Toll-like receptor 6 (TLR6) is one of a series of highly conserved innate immune receptors. We resequenced TLR6 in DNA samples from 24 African Americans, 23 European Americans, and 24 Hispanic Americans, identifying 53 SNPs, 22 with an allele frequency >5%. Significant differences in SNP frequencies among the three populations were noted. In all, 11 SNPs caused amino-acid changes, including one with a frequency >5% in all three populations. Utilizing this SNP (Ser249Pro), we performed exploratory nested case–control disease-association studies, including one involving 56 African Americans with asthma and 93 African American controls. The minor allele of this SNP was associated with decreased risk for asthma (odds ratio 0.38, 95% CI 0.16–0.87, P=0.01), an effect consistent with the known biology of the toll-like receptors. Although replication of this finding in other, larger samples is needed, variation in TLR6 may have relevance to the pathogenesis of immunologically mediated diseases.
Toll-like receptor 6 (TLR6) is a member of the Toll-like receptor family, a highly conserved series of ancient innate immune pattern recognition receptors. Antigen-presenting cells, including tissue macrophages, blood monocytes, and dendritic cells, respond to microbial antigens via these receptors, thereby activating genes that mediate innate immune responses and initiate transition from innate to acquired immunity.1, 2, 3
TLR6 (NM_006068) was first described by Takeuchi et al4 in 1999 as a 91.9-kDa, 796 amino-acid polypeptide. The gene for TLR6 has been mapped to human chromosome 4p13. It has a single exon, and contains an N-terminal signal peptide, 20 tandemly repeated extracellular leucine-rich motifs, and a cytoplasmic domain homologous to interleukin-1 receptor (IL1R), similar to other TLRs.4 To date, neither extensive description of variation within TLR6 nor association of the diversity within this gene with disease has been reported.
Here we describe variation of the TLR6 gene, focusing on single-nucleotide polymorphisms (SNPs) discovery using DNA from three distinct ethnic samples. Additionally, we discuss the results from one of our exploratory case–control association analyses between this gene and clinical diagnoses of asthma, myocardial infarction, deep venous thrombosis, and chronic obstructive pulmonary disease. These diseases were selected since variation in the innate immunity system may play a role in their development.
Results and discussion
Complete sequencing and genotyping protocols and primers utilized are available on the Innate Immunity PGA website, http://www.innateimmunity.net. Detailed descriptions of linkage disequilibrium patterns within TLR6, case–control association populations, statistical methodology, and association results not reported here are also available on that site.
Unrelated DNA samples from the Coriell Institute (n=24 African American, n=23 European American) and the University of Arizona (n=24 Hispanic American) were resequenced utilizing an ABI 3100 Sequence Detector using the default sequencing protocol. PCR amplicons and primers were designed using Primer3 (Whitehead Institute) and ranged from 700 to 900 bases in length, with contiguous amplicons overlapping by ∼200 bases. The region resequenced is ∼7753 base pairs in length, corresponding to chromosome 4 from 38723088–38730841 in the July 2003 Golden Path freeze (http://genome.ucsc.edu), and includes the single TLR6 exon and ∼2.5 kb 5′ and 3′ contiguous regions. Phred, Phrap, Consed, and Polyphred (University of Washington) were used for assembly, editing, and identification of putative SNPs, using a quality threshold of 20. Genotypes for each SNP were visually confirmed by inspecting the chromatograms. Table 1 shows the 53 SNPs detected in the three populations. SNP positions were calculated relative to the A base of the TLR6 ATG start codon. Flanking regions for the SNPs are noted in Web Table A (see Supplementary Information). Overall, 22 SNPs had an allelic frequency of ⩾5%. In all, 16 of these were seen in all the three populations. A total of 11 SNPs encoded for changes in amino-acid sequence, one (Ser249Pro) with an allelic frequency >5%.
All of the SNPs shown in Table 1 were in Hardy–Weinberg equilibrium (HWE)5 among European Americans. Two SNPs (3639, 4128) in African Americans and three SNPs (−1633, 4116, and 4223) in Hispanic Americans were not in HWE (P<0.03). Each SNP passed the stringent criteria for quality control, making genotyping error unlikely. Therefore, these are likely chance findings, since 28 tests were performed in each of three samples.
As reported in other human genes,6, 7, 8 more population-specific polymorphisms were identified in African Americans (n=18), compared to European Americans (n=2) and Hispanic Americans (n=5). Of the 53 SNPs, seven were previously noted in the dbSNP public database (http://www.ncbi.nlm.nih.gov/SNP)—two in the promoter, two in the exon, and three in the 3′UTR. These included SNPs at positions −502, −410, 1082, 1262, 4116, 4223, and 4280, corresponding precisely to dbSNP entries rs1039559, rs1039560, rs3821985, rs3775073, rs2890664, rs2381289, and rs2381290.
We evaluated Ser249Pro in case–control fashion on subjects with and without asthma utilizing banked DNA samples from the Normative Aging Study, an ongoing longitudinal cohort of 2180 individuals, initially aged 45–60.9, 10 In all, 56 asthma cases and 97 controls matched by age, gender, and African American heritage were examined from this cohort. Genotyping was done using the SNaPshot primer extension kit (Applied Biosystems) as described by the manufacturer, except that the SNaPshot mix was diluted 1:1 with HalfBD BigDye sequencing dilution buffer (Genpak). Primers included PCR (reverse: IndexTermGGCATTTCCAAGTCGTTTC, forward: IndexTermAATTTTTATCAGAACTCACCAGAGG) and internal SnaPshot: IndexTermATTAAATTTTTATCAGAACTCACCAGAGGT. Allele determination was carried out using ABI Prism Genotyper software after separation on an ABI 3100 Sequence Detector in the presence of a fluorescently labelled size standard. Tables 2 and 3 demonstrate the allelic and genotypic association of the C → T transition at this position. There were far fewer T alleles in African American asthmatics vs controls (odds ratio (OR) 0.38, 95% CI 0.16–0.87, P=0.01). In the evaluation of genotypes, five of 97 African American subjects were homozygous TT compared to one of 56 cases (Fisher's exact P=0.05).
The association of the Ser249Pro in European Americans with the diagnoses of asthma, chronic obstructive pulmonary disease, myocardial infarction, and deep venous thrombosis was also performed utilizing banