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Transforming growth factor beta-1 (TGFB1) may influence asthma by modulating allergic airway inflammation and airway remodeling. The role of single nucleotide polymorphisms (SNPs) of TGFB1 in asthma remains inconclusive. We examined TGFB1 SNPs in relation to asthma risk and degree of atopy among 546 case-parent triads, consisting of asthmatics aged 4 to 17 years and their parents in Mexico City. Atopy to 24 aeroallergens was determined by skin prick tests. We genotyped five TGFB1 SNPs, including two known functional SNPs [C-509T (rs1800469), T869C (rs1982073)] and three others (rs7258445, rs1800472, rs8179181), using TaqMan and Masscode assays. We analyzed the data using log-linear and polytomous logistic methods. Three associated SNPs, including the two known functional SNPs, were statistically significantly related to asthma risk. Individuals carrying the T allele of C-509T had an increased risk of asthma [relative risk (RR) = 1.42, 95% confidence interval (CI) = 1.08–1.87 for one copy; RR (95%CI) = 1.95 (1.36–2.78) for two copies]. For T869C, the RRs (95%CI) were 1.47 (1.09–1.98) for one and 2.00 (1.38–2.90) for two copies of the C allele. Similar results were found for rs7258445. The haplotype containing all three risk alleles conferred an increased risk of asthma (RR = 1.48, 95% CI = 1.11–1.95 for one copy; RR = 1.77, 95% CI = 1.22–2.57 for two copies). These three SNPs were also related to the degree of atopy. This largest study to date of genetic variation in TGFB1 and asthma and atopy adds to increasing evidence for a role in these disorders.
Transforming growth factor beta-1 (TGFB1) is a multifunctional cytokine with pro-inflammatory effects in some settings and anti-inflammatory effects in others. It is expressed in many cell types including inflammatory cells and structural cells, such as airway epithelial and smooth muscle cells (Duvernelle et al. 2003). TGFB1 levels in bronchoalveolar lavage (BAL) fluid are higher in asthma patients and increase further in response to allergen exposure compared with healthy control subjects (Redington et al. 1997). TGFB1 may modulate the development of allergic inflammation and airway remodeling in asthma (Duvernelle et al. 2003).
Several single nucleotide polymorphisms (SNPs) in TGFB1 have been studied for association with asthma and atopy, including G-800A, C-509T, T869C (SNP in codon 10) and G915C (SNP in codon 25). The C-509T SNP in the TGFB1 promoter and T869C in codon 10 of exon 1 appear to influence TGFB1 blood levels (Grainger et al. 1999; Suthanthiran et al. 2000) and gene expression in the lung (Silverman et al. 2004). Associations of C-509T and other TGFB1 SNPs with wheezing illness in infants (Hoffjan et al. 2004), asthma diagnosis (Mak et al. 2006; Silverman et al. 2004), severity (Pulleyn et al. 2001) and increased IgE in asthmatic children (Hobbs et al. 1998) have been found in some studies but not all (Buckova et al. 2001; Heinzmann et al. 2005). Few data have addressed TGFB1 haplotypes and the risk of asthma or atopy.
We used the case-parent triad design to study genetic variation in TGFB1 in relation to asthma risk and degree of atopy in allergic asthmatic children in Mexico City.
Using the case-parent triad design (Weinberg et al. 1998; Wilcox et al. 1998), we recruited nuclear families consisting of a case and both parents. Cases were children aged 4–17 years with asthma, diagnosed by a pediatric allergist at the allergy referral clinic of a large public pediatric hospital in central Mexico City (Hospital Infantil de México Federico Gómez) between June 1998 and November 2003. Children and parents provided blood samples as sources of DNA. The study population included 546 complete case-parent triads. A questionnaire on the child’s symptoms and risk factors for asthma including environmental tobacco smoking was completed during interviews with 541 parents.
A pediatric allergist based the diagnosis of asthma on clinical symptoms and response to treatment (BTS/SIGN 2003). The severity of asthma among the cases was rated by a pediatric allergist according to symptoms in the Global Initiative on Asthma schema as mild (intermittent or persistent), moderate, and severe (NHLBI 1998). Severity data were available for 520 cases. Pulmonary function was measured using the EasyOne spirometer (ndd Medical Technologies, Andover, MA, USA) for 415 subjects according to American Thoracic Society specifications (American Thoracic Society 1995). The best test out of three technically acceptable tests was selected. Spirometric prediction equations from a Mexico City childhood population were used to calculate the percent predicted forced expiratory volume in one second (FEV1) (Perez-Padilla et al. 2003). Children were asked to hold asthma medications on the morning of the test.
