gene and asthma or asthma severity. Gene fragment analysis of the promoter polymorphism revealed novel, conserved repeat numbers in our population, and no new polymorphisms were found in the coding region of the gene. CONCLUSION: These findings in a large, well characterized asthma population, reveal that, while FLAP is an important enzyme in cys-LTs biosynthesis, polymorphisms in the ALOX5AP gene are not likely to be functionally associated with the asthma phenotype.     

Leukotriene-related gene polymorphisms in patients with aspirin-intolerant urticaria and aspirin-intolerant asthma: differing contributions of ALOX5 polymorphism in Korean population

The pathogenesis of aspirin (acetylsalicylic acid, ASA)-intolerant urticaria (AIU) is still poorly understood but it has recently been suggested that it is associated with the overproduction of leukotriene (LT). This is supported by evidence that cyclooxygenase 2 inhibitor is given safely to patients with AIU. The present study was designed to investigate the role of genetic polymorphism of LT related genes in the pathogenesis of AIU via a case-control study. We screened single nucleotide polymorphisms (SNPs) in genes encoding enzymes involved in leukotriene synthesis in the Korean population with AIU (n = 101), ASA-intolerant asthma (AIA, n = 95) and normal healthy controls (n = 123). Genotype was determined by primer extension reactions using the SNapShot ddNTP primer extension kit. Among 8 SNPs of four LT related genes, the polymorphism of ALOX5 at positions of -1708 G > A showed significant difference in genotype frequency between AIU and AIA (p = 0.01). Furthermore, there were significant differences observed in the frequencies of two ALOX5 haplotypes between the AIU group and AIA group (p < 0.05). However, there were no differences in allele, genotype, or haplotype frequencies of ALOX5 between the AIU group and the normal control group. These results suggested that ALOX5 has a differing contribution in two major clinical pathogenesis related to ASA-sensitivity.     

Involvement of the cysteinyl-leukotrienes in allergen-induced airway eosinophilia and hyperresponsiveness in the mouse

The leukotriene modifiers are a novel generation of therapeutic agents in the treatment of allergic asthma. However, the mechanisms by which the cysteinyl (cys) leukotrienes (LTs) participate in allergen-induced airway eosinophilia and airway hyperresponsiveness (AHR) are still unclear. In the present study, we have investigated the role of cys-LTs in ovalbumin (OVA)-induced airway responses in a murine model of asthma. Montelukast (3 or 10 mg/kg), a selective cys-LT1 receptor antagonist, reduced airway eosinophilia and AHR after OVA challenge. The levels of interleukin (IL)-5 and eotaxin in the bronchoalveolar lavage fluid (BALF) from montelukast-treated (3 mg/kg) mice were unaffected, although a decrease in IL-5 was observed with a dose of 10 mg/kg. LTD4 (50 ng) instilled intranasally to immunized mice augmented macrophages in the BALF, but in conjunction with OVA challenge it caused BALF eosinophilia and neutrophilia when given before challenge and BALF neutrophilia but not eosinophilia when given 2 h after challenge. However, there were no increases of IL-5 or eotaxin in BALF following LTD4 treatment. Repeated instillations of LTD4 to immunized mice, mimicking allergen challenge, did not induce AHR but in conjunction with OVA challenge LTD4 enhanced AHR. These results indicate that allergen-induced eosinophilia and AHR are in part mediated by the cys-LT1 receptor, and that, although LTD4 alone has no effect on airway eosinophilia, in conjunction with antigenic stimulation it potentiates the degree of airway inflammation and AHR.     

Serum interleukin-5 in aspirin-induced asthma

BACKGROUND: Eosinophilopoetic cytokine IL-5 enhances cysteinyl-leukotriene (cys-LT) synthesis in eosinophils in vitro. In patients with aspirin-induced asthma (AIA) bronchial biopsies revealed eosinophil infiltration and a marked increase in IL-5 positive cells. OBJECTIVE: We wondered whether in AIA patients the bronchial IL-5 increase is reflected in peripheral blood, and if so, whether it is related to overproduction of cys-LT. METHODS: In 11 stable patients with AIA, 32 with ATA (aspirin-tolerant asthma) and in 16 controls we measured serum IL-5 concentrations and urinary LTE4, believed to reflect global cys-LT production. RESULTS: Serum IL-5 was detectable in 12 of 43 asthmatics, but in none of the control subjects. It was highest in the ATA group and differed significantly from the controls. There was no significant difference in IL-5 levels between: (i) the asthmatic groups studied, and (ii) AIA patients and controls. No relationship was found between serum IL-5 and urinary cys-LT. CONCLUSION: Overexpression of IL-5 reported in the airways of aspirin-sensitive patients with asthma was not reflected in their blood. If IL-5 affects cys-LT production, it is rather in the bronchi of the patients than in the blood.     

