BACKGROUND:Although previous studies suggest that exposure to traffic-related pollution during childhood increases the risk of childhood overweight or obesity (COWO), the role of early life exposure to fine particulate matter (aerodynamic diameter <2.5 μm; PM2.5) and its joint effect with the mother’s prepregnancy body mass index (MPBMI) on COWO remain unclear. OBJECTIVES:The present study was conducted to examine the individual and joint effects of ambient PM2.5 exposures and MPBMI on the risk of COWO. METHODS:We estimated exposures to ambient PM2.5in utero and during the first 2 y of life (F2YL), using data from the U.S. Environmental Protection Agency’s (EPA’s) Air Quality System matched to residential address, in 1,446 mother–infant pairs who were recruited at birth from 1998 and followed up prospectively through 2012 at the Boston Medical Center in Massachusetts. We quantified the individual and joint effects of PM2.5 exposure with MPBMI on COWO, defined as the child’s age- and sex-specific BMI z-score ≥85th percentile at the last well-child care visit between 2 and 9 y of age. Additivity was assessed by estimating the reduced excess risk due to interaction. RESULTS:Comparing the highest and lowest quartiles of PM2.5, the adjusted relative risks (RRs) [95% confidence intervals (CIs)] of COWO were 1.3 (95% CI: 1.1, 1.5), 1.2 (95% CI: 1.0, 1.4), 1.2 (95% CI: 1.0, 1.4), 1.3 (95% CI: 1.1, 1.6), 1.3 (95% CI: 1.1, 1.5) and 1.3 (1.1, 1.5) during preconception; the first, second, and third trimesters; the entire period of pregnancy; and F2YL, respectively. Spline regression showed a dose–response relationship between PM2.5 levels and COWO after a threshold near the median exposure (10.46 μg/m3–10.89 μg/m3). Compared with their counterparts, children of obese mothers exposed to high levels of PM2.5 had the highest risk of COWO [RR≥2.0, relative excess risk due to interaction (RERI) not significant]. CONCLUSIONS:In the present study, we observed that early life exposure to PM2.5 may play an important role in the early life origins of COWO and may increase the risk of COWO in children of mothers who were overweight or obese before pregnancy beyond the risk that can be attributed to MPBMI alone. Our findings emphasize the clinical and public health policy relevance of early life PM2.5 exposure. https://doi.org/10.1289/EHP261

IMPORTANCE:Previous reports have linked maternal prepregnancy obesity with low folate concentrations and child overweight or obesity (OWO) in separate studies. To our knowledge, the role of maternal folate concentrations, alone or in combination with maternal OWO, in child metabolic health has not been examined in a prospective birth cohort.OBJECTIVE:To test the hypotheses that maternal folate concentrations can significantly affect child metabolic health and that sufficient maternal folate concentrations can mitigate prepregnancy obesity-induced child metabolic risk.DESIGN, SETTING, AND PARTICIPANTS:This prospective birth cohort study was conducted at the Boston Medical Center, Boston, Massachusetts. It included 1517 mother-child dyads recruited at birth from 1998 to 2012 and followed up prospectively up to 9 years from 2003 to 2014.MAIN OUTCOMES AND MEASURES:Child body mass index z score calculated according to US reference data, OWO defined as a body mass index in the 85th percentile or greater for age and sex, and metabolic biomarkers (leptin, insulin, and adiponectin).RESULTS:The mean (SD) age was 28.6 (6.5) years for mothers and 6.2 (2.4) years for the children. An L-shaped association between maternal folate concentrations and child OWO was observed: the risk for OWO was higher among those in the lowest quartile (Q1) as compared with those in Q2 through Q4, with an odds ratio of 1.45 (95% CI, 1.13-1.87). The highest risk for child OWO was found among children of obese mothers with low folate concentrations (odds ratio, 3.05; 95% CI, 1.91-4.86) compared with children of normal-weight mothers with folate concentrations in Q2 through Q4 after accounting for multiple covariables. Among children of obese mothers, their risk for OWO was associated with a 43% reduction (odds ratio, 0.57; 95% CI, 0.34-0.95) if their mothers had folate concentrations in Q2 through Q4 compared with Q1. Similar patterns were observed for child metabolic biomarkers.CONCLUSIONS AND RELEVANCE:In this urban low-income prospective birth cohort, we demonstrated an L-shaped association between maternal plasma folate concentrations and child OWO and the benefit of sufficient folate concentrations, especially among obese mothers. The threshold concentration identified in this study exceeded the clinical definition of folate deficiency, which was primarily based on the hematological effect of folate. Our findings underscore the need to establish optimal rather than minimal folate concentrations for preventing adverse metabolic outcomes in the offspring.

This study aimed to investigate the optimal degree of weight gain across the gestational spectrum in 1971 children enrolled at birth and followed up to age 7 years. Weight gain in infancy was categorized into four groups based on weight gain z-scores: slow (<−0.67), on track (−0.67 to 0.67), rapid (0.67 to 1.28), and extremely rapid (>1.28). Underweight and overweight or obesity (OWO) were defined as a body mass index ≤5th and ≥85th percentile, respectively, for age and gender. In our population, OWO was far more common than underweight (39.7% vs. 3.6%). Weight gain tracked strongly from age 4 to 24 months, and was positively associated with OWO and an unfavorable pattern of metabolic biomarkers, although the degree of weight gain for the risk was different across gestational categories. Extremely rapid weight gain led to a particularly high risk of OWO among children born early term and late preterm: odds ratio: 3.3 (95% confidence interval: 1.9 to 5.5) and 3.7 (1.8 to 7.5), respectively, as compared to those with on track weight gain. Our findings suggest that monitoring and ensuring optimal weight gain across the entire gestational spectrum beginning from birth represents a first step towards primary prevention of childhood obesity.

