Genetic Analyses For Epidemiology Of Respiratory Disease

这个项目描述了斯蒂芬妮·伦敦的实验室在呼吸生物学实验室中的作用，以支持她的流行病学研究。该实验室正在对伦敦博士的呼吸道疾病流行病学研究中的样本进行分析。在过去的一年里，我们专注于墨西哥城儿童哮喘的研究(在题为儿童呼吸健康中的遗传和环境因素的项目中描述)。该实验室使用聚合酶链式反应技术来鉴定这些样本中的遗传多态。使用的方法包括RFLP-PCR、ARMS-PCR和Real-Time PCR(TaqMan)。我们还将DNA测序核心用于两个目的。第一个问题是确定在我们的墨西哥人口中，没有根据频率表征的多态是否足够普遍，值得我们考虑。我们对一组20个质量控制样本进行了测试。测序核心的另一个用途是为我们正在使用的任何一种基因分型方法产生已知基因型的对照。 今年，我们重点关注墨西哥城儿童哮喘的病例-父母三合会研究。在这项研究中，数据收集接近完成，我们将有500例父母三合会加上三分之二的三合会的兄弟姐妹。我们还有大约100个残缺不全的三合会，失踪的父亲。我们一直在进行基因分析，而不是等待研究完成。我们墨西哥城的人口是独一无二的，暴露在北美最高的臭氧水平中。虽然臭氧长期以来一直被认为会加重哮喘，但越来越多的证据表明臭氧在哮喘发病率中起到了作用。对小鼠的连锁研究表明，与哮喘发病机制相关的肺对臭氧的反应方面受到基因控制。因此，检测遗传易感性的能力可能有助于阐明臭氧在哮喘和呼吸损伤中的作用。由于我们墨西哥城的人口高度暴露在臭氧中，我们主要集中在既可能与哮喘发病有关，又已知或怀疑与臭氧的途径或反应有关的基因。这些基因主要参与对氧化应激和炎症的反应。DNA提取是由合同实验室Bioserve完成的。这是一个劳动密集型的过程，部分原因是数据库的处理和验证。 在过去的一年里，我们将重点从单个SNPs转移到单倍型分析上。这就需要对每个基因进行更高的基因分型要求。为了获得这方面的专业知识，我们参加了北卡罗来纳大学关于单倍型分析的期刊俱乐部，还用实验室资金把技术人员李慧玲送到华盛顿大学参加了一门关于SNP选择和单倍型分析的课程。 在下一财年，我们还将开始与SAPALDIA研究合作，包括对5000个样本进行基因分型，这些样本来自具有良好特征的空气污染队列中的受试者。我们将研究可能会改变空气污染对呼吸和心血管影响的基因。

This project describes the role of the lab of Stephanie London in the Laboratory of Respiratory Biology in support of her epidemiologic studies. The laboratory is engaged in analysis of samples from Dr. London's epidemiologic studies of respiratory disease. In the past year, we have focused on the study of childhood asthma in Mexico City (described under project entitled Genetic And Environmental Factors In Childhood Respiratory Health). The laboratory uses PCR technology to identify genetic polymorphisms in these samples. The methods used include RFLP-PCR, ARMS-PCR and real-time PCR (TaqMan). We also use the DNA sequencing core for two purposes. The first is identifying whether polymorphisms which have not been characterized with respect to their frequency are common enough in our Mexican population to warrant our consideration. We do this on a set of 20 quality control samples. The other use of the sequencing core is to generate controls of known genotype for whichever genotyping method that we are using. This year we have focused on the case-parent triad study of childhood asthma in Mexico City. Data collection is nearly complete in this study and we will have 500 case parent triads plus siblings on two-thirds of triads. We also have about 100 incomplete triads, missing the father. We have been conducting genetic analyses rather than waiting for the study to complete. Our Mexico City population is unique in being exposed to the highest ozone levels in North America. Although ozone has long been known to exacerbate asthma, evidence for a role of ozone in incidence of asthma is increasing. Linkage studies in mice indicate that aspects of pulmonary response to ozone that are relevant to asthma pathogenesis are under genetic control. Thus the ability to examine genetic susceptibility may help clarify the role on ozone in asthma and respiratory impairment. Because of the high exposure of our Mexico City population to ozone, we have primarily concentrated on genes that are both plausibly involved in asthma pathogenesis and known or suspected to be involved in pathways or response to ozone. These genes are predominantly involved in response to oxidative stress and inflammation. DNA extraction is done by the contract laboratory Bioserve. This is a labor intensive process in part because of the database processing and verification. In the past year, we have shifted our focus from individual SNPs to haplotype analysis. This entails greater genotyping requirement for each gene. To gain this expertise, we have been attending a journal club on haplotype analysis at UNC and also used lab funds to send the technician, Huiling Li, to a course on SNPs selection and haplotype analysis at University of Washington. In the next fiscal year, we will also be beginning a collaboration with the SAPALDIA study which involves the genotyping of 5,000 samples from subjects in a very well characterized air pollution cohort. We will be looking at genes that may modify respiratory and cardiovascular effects of air pollution.