Association of the serotonin transporter gene with sudden infant death syndrome: a haplotype analysis

Serotonergic receptor binding in the arcuate nucleus, n. raphé obscurus, and other medullary regions is decreased in sudden infant death syndrome (SIDS) cases. Further, an insertion/deletion polymorphism in the promoter region of the serotonin transporter protein (5-HTT) gene has recently been associated with risk of SIDS. This polymorphism differentially regulates 5-HTT expression, with the long allele (L), the SIDS-associated allele, being a more effective promoter than the short allele (S). To further elucidate the role of the 5-HTT gene in SIDS, we investigated the 5-HTT intron 2 polymorphism, which also differentially regulates 5-HTT expression with the 12 repeat allele being the more effective promoter. In a cohort of 90 SIDS cases (44 African-American and 46 Caucasian) and gender/ethnicity-matched controls, significant positive associations were found between SIDS and the intron 2 genotype distribution (P-value = 0.041) among African-American SIDS vs. African-American controls, specifically with the 12/12 genotype (P-value = 0.03), and with the 12 repeat allele (P-value=0.018). The frequency of the 12/12 genotype and 12-repeat allele was significantly different (P < 0.001) between the African-American and Caucasian SIDS cases. Furthermore, the promoter and intron 2 loci were in significant linkage disequilibrium, and the L-12 haplotype was significantly associated with SIDS in the African-American (P = 0.002) but not Caucasian (P = 0.117) subgroups. These results indicate a relationship between SIDS and the 12-repeat allele of the intron 2 variable number tandem repeat of the 5-HTT gene in African-Americans, and a significant role of the haplotype containing the 12-repeat allele and the promoter L-allele in defining SIDS risk in African-Americans. These data, if confirmed in larger studies, may begin to explain the differences in SIDS incidence by ethnicity, suggest a role for levels of 5-HTT expression in generation of SIDS susceptibility, and provide an important tool for identifying at-risk individuals and estimating the risk of recurrence.     

Genetic markers for differentiating aspirin-hypersensitivity

Aspirin-induced asthma (AIA) and aspirin-induced urticaria/ angioedema (AIU) are two major aspirin-related allergies. We summarize recent findings related to their molecular genetic mechanisms in order to identify genetic susceptibility markers for differentiating AIU and AIA. The overproduction of cysteinyl leukotriene has been suggested as a mechanism in both AIU and AIA. Increased expression of CYSLTR1 with CYLSTR1 and CYSLTR2 polymorphisms are new findings in AIA, while the ALOX5 promoter polymorphism has been noted in AIU. An HLA study suggested that DPB1*0301 is a strong genetic marker for AIA, and that HLA DRB1*1302 and DQB1*0609 are markers for AIU susceptibility. Several single nucleotide polymorphisms (SNPs) in the promoters of EP2, TBX21, COX-2, Fc epsilon RIbeta, and TBXA2R were associated with AIA, while an Fc epsilon RIalpha promoter polymorphism was associated with AIU. The functional studies of the key genes involved in AIA and AIU are summarized. The identification and functional study of genetic markers for AIA and AIU susceptibility would further elucidate the pathogenic mechanisms and facilitate the development of early diagnostic markers to establish therapeutic targets.     