IgE-mediated cow's milk allergy (CMA), which affects 2% to 3% of young children, is among the most common types of food allergy. The molecular mechanisms underlying the development, persistence, and resolution of CMA remain largely unknown. Several recent studies have suggested that epigenetic alterations are involved in allergy development.1Hong X. Wang X. Early life precursors, epigenetics, and the development of food allergy.Semin Immunopathol. 2012; 34: 655-669Crossref PubMed Scopus (40) Google Scholar, 2Su R.C. Becker A.B. Kozyrskyj A.L. Hayglass K.T. Epigenetic regulation of established human type 1 versus type 2 cytokine responses.J Allergy Clin Immunol. 2008; 121: 57-63.e3Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar DNA methylation (DNAm), a type of epigenetic mechanism, regulates gene expression. Alterations in DNAm play a key role in T-cell differentiation and in maintaining TH1/TH2 balance,1Hong X. Wang X. Early life precursors, epigenetics, and the development of food allergy.Semin Immunopathol. 2012; 34: 655-669Crossref PubMed Scopus (40) Google Scholar, 2Su R.C. Becker A.B. Kozyrskyj A.L. Hayglass K.T. Epigenetic regulation of established human type 1 versus type 2 cytokine responses.J Allergy Clin Immunol. 2008; 121: 57-63.e3Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar and thus may offer a potential mechanistic explanation for the natural history of CMA. However, to date, research on the epigenetics of food allergy,3Martino D. Dang T. Sexton-Oates A. Prescott S. Tang M.L. Dharmage S. et al.Blood DNA methylation biomarkers predict clinical reactivity in food-sensitized infants.J Allergy Clin Immunol. 2015; 135: 1319-1328.e12Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar or CMA in particular,4Berni Canani R. Paparo L. Nocerino R. Cosenza L. Pezzella V. Di Costanzo M. et al.Differences in DNA methylation profile of Th1 and Th2 cytokine genes are associated with tolerance acquisition in children with IgE-mediated cow's milk allergy.Clin Epigenetics. 2015; 7: 38Crossref PubMed Scopus (51) Google Scholar has been very limited. Most of the available studies are based on small sample sizes (<60 children), and were not replicated in an independent sample. We performed the first epigenome-wide association study of CMA in US children using a 2-stage approach. In the discovery stage, we measured DNAm at 485,512 genomic loci in whole blood samples from 106 white children with CMA (cases) and 76 nonallergic and nonatopic whites (controls) using the Illumina HumanMethylation450 arrays. All the cases and controls were enrolled by the Chicago Food Allergy Study using identical protocols. We then sought to validate the top significant DNAm loci in 2 independent replication samples including (1) childhood whole blood samples of 25 white children (5 CMA cases and 20 controls) from the same Chicago Food Allergy Study (Chicago replication sample) and (2) cord blood samples from 140 black children (8 cases and 132 controls) from the Boston Birth Cohort (Boston replication sample) (see this article's Methods section in the Online Repository at www.jacionline.org). Population characteristics of the discovery and replication samples are presented in Tables E1 and E2 in this article's Online Repository at www.jacionline.org, respectively. CMA cases were defined if the children met the following criteria: (1) a convincing history of symptoms indicative of an allergic reaction within 2 hours of ingestion of cow's milk and (2) clear evidence of sensitization defined as having a specific IgE level of 0.35 kU/L or more to cow's milk and/or a positive skin prick test result to cow's milk with a mean wheal diameter of 3 mm or more greater than that with the saline control. After quality control steps (see this article's Methods section), 435,642 CpG sites were available for subsequent data analyses. We fit a linear regression model for ComBat-transformed M value at each CpG site as a function of CMA status, adjusting for potential confounders (see this article's Methods section). Bonferroni correction was applied to account for multiple testing (P < 1.15 × 10−7). In the discovery stage, we identified 575 significant autosomal differentially methylated positions (DMPs) (469 located within 385 genes and 106 intergenic): 568 hypomethylated and 7 hypermethylated in CMA cases compared with controls (P < 1.15 × 10−7; Fig 1). Most of these 575 DMPs showed a small DNAm change between cases and controls, and only 5 DMPs had an absolute mean DNAm difference of 5% or more (Fig 1 and Table I). On the X-chromosome, DMP cg16737869 met our P value significance threshold, but with a modest DNAm change between cases and controls (2%).Table IThe top significant differentially methylated positions and their associations with CMA in the discovery and replication samplesDMPsCHRGeneLocationChicago discoveryChicago replicationBoston replicationβdiff∗Mean methylation difference (%) between cases and controls.P value†P value was calculated on the basis of linear regression models with ComBat-transformed M value at each DMP as the outcome, adjusted for age, sex, breast-feeding, parental history of food allergy, estimated cell composition, and genetic ancestry.βdiff∗Mean methylation difference (%) between cases and controls.P value‡P value was calculated on the basis of linear regression model with ComBat-transformed M value at each DMP as the outcome, adjusted for cell composition and age. Further adjustment for sex and parental history of allergy did not significantly change the associations.βdiff∗Mean methylation difference (%) between cases and controls.P value§P value was calculated on the basis of linear regression model with ComBat-transformed M value at each DMP as the outcome, adjusted for cell composition, sex, parental history of food allergy, and gestational age group.DMPs with absolute DNAm difference of ≥5% in cases vs controls cg1177032313NDFIP2Intron−5.72.0 × 10−15−4.0.005FDR < 5%.−0.9.811 cg1640945214EVL3′UTR−5.45.6 × 10−14−7.3.042−1.4.322 cg1855084714EVL3′UTR−5.39.6 × 10−14−7.4.034FDR < 5%.−1.9.187 cg093775318TRAPPC9Intron−5.17.0 × 10−14−7.8.004FDR < 5%.−1.5.260 cg150908996RPS6KA2Exon−5.13.6 × 10−08−7.1.384−2.1.811DMPs with annotated genes relevant to TH1-TH2 pathway cg163861582IL1RL1TSS1500−3.81.7 × 10−13−8.8.007FDR < 5%.−2.4.041 cg084042253IL5RA5′UTR−3.79.3 × 10−10−5.5.062−2.2.001FDR < 5%. cg133161482STAT4Intron−3.26.3 × 10−09−5.2.013FDR < 5%.−0.4.301 cg267872395IL4TSS1500−2.66.7 × 10−09−4.8.006FDR < 5%.−1.5.042 cg0604087217CCL18Intron−3.24.0 × 10−09−5.2.018FDR < 5%.−0.6.256CHR, Chromosome; TSS, transcription start site; UTR, untranslated region.∗ Mean methylation difference (%) between cases and controls.† P value was calculated on the basis of linear regression models with ComBat-transformed M value at each DMP as the outcome, adjusted for age, sex, breast-feeding, parental history of food allergy, estimated cell composition, and genetic ancestry.‡ P value was calculated on the basis of linear regression model with ComBat-transformed M value at each DMP as the outcome, adjusted for cell composition and age. Further adjustment for sex and parental history of allergy did not significantly change the associations.§ P value was calculated on the basis of linear regression model with ComBat-transformed M value at each DMP as the outcome, adjusted for cell composition, sex, parental history of food allergy, and gestational age group.|| FDR < 5%. Open table in a new tab CHR, Chromosome; TSS, transcription start site; UTR, untranslated region. In parallel with individual DMP analyses, we performed a genome-wide screen to identify differentially methylated regions (DMRs) using a “bump hunting” approach. A marginally significant DMR in the 3′ untranslated region of the Enah/Vasp-like (EVL) gene was identified, which was hypomethylated in CMA cases compared with controls (family-wise error rate = 0.085; see Fig E1, A, in this article's Online Repository at www.jacionline.org). This DMR included 5 highly correlated DMPs (r > 0.8; Fig E1, B) that were all significantly associated with CMA at P < 1.15 × 10−7, 2 of which having an absolute mean DNAm difference of more than 5% between cases and controls. We then performed KEGG pathway-enrichment analysis using WebGestalt on the 386 annotated genes that contained at least 1 significant DMP associated with CMA. Three enriched pathways were identified—“Butirosin and neomycin biosynthesis,” “Starch & sucrose metabolism,” and “Fructose and mannose metabolism”—all of which shared 3 genes (hexokinase 1-3) that are critical to glucose metabolism (see Table E3 in this article's Online Repository at www.jacionline.org). This result supports our previous findings on the significant associations between gestational diabetes and increased risk of food sensitization.5Kumar R. Ouyang F. Story R.E. Pongracic J.A. Hong X. Wang G. et al.Gestational diabetes, atopic dermatitis, and allergen sensitization in early childhood.J Allergy Clin Immunol. 2009; 124 (e1-4): 1031-1038Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Among the 576 DMPs associated with CMA, 10 loci meeting the following criteria were selected for replication: (1) significant DMPs with an absolute DNAm difference of more than 5% (n = 5) and/or (2) significant DMPs that annotated to genes relevant to the TH1-TH2 pathway (n = 5) (Table I). In the Chicago replication sample, 7 DMPs were validated for their associations with CMA at a false-discovery rate (FDR) of less than 0.05 (Table I). In the Boston replication sample, only DMP cg08404225 in the IL5RA gene was significantly hypomethylated in CMA cases relative to the normal controls after FDR correction (FDR = 0.01). The associations between the 8 validated DMPs and CMA in the replication samples were in the same directions as were identified in the discovery sample (Table I; see Fig E2 in this article's Online Repository at www.jacionline.org). The biological significance of the 8 validated DMPs was explored with a Web tool, EpiExplorer. As shown in Table E4 in this article's Online Repository at www.jacionline.org, DMP cg11770323 overlapped with the DNaseI hypersensitive site; DMP cg18550847 was within a CpG island; and DMPs cg13316148, cg08404225, and cg26787239 overlapped with the enhancers and with lamina-associated domains. We also found that DMPs cg16386158, cg13316148, cg26787239, and cg11770323 colocalized with the binding sites of CCCTC-binding factor (CTCF) (see Table E4). Overall, these data indicate that DNAm alteration of these loci may be biologically relevant, but further validation is needed. This study not only confirmed that DNAm alterations in TH1-TH2 balance (ie, those annotated to IL1RL1, IL5RA, IL4, CCL18, and STAT4 genes) are associated with CMA, it also revealed some novel candidates for CMA. Among the 8 validated DMPs, 3 DMPs, all with an absolute mean DNAm difference of more than 5% between cases and controls, were annotated to NDFIP2 (cg11770323), EVL (cg18550847), and TRAPPC9 (cg09377531), respectively. These 3 genes have not previously been implicated in allergy, but their role may be biologically plausible. NDFIP2, one of the IL4–regulated genes, can promote IFN-γ production via polarized human TH1 lymphocytes.6Lund R.J. Loytomaki M. Naumanen T. Dixon C. Chen Z. Ahlfors H. et al.Genome-wide identification of novel genes involved in early Th1 and Th2 cell differentiation.J Immunol. 2007; 178: 3648-3660Crossref PubMed Scopus (67) Google Scholar NDFIP2 can also activate E3 ubiquitin ligases, which may play an important role in preventing allergic diseases. EVL is one of the IL-13–regulated genes.7Scotton C.J. Martinez F.O. Smelt M.J. Sironi M. Locati M. Mantovani A. et al.Transcriptional profiling reveals complex regulation of the monocyte IL-1 beta system by IL-13.J Immunol. 2005; 174: 834-845Crossref PubMed Scopus (117) Google Scholar The analyses from GIANT8Greene C.S. Krishnan A. Wong A.K. Ricciotti E. Zelaya R.A. Himmelstein D.S. et al.Understanding multicellular function and disease with human tissue-specific networks.Nat Genet. 2015; 47: 569-576Crossref PubMed Scopus (485) Google Scholar showed that the EVL and IL4 genes are highly coexpressed in basophils. TRAPPC9 encodes a protein that likely plays a role in nuclear factor kappa B signaling, a pathway that may be relevant to the immune system. Further studies are needed to explore the role of these annotated genes in CMA. The 2 replication analyses in this study, although with limited sample sizes, have provided additional information. Replication in the Chicago sample, where the cases were all allergic to both peanut and cow's milk and the non-CMA controls were all allergic to peanut only, suggested that the replicated DNAm associations may be specific to CMA, whereas replication in the Boston sample, where DNAm was measured in cord blood from black samples, indicate that the replicated DNAm alterations between cases and controls may occur in utero and may be common across race groups. Several limitations are noted. First, we used whole blood DNA to obtain DNAm profiling. Second, questions remain regarding how to interpret small DNAm differences between CMA and controls, mostly less than 5%. One possibility is that such small changes in whole blood may be reflective of larger changes in a specific cell type, as demonstrated in a previous study.9Liang L. Willis-Owen S.A. Laprise C. Wong K.C. Davies G.A. Hudson T.J. et al.An epigenome-wide association study of total serum immunoglobulin E concentration.Nature. 2015; 520: 670-674Crossref PubMed Scopus (160) Google Scholar Third, DNAm levels of the identified DMPs were not validated using other techniques such as pyrosequencing, and no functional assays were conducted in this study. However, previous studies have demonstrated that the 450K array correlates well with direct pyrosequencing.10Roessler J. Ammerpohl O. Gutwein J. Hasemeier B. Anwar S.L. Kreipe H. et al.Quantitative cross-validation and content analysis of the 450k DNA methylation array from Illumina, Inc.BMC Res Notes. 2012; 5: 210Crossref PubMed Scopus (89) Google Scholar In conclusion, this was the first epigenome-wide association study of CMA in US children and it clearly demonstrated that methylation alterations in specific gene loci are associated with CMA. Findings from this study warrant additional validation and functional studies, and, if confirmed, may help to improve our understanding of the epigenetic mechanisms underlying the development and resolution of CMA and offer novel targets for prediction, prevention, and treatment of CMA. We gratefully acknowledge the individuals and families who participated in the Chicago Food Allergy Study and in the Boston Birth Cohort. For our epigenome-wide screen, we obtained blood from 106 white children with CMA and 77 nonallergic white controls as defined below from the Chicago Food Allergy Study (the discovery sample), which has been previously described in detail.E1Tsai H.J. Kumar R. Pongracic J. Liu X. Story R. Yu Y. et al.Familial aggregation of food allergy and sensitization to food allergens: a family-based study.Clin Exp Allergy. 2009; 39: 101-109Crossref PubMed Scopus (104) Google Scholar, E2Hong X. Hao K. Ladd-Acosta C. Hansen K.D. Tsai H.J. Liu X. et al.Genome-wide association study identifies peanut allergy-specific loci and evidence of epigenetic mediation in US children.Nat Commun. 2015; 6: 6304Crossref PubMed Scopus (163) Google Scholar Briefly, eligible families had at least 1 biological child with food allergy (FA) and were willing to participate in the study. For each enrolled participant, the following procedures were completed: (1) questionnaire interview by trained research staff to obtain information on home environment, diet, lifestyle, and history of FA and other allergic diseases; (2) allergy skin prick testing (SPT) to 9 food allergens and 6 aeroallergens; and (3) collection of venous blood samples. For each child, we also collected a detailed history of the clinical allergic reaction associated with ingestion of specific foods. The Institutional Review Board of Ann & Robert H. Lurie Children's Hospital of Chicago and Johns Hopkins Bloomberg School of Public Health approved the study protocol. For replication purposes, we obtained 2 independent samples. The first sample consisted of 5 white cases with allergy to both cow's milk and peanut and 20 positive controls with allergy to peanut but not to cow's milk (see detailed description below for phenotypic classification) enrolled from the same Chicago Food Allergy Study (the Chicago replication sample). These subjects were independent of the discovery sample. The second sample consisted of 140 black children from the prospective Boston Birth Cohort (BBC)E3Hong X. Wang G. Liu X. Kumar R. Tsai H.J. Arguelles L. et al.Gene polymorphisms, breast-feeding, and development of food sensitization in early childhood.J Allergy Clin Immunol. 2011; 128: 374-381.e2Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar including 8 cases with CMA and 132 normal controls (the Boston replication sample). Unlike the samples obtained from the Chicago Food Allergy Study, the Boston sample had DNAm measured in cord blood (before clinical expression of disease). The institutional review boards at the Boston Medical Center and Johns Hopkins Bloomberg School of Public Health approved the BBC study protocol. As reported previously,E2Hong X. Hao K. Ladd-Acosta C. Hansen K.D. Tsai H.J. Liu X. et al.Genome-wide association study identifies peanut allergy-specific loci and evidence of epigenetic mediation in US children.Nat Commun. 2015; 6: 6304Crossref PubMed Scopus (163) Google Scholar, E4Hong X. Caruso D. Kumar R. Liu R. Liu X. Wang G. et al.IgE, but not IgG4, antibodies to Ara h 2 distinguish peanut allergy from asymptomatic peanut sensitization.Allergy. 2012; 67: 1538-1546PubMed Google Scholar we defined CMA cases in this study using the following stringent criteria: (1) a convincing history of symptoms indicative of an allergic reaction within 2 hours of ingestion of cow's milk and (2) clear evidence of sensitization, defined as having a specific IgE (sIgE) level of 0.35 kU/L or more to cow's milk and/or a positive SPT result to cow's milk with a mean wheal diameter (MWD) of 3 mm or more greater than that with the saline control. Normal controls were defined as a child who had neither a clinical allergic reaction nor evidence of sensitization to any of the common foods (peanut, egg white, cow's milk, soy, wheat, walnut, fish, shellfish, and sesame seed). In the Chicago replication sample, positive controls were defined as a child who had neither a clinical allergic reaction nor evidence of sensitization to cow's milk, but was allergic to peanut. In the Chicago Food Allergy Study, SPT to 9 food allergens (cow's milk, egg white, soybean, wheat, peanut, English walnut, sesame seed, fish mix [cod, flounder, halibut, mackerel, tuna], and shellfish mix [clam, crab, oyster, scallops, shrimp]) and 6 aeroallergens (2 dust mites [Dermatohagoides teronyssinus and Dermatophagoides farina], cat hair, dog epithelia, cockroach mix, and Alternaria tenius), plus negative (50% glycerinated saline) and positive (histamine, 1.0 mg/mL) controls (Greer, Lenoir, NC), was performed using a Multi-Test II device (Lincoln Diagnostics, Decatur, Ill) as previously described in detail.E2Hong X. Hao K. Ladd-Acosta C. Hansen K.D. Tsai H.J. Liu X. et al.Genome-wide association study identifies peanut allergy-specific loci and evidence of epigenetic mediation in US children.Nat Commun. 