文摘
Cyclooxygenase-1 (COX-1)调节前列腺素的生物合成,这是哮喘的重要介质。可能COX-1协会的基因多态性与哮喘没有被调查。
20 COX-1多态性的等位基因频率是决定在一个随机的澳大利亚白人人口使用MassARRAY技术。信息和潜在功能的启动子(c。8592 c > T, c.1676C (c > T)和编码区域。22C>T, c.50C>T) polymorphisms were investigated in carefully phenotyped patients with mild (n = 316), moderate (n = 241), severe (n = 86) or aspirin-intolerant asthma (AIA) (n = 58), and in nonasthmatic subjects (n = 477).
没有等位基因,基因型或haplotypic协会这四个多态性和哮喘和哮喘严重程度之间的关系。代表的c。50TT genotype among AIA patients (3.4%) compared with aspirin-tolerant patients (0.8%), and a global haplotype association with AIA did not reach statistical significance. The c.22TT genotype was less frequent among atopic (0.1%) rather than nonatopic individuals (1.2%; odds ratio = 9.05, 95% confidence interval 1.01–81.29).
总之,目前的调查cyclooxygenase-1多态性在哮喘似乎表明他们不发挥实质性作用遗传预期哮喘和哮喘严重程度。然而,c。22TT genotype confers a small protective effect against atopy. Potential associations with aspirin-intolerant asthma were identified and warrant further investigation in a larger population of aspirin-intolerant asthma patients.
前列腺素(后卫)是重要的脂质介质,促炎和抗炎和bronchoprotective角色在人类的航空公司1- - - - - -3。环氧合酶(COX)是关键酶,一起修复合成酶,调节后卫从花生四烯酸的合成4,5。描述了三种亚型的考克斯,COX-1和COX-3拼接变种相同的基因6,而cox - 2是由一个不同的基因编码。cox - 2是一种诱导酶的合成负责动力分配在各种炎症状态7。COX-1历来被视为持续表达的酶产生动力和信息信号、凝血、肾脏功能和维护,组织内稳态和胃肠道的完整性4,7。然而,COX-1也被认为在病理生理过程中发挥作用,包括炎症、关节炎的疾病和癌症,尽管有证据表明COX-1表达式可能监管,对机制所知甚少8,9。COX-1和cox - 2都是既定的气道上皮细胞的表达10,11和铂族元素2由这些酶可能在哮喘患者的气道保护功能,类似于COX-1-derived前列腺素类在胃肠道和脉管系统12。
Aspirin-intolerant哮喘(AIA)是一种特定亚型的哮喘的特点是慢性鼻窦炎和鼻息肉病,症状发展人生的第三个十年后,在摄入阿司匹林和其他非甾体类抗炎药(非甾体抗炎药)13。阿司匹林和非甾体抗炎药,引发哮喘3 - 20%的成年患者14- - - - - -16COX-1的有力不可逆抑制剂,而具体的cox - 2抑制剂不出现沉淀哮喘发作17。因此,抑制COX-1可能是友邦保险的发病机制的主要因素13。
COX-1 (PG-endoperoxide合酶1 (PTGS1))基因位于染色体9问,和由11个外显子的长度22 kb。主要的转录起始站点位于136年基地ATG起始密码子的上游18。多个启动子的转录因子结合位点已确定,包括两个Sp1绑定网站似乎是重要的基底基因转录19。COX-1基因的有限调查发现13编码区域和五intronic多态性在48个非洲裔和47个白种人健康人20.,以及一个额外的编码区和三个启动子多态性分布在38个白人血统的健康人21。
虽然目前的作者最近报道,cox - 2基因启动子的功能多态性与哮喘无关22另一项研究报道,性协会和功能多态性在哮喘的影响23。COX-1基因的单核苷酸多态性(SNPs)也可能对COX-1表达式和PG生物合成,产生深远的影响,直接或间接地影响发病机理,发展和哮喘的严重程度和特异反应性以及反应治疗。因此,在这项研究中,一个大,well-phenotyped澳大利亚白人人口调查以确定是否有等位基因,基因型或haplotypic COX-1基因多态性之间的关联和哮喘,哮喘严重程度、阿斯匹林不耐受或特异反应性。最好的目前作者的知识,这是第一个详细调查之间的潜在关联COX-1基因的变异和哮喘。
方法
主题
COX-1多态性的频率在普通人群中运用随机确定血液样本收集从176年澳大利亚白种人捐赠者在红十字会血液银行在珀斯,西澳大利亚州。协会研究包括477名健康控制个人和643名哮喘患者,其中316例有轻微,中等241和86有严重的哮喘,以及与友邦58人。哮喘被定义为被诊断出的哮喘。尽管没有可逆性或hyper-responsiveness测试进行招聘的时候,许多患者有这样的测试执行。所有受试者无关,年龄在20 - 89岁。参与者被随机寄出,除了患者更严重的哮喘和友邦,也招募了通过医生推荐。所有科目给书面知情同意和被呼吸道员工亲自采访经验的哮喘和慢性阻塞性肺疾病患者在日常生活中。受试者完成一份详细的问卷调查表对哮喘、呼吸健康和其他与健康有关的问题,他们的肺功能评估肺量测定法并提供血液样本(10毫升)进行基因分型分析。Nonasthmatic科目没有哮喘史、慢性呼吸道疾病或其他严重疾病,但它是可能的,其中一些可能有鼻(过敏性鼻炎)或皮肤(特应性湿疹)特异反应性的表现,没有宣布。这项协议是人类研究伦理委员会批准的查尔斯爵士Gairdner医院(佩斯),查尔斯王子医院(布里斯班),阿尔弗雷德医院(澳大利亚墨尔本)。
分类的临床表型
患者分为轻度,中度或重度哮喘,根据指定的标准澳大利亚国立哮喘委员会和美国国立卫生研究院24,25。这些标准,详细描述了26,包括肺功能,使用口服糖皮质激素在过去12个月里,每天吸入皮质类固醇剂量,救援药物的使用频率、频率白天症状、夜间觉醒频率由于哮喘,计划外访问全科医生在过去12个月中和哮喘住院在过去的12个月。哮喘患者也被归类为友邦保险或aspirin-tolerant哮喘(ATA)使用一个单独的问卷调查,此前曾被用于评估哮喘患者的阿斯匹林不耐受的患病率16。27 58例,友邦保险的诊断已经证实了阿司匹林的挑战,剩下的31名患者有很强的阿斯匹林/非甾体消炎药诱发哮喘、历史与传统临床特征发生后不久在多个场合摄入的药物。
特异反应性测试
过敏性状态的评估是基于皮反应(鞭痕直径≥3毫米)至少五个常见的气源性致敏原之一,包括猫、狗、屋尘螨、霉菌混合(链格孢属清塞音,曲霉属真菌种虫害混合,枝孢属spp。青霉菌种虫害混合)和草花粉混合(肯塔基蓝、果园、红,盖,甜美的春天的,草地羊茅,常年黑麦;美国Hollister-Stier,斯波坎,佤邦)。
选择多态性
总共有20个多态性位于启动子和编码区域COX-1 (PTGS1)基因被确定通过搜索国家生物技术信息中心(NCBI)网站(www.ncbi.nlm.nih.gov / SNP)和SNP数据库27。基因的多态性的位置被证实使用基本的局部比对搜索工具程序28对齐dbSNP集群COX-1 DNA序列(序列加入基因库。AF440204)。随机澳大利亚白人受试者(n = 176)最初是为这些20个snp基因分型。选择snp基因分型结果为进一步在哮喘和nonasthmatic主题是基于以下标准:1)随机频率> 5%白人,和2)单核苷酸多态性了潜在功能的后果,由于他们在转录因子结合位点的位置,或者因为他们导致氨基酸的变化可能会改变蛋白质的功能。总共4的10个snp检测的随机满足这些标准和白人受试者,因此,选择基因分型的哮喘和nonasthmatic科目。
基因型分析
DNA提取外周血白细胞使用DNA提取工具包(试剂盒、希尔登,德国),根据制造商的指示。的pcr c。-1676 c > T, c。-8592 c > T, c。22 c > T和c。50C>T polymorphisms was performed using the MassARRAY system (Sequenom Inc., San Diego, CA, USA), commercially available at the Australian Genome Research Facility (Brisbane, Australia). Briefly, following PCR amplification, primer extension products were analysed by chip-based matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. Primers were designed using Sequenom software, and the extension reaction produced allele-specific products with masses differing by ∼300 Da, or approximately one single nucleotide. Primer extension and PCR were performed according to the manufacturer's instructions, using Thermosequenase (Sequenom) and HotStarTaq DNA polymerase (Qiagen). After desalting of the reaction products (SpectroCLEAN; Sequenom), ∼10 nL were loaded into a SpectroCHIP (Sequenom) and analysed in the MALDI-TOF MassARRAY system in the fully automated mode. Genotypes were automatically identified by the SpectroTYPER software (Sequenom), and only conservative and moderate calls, as defined by the software, were accepted for this study. As the Sequenom analysis was unsuccessful in some samples, there was minor variation in the numbers of subjects investigated for the different polymorphisms.
