mice [35]. Despite progress made in experimental studies, fundamental questions related to asthma risk among human populations remain; these can only be addressed by cohort studies using specimens linked to well-annotated clinical and environ- mental exposure data. Such cohort-driven epigenetic research has the potential to address key questions, such as those concerning the influence of early life factors, environmental exposure, other disease states, and lifestyle factors on susceptibility to asthma. In this proof-of-principle study, we developed an innovative ap- proach to population-based studies of environmental epigenetics, which could easily be implemented in future large-scale investi- gations. A range of approaches is available to obtain quantitative and qualitative information on genomic DNA methylation changes [27]. We chose methylation sensitive restriction fingerprinting ( MSRF; [19,20] ) as a tool for the discovery of PAH-reprogram- mable genes in UCWBC due to its high sensitivity, requirement for very small amounts of starting materials (,100 ng), and the ease in visual identification of differentially methylated sequences among a moderately large number of samples. Using only four arbitrary primers and six permutations of MSRF analyses on UCWBC samples from the high and low PAH exposure groups, we discovered 31 differentially methylated sequences. In future studies more arbitrary primers could be used to cover a larger portion of the genome in order to uncover more candidates [27]. In this study we had overcome another technical hurdle related to the quantification of gene expression in UCWBC. This task is challenging because the amount of cellular components in umbilical cord blood is usually very limited and prohibitive for high quality RNA isolation. To circumvent this problem, in a subset of samples, we assessed the concordance between the extents of methylation in the promoter-CGI(s) of each gene (using UCWBC DNA samples) with its respective transcript expression levels in RNA samples isolated from matched FPT samples. This choice was based on the logic that both UCWBC and FPT are fetal tissues are subjected to similar environmental exposure through the mother. Our results demonstrated good concordance for at least the ACSL3 gene. In future design of such an epigenetic epidemiology experiment, preference should be given to improved techniques for the isolation of RNA and DNA from the UCWBC. ACSL3 belongs to the acyl-CoA synthetase long chain (ACSL) family of genes which encodes key enzymes in fatty acid metabolism [36]. ACSL3 is expressed in lung and thymic tissue [37,38]. Intracellular conversion of long chain fatty acids to acyl- CoA is required for energy production. ACSL catalyzes the initial step in this conversion yielding acyl-CoAs, which are used both in the synthesis of cellular lipids and in degradation via beta- oxidation for energy production [39,40]. Moreover, acyl-CoA synthetase can catalyze the ATP-dependent production of arachidonoyl-CoA that is a substrate for lysophosphatidyl acyltransferase involved in arachidonic acid-containing phospho- lipid remodeling in proliferating T cells [41]. Lipid metabolism influences membrane proteins, including ion channels. In the lung, ACSL3 is found most abundantly in microsomal and cell membrane fractions, with mitochondria as the next richest source [42]. Inhibition of ACS enzyme activity has been shown to cause cell death in p53-defective lung cancer cells [43]. Thus hypermethylation of this gene in T helper cells or lung tissues is expected to diminish fatty acid utilization and beta-oxidation- energy production, and possibly influence membrane phospholip- id composition. Whether these functional changes directly affect asthma is unknown. However, several epidemiological studies show that fatty acid composition in milk [44,45], diet [46,47], and umbilical cord blood [48] affects the development of allergy and PAH, Epigenetic and Asthma other inflammatory diseases including asthma. For example, dietary omega-3 (n-3) fatty acids having a variety of anti- inflammatory and immune-modulating effects may affect allergy and asthma. Further mechanistic studies in in vitro and in vivo experimental models are needed to decipher the role of ACSL3 in childhood asthma. Interestingly, ACSL3 is located in 2q36.1 which has recently been shown to be associated with regions of the asthma susceptibility loci, in specific populations [49–50] Finally, the current finding of a putative epigenetic marker that is associated with PAH exposure and asthma adds to other evidence from the CCCEH cohort that PAHs increase risk of respiratory symptoms and probable asthma. At age 2 years, significantly more difficulty breathing and more probable asthma were reported among children jointly exposed prenatally to PAHs and postnatally to environmental tobacco smoke (ETS) (OR for probable asthma = 7.52, 95% CI 1.71–33.11, p,0.01) [3]. These relationships were reexamined through age 5 years and similar trends were detected, with an observed interaction between high prenatal PAH exposure and postnatal ETS on wheeze and probable asthma at age 5 (OR = 5.48, 95% CI 1.17–25.58, p,0.01) (unpublished data). A parallel cohort study in Krakow, Poland has found a significant relationship between prenatal PAH exposure and respiratory symptoms [51]. In a subset of 333 subjects, PAH exposure measured during pregnancy by personal air monitors was associated with an increased risk for wheezing without cold (RR 3.8; 95% CI: 1.2–12.4), during the course of the infant’s first year of life [51]. In conclusion, no previous studies have examined the effects of prenatal exposure to ambient air pollutants on DNA methylation patterns in genes potentially associated with the asthma phenotype of the offspring. This is an important gap in our understanding of asthma pathogenesis. Given the potential importance of epigenetic mechanisms in the etiology of childhood asthma, research is needed to identify an epigenetic profile related to PAH-associated childhood asthma and determine whether reprogramming events associated with transplacental exposure to PAHs increase risk of childhood asthma. The current report of ACSL3 as a candidate biomarker of PAH exposure and a putative predictor of PAH- associated childhood asthma represents an important first step in this area of research. Finally, the novel design of this study could serve as a prototype approach for future discoveries of additional exposure biomarkers or surrogate disease endpoints in other birth cohort studies. Materials and Methods Study Population Study subjects were nonsmoking Dominican and African American women and their children residing in Washington Heights, Central Harlem and the South Bronx participating in the CCCEH cohort study previously described [4–6]. Written informed consent was obtained from all subjects following procedures approved by the Institutional Review Board of the New-York Presbyterian Medical Center. Full enrollment into the study required completion of prenatal air monitoring and collection of a maternal and/or umbilical cord blood sample at delivery. As of November 1, 2007, 74% of the more than 600 fully enrolled subjects had been retained in the cohort through at least five years. During pregnancy and following delivery, a trained bilingual research team administered repeat questionnaires eliciting environmental and respiratory health histories [4]. There were no differences in demographics of subjects fully enrolled and those lost to follow-up [4–6]. Based on parental report of a doctor’s diagnosis of asthma or probable asthma before the age of five, PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4488