Atopy was determined using skin prick tests. The following battery of 24 aeroallergens (IPI ASAC, Mexico) common in Mexico City was used: Aspergillus fumigatus, Alternaria, Mucor, Blattella germanica, Periplaneta americana, Penicillium, cat, dog, horse, Dermatophagoides (pteronyssinus and farinae), Ambrosia, Artemisia ludoviciana, Cynodon dactylon, Chenopodium album, Quercus robur, Fraxinus, Helianthus annus, Ligustrum vulgare, Lolium perenne, Plantago lanceolata, Rumex crispus, Schinus molle, Salsola, and Phleum pratense. Histamine was used as the positive control and glycerin as the negative control. Children were considered atopic if the diameter of the skin reaction to at least one allergen exceeded 4 mm. The test was considered valid if the reaction to histamine was ≥ 6 mm according to the grading of the skin prick test recommended by Aas and Berlin (Aas and Belin 1973). Skin test data were available on 496 (of 546) triads with complete genotyping data.
We obtained measurements of ambient ozone from the Mexican government’s air monitoring stations. Ozone levels were measured via UV photometry (analyzer-model 400, API). The residence of each child who participated in this study was located using a map, and each child was assigned to the monitoring station that was closest to his or her residence (Romieu et al. 2002). The ozone exposure data were collected for the year prior to the time of entry into the study. The parameter we used was the annual average of the daily maximum eight hour averages. We dichotomized this variable at the median of 67 ppb for stratified analyses.
The Institutional Review Boards of the Mexican National Institute of Public Health, the Hospital Infantil de México Federico Gómez and the U.S. National Institute of Environmental Health Sciences approved the protocol. Parents provided consent for the child’s participation and children provided assent.
We selected tagging SNPs based on linkage disequilibrium criteria and also aimed to include SNPs with functional importance or association with asthma or related traits from the literature. Tagging SNP selection was based on resequencing data from Caucasians from the Innate Immunity Program for Genomic Applications (http://innateimmunity.net) and genotyping data in Mexican-Americans from SNP 500 (http://snp500cancer.nci.nih.gov). We used the ldSelect Version 1.0 (Carlson et al. 2004) with the default of r2 = 0.64 to identify tagging SNPs. We gave priority to bins with at least two SNPs (de Bakker et al. 2005). All SNPs with demonstrated functional significance were included even if they had r2 values above 0.64 with another tagging SNP. In this manner, we selected five tagging SNPs: C-509T, T869C, rs7258445, rs1800472, and rs8179181. Our selection of five SNPs covers both of the common haplotypes (frequency > 10%) that occur in Caucasians as identified by the software program PHASE (Stephens et al. 2001). There is increasing documentation that tagging SNPs selected from Caucasians often provide excellent coverage in other non-African populations (Gonzalez-Neira et al. 2006; Tenesa and Dunlop 2006).
Peripheral blood lymphocytes were isolated, and DNA was extracted using Gentra Puregene kits (Gentra Systems, Minneapolis, MN). Primers and probes for four SNPs (C-509T, T869C, rs1800472, and rs8179181) were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA). We genotyped these four SNPs using TaqMan 5′ exonuclease assay on GeneAmp PCR Systems according to the manufacturer’s instruction. SNP rs7258445 was genotyped using the Masscode™ system (Qiagen Genomics, Inc, Bothell, WA) by BioServe Biotechnologies, Ltd. (BioServe Biotechnologies, Ltd., Laurel, MD, http://www.bioserve.com). Assays were done by researchers who were blinded to parent or child status of samples. For both TaqMan assay and Masscode™ assay, the genotype calling rate is over 95% with 100% matching for quality control samples and blind replicates.
We ascertained non-parentage with a set of short-tandem repeats (AmpFLSTR Profiler Plus; Applied Biosystems, Foster City, CA) analyzed using Pedcheck software (O’Connell and Weeks 1998). A total of 546 complete triads had genotyping data for at least one SNP.
Linkage disequilibrium between pairs of SNPs was measured by computing the squared correlation coefficient (r2) (Devlin and Risch 1995).