2015; 6: 6304Crossref PubMed Scopus (163) Google Scholar, E4Hong X. Caruso D. Kumar R. Liu R. Liu X. Wang G. et al.IgE, but not IgG4, antibodies to Ara h 2 distinguish peanut allergy from asymptomatic peanut sensitization.Allergy. 2012; 67: 1538-1546PubMed Google Scholar Data were excluded if the MWD for the negative control was 3 mm or more, or if the MWD for the positive control was less than 3 mm, or if the difference of the positive minus the negative control was less than 3 mm. No SPT measurements were performed in the BBC. In the Chicago Food Allergy Study, sIgE levels for 9 food allergens (egg white, sesame, peanut, soy, milk, shellfish, walnut, cod fish, and wheat) and 6 aeroallergens (Dermatophagoides pteronyssinus and Dermatophagoides farinae, cat dander, dog dander, German cockroach, and Alternaria alternata) were measured using the Phadia ImmunoCAP system (Phadia US, Inc, Portage, Mich) by the Clinical Immunology Laboratory at Lurie Children's, a Clinical Laboratory Improvement Amendment certified laboratory for the ImmunoCAP assay. The lower detection limit for each sIgE was 0.1 kUA/L and the reporting range was 0.1 to 100 kUA/L. In the BBC, the sIgE level for 8 food allergens (egg white, peanut, soy, milk, shrimp, walnut, cod fish, and wheat) was measured at Quest Diagnostics using the Phadia ImmunoCAP system. The lower detection limit for food sIgE level was 0.35 kUA/L. In the discovery stage, case and control DNA samples (50 ng/uL) were evenly distributed across each plate to minimize batch effects, which were then shipped to the Northwestern University Genomics Core for DNAm profiling. Briefly, 0.5 μg of genomic DNA was bisulfite-converted using the EZ-96 DNA Methylation Gold Kit, and DNAm levels at 485,512 loci were measured using the Infinium HumanMethylation450 BeadChips (450K), according to the manufacturer's instructions. A raw intensity file (.idat) for each sample was processed, and several quality control steps were performed using the “minfi”E5Aryee M.J. Jaffe A.E. Corrada-Bravo H. Ladd-Acosta C. Feinberg A.P. Hansen K.D. et al.Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays.Bioinformatics. 2014; 30: 1363-1369Crossref PubMed Scopus (2063) Google Scholar Bioconductor package, as we reported previously.E2Hong X. Hao K. Ladd-Acosta C. Hansen K.D. Tsai H.J. Liu X. et al.Genome-wide association study identifies peanut allergy-specific loci and evidence of epigenetic mediation in US children.Nat Commun. 2015; 6: 6304Crossref PubMed Scopus (163) Google Scholar First, we examined the 450K control probes to assess bisulfite conversion, extension, hybridization, staining, specificity, and others. Second, we computed the median for both Meth and Unmeth signals for each array and displayed them in a scatter plot. This approach clearly identified one outlier sample with a median log2 intensity value of less than 10.5, which was removed from further analyses. Third, we removed 748 loci that had a detection P value (a measure of probe performance) of more than .01 in 10% or more of the samples. Fourth, we removed 21,524 CpG sites that had an annotated single nucleotide polymorphism (minor allele frequency of >0.01) at the measured and/or neighboring locus (±1bp depending on probe strand orientation), and removed 27,513 CpG sites previously reported to be cross-reactive.E6Chen Y.A. Lemire M. Choufani S. Butcher D.T. Grafodatskaya D. Zanke B.W. et al.Discovery of cross-reactive probes and polymorphic CpGs in the Illumina Infinium HumanMethylation450 microarray.Epigenetics. 2013; 8: 203-209Crossref PubMed Scopus (946) Google Scholar After these quality control steps, DNAm of 435,642 sites (including 424,420 autosomal sites and 11,222 X-chromosome sites) from 182 samples were available for subsequent data analyses. Using the minfi package,E5Aryee M.J. Jaffe A.E. Corrada-Bravo H. Ladd-Acosta C. Feinberg A.P. Hansen K.D. et al.Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays.Bioinformatics. 2014; 30: 1363-1369Crossref PubMed Scopus (2063) Google Scholar a stratified quantile normalization procedure was applied to the raw data and normalized beta values (β), ranging from 0 to 1 for 0% to 100% methylated, were obtained. M values (logit transformed β) were also computed. Both the β and M values were ComBat-transformedE7Johnson W.E. Li C. Rabinovic A. Adjusting batch effects in microarray expression data using empirical Bayes methods.Biostatistics. 2007; 8: 118-127Crossref PubMed Scopus (4059) Google Scholar using the “sva” packageE8Leek J.T. Johnson W.E. Parker H.S. Jaffe A.E. Storey J.D. The sva package for removing batch effects and other unwanted variation in high-throughput experiments.Bioinformatics. 2012; 28: 882-883Crossref PubMed Scopus (2290) Google Scholar with the array number as the surrogate for the batches. ComBat-transformed M values were used for downstream statistical analyses because they represent a more normal distribution than do β values. ComBat-transformed β values were used for plotting purposes because they are more intuitive. The same protocols for DNAm measurement and data cleaning were applied to the 2 replication samples. To account for potential differences in the proportions of cells that comprised the blood,E10Houseman E.A. Accomando W.P. Koestler D.C. Christensen B.C. Marsit C.J. Nelson H.H. et al.DNA methylation arrays as surrogate measures of cell mixture distribution.BMC Bioinformatics. 