Sudden infant death syndrome: association with a promoter polymorphism of the serotonin transporter gene

Serotonergic receptor binding in the arcuate nucleus, n. raphé obscurus, and other medullary regions is decreased in sudden infant death syndrome (SIDS) cases. Further, a variable tandem repeat sequence polymorphism in the promoter region of the serotonin transporter protein (5-HTT) gene has recently been associated with risk of SIDS in a Japanese cohort. This polymorphism differentially regulates 5-HTT expression, with the long allele (L), the SIDS-associated allele, being a more effective promoter than the short allele (S). We therefore investigated the 5-HTT promoter polymorphism in a cohort of 87 SIDS cases (43 African American and 44 Caucasian) and gender/ethnicity-matched controls. Significant positive associations were found between SIDS and the 5-HTT genotype distribution (P = 0.022), specifically with the L/L genotype (P = 0.048), and between SIDS and the 5-HTT L allele (P = 0.005). There was also a significant negative association between SIDS and the S/S genotype (P = 0.011). The comparisons were repeated in the African American and Caucasian subgroups. The data patterns were consistent in the subgroups, i.e., the L/L genotype and L allele were increased in the cases, but not all subgroup comparisons were statistically significant. These results indicate a relationship between SIDS and the L allele of the 5-HTT gene in African Americans and Caucasians, and if confirmed, will provide an important tool for identifying at-risk individuals and estimating the risk of recurrence.     

Monoamine oxidase A gene promoter polymorphism affects novelty seeking and reward dependence in healthy study participants

It has been suggested that monoamine oxidase A plays an important role in the characterization of personality. Previous studies on the association between the polymorphism of variable number tandem repeat in the promoter region of the monoamine oxidase A gene and personality traits have, however, been unproductive. In the present study, the association between the monoamine oxidase A variable number tandem repeat polymorphism and personality traits assessed by the Temperament and Character Inventory was examined in 324 Japanese volunteers without psychiatric disorders. The low activity allele with three repeats (allele 3) and high activity allele with four repeats (allele 4) were determined by a polymerase chain reaction method. The carriers of allele 3 in males and the homozygotes of allele 3 in females were classified as the low activity group, the heterozygotes of alleles 3 and 4 in females as the medium activity group, and the carriers of allele 4 in males and the homozygotes of allele 4 in females as the high activity group. One-way analysis of variance showed that the scores of novelty seeking (P=0.006) and reward dependence (P=0.013) were significantly higher in the high activity group than in the low activity group. Multiple regression analysis demonstrated that the excess in the high activity allele was significantly associated with higher scores of novelty seeking (P=0.004) and reward dependence (P=0.003). The present study thus suggests that the monoamine oxidase A variable number tandem repeat polymorphism affects novelty seeking and reward dependence in healthy study participants.     

Determination of ancestral allele for possible human cancer-associated polymorphisms

To determine ancestral allele in possible cancer-associated polymorphisms, DNA samples from 10 chimpanzees (Pan troglodytes) were sequenced for alleles corresponding to 17 polymorphisms: 8 short tandem repeats [IL1RN (alias IL-1RA) variable number tandem repeat (VNTR); TYMS (previously TS) VNTR; AR CAG repeat; dinucleotide repeats of UGT1A1, IGF1, IFNG (alias IFN-gamma), ESR1 (alias ER-alpha), and EGFR] and 9 single nucleotide polymorphisms (MMP1-1607 1G/2G, MMP3-1171 5A/6A, OGG1 Ser326Cys, ALDH2 Gly487Lys, TP53 Arg72Pro, ABCG2 Gln141Lys, MGMT Leu84Phe, SOD2 Ala-9Val, and MTHFR Ala222Val). No chimpanzee polymorphism corresponded to human IL1RN VNTR; the ancestral allele was a repeat lost in humans. Dinucleotide repeat polymorphisms of IGF1, IFNG, ESR1, and EGFR were shared by chimpanzees, but the length of repeats tended to be longer in humans than in chimpanzees. This tendency was particularly evident for IGF1. All of the SNPs tested are human-specific nucleotide changes. The ancestral allele 7A was shown to be lost in MMP3-1171 5A/6A. Thus, all of the possible cancer-associated polymorphisms tested have human-specific alleles, and the ancestral allele is lost in three polymorphisms (IL1RN VNTR, UGT1A1 CA repeat, and MMP3-1171 5A/6A), suggesting a possible involvement of human-specific alleles in cancer susceptibility.     

COL1A1基因PCOL2和Sp1结合位点多态与先天性髋脱位的关联分析

目的 研究Ⅰ型胶原a1链(collagen type Ⅰ alpha 1，COL1A1)基因PCOL2和Sp1结合位点多态性在中国北方人群中的分布，并探讨其与先天性髋脱位(congenital dislocation of the hip，CDH)的关系。方法 在81个CDH核心家系的243名成员中，采用聚合酶链反应-限制性片段长度多态性分析方法，对位于COLIAI基因启动子内的PCOL2多态(-1997G／T)和转录调控区第1内含子内的Sp1多态(1546G／T)进行基因分型，并进行传递不平衡检验。结果COL1A1，基因PCOL2结合位点G／T多态与CDH不存在明显关联(P=0．537)，中国人PCOL2多态位点的基因型和等位基因频率分布与西班牙白人群和美国白人群差异存在统计学意义；Sp1结合位点只检测到SS基因型，未检测到Ss或ss基因型。结论 COL1A1基因PCOL2和Sp1结合位点多态存在种族差异，PCOL2和Sp1结合位点多态可能与中国人CDH的发病风险无关。     

Molecular characterisation and polymorphism of MinLm1, a minisatellite from the phytopathogenic ascomycete Leptosphaeria maculans

A sequence-characterised amplified region marker was identified in the phytopathogenic fungus Leptosphaeria maculans, which generated a single-banding pattern corresponding to six alleles showing size polymorphism between L. maculans field isolates. The size polymorphism was due to 2-7 tandem repeats of the 23-bp motif 5' TCTTACTTACATACACACCTCCC 3'. The repeated sequence, termed MinLm1, shares many features specific to minisatellites, e.g. a very strong G/C strand asymmetry, the presence of 6-bp direct repeats at both ends of the sequence and its occurrence in a region rich in microsatellites such as (CT)n, (ATG)n, (GTG)n and (CAT)n. MinLm1 shows a very high degree of conservation of the bases from one repeat to another and from one isolate to another (percent match range: 99.6-100%), whatever their geographical or temporal relatedness. MinLm1 is a single-locus minisatellite located on chromosomes sized 2.79 Mb and 2.48 Mb, of L. maculans isolates a.2 and H5, respectively. In agricultural populations of L. maculans, two alleles of MinLm1 were prevalent, corresponding to 2x and 5x repeats of the core motif. Differences in allele frequencies were observed in some cropping conditions, suggesting that MinLm1 is an informative marker for epidemiological studies of the pathogen.     

Genetic mechanism of aspirin-induced urticaria/angioedema

PURPOSE OF REVIEW: Aspirin-induced urticaria/angioedema is a major aspirin-related hypersensitivity often associated with aspirin-intolerant asthma. Genetic studies on aspirin-intolerant asthma have shown chronic overproduction of cysteinyl leukotrienes. The genetic analysis of aspirin-induced urticaria/angioedema is limited, however. RECENT FINDINGS: A recent study on HLA genotypes has suggested that the HLA alleles DRB11302 and DQB10609 may be genetic markers for aspirin-induced urticaria/angioedema. A polymorphism study that examined nine single-nucleotide polymorphisms of five leukotriene-related genes [ALOX5 (encoding 5-lipoxygenase), ALOX5AP (5-lipoxygenase-activating protein), PTGS2 (cyclooxygenase 2), LTC4S (leukotriene C4 synthase), and CYSLTR1 (cysteinyl leukotriene receptor 1)] found that promoter polymorphisms of ALOX5 (-1708A>G) and CYSLTR1 (-634C>T) were significantly different between aspirin-intolerant asthma and aspirin-induced urticaria/angioedema, suggesting different contributions to the lipoxygenase pathway. A second polymorphism study, conducted on histamine-related genes, did not find any significant associations with aspirin-induced urticaria/angioedema for the genes HNMT (encoding histamine N-methyltransferase), HRH1 or HRH2 (encoding histamine receptor types 1 and 2 respectively), or the gene encoding high-affinity IgE receptor Ibeta (FcepsilonRIbeta); however, the FcepsilonRIalpha gene promoter polymorphism was significantly associated with aspirin-induced urticaria/angioedema. This finding has been supported by in vitro functional studies. SUMMARY: The HLA alleles DRB11302 and DQB10609, and the ALOX5 and FcepsilonRIalpha promoter polymorphisms, may contribute to the pathogenesis of aspirin-induced urticaria/angioedema. Further investigation to identify candidate genetic markers would help to elucidate the pathogenic mechanism of this condition.     