结果
启动子和外显子的snp COX-1基因
共有20个多态性,六子和14(12产生的,两个同义突变)的编码区基因,确定在NCBI和snp数据库27。13其实snp的研究在最近的一项研究中,非洲裔和白人健康个体20.,9个研究在当下随机白人人口,但是四个同义突变。之前10个snp,轻微的等位基因的频率很低没有检测到任何的176个随机白种人的科目。10的多态性检测,基因的位置,假定的转录因子结合位点的影响31日或产生的氨基酸替换,等位基因频率在176年随机澳大利亚白种人个人如表1所示⇓。六个snp,轻微的等位基因频率< 5%,这些相对罕见突变没有任何进一步的研究。剩下的多态性,c。-1676 c > T和c。- - - - - -8592C>T in the promoter, and c.22C>T and c.50C>T in the coding region, were investigated in the asthmatic and non-asthmatic populations.
关联分析
人口和哮喘的表型特征和控制人口表2所示⇓。特异反应性是更大的在哮喘的患病率比nonasthmatic主题(p < 0.0001)。虽然使用了八个不同的标准,最终分类的疾病严重程度似乎意味着数据密切相关肺功能和药物使用。友邦保险的病人中,21个被划分温和,25中度和12重度哮喘。
c的基因型和等位基因频率。-1676 c > T, c。-8592 c > T, c。22 c > T和c。50C>T polymorphisms in all asthma patients, and when categorised by asthma severity, as well as the frequencies in nonasthmatic subjects, are presented in table 3⇓。所有四个snp在哈迪温伯格平衡在目前的人口(p≥0.17)。小c等位基因频率∼21%。-1676 c > T和c。- - - - - -8592C>T and between 6.4 and 7.8% for c.22C>T and c.50C>T, in asthmatic and nonasthmatic subjects. There were no statistically significant differences between the asthmatic and nonasthmatic subjects for either the genotype or allele frequencies of these four polymorphisms. With a total of 457 control subjects, and based on the genotype and allele frequencies in table 3⇓,α值= 0.05和90%,最小显著优势比(ORs)基因型对哮喘是2.05 c。-8592 tt, 2.2 c。- - - - - -1676CC, and 4.25 for c.22TT and c.50TT. Similarly, the minimum significant ORs for allelic associations would have been 1.61 for c.–8592T and c.–1676C, and 1.91 for c.50T and 2.0 for c.22T. Although the c.22T allele and the c.22CT genotype were slightly over-represented among patients with moderate and severe asthma, these differences were not statistically significant (p>0.17) and nor were the other polymorphisms associated with asthma severity. With 83 severe asthmatic patients, the minimum significant ORs for allelic associations would have been 2.8 for c.–1676C and c.–8592T, and 3.6 for c.50T and 3.8 for c.22T.
友邦保险的病人似乎较低频率的c。- - - - - -8592T allele (17.0%) compared with ATA patients (21.6%), although the difference was not statistically significant. An apparent over-representation of the c.50TT genotype among AIA patients (3.4%) compared with ATA patients (0.8%) also did not reach statistical significance after adjustment for multiple testing (p = 0.13). For 58 AIA patients, the minimum significant OR for association of the c.50TT genotype with AIA would have been 12.5.
所有受试者在这项研究最初招募哮喘或nonasthmatic,和其异位的地位被皮然后评估测试的时候加入了这项研究。因为这些科目应该大约代表一个随机招募过敏性和nonatopic科目,对特异反应性作为一个独立的表型也被评估。一个明显的c中所占比例较小。22T allele among atopic (6.5%) compared with nonatopic subjects (8.4%) was not statistically significant (minimum significant OR = 2.2). The c.22TT genotype was, however, significantly more frequent among nonatopic (1.2%) than atopic individuals (0.1%; OR = 9.05, 95% confidence interval 1.01–81.29; p = 0.034). This association was weak and its significance would be reduced by adjustment for multiple testing.
单体型协会
强大的连锁不平衡存在四种信息SNPs (Lewontin D≥0.84)为这些多态性和单体型协会如表4所示⇓。全球单体型分析显示没有联系与哮喘(p = 0.46),哮喘严重程度(p = 0.28)或AIA (p = 0.13)。的野生型CTCC单体型最常见,出现在57%的人口。另外两个主要单TTCC(21%)和预备(8%)。这些单都是单独与哮喘相关(p≥0.32)或哮喘严重程度(p≥0.19)。最强大的协会是友邦保险和TTCC之间单体型,但这不是重要的修正后为多个测试(p = 0.09),可能是因为样本量相对较小的友邦保险的病人。
讨论
这是第一个全面研究探索潜在的关联COX-1多态性与哮喘、疾病严重程度、友邦保险和特异反应性。20之前报道多态性的等位基因频率在启动子和基因的编码区被确定在一个随机的白人人口。之前报道的20个snp, 10没有发现在这个人口。四个(c。158 g > A, c。204 c > T, c。554A>C and c.1022A>G) have been previously reported to have variant allele frequencies of zero in Caucasians and 1–2% in African-Americans20.。另两个snp没有发现在目前的白人人口(c。688G>A, c.1428C>T) had previously reported variant allele frequencies of 1% in Caucasians20.,虽然没有发布信息频率的其他四个单核苷酸多态性(c。> 1076 G、c。> 1327 G、c。1399年A>G, c.–9169C>G) that were not detected in the present Caucasian population.
对于大多数的10个多态性出现在随机的白人人口,变异等位基因频率很低,除了c。22 t和c。50T alleles, which both had frequencies of 9%. The c.50C>T and c.22C>T SNPs were not only relatively frequent but also resulted in amino acid substitutions that are likely to affect COX-1 protein function20.。c。22C>T polymorphism results in the substitution of a positively charged arginine with an aromatic tryptophan residue (p.R8W), while the c.50C>T polymorphism results in the substitution of a neutral proline with a hydrophobic leucine residue (p.P17L).
c。22C>T and c.50C>T polymorphisms may also be functionally significant at the level of gene transcription, since the exonic splicing enhancer (ESE) finder program of Cartegniet al。32预测,这些多态性可能改变公认的拼接的绑定(SR蛋白)COX-1基因因素。相比之下,尽管c。-1676 c > T和c。- - - - - -8592C>T promoter polymorphisms were relatively frequent in the present populations, neither the transcription factor binding sites putatively deleted nor those created by these polymorphisms had any obvious relevance to asthma. Furthermore, these promoter polymorphisms did not affect the two Sp1 binding sites that have been reported to be essential for COX-1 transcription19。
c。22C>T and c.50C>T SNPs, as well as the two promoter polymorphisms (c.–1676C>T and c.–8592C>T), were selected for detailed investigation in the current asthmatic and nonasthmatic populations. There were no allelic or genotypic associations between any of these polymorphisms and asthma or asthma severity. It could be argued that with allele frequencies of <9% for c.22C>T and c.50C>T, large populations would have to be studied in order to find an association. However, the populations of asthmatic and nonasthmatic individuals were bigger than in most other studies, and power calculations indicated that for most of the associations tested, the sample sizes in the present study had a power of 90% to detect ORs of 1.5–3 as being statistically significant. Therefore, the study had reasonably adequate power to detect associations with asthma or asthma severity, and, despite the potential functional consequences of these COX-1 polymorphisms, any large effect on enzyme expression or activity in such a way as to influence the development or severity of asthma can be excluded.