2012; 13: 86Crossref PubMed Scopus (1900) Google Scholar, E11Reinius L.E. Acevedo N. Joerink M. Pershagen G. Dahlen S.E. Greco D. et al.Differential DNA methylation in purified human blood cells: implications for cell lineage and studies on disease susceptibility.PLoS One. 2012; 7: e41361Crossref PubMed Scopus (708) Google Scholar, E9Jaffe A.E. Irizarry R.A. Accounting for cellular heterogeneity is critical in epigenome-wide association studies.Genome Biol. 2014; 15: R31Crossref PubMed Scopus (648) Google Scholar we empirically estimated the proportion of CD8+ T cells, CD4+ T cells, natural killer cells, B cells, monocytes, and granulocytes as previously described by Houseman et alE10Houseman E.A. Accomando W.P. Koestler D.C. Christensen B.C. Marsit C.J. Nelson H.H. et al.DNA methylation arrays as surrogate measures of cell mixture distribution.BMC Bioinformatics. 2012; 13: 86Crossref PubMed Scopus (1900) Google Scholar for all the discovery and replication samples, using the minfi package.E5Aryee M.J. Jaffe A.E. Corrada-Bravo H. Ladd-Acosta C. Feinberg A.P. Hansen K.D. et al.Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays.Bioinformatics. 2014; 30: 1363-1369Crossref PubMed Scopus (2063) Google Scholar To identify DMPs associated with CMA in the discovery sample, we used the “limma” packageE12Smyth G.K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments.Stat Appl Genet Mol Biol. 2004; 3: Article3Crossref PubMed Scopus (9158) Google Scholar to fit a linear regression model for ComBat-transformed M value at each CpG site as a function of CMA status (1 = case, 0 = control), adjusting for potential confounders including sex, age group (coded as 0 = <2 years, 1 = 2-6 years, 2 = 6-10 years, 3 = ≥10 years), breast-feeding history (yes/no), parental history of FA (0 = none, 1 = one parent, 2 = both parents with FA), cell-type proportions for granulocytes, monocytes, B cells, natural killer cells, CD4+ T cells, and CD8+ T cells, as well as genetic ancestry (represented by the first 2 principal components based on genome-wide single nucleotide polymorphism data, as we reported previouslyE2Hong X. Hao K. Ladd-Acosta C. Hansen K.D. Tsai H.J. Liu X. et al.Genome-wide association study identifies peanut allergy-specific loci and evidence of epigenetic mediation in US children.Nat Commun. 2015; 6: 6304Crossref PubMed Scopus (163) Google Scholar). Bonferroni correction was applied to account for multiple testing (P < 1.15 × 10−7). For CpG sites on the X-chromosome, sex-specific analyses were also performed. As a complementary approach to single-site DMP analyses, we searched for DMRs associated with CMA using the bumphunting() functionE13Jaffe A.E. Murakami P. Lee H. Leek J.T. Fallin M.D. Feinberg A.P. et al.Bump hunting to identify differentially methylated regions in epigenetic epidemiology studies.Int J Epidemiol. 2012; 41: 200-209Crossref PubMed Scopus (425) Google Scholar in the minfi package. Briefly, each locus was a priori assigned to a region such that any 2 neighboring loci separated by 300bp or less were assigned to the same region. Next, for each CpG site, we estimated the difference in average combat-transformed M value between CMA cases and controls, adjusting for the same covariates as described above. Significance was assigned with the application of a bootstrapping method (n = 1000 times). CMA candidate regions with a family-wise error of less than .05 were considered significant. All statistical analyses were performed using R-3.1.1 and Bioconductor 2.12. We then performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify biological pathways with enrichment of genes that exhibited differential DNAm between 106 CMA cases and 76 controls, using WebGestaltE14Wang J. Duncan D. Shi Z. Zhang B. WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013.Nucleic Acids Res. 2013; 41: W77-W83Crossref PubMed Scopus (1187) Google Scholar and with the approximately 20,000 annotated genes of the studied 435,642 CpG sites as the background. We used a Bonferroni-adjusted P value of less than .05 as our significance threshold. We decided to validate a subset of identified CMA-associated DMPs in our replication analyses because of the small number of samples. These subsets were selected on the basis of 1 or more of the following criteria: (1) significant DMPs (P < 1.15 × 10−7) with an absolute DNAm difference of more than 5% and/or (2) significant DMPs (P < 1.15 × 10−7) that annotated to genes relevant to the TH1-TH2 pathway, a critic

BACKGROUND:Prenatal exposure to ambient PM2.5, (i.e., fine particulate matter, aerodynamic diameter ≤ 2.5 μm) has been associated with preterm birth and low birth weight. The association between prenatal PM2.5 exposure and intrauterine inflammation (IUI), an important risk factor for preterm birth and neurodevelopmental outcomes, has not been evaluated. OBJECTIVES:We aimed to investigate the association between maternal exposure to PM2.5 and IUI in the Boston Birth Cohort, a predominantly urban low-income minority population. METHODS:This analysis included 5,059 mother-infant pairs in the Boston Birth Cohort. IUI was assessed based on intrapartum fever and placenta pathology. PM2.5 exposure was assigned using data from the U.S. EPA's Air Quality System. Odds ratios (OR) and 95% confidence intervals (CI) quantified the association of maternal PM2.5 exposure during preconception and various periods of pregnancy with IUI. RESULTS:Comparing the highest with the lowest PM2.5 exposure quartiles, the multi-adjusted association with IUI was significant for all exposure periods considered, including 3 months before conception (OR = 1.52; 95% CI: 1.22, 1.89), first trimester (OR = 1.93; 95% CI: 1.55, 2.40), second trimester (OR = 1.67; 95% CI: 1.35, 2.08), third trimester (OR = 1.53; 95% CI: 1.24, 1.90), and whole pregnancy (OR = 1.92; 95% CI: 1.55, 2.37). CONCLUSIONS:Despite relatively low exposures, our results suggest a monotonic positive relationship between PM2.5 exposure during preconception and pregnancy and IUI. IUI may be a sensitive biomarker for assessing early biological effect of PM2.5 exposure on the developing fetus. CITATION:Nachman RM, Mao G, Zhang X, Hong X, Chen Z, Soria CS, He H, Wang G, Caruso D, Pearson C, Biswal S, Zuckerman B, Wills-Karp M, Wang X. 2016. Intrauterine inflammation and maternal exposure to ambient PM2.5 during preconception and specific periods of pregnancy: the Boston Birth Cohort. Environ Health Perspect 124:1608-1615; http://dx.doi.org/10.1289/EHP243.

