This project describes the role of Stephanie London's lab in LPP 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 previous two years, we concentrated on the LA lung cancer study (described under project entitled Genetic and environmental factors in cancer). We have now run low on DNA in the LA lung samples and will need to subject remaining samples to whole genome amplification in order to continue with that work. 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 10 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. Although data collection is still ongoing in this study, we have conducted genotyping analyses on the 220 complete triads that we have with extracted DNA in hand. 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. We are continuously updating our sample set and expect to have samples from the complete study of 500 triads by early 2005. DNA extraction is done by the contract laboratory Bioserve. This is a labor intensive process in part because of the database processing and verification. We have begun to shift our focus from characterizing one or two functional SNPs in each gene to doing sufficient genotyping for each gene to construct haplotypes. This entails greater genotyping requirement for each gene. This is the reason for our recent upgrade of our TaqMan machine to one with a 384 well plate format which allows higher throughput and saves on both DNA and reagants.
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