Published ahead of print on September 25, 2002, doi:10.1164/rccm.200207-634OC
© 2002 American Thoracic Society
Polymorphisms in Toll-Like Receptor 4 Are Not Associated with Asthma or Atopy-related PhenotypesChanning Laboratory, Brigham and Women's Hospital, Harvard Medical School; Department of Biostatistics, Harvard School of Public Health; Hematology Division, Brigham and Women's Hospital; Harvard Partners Center for Genetics and Genomics, Boston, Massachusetts; Arizona Respiratory Center, University of Arizona, Tucson, Arizona; Community Genomic Medicine Center, University of Montreal, Chicoutimi Hospital; University of Quebec at Chicoutimi, Departments of Fundamental Sciences and Human Sciences, Chicoutimi; Research Institute of the McGill University Health Centre; and Departments of Human Genetics and Medicine, McGill University, Montreal Genome Centre, McGill University Health Centre, Montreal, Quebec, Canada Correspondence and requests for reprints should be addressed to Benjamin A. Raby, M.D., Channing Laboratory, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115. E-mail: benjamin.raby{at}channing.harvard.edu
Toll-like receptor 4 (TLR4) is the principal receptor for bacterial endotoxin recognition, and functional variants in the gene confer endotoxin-hyporesponsiveness in humans. Furthermore, there is evidence that endotoxin exposure during early life is protective against the development of atopy and asthma, although this relationship remains poorly understood. It is therefore possible that genetic variation in the TLR4 locus contributes to asthma susceptibility. In this study we characterize the genetic diversity in the TLR4 locus and test for association between the common genetic variants and asthma-related phenotypes. In a cohort of 90 ethnically diverse subjects, we resequenced the TLR4 locus and identified a total of 29 single nucleotide polymorphisms. We assessed five common polymorphisms for evidence of association with asthma in two large family-based cohorts: a heterogeneous North American cohort (589 families), and a more homogenous population from northeastern Quebec, Canada (167 families). Using the transmission-disequilibrium test, we found no evidence of association for any of the polymorphisms tested, including two functional variants. Furthermore, we found no evidence for association between the TLR4 variants and four quantitative intermediate asthma- and atopy-related phenotypes. Based on these results, we found no evidence that genetic variation in TLR4 contributes to asthma susceptibility.
Key Words: asthma • genetics • polymorphism • toll-like receptor 4 • genetic association
The toll-like receptor 4 (TLR4) is the principal receptor for bacterial endotoxin in mice and humans (1, 2) and plays a critical role in the innate immune response to both gram-negative pathogens and respiratory syncytial virus (3). Through interactions with both CD14 antigen and lipopolysaccharide (LPS) binding protein (LBP), endotoxin binds to membrane-bound TLR4 and initiates a complex intracellular signaling pathway, resulting in the activation of several nuclear transcription factors, including nuclear factor (NF)-  B. Significant interindividual variability in response to endotoxin has long been recognized (4, 5). Arbour and colleagues have recently demonstrated that common polymorphisms in the coding region of the TLR4 gene are responsible for a substantial portion of this variability (6). Specifically, a substitution of glycine for asparginine at amino acid position 259 (D259G) results in reduction in cell surface expression of TLR4 and subsequent disruption of LPS-mediated signaling. Furthermore, subjects heterozygous for D259G demonstrate a blunted bronchoconstrictor response to inhaled LPS, suggesting a dominant genetic effect. A second amino acid variant, a substitution of threonine for isoleucine at position 359 (T359I), which is in tight linkage disequilibrium (LD) with D259G, also demonstrated in vitro LPS-hyporesponsive effects. There is evidence that these functional variants are important determinants of health and disease, including sepsis susceptibility and atherogenesis (7, 8). Smirnova and colleagues have resequenced the complete coding region of TLR4 in 348 individuals of diverse ethnic origin and identified 10 additional amino acid variants of extremely low frequency (9). Although functional data regarding these rare variants is currently unavailable, this excess number of rare variants suggests that weakly purifying selective forces influence variation at the TLR4 locus, further suggesting that these variants confer detrimental functional properties (9). Exposure to bacterial endotoxin has received considerable attention as a potential risk factor for the development of asthma and allergic disease (10). Several groups have demonstrated that early-life exposure to endotoxin has a protective effect on the development of atopic phenotypes (11, 12). For instance, in a study of children with a history of recurrent wheeze, house-dust endotoxin levels were significantly higher in the homes of infants that did not develop allergen sensitization, as compared with levels in the homes of those infants who did become sensitized (12). The global incidence of asthma and atopic disease is rising, particularly among industrialized nations (13), and it has been suggested that this phenomenon may be due in part to increased environmental cleanliness, reflecting a decline in early-life exposure to environmental endotoxin (14, 15). In contrast, a recent prospective evaluation of household endotoxin exposure in a birth cohort of 499 infants at high risk of developing asthma demonstrated that higher household endotoxin levels were associated with an increased risk of persistent wheeze during the first year of life (16). Household endotoxin level is also significantly associated with asthma severity among dust mite-sensitive children (17). The role of endotoxin in the development and expression of asthma remains unclear (15, 18), perhaps in part due to interindividual variability in endotoxin responsiveness. The relative importance of genetic variation in TLR4 in the development of asthma or atopy remains unknown. The purpose of this study was to evaluate the role of TLR4 as an asthma-susceptibility gene. Using DNA obtained from 90 ethnically diverse subjects, including 19 self-reported subjects with asthma, we resequenced the TLR4 locus and characterized the genetic variation. We subsequently evaluated the more common variants for evidence of association with a diagnosis of asthma or asthma-related phenotypes using a family-based genetic approach in two large, independently ascertained cohorts.