Although previous reports have linked DNA damage with both transmissions across generations as well as our own survival, it is unknown how to reverse the lesion. Based on the data from a Randomized, Double-blind, Placebo Controlled Clinical Trial, this study aimed to assess the efficacy of folic acid supplementation (FAS) on DNA oxidative damage reversal. In this randomized clinical trial (RCT), a total of 450 participants were enrolled and randomly assigned to 3 groups to receive folic acid (FA) 0.4 mg/day (low-FA), 0.8 mg/day (high-FA), or placebo (control) for 8 weeks. The urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and creatinine (Cr) concentration at pre- and post-FAS were measured with modified enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. A multivariate general linear model was applied to assess the individual effects of FAS and the joint effects between FAS and hypercholesterolemia on oxidative DNA damage improvement. This clinical trial was registered with ClinicalTrials.gov, number NCT02235948. Of the 438 subjects that received FA fortification or placebo, the median (first quartile, third quartile) of urinary 8-OHdG/Cr for placebo, low-FA, and high-FA groups were 58.19 (43.90, 82.26), 53.51 (38.97, 72.74), 54.73 (39.58, 76.63) ng/mg at baseline and 57.77 (44.35, 81.33), 51.73 (38.20, 71.30), and 50.65 (37.64, 76.17) ng/mg at the 56th day, respectively. A significant decrease of urinary 8-OHdG was observed after 56 days FA fortification (P < 0.001). Compared with the placebo, after adjusting for some potential confounding factors, including the baseline urinary 8-OHdG/Cr, the urinary 8-OHdG/Cr concentration significantly decreased after 56 days FAS [β (95% confidence interval) = −0.88 (−1.62, −0.14) and P = 0.020 for low-FA; and β (95% confidence interval) = −2.68 (−3.42, −1.94) and P < 0.001 for high-FA] in a dose-response fashion (Ptrend < 0.001). Test of interaction between hypercholesterolemia and FA supplementation on urinary 8-OHdG reduction was significant (P = 0.001). The present study demonstrates that FA fortification is independently linked to the reduction of urinary 8-OHdG/Cr in a dose-related pattern, which suggests that FA is beneficial to protect against oxidative damage to DNA. This effect is apparently stronger in those with hypercholesterolemia. The authors provide a new insight into the prevention and reversal of oxidative DNA damage.

Food allergy (FA) affects 2%-10% of US children and is a growing clinical and public health problem. Here we conduct the first genome-wide association study of well-defined FA, including specific subtypes (peanut, milk and egg) in 2,759 US participants (1,315 children and 1,444 parents) from the Chicago Food Allergy Study, and identify peanut allergy (PA)-specific loci in the HLA-DR and -DQ gene region at 6p21.32, tagged by rs7192 (P=5.5 × 10(-8)) and rs9275596 (P=6.8 × 10(-10)), in 2,197 participants of European ancestry. We replicate these associations in an independent sample of European ancestry. These associations are further supported by meta-analyses across the discovery and replication samples. Both single-nucleotide polymorphisms (SNPs) are associated with differential DNA methylation levels at multiple CpG sites (P<5 × 10(-8)), and differential DNA methylation of the HLA-DQB1 and HLA-DRB1 genes partially mediate the identified SNP-PA associations. This study suggests that the HLA-DR and -DQ gene region probably poses significant genetic risk for PA.

Mandatory folic acid fortification of grain products was implemented in the U.S. in 1998 and routine clinical recommendation on prenatal folic acid supplementation for pregnant women began as early as 1992. Yet there is a dearth of current data on folate, vitamin B12 and homocysteine (Hcy) levels in the U.S. urban minority mothers.Blood samples (obtained 2‐3 days post‐delivery) from 2,057 mothers 16‐51 y old enrolled in the Boston Birth cohort (during 1999‐2012) were analyzed for plasma folate, plasma vitamin B12 and Hcy levels. The percentage of self‐reported prenatal multi‐vitamin use during 3 month preconception, 1st, 2nd and 3rd trimester were 4.4%, 86.2%, 90.4%, and 88.5%, respectively. The plasma folate and B12 levels were significantly associated with prenatal multi‐vitamin intake in the 2nd and 3rd trimesters. Only 1.6% of the total samples had folate deficiency (<7 nmol/l) and 2.2% vitamin B12 deficiency (<150 pmol/l); but 20.8% had elevated Hcy levels (> 10 μmol/L). Consistent with the NHANES 2003‐06 data, black women had the lowest geometric mean of folate levels: 26.8 nmol/L and the highest vitamin B12 level: 282.5 pmol/L. Black women also had the highest Hcy: 7.9 μmol/L. The highest quartile of B12 was a significant predictor of elevated Hcy, after adjusting for age, race, smoking, alcohol, education and folate levels (p=0.001).This study underscores the need to improve preconception multivitamin intake and optimize B‐vitamin status in black mothers. The significantly higher percentage of elevated Hcy in black mothers (22.7%) compared to other racial/ethnic groups warrants further investigation.Research Support: In part by NICHD (R01 HD041702)

Although previous reports have linked DNA damage with both transmissions across generations as well as our own survival, it is unknown how to reverse the lesion. Based on the data from a Randomized, Double-blind, Placebo Controlled Clinical Trial, this study aimed to assess the efficacy of folic acid supplementation (FAS) on DNA oxidative damage reversal.In this randomized clinical trial (RCT), a total of 450 participants were enrolled and randomly assigned to 3 groups to receive folic acid (FA) 0.4mg/day (low-FA), 0.8mg/day (high-FA), or placebo (control) for 8 weeks. The urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and creatinine (Cr) concentration at pre- and post-FAS were measured with modified enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. A multivariate general linear model was applied to assess the individual effects of FAS and the joint effects between FAS and hypercholesterolemia on oxidative DNA damage improvement. This clinical trial was registered with ClinicalTrials.gov, number NCT02235948.Of the 438 subjects that received FA fortification or placebo, the median (first quartile, third quartile) of urinary 8-OHdG/Cr for placebo, low-FA, and high-FA groups were 58.19 (43.90, 82.26), 53.51 (38.97, 72.74), 54.73 (39.58, 76.63) ng/mg at baseline and 57.77 (44.35, 81.33), 51.73 (38.20, 71.30), and 50.65 (37.64, 76.17) ng/mg at the 56th day, respectively. A significant decrease of urinary 8-OHdG was observed after 56 days FA fortification (P<0.001). Compared with the placebo, after adjusting for some potential confounding factors, including the baseline urinary 8-OHdG/Cr, the urinary 8-OHdG/Cr concentration significantly decreased after 56 days FAS [ (95% confidence interval)=-0.88 (-1.62, -0.14) and P=0.020 for low-FA; and (95% confidence interval)=-2.68 (-3.42, -1.94) and P<0.001 for high-FA] in a dose-response fashion (P-trend<0.001). Test of interaction between hypercholesterolemia and FA supplementation on urinary 8-OHdG reduction was significant (P=0.001).The present study demonstrates that FA fortification is independently linked to the reduction of urinary 8-OHdG/Cr in a dose-related pattern, which suggests that FA is beneficial to protect against oxidative damage to DNA. This effect is apparently stronger in those with hypercholesterolemia. The authors provide a new insight into the prevention and reversal of oxidative DNA damage.