ALOX5 variants associated with susceptibility to human pulmonary tuberculosis

The 5-lipoxygenase (ALOX5)-derived lipid mediators leukotrienes and lipoxins have regulatory functions in inflammation by modulating activities of immune cells and cytokine production. Recently, it was shown in ALOX5-/- mice that host control of Mycobacterium tuberculosis is regulated by 5-lipoxygenase (5-LO). ALOX5 polymorphisms were genotyped in 1916 sputum-positive patients with pulmonary tuberculosis (TB) from Ghana and in 2269 exposed, apparently healthy controls. Polymorphisms of a variable number of tandem repeats (VNTR) of the ALOX5 promoter and of the exonic non-synonymous variant g.760G>A were analysed by fragment length determination and fluorescence resonance energy transfer, respectively, and DNA sequencing. Mycobacterial lineages of >1400 isolates were differentiated biochemically and genetically. Carriers of one variant (n repeats not equal 5) and one wild-type VNTR allele (n = 5) or of the exonic allele g.760A had a higher risk of TB [P(corrected) = 0.026, odds ratio (OR) 1.19 (95% CI 1.04-1.37) and P(corrected) = 0.026, OR 1.21 (95% CI 1.04-1.41), respectively]. The association of the exonic variant was stronger in infections caused by the mycobacterial lineage M. africanum West-African 2 [P(corrected) = 0.024, OR 1.70; (95% CI 1.2-2.6)]. Determination of haplotypes revealed the strongest associaton with TB for the 'non-5/760A' haplotype compared with the 'non-5/760G' haplotype (P = 0.003, OR 1.50). Our observation of an association of ALOX5 variants with susceptibility to TB contributes evidence of the importance of 5-LO products to the regulation of immune responses to M. tuberculosis.     

A polymorphism of MS4A2 (−109T>C) encoding the β-chain of the high-affinity immunoglobulin E receptor (FcɛR1β) is associated with a susceptibility to aspirin-intolerant asthma

BACKGROUND AND OBJECTIVE: The MS4A2 gene, the beta chain of the high-affinity receptor for immunoglobulin (Ig)E, has previously been linked to atopy and asthma. The beta-chain of FcepsilonR1 enhances receptor maturation and signal transduction capacity, leading to the release of proinflammatory mediators and cytokines that can exacerbate the symptom of asthma. This study was performed to evaluate whether two genetic polymorphisms of the FcepsilonR1beta gene (FcepsilonR1beta-109T > C and FcepsilonR1beta E237G) are associated with aspirin-intolerant asthma (AIA). The MS4A2 gene polymorphisms (FcepsilonR1beta-109T > C and FcepsilonR1beta E237G) were determined by SNP-IT assays in patients with AIA (N = 164), aspirin-tolerant asthma (ATA, N = 144) and normal controls (NC, N = 264) recruited from a Korean population. RESULTS: The genotype frequencies of FcepsilonR1beta-109T > C and E237G polymorphisms were not significantly associated with the pathogenesis of AIA. However, FcepsilonR1beta-109T > C polymorphism was significantly associated with the presence of specific IgE to Staphylococcal enterotoxin B (SEB); the number of subjects carrying both homozygous TT genotype of FcepsilonR1beta-109T > C and specific IgE to SEB was significantly higher in the AIA group when compared with the other control groups (P = 0.01, odds ratio (OR) = 7.723, 95% confidence interval (CI) = 1.327-39.860 for AIA vs. ATA; P = 0.02, OR = 6.364, 95% CI = 1.149 approximately 35.229 for AIA vs. NC). In addition, luciferase reporter assays also showed that the FcepsilonR1beta-109T allele was associated with higher promoter activity of MS4A2 in both RBL-2H3 and A549 cell lines. CONCLUSION: FcepsilonR1beta-109T > C polymorphism may increase expression of MS4A2 by mast cells, leading to enhanced release of proinflammatory mediators in the asthmatic airway, contributing to increased susceptibility to AIA.