然而,一个弱c基因型的协会之间的观察。22C>T (p.R8W) and atopy, suggesting that changes in the signal peptide of COX-1, which targets the protein for translocation into the endoplasmic reticulum33可能与过敏反应的某些方面联系在一起。目前的结果表明,较小的p。8W variant of COX-1 may provide a small protective effect against the development of atopy, which was defined in this study as the presence of at least one positive specific immunoglobulin E antibody response to inhaled allergens (positive skin-prick test). SNPs in a number of genes, including interleukin (IL)-4, IL-13, IL-4RA34,CD1435toll样受体4 (TLR4)36和巨噬细胞迁移抑制因子37,已经与过敏性表型有关,虽然不一定与哮喘。当前作者最近也证明了一个c。- - - - - -159C>T polymorphism in the CD14 gene was very weakly associated with atopy, but not with asthma38。然而,哮喘和特异反应性共享一个共同的病理生理背景和特定的多态性,如肿瘤坏死factor-α基因与哮喘和特异反应性有关39。明显的弱特异反应性和c之间的联系。22C>T SNP may be the result of linkage disequilibrium with another polymorphism that is causally related to atopy, and the COX-1 gene is located in the same chromosomal region (9q32) as the TLR4 gene, which contains a SNP that has been linked with the severity of atopy36。尽管过敏性敏感性可能随着年龄的增长,减少过敏性和nonatopic科目在目前的研究跨越了一个类似的年龄范围,和没有年龄的调整都是在评估协会与当前过敏性表型的科目。然而,c之间的弱关系。22C>T polymorphism and atopy, and the possible influence of age, require further investigation.
阿司匹林COX-1不可逆转地灭活由乙酰化一个活跃的网站丝氨酸残基在530位置,蛋白质的羧基末端附近40。丝氨酸- 530是在第11外显子编码,但是搜索的数据库没有识别出任何产生的多态性在第11外显子,很可能会影响阿司匹林的药理作用。在目前的研究中,没有统计上显著的友邦和c之间的关联。22 c > T或c。50C>T polymorphisms. Nevertheless, it is interesting that there was an over-representation of the c.50TT genotype (p.17LL) among AIA patients, as well as a haplotype association with AIA that, although possibly real, may not have reached statistical significance due to the relatively small sample size of AIA patients. The possibility of a link between p.P17L and the pharmacogenetics of aspirin is also supported by a recent report of the interaction between aspirin use and this polymorphism in relation to the risk of colorectal polyps41。
血小板对阿司匹林COX-1相关基因型在38个健康的美国白人受试者的研究21。在这个研究中,一个启动子多态性(c - 842 a > G)并不确定在目前筛查分析似乎完全与c连锁不平衡。50 c > T多态性。抑制PGF2α阿司匹林是更大的在血小板形成杂合的c。- - - - - -842A>G/50C>T haplotype compared with platelets from wild-type homozygotes. In the only other study that has investigated COX-1 polymorphisms in relation to disease, linkage analysis showed that the c.22C>T and c.50C>T SNPs were not associated with an increased incidence of thrombosis in a large Vermont kindred population42。
目前的研究已经确定了20个多态性的等位基因频率在启动子和cyclooxygenase-1基因的编码区随机澳大利亚白种人的主题,和四个常见的多态性进行了研究进一步与哮喘和特异反应性。这项研究中,充分的动力,没有发现任何关联的四个人多态性及其单体型与哮喘和哮喘严重程度,表明尽管其潜在功能的重要性,这些启动子和编码区多态性cyclooxygenase-1基因不太可能在哮喘中发挥作用。然而,这项研究的结果表明,c。22TT genotype may confer a small protective effect against atopy. In addition, a possible association between the c.50TT genotype and aspirin-intolerant asthma, as well as a possible haplotype association with aspirin-intolerant asthma, were identified, and may warrant further investigation in a larger population of aspirin-intolerant asthma patients.
确认
作者要感谢参与本研究的患者,f .失去和员工在哮喘和过敏研究所和澳大利亚红十字会血液服务(澳大利亚珀斯)。作者还要感谢k方(查尔斯王子医院,布里斯班,澳大利亚),r . O 'Hehir和f .梭(澳大利亚墨尔本阿尔弗雷德医院)辅助临床信息的收集和血液样本。
- 收到了2004年12月9日。
- 接受2005年4月23日。
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