To analyze associations with individual SNPs, we used a log-linear-based method implemented in SAS version 9.1 for Windows (SAS Institute, Cary, NC) and STATA version 8.0 (Stata Press, College Station, TX). The analysis of triad data with the log-linear model eliminates potential bias due to genetic population stratification (Weinberg et al. 1998). The log-linear method is a generalization of the transmission disequilibrium test (TDT) that has the advantage of providing estimates of the magnitude of associations rather than simply tests of significance. Similar to TDT-based methods such as the family based association test (FBAT) (Horvath et al. 2001; Horvath et al. 2004), the log-linear model stratifies on parental mating type, eliminating potential bias due to population stratification (Lake and Laird 2004; Weinberg et al. 1998). The log-linear model thus gives similar P values to TDT-based methods such as FBAT. We calculated relative risks for individual SNPs without restricting to a specific genetic model and also under dominant and recessive genetic models. The log-linear models are inherently immune to confounding by demographic or lifestyle factors such as parental smoking. However, we did examine effect modification by gender, asthma severity, parental smoking, and level of ozone exposure (annual average of the eight hour maximum). We calculated likelihood ratio tests and relative risks for the joint effects of genotype and parental smoking, using the method of Umbach and Weinberg (Umbach and Weinberg 2000).
To examine whether TGFB1 polymorphisms influenced the degree of atopy, as assessed by the number of positive skin tests out of 24 performed, we tested for linkage and association with each of the five SNPs in TGFB1 by using the polytomous logistic method of Kistner and Weinberg (Kistner and Weinberg 2004). We also used this method to analyze the relation between TGFB1 SNPs and lung function, as assessed by percent predicted FEV1. The P values were calculated from likelihood ratio tests.
We analyzed associations with haplotypes formed by the five TGFB1 SNPs, as opposed to single SNPs, using HAPLIN Version 2.0 (http://www.uib.no/smis/gjessing/genetics/software/haplin) running under S-PLUS Version 6.2 for Windows (Seattle, Washington). HAPLIN is a direct extension of the log-linear models for a single diallelic locus (Weinberg et al. 1998), estimating single- and double-dose effects of haplotypes, rather than simply tests of significance, using maximum likelihood (Gjessing and Lie 2006). The HAPLIN analysis includes 533 complete triads for TGFB1.
We present P values computed using the above methods. Although we did only hypothesis based testing, we used two approaches to address multiple comparisons: calculating a threshold P value based on the pairwise linkage disequilibrium between SNPs (Nyholt 2004) and the false discovery rate (FDR) (Storey and Tibshirani 2003).
Clinical characteristics of the asthmatic children with genotyping data are presented in Table 1. Among our 546 cases, the mean age was 9.0 years [standard deviation (SD) 2.4 years; range 4–17 years] and 59.3% were male. Most had mild (71.1%) as opposed to moderate or severe asthma (28.9%). Nearly all cases (98.0%) had used medication for asthma in the past 12 months. Wheezing in the past 12 months was reported by 90.1%, and chronic dry cough was reported by 64.8%. For 73.2% of the cases, asthma symptoms had interfered with daily activities or school attendance in the past 12 months. Exacerbation of asthma symptoms by exercise was reported by 72.1%. Among the 415 cases with spirometry data available, the mean FEV1 percent predicted was 90.4% (SD = 16.7). Atopy to aeroallergens, defined as at least one positive skin test, was present in 91.7% of cases with skin test data, and 51.4% of cases had five or more positive skin tests. The highest rates of skin test positivity were seen for dust mite (69.4%) and cockroach (42.3%). A smoking parent was present for 51.8% of cases; only 4.9% of mothers reported smoking during pregnancy. This figure is virtually identical to the 4.7% smoking prevalence found among pregnant women in Mexico (Sanchez-Zamorano et al. 2004) and the 4.2% seen among Mexican-born pregnant women in California (Harley and Eskenazi 2006).
For the five SNPs genotyped for TGFB1, the NCBI SNP ID, the SNP location in the gene, and minor allele frequency are presented in Table 2. The r2 value between the two functional SNPs that we studied (C-509T and T869C) was 0.87; whereas the r2 value between C-509T and rs7258445 was 0.49 in our population. The r2 value between T869C and rs7258445 was 0.36. All other r2 values were below 0.36.