Populations Three independent cohorts were examined in this study: a single nucleotide polymorphism (SNP) discovery cohort (90 subjects), an ethnically heterogeneous North American cohort of subjects with asthma (1,767 subjects), and a more homogeneous asthmatic cohort from northeastern Quebec (591 subjects). Respective institutional review boards for human studies approved the study protocols, and informed consent and/or ascent was obtained in all three cohorts. Methods of subject ascertainment and phenotypic characterization have been previously reported (19–21). Briefly, SNP discovery was performed using DNA from 19 individuals with a self-reported diagnosis of asthma (of diverse ethnicity) and from a group of 71 apparently healthy and unrelated individuals: 24 black Americans, 23 white Americans (DNA obtained from Coriell, Camden, NJ), and 24 Hispanic Americans (DNA obtained through the Arizona Respiratory Center, University of Arizona, Tucson, Arizona). The Childhood Asthma Management Program (CAMP) is a multicenter, randomized, double-masked, placebo-controlled clinical trial designed to investigate the long-term effects of inhaled antiinflammatory medications in children with mild to moderate asthma (21). Results of the original clinical trial have been previously reported (22). Of the 1,041 children enrolled in the original clinical trial, DNA samples were obtained from 968 participating children and 1,518 of their parents. Of 650 complete parent-child trios available for genotyping, 480 were of white American descent, 63 of black American descent, and 46 of Hispanic American descent. These 589 families are included in the analysis presented here. A diagnosis of asthma was established based on methacholine hyperreactivity (PC20 no greater than 12.5 mg/ml), and one or more of the following criteria for at least 6 months in the year before recruitment: (1) asthma symptoms at least two times per week; (2) at least two usages per week of an inhaled bronchodilator; and (3) daily asthma medication. Spirometry was performed according to American Thoracic Society recommendations using a volume-displacement spirometer, and airway responsiveness was assessed by methacholine challenge using the Wright nebulizer tidal breathing technique (21). Serum eosinophil counts were performed using center-specific methods. Skin-prick tests were performed for a standard panel of 10 common allergens as well as locally relevant allergens. Total serum immunoglobulin (Ig) E was measured using radioimmunoabsorbent assays from blood samples collected during the screening sessions of the CAMP study. The Quebec cohort consists of 167 corticosteroid-dependent probands with asthma and their families (591 individuals) recruited from the Saguenay-Lac-St-Jean (SLSJ) region of northeastern Quebec, Canada. Several thousand individuals settled this region about 350–400 years ago. The population grew at a high rate and with little admixture and is considered an example of a young founder population (23, 24). All subjects completed a general questionnaire modified from the standard respiratory questionnaire (25), including questions concerning family history of asthma and/or atopy. For inclusion, probands were required to fulfill at least two of the following three criteria: (1) a minimum of three clinic visits for acute asthma within 1 year; (2) two or more asthma-related hospital admissions within 1 year; or (3) steroid dependency, as defined by either 6 months of oral, or 1 year of inhaled corticosteroid use. Expiratory flow was measured with a Morgan spirometer (Morgan Spiro 232; P.K. Morgan Ltd, Kent, England) according to American Thoracic Society recommendations (26), and methacholine-based bronchoprovocation tests were performed according to the method described by Juniper and colleagues (27). Skin-prick tests were performed against a panel of 26 inhalant allergens, and blood was obtained for measurement of total serum IgE levels.