Plasma homocysteine (Hcy) is a modifiable, independent risk factor for cardiovascular disease (CVD) and is affected by both environmental and genetic factors. This study aimed to describe the gender- and age-specific distribution of Hcy concentration for 1117 subjects aged 10–66 years, a subset of a community-based rural Chinese twin cohort. In addition, we examined environmental and genetic contributions to variances in Hcy concentration by gender and age groups. We found that the distribution pattern for Hcy varied by both age and gender. Males had higher Hcy than females across all ages. Elevated Hcy was found in 43% of male adults and 13% of female adults. Moreover, nearly one fifth of children had elevated Hcy. Genetic factors could explain 52%, 36% and 69% of the variation in Hcy concentration among children, male adults and female adults, respectively. The MTHFR C677T variant was significantly associated with Hcy concentrations. Smokers with the TT genotype had the highest Hcy levels. Overall, our results indicate that elevated Hcy is prevalent in the children and adults in this rural Chinese population. The early identification of elevated Hcy will offer a window of opportunity for the primary prevention of CVD and metabolic syndrome.

IMPORTANCEAlthough previous reports have linked preterm birth with insulin resistance in children and adults, it is not known whether altered insulin homeostasis is detectable at birth and tracks from birth through childhood.OBJECTIVETo investigate whether preterm birth is associated with elevated plasma insulin levels at birth and whether this association persists into early childhood.DESIGN, SETTING, AND PARTICIPANTSA prospective birth cohort of 1358 children recruited at birth from 1998 to 2010 and followed-up with prospectively from 2005 to 2012 at the Boston Medical Center in Massachusetts.MAIN OUTCOMES AND MEASURESRandom plasma insulin levels were measured at 2 time points: at birth (cord blood) and in early childhood (venous blood). The median age was 1.4 years (interquartile range [IQR], 0.8-3.3) among 4 gestational age groups: full term (≥39 wk), early term (37-38 wk), late preterm (34-36 wk), and early preterm (<34 wk).RESULTSThe geometric mean of insulin levels at birth were 9.2 µIU/mL (95% CI, 8.4-10.0) for full term; 10.3 µIU/mL (95% CI, 9.3-11.5) for early term; 13.2 µIU/mL (95% CI, 11.8-14.8) for late preterm; and 18.9 µIU/mL (95% CI, 16.6-21.4) for early preterm. In early childhood, these levels were 11.2 µIU/mL (95% CI, 10.3-12.0) for full term; 12.4 µIU/mL (95% CI, 11.3-13.6) for early term; 13.3 µIU/mL (95% CI, 11.9-14.8) for late preterm; and 14.6 µIU/mL (95% CI, 12.6-16.9) for early preterm. Insulin levels at birth were higher by 1.13-fold (95% CI, 0.97-1.28) for early term, 1.45-fold (95% CI, 1.25-1.65) for late preterm, and 2.05-fold (95% CI, 1.69-2.42) for early preterm than for those born full term. In early childhood, random plasma insulin levels were 1.12-fold (95% CI, 0.99-1.25) higher for early term, 1.19-fold (95% CI, 1.02-1.35) for late preterm, and 1.31-fold (95% CI, 1.10-1.52) for early preterm than those born full term. The association was attenuated after adjustment for postnatal weight gain and was not significant after adjustment for insulin levels at birth. Infants ranked in the top insulin tertile at birth were more likely to remain in the top tertile (41.2%) compared with children ranked in the lowest tertile (28.6%) in early childhood.CONCLUSIONS AND RELEVANCEThere was an inverse association between gestational age and elevated plasma insulin levels at birth and in early childhood. The implications for future development of insulin resistance and type 2 diabetes warrant further investigation.

There is an emerging hypothesis that exposure to cadmium (Cd), mercury (Hg), lead (Pb), and selenium (Se) in utero and early childhood could have long-term health consequences. However, there are sparse data on early life exposures to these elements in US populations, particularly in urban minority samples. This study measured levels of Cd, Hg, Pb, and Se in 50 paired maternal, umbilical cord, and postnatal blood samples from the Boston Birth Cohort (BBC). Maternal exposure to Cd, Hg, Pb, and Se was 100% detectable in red blood cells (RBCs), and there was a high degree of maternal–fetal transfer of Hg, Pb, and Se. In particular, we found that Hg levels in cord RBCs were 1.5 times higher than those found in the mothers. This study also investigated changes in concentrations of Cd, Hg, Pb, and Se during the first few years of life. We found decreased levels of Hg and Se but elevated Pb levels in early childhood. Finally, this study investigated the association between metal burden and preterm birth and low birthweight. We found significantly higher levels of Hg in maternal and cord plasma and RBCs in preterm or low birthweight births, compared with term or normal birthweight births. In conclusion, this study showed that maternal exposure to these elements was widespread in the BBC, and maternal–fetal transfer was a major source of early life exposure to Hg, Pb, and Se. Our results also suggest that RBCs are better than plasma at reflecting the trans-placental transfer of Hg, Pb, and Se from the mother to the fetus. Our study findings remain to be confirmed in larger studies, and the implications for early screening and interventions of preconception and pregnant mothers and newborns warrant further investigation.

Metabolic syndrome (MetS) affects more than 47 million people in the U.S. Even more alarming, MetS, once regarded as an “adult problem”, has become increasingly common in children. To date, most related research and intervention efforts have occurred in the adult medicine arena, with limited understanding of the root causes and lengthy latency of MetS. This review highlights new science on the early life origins of MetS, with a particular focus on exposure to two groups of environmental toxicants: endocrine disrupting chemicals (EDCs) and metals during the prenatal and early postnatal periods, and their specific effects and important differences in the development of MetS. It also summarizes available data on epigenetic effects, including the role of EDCs in the androgen/estrogen pathways. Emerging evidence supports the link between exposures to environmental toxicants during early life and the development of MetS later in life. Additional research is needed to address important research gaps in this area, including prospective birth cohort studies to delineate temporal and dose–response relationships, important differences in the effects of various environmental toxicants and their joint effects on MetS, as well as epigenetic mechanisms underlying the effects of specific toxicants such as EDCs and metals.