The frequency distributions of the 15 possible mating types of triads for the polymorphisms of TGFB1 are shown in Table 3. The log-linear analysis of individual SNPs in TGFB1 in relation to asthma is shown in Table 4. Carrying either one copy or two copies of the T allele of C-509T, the C allele of T869C, or the C allele of rs7258445 was statistically significantly associated with increased risk of asthma. For C-509T, the relative risks (RR) with 95% confidence interval (CI) were 1.42 (1.08–1.87) for carrying one copy of T allele and 1.95 (1.36–2.78) for two copies of T. Individuals with either one copy or two copies of C-509T T allele had a RR of 1.47 (95% CI = 1.12–1.94, P = 0.0052). For T869C and rs7258445, the results were similar (Table 4). The results did not differ appreciably by gender, parental smoking, asthma severity, or level of ozone exposure stratified at the median (data not shown).
Addressing the potential issue of multiple comparisons, we calculated a threshold level of 0.012 using the SNP spectral decomposition approach (Nyholt 2004). In addition, the false discovery rate for the effect estimates for C-509T, T869C, or rs7258445 was less than 5%. We did not observe a relationship between the TGFB1 SNPs and FEV1 (data not shown).
There were seven haplotypes with frequency over 1% formed by C-509T, T869C, rs7258445, rs1800472, and rs8179181 in order. The most frequent haplotype, TCCCC, contains the increased risk alleles for C-509T, T869C, and rs7258445, and was statistically significantly associated with increased risk of asthma (RR = 1.48, 95% CI = 1.11–1.95 for one copy, FDR = 0.036; RR = 1.77, 95% CI = 1.22–2.57 for two copies, FDR = 0.024). Conversely, homozygotes of haplotype CTTCC were at reduced risk of asthma (Table 5).
We examined the relationship between individual SNPs of TGFB1 and the degree of atopy to aeroallergens, assessed by the number of positive skin tests out of a battery of 24 tests. To select the appropriate genetic model for analyses of these data, we examined the mean number of positive skin tests by the number of copies of the variant alleles for the five TGFB1 SNPs (Table 6) and also examined estimates from a two degrees of freedom test. The results suggested that a dominant model was appropriate for all SNPs. For C-509T, carrying one or two copies of the T allele conferred increased odds of 1.69 (95% CI = 1.13–2.54, P = 0.008, FDR = 0.016) for an increase in the number of positive skin tests of five or more. We found a similar result for T869C, with increased odds of 1.70 (95% CI = 1.11–2.61, P = 0.010, FDR = 0.016). For rs7258445, the C allele was also associated with degree of atopy but with lower statistical significance (OR = 1.73, 95% CI = 0.97–3.05, P = 0.047, FDR = 0.047). We also looked at the effects of TGFB1 SNPs on the mean number of positive tests to perennial versus seasonal allergens and found no appreciable difference (data not shown).
We found that polymorphisms in TGFB1 were associated with asthma and atopy in Mexican children. This is the largest study of TGFB1 polymorphisms in asthma and atopy. The T allele of C-509T, the C allele of T869C, and the C allele of rs7258445 increased the risk of asthma; two of these SNPs are functional. Haplotype analysis results are consistent with the single SNP analyses; the haplotype containing the T allele of C-509T, the C allele of T869C, and the C allele of rs7258445 was related to increased risk of asthma. We also found the same three alleles to be positively associated with the degree of atopy, as assessed by the number of positive skin tests.
We examined five SNPs in TGFB1 based on linkage disequilibrium criteria and functional data. Previous association studies of TGFB1 have mainly focused on SNPs in the promoter [G-800A, C-509T] and in exon 1 [T869C (SNP in codon 10), G915C (SNP in codon 25)]. We evaluated tagging SNPs throughout the gene. An efficient way of selecting tagging SNPs without losing power is to give priority to SNPs with the largest number of other SNPs in high linkage disequilibrium (de Bakker et al. 2005). We covered all the common SNPs (minor allele frequency ≥ 10%) in the promoter region and the exons. We chose C-509T because of strong evidence that it influences TGFB1 function, promoter activity, and circulating TGFB1 level (Awad et al. 1998; Grainger et al. 1999; Silverman et al. 2004). We also included T869C in codon 10, despite an r2 of 0.87 with C-509T in our Mexican population because it is also functional. The T869C T to C substitution leads to an amino acid change from leucine to proline in the signal peptide resulting in increased secretion of TGFB1 protein in vitro (Dunning et al. 2003) and increased circulating TGFB1 concentration (Yamada et al. 2001; Yokota et al. 2000). In addition, it has been associated with atopic asthma (Mak et al. 2006) and cystic fibrosis severity (Drumm et al. 2005). A polymorphism in codon 25 leads to higher levels of TGFB1 production (Awad et al. 1998), but it was not found in a Hispanic polymorphism discovery set that includes Mexicans (http://snp500cancer.nci.nih.gov) and thus we did not study it.