Molecular Methods SNP genotyping in the CAMP cohort was performed by single-base extension minisequencing (31). The minisequencing reaction was performed in multiplex using the SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA). Reactions were performed according to manufacturer's protocol with the exception that the SnaPshot mix was diluted 1:1 with HalfBD BigDye sequencing dilution buffer (Genetix, New York, NY). Reaction products were separated on an ABI 3100 capillary electrophoresis system. Allele determination was performed using ABI Prism Genotyper v.3.7 software (Applied Biosystems). SNP genotyping in the SLSJ cohort was performed using the TaqMan 5' exonuclease assay (32). Major and minor allele probes were labeled with 5' FAM and 5' TET fluorophore as reporters (Research Genetics, Burlington, ON, Canada), respectively. Probe fluorescence signal detection was performed using the ABI Prism 7700 Sequence Detector System (Applied Biosystems) per manufacturer's specifications.
Statistical Analysis Genotype data was assessed for errors using the PEDCHECK (39) program.
Evidence of association with the binary traits asthma (proband definition) and skin prick test reactivity (defined as at least one positive skin prick test Oracle v8i, SAS v6.12 and Arlequin v2.0 (http://anthro.unige.ch/arlequin) (43) were used to manage and analyze the data. p Values were derived by empirical simulation for the LD analysis, transmission/disequilibrium test tests and quantitative trait analysis. Statistical significance was defined at the 5% level.
Power calculations.
Comparative Sequence Analysis The complete coding region of TLR4, intron/exon boundaries, and 200 bases of surrounding intronic sequence, and approximately 1.5 kb of 5' genomic DNA were resequenced from DNA obtained from 90 individuals: 24 blacks, 23 whites, 24 Hispanics, and 19 self-reported individuals with asthma. A total of 29 SNPs were identified, 17 of which are novel (Figure 1 and Table 1). The majority of SNPs identified were rare variants: 13 variants were observed only once among the aggregate 180 chromosomes (minor allele frequency 0.006), and only 5 variants were observed in all 3 ethnic groups. TLR4 displays substantial genetic diversity across ethnic groups. Blacks demonstrated the greatest degree of genetic diversity (21 variants), whereas whites demonstrated the least (9 variants). Furthermore, six SNPs seen at high frequency (>10%) in the black cohort were not observed, or were observed at very low frequency, in the Hispanic and white cohorts. Most notably, the D259G variant, a functional polymorphism known to be responsible for endotoxin hyporesponsiveness (6), was observed at a frequency of 15% among DNA samples from blacks, compared with 4% in the samples from whites and Hispanics (p = 0.04). Seven nonsynonymous SNPs were identified, although only two (D259G and T359I) were observed in all four population samples. Nine variants were identified in the 5' genomic region, including three of modest frequency (-6142A>G, -5749T>C, and -5723T>C). Three rare variants were observed in the 3' untranslated region among blacks.
Intragenic Linkage Disequilibrium and Haplotype Structure of the TLR4 Locus Hardy-Weinberg equilibrium was confirmed for all loci in the black, white, and asthmatic cohorts. Among the Hispanic cohort, three loci (-495, 45,982 and 46,282) were not in Hardy-Weinberg equilibrium (empirical p value = 0.02 at each locus). This distortion of Hardy-Weinberg equilibrium among Hispanics was due to the presence of one individual who was homozygous for the minor allele at all three loci, while the remaining 23 Hispanics surveyed were monomorphic for the wild-type alleles. The sequence tracings for this individual were of high quality (Phred quality scores greater than 30), and allele calls were confirmed by manual inspection of the tracings. Given the relatively small sample size of the individual ethnic groups, the significance of this finding is unclear. It is notable that among 94 Hispanic parents of probands with asthma in the CAMP cohort (who were not included in our association studies due to the small number of Hispanic probands), loci 45,982 and 46,282 were both polymorphic and in Hardy-Weinberg equilibrium—the minor allele at both loci were observed in the heterozygous form only (minor allele frequencies of 0.048 and 0.044, respectively). These observations suggest that the three loci are polymorphic among Hispanics, with similar frequency distributions to those of the other populations in this study. Pairwise linkage disequilibrium was measured by r2 among the 12 SNPs with a frequency greater than 5% in at least one ethnic group (data not shown). White and Hispanic LD patterns were identical at all loci across TLR4. In contrast, LD patterns in the black cohort were quite distinct from those of both whites and Hispanics. Interestingly, no pair of loci was in significant LD among all three ethnic groups. Specifically, the two nonsynonymous functional variants (D259G and T359I), which are in strong LD among whites in our study and in white groups reported elsewhere (6), are in only moderate LD in the black cohort. Haplotypes were constructed using the 8 SNPs with minor allele frequencies of at least 10% in one or more populations (Table 2). A total of 17 haplotypes were identified—a small fraction of the 256 (28) possible haplotypes (assuming complete linkage equilibrium). This restricted haplotype diversity is similar to that reported at other loci across the genome (45, 46). Diversity was further restricted within ethnic groups, with 11, 6, and 7 haplotypes observed in the black, white, and Hispanic cohorts, respectively. Notably, among whites and Hispanics, the three most common haplotypes represent 93.5% and 89.7% of their total haplotype diversity, respectively. Paralleling the LD patterns noted above, the haplotype profiles of the Hispanic and white cohorts were very similar, with the three most common haplotypes sharing nearly identical distributions. Haplotype distribution among the 19 subjects with asthma was similar to that seen among the whites, with minor differences: three haplotypes (4, 6, and 12) were observed only among the asthmatic cohort, and two haplotypes (5 and 11) were only observed among whites, all at low frequency. In addition, haplotype 2 was observed at a frequency greater than twice that seen among the white or Hispanic cohorts (p = 0.07).
Family-based Association Analysis of TLR4 with Asthma and Asthma-related Phenotypes To investigate the hypothesis that genetic variation in TLR4 is important in the pathobiology of asthma, we genotyped five allelic variants (-6142A>G, -5749T>C, -5723T>C, 45,982 [D259G], and 46,282 [T359I]) in two independently ascertained populations of families with asthma and assessed these for evidence of allelic association to both quantitative and qualitative asthma-related phenotypes. The SNPs genotyped were selected either because of their role in resolving most common haplotypes among whites (-6,142A>G and -5,723T>C, D259G), their functional significance as endotoxin-hyporesponsive variants (D259G and T359I), or increased frequency in the asthma-sequencing cohort (-5,749T>C). Evidence for association with the asthma phenotype was assessed using the transmission/disequilibrium test (Table 3). No allele demonstrated significant association with asthma. Among the white families in the CAMP cohort, position -5,723 demonstrated weak evidence of transmission distortion (ratio of transmitted to untransmitted C alleles = 96:124,  2 = 3.56, 1 degree of freedom, unadjusted p value = 0.06). This distortion was not statistically significant (particularly after any correction for multiple testing) and was not observed among black, Hispanic, or SLSJ families. Importantly, the common endotoxin-hyporesponsiveness variants (SNP 45,982 and 46,282 in Table 3) showed no evidence of association with the asthma phenotype. No evidence of association was observed between any of the SNPs and skin prick test reactivity (data not shown).
To exclude the possibility of haplotype association (due either to the combined interaction of adjacent variants or to linkage disequilibrium between an untyped disease-susceptibility allele and TLR4 haplotype), we compared the estimated number of observed 5-allele haplotype transmissions to the expected number, as derived from the estimated parental haplotypes, using the TRANSMIT program (Version 2.5) (38). The 5 SNPs selected allow for 98.5% discrimination of the observed white haplotypes, with one rare haplotype (number 11 in Table 2) being indistinguishable from haplotype 1. Table 4 shows the results for the individual haplotype analysis in both the white CAMP and SLSJ families. We detected no significant association between the asthma phenotype and TLR4 haplotypes. No haplotype association was observed in either the Hispanic or black trios (data not shown), although the genotyped loci do not accurately discriminate between the common haplotypes in the latter population (Table 2). Of note, haplotype 2 (C and E in Table 4), which was observed at higher frequency among subjects with asthma than among whites and Hispanics in the sequencing cohorts, demonstrated balanced transmission in all family-based cohorts.
Four quantitative asthma-related traits (percent predicted postbronchodilator FEV1, airway hyperresponsiveness to methacholine [log-transformed PC20], total serum IgE levels, and total serum eosinophils [both log-transformed]) were assessed for evidence of association to TLR4. In the SLSJ cohort, a weak association was noted between the -6142A>G locus and serum eosinophil levels (T score = 1.31, unadjusted p value = 0.04); there was no such finding in the CAMP cohorts. No associations were observed between the remaining phenotypes and any of the five SNPs assessed, including the functional variants conferring endotoxin hyporesponsiveness (data not shown).
Power Calculations
This study represents the first evaluation of the TLR4 gene as an asthma susceptibility locus. Using a large sample of both asthmatic and unaffected individuals, we identified the common genetic variation at this locus, assessed the extent of linkage disequilibrium across the gene, and characterized the common haplotype structure in several ethnically diverse populations. Using a family-based approach, we evaluated the common allelic variants for evidence of association to the asthma phenotype using two large, independently ascertained cohorts. We found no evidence to support a significant association between TLR4 polymorphisms and a diagnosis of asthma. The common -5723T variant demonstrated weak evidence for preferential transmission to probands with asthma in the CAMP families of white origin, but not at a statistically significant level. In addition, this preferential transmission was not observed among the black, Hispanic, or SLSJ families. Importantly, we found no evidence of significant association between endotoxin-hyporesponsiveness TLR4 variants and asthma, and found no evidence of association between asthma and the common TLR4 haplotypes. Finally, we found no evidence for association between TLR4 genetic variation and several asthma and atopy-related quantitative traits. These findings suggest that common genetic variation at the TLR4 locus, including variants conferring endotoxin-hyporesponsiveness, is not an important determinant of asthma susceptibility. The functional variants in TLR4 that predict endotoxin hyporesponsiveness do not appear to affect asthma susceptibility, despite the suggestion by several epidemiologic studies that lack of exposure to endotoxin is a risk factor for the development of atopic and asthmatic phenotypes (11, 12). Although our findings may raise doubt regarding the role of bacterial endotoxin in the pathobiology of asthma and atopy, we do not claim that the possibility of such a relationship can be dismissed on the basis of our results. Although variants D259G and T359I are important determinants of responsiveness to bacterial endotoxin, it is clear that the TLR4 locus itself is not the sole determinant. In Arbour and colleagues' study of 83 subjects, 259Gly was observed among only 7 of 31 patients who were endotoxin hyporesponsive, and several subjects demonstrated normal response despite harboring this variant (6). Furthermore, several inbred mouse strains that have mutations at the TLR4 locus maintain normal LPS responsiveness (47). This suggests that additional genetic determinants, perhaps involving other important genes in the endotoxin-signaling pathway, contribute to the endotoxin-responsiveness phenotype. Variation at these other loci may indeed play important roles in the development of asthma. However, at this time there is little evidence to support this hypothesis. Baldini and colleagues demonstrated a positive association between variation in the promoter region of CD14 and IgE levels among skin prick test-positive subjects (48). This association has been replicated in a second cohort (49). However, neither group found evidence for similar associations with asthma, despite the second cohort having been ascertained through probands with asthma. In addition, a third study found no association between the CD14 promoter polymorphism and the diagnosis of asthma in a cohort from Iceland (50). These studies suggest that variants in the endotoxin-responsiveness pathway may play a role in the development of atopy, but not of asthma. Our study has several important strengths. First, we surveyed the 5' genomic and coding regions of TLR4 in a large and diverse cohort of both healthy individuals and those with asthma. We estimate that our survey of 180 chromosomes (90 individuals) enables us to detect all common polymorphisms (allele frequency > 5%) and many less-common SNPs with greater than 95% confidence (51). Furthermore, implementing a bidirectional sequencing strategy for all amplicons reduced the likelihood of false-negative SNP identification. It is therefore unlikely that we failed to identify important coding or regulatory SNPs in TLR4 that may contribute to asthma susceptibility. We assessed five polymorphisms in the TLR4 locus in two large, extensively phenotyped North American cohorts ascertained on a diagnosis of asthma. The use of large cohorts provided us power to detect modest gene effects, including those from SNPs with population frequencies of 5% (the frequency of the endotoxin-hyporesponsiveness SNPs). In addition, our family-based study design eliminated the possibility of spurious associations on the basis of population stratification. The children comprising the CAMP cohort are likely representative of subjects with mild to moderate asthma of the general North American population, lending to the generalizability of our results. The SLSJ cohort provides important independent and complementary information in our evaluation of the TLR4 locus. As an example of a young founder population (23, 24), this cohort likely exhibits reduced locus and allelic heterogeneity, thus improving the likelihood of identifying associations that might otherwise be difficult to detect. As noted by Smirnova and colleagues, these features of young founder populations are particularly useful in evaluating genes with multiple rare coding variants (9). Our analysis included the evaluation of both individual SNPs and their common 5-locus haplotypes. Haplotype analysis is increasingly recognized as a powerful tool in the evaluation of complex trait genetics for several reasons. First, haplotype analysis can provide evidence for association with disease when the haplotype is in linkage disequilibrium with an unidentified or untyped disease-causing variant. It has been suggested that haplotype-associated LD is more constant and predictable than that associated with individual SNPs alone (45), thereby improving the likelihood of detecting the association between the marker haplotype and the disease locus if they are indeed associated. Second, it has been recognized that the combination of several allelic variants within a gene, present together physically on the same chromosome, may act in concert as a ‘meta-allele’ to produce phenotypic effects distinct from those produced by the individual SNPs alone (52, 53). Finally, along with qualitative analysis of the dichotomous asthma phenotype, we extended our analysis to several important quantitative phenotypes, including measures of lung function and airway responsiveness, as well as serum IgE and eosinophil levels. Our failure to identify any association with these intermediate phenotypes further suggests that the TLR4 locus is not an important independent disease modifier of either asthma or atopic disease. Several limitations should be considered when interpreting our study's results. First, although we assessed many of the common polymorphisms so far identified, there remain several less common variants, including five rare nonsynonymous SNPs (Q51R, L365F, E434K, Q470H, and Q884K) and variants restricted to only one ethnic group that we did not assess for association with asthma. Although it is possible that one or more of these variants may be associated with asthma susceptibility, their low minor allele frequencies preclude sufficient power in our cohorts to detect such an association. It has been suggested that association studies, including family-based association studies, are not suitable for the evaluation of loci like TLR4 with many rare coding variants due to the increased allelic heterogeneity, and that linkage-based studies are preferable (9). Although our study design does not allow us to exclude these rare variants as asthma-susceptibility variants, it is noteworthy that of the numerous genome-wide linkage-based surveys for asthma susceptibility loci published to date, none have demonstrated evidence of linkage to the TLR4 region of chromosome 9 (54). In addition, none of the rare amino acid variants were observed among the 19 subjects with asthma who were resequenced, suggesting that the prevalence of these alleles is very low among subjects with asthma. Together, these findings suggest that rare variants at the TLR4 locus are not important determinants of asthma susceptibility. Another limitation of our study is that we did not complete sequencing on all intervening intronic sequences, and it is conceivable that we failed to identify important intronic variation that regulates gene expression. Finally, we do not have information regarding environmental endotoxin exposure, preventing us from examining a potentially important gene/environment interaction. It is certainly possible that TLR4 polymorphisms influence asthma risk in populations exposed to threshold levels of endotoxin, or that the functional variants of the TLR4 locus modify the endotoxin-atopy relationship in a highly complex manner, including distinct temporal and dose-dependent effects. Assessment of TLR4 genotype in additional cohorts with available longitudinal environmental endotoxin exposure data would be useful to clarify this issue. In conclusion, we have performed a comprehensive evaluation of the TLR4 locus, identified 29 polymorphic sites (17 novel sites), and characterized the haplotype structure. These data should be useful for further evaluation of the role of the TLR4 locus in many complex disease states in which bacterial endotoxin may play a role, including sepsis susceptibility, organic dust syndrome, atherosclerosis, and occupational asthma. We evaluated the most common TLR4 SNPs in two large cohorts of subjects with asthma and found no evidence to support the notion that TLR4 is an asthma-susceptibility gene. Further investigation in additional cohorts of subjects with asthma of both this locus and other loci in the endotoxin signaling pathway would be helpful to better define the role of endotoxin exposure in the development of asthma and allergy.
The authors thank all the families for their enthusiastic participation in this study. They acknowledge the CAMP investigators and research team for collection of CAMP genetic ancillary study data. They also acknowledge the following individuals for the collection of the SLSJ cohort data: D. Gagné, H. Archibald, M. Laforte, J. P. Leblanc, C. Allard, J. Millot, and F. Deschesnes.
Supported by research grants and contracts from the National Institutes of Health and the National Heart, Lung, and Blood Institute (2 T32 HL07427-22, N01HR16049, P01 HL67664, 1 U01 HL66795-01) and from the Canadian Institutes of Health Research. B.A.R. and T.J.H. are recipients of Clinician Scientist Awards from the Canadian Institutes of Health Research. C.L. was supported by the Fonds de Recherche sur la Nature et les Technologies du Québec. C.G. is a Chercheur Boursier of the Fonds pour la Recherche en Santé du Québec. Received in original form July 1, 2002; accepted in final form September 23, 2002
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ABSTRACT
INTRODUCTION
3 mm diameter) was evaluated using the transmission/disequilibrium test (