We used resequencing data generated from Caucasians and Mexican-Americans to make our selection of tagging SNPs. There is increasing documentation that tagging SNPs selected from Caucasians provide excellent coverage in other non-African populations (Gonzalez-Neira et al. 2006; Tenesa and Dunlop 2006). Tenesa et al. demonstrated that power does not depend on which population is used to select tagging SNPs, especially in non-African populations (Tenesa and Dunlop 2006). Mexicans are predominantly a mix of Caucasian and Native American ancestry with a minimal African component (Choudhry et al. 2006). Mexicans from Mexico City and Mexican-Americans should be reasonably similar.
There have been seven studies of TGFB1 C-509T and asthma. Positive associations with the T allele were found in four studies (Mak et al. 2006; Nagpal et al. 2005; Pulleyn et al. 2001; Silverman et al. 2004) and no association in three studies (Buckova et al. 2001; Heinzmann et al. 2005; Hobbs et al. 1998). It is notable that the two largest studies, ours (n = 546) and that of Silverman et al. (n = 527) (Silverman et al. 2004), both reported positive associations.
We also found suggestive evidence of a positive association between the T allele C-509T and higher degree of atopy. The T allele of C-509T was associated with elevated total serum IgE in two studies (Hobbs et al. 1998; Nagpal et al. 2005), and no association was seen in three studies (Buckova et al. 2001; Meng et al. 2005; Silverman et al. 2004). The C allele of the SNP T869C was associated with increased risk of asthma and higher degree of atopy in our study. Mak et al. found that the CC genotype was related to increased risk of atopic asthma (Mak et al. 2006), consistent with our finding. The C allele of T869C enhances TGFB1 secretion in vitro independent of any effect of the C-509T polymorphism (Dunning et al. 2003). There are no published results on the relationship between T869C and the degree of atopy.
We found that the rs7258445 C allele was associated with increased risk of asthma and higher degree of atopy in our population. There are no prior published data on this SNP in relation to asthma or atopy. SNP rs7258445 is in an Alu repeat region. Alu-SNPs may harbor potential sites associated with disease (Ng and Xue 2006) and Alu sequences can influence gene expression (Batzer and Deininger 2002). However, the association could simply reflect the r2 of 0.49 with C-509T which is clearly functional. Based on our haplotype analysis, the association with this SNP is probably not independent of the association with C-509T.
One other study (Nagpal et al. 2005) has examined TGFB1 haplotypes in relation to asthma. Nagpal et al. studied the influences of haplotypes of three polymorphisms [(CT)n(CA)m repeat, G-800A and C-509T] on TGFB1 serum level and asthma risk in an Indian population (Nagpal et al. 2005). They found the haplotype 23_G_T, containing the T allele of C-509T, to be associated with higher TGFB1 serum levels and increased asthma risk, whereas 22_G_C, containing the C allele of C-509T, was associated with lower serum levels of TGFB1 and lower risk of asthma. These results are similar to ours where the most common haplotype TCCCC, containing the risk alleles from C-509T, T869C and rs7258445, was associated with statistically significantly increased asthma risk. As expected, the haplotype CTTCC, with the lower risk alleles at these three sites, was inversely associated with asthma in our data.
The associations that we found between TGFB1 polymorphisms and increased risk of asthma and atopy may result from increased TGFB1 gene expression. TGFB1 is overexpressed in airways of asthmatics compared with healthy controls (Minshall et al. 1997; Ohno et al. 1996). In particular, the T allele of C-509T is associated with increased gene expression (Silverman et al. 2004), and the C allele of T869C results in increased TGFB1 secretion (Dunning et al. 2003). Both C-509T and T869C may be associated with increased asthma risk based on enhanced TGFB1 function.
TGFB1 has been implicated in asthma pathogenesis by various mechanisms (Duvernelle et al. 2003). Airway remodeling appears to be important in the pathogenesis of childhood asthma. TGFB1 is a potent profibrogenic factor with major involvement in the initiation and persistence of airway remodeling in asthma (Boxall et al. 2006). Many cells express TGFB1, including fibroblasts, smooth muscle, epithelial and inflammatory cells such as eosinophils, lymphocytes and mast cells (Duvernelle et al. 2003). Minshall and coworkers demonstrated increased TGFB1 mRNA and protein expression in eosinophils in the airways of asthmatic patients, resulting in subepithelial fibrosis (Minshall et al. 1997; Ohno et al. 1996). TGFB1 plays a role in synthesis and deposition of extracellular matrix proteins. TGFB1 can increase collagen and fibronectin production in asthmatic airways which may contribute to fibrosis and remodeling (Burgess et al. 2006; Coutts et al. 2001; Nomura et al. 2002; Romberger et al. 1992). TGFB1 has been shown to be a critical mediator of lung fibrosis in animal models, and a number of studies have shown that antagonizing TGFB1 prevents the development of tissue fibrosis (Noble 2003).
Recent evidence suggests a role for TGFB1 in airway hyperresponsiveness. Specifically, TGFB1 influences airway responsiveness to bradykinin (Bronner 2005). In human airway smooth muscle cells in culture, TGFB1 has been shown to increase the expression of bradykinin 2 receptors (Kim et al. 2005) and decrease the number of β-adrenergic receptors (Nogami et al. 1994). Based on these observations, higher levels of TGFB1 might be related to higher levels of airway responsiveness.
TGFB1 may also influence asthma and atopy etiology via its role in T-cell regulation. TGFB1 blocks Th1 differentiation by means of inhibition of expression of T-bet (T-box expressed in T cells) (Gorelik et al. 2002). This reduced Th1 differentiation can lead to the increased production of Th2 cytokines such as IL-4 and IL-13, which are important in the allergic asthmatic response to inhaled allergen (Finotto and Glimcher 2004).
Our asthma cases were diagnosed by pediatric allergists at a pediatric allergy specialty clinic of a large public hospital. Referral to this pediatric allergy clinic is a tertiary referral, and thus the children in our study had already been seen by a generalist and a pediatrician over time for recurrent asthma symptoms. Diagnoses were made on clinical grounds according to previous guidelines (BTS/SIGN 2003). We did not have tests of bronchial hyperreactivity. The physician diagnosis of asthma is a valid outcome compared to objective measurements (Jenkins et al. 1996). Of note, Kauppi et al found that 87% of self-reports of asthma diagnosis among Finnish adults were confirmed by objective data (Kauppi et al. 1998). Phenotyping errors result in reduction in the power of genetic association studies (Edwards et al. 2005). The resulting non-differential misclassification may also bias results toward the null. Misclassification with chronic obstructive pulmonary disease, a potential problem in adults, is a concern lacking in children. We had objective data on atopy; skin prick tests revealed that the vast majority of these children with asthma (92%) to be atopic to aeroallergens.
Associations of TGFB1 polymorphisms with asthma and atopy were not observed in all studies. This might reflect differences in study size, asthma classification, or populations. Phenotyping differences can contribute to lack of replication (Zheng and Tian 2005). We studied allergic asthmatic children, whereas some others studied adult asthma patients. Ours is the largest study (546 cases) after that of Silverman et al. (527 cases) (Silverman et al. 2004) which found similar results; other studies had 30 to 231 cases and may have been underpowered. In examining reasons for non-replication in genetic association studies, Lohmueller et al. found that small sample size was the major factor (Lohmueller et al. 2003).
In summary, we found that three SNPs in TGFB1, including the functional SNPs C-509T and T869C, influence childhood asthma and the degree of atopy in a Mexican population. These data add to increasing mechanistic and epidemiologic evidence for a role of TGFB1 in asthma and atopy.
This study was supported by the Division of Intramural Research, National Institute of Environmental Health Sciences (Z01 ES49019), National Institutes of Health, USA, and the National Council of Science and Technology (Grant 26206-M), Mexico. Dr. Romieu is supported by the National Center for Environmental Health at the Centers for Disease Control, USA. The authors would like to acknowledge the children and parents who participated in this study; Stephan Chanock, MD, National Cancer Institute, NIH, DHHS, for determination of short tandem repeats for parentage testing; Grace Chiu, Ph.D. (Westat, Research Triangle Park, NC) for data management and analyses; and Susan Baker, Sarah Lein and Sheri McReynolds (Social & Scientific Systems, Inc.; Research Triangle Park, NC) for specimen handling and database management. The authors wish to thank Dulce Ramirez for participation in the fieldwork and Drs. Clarice Weinberg and John Sundy for critical review of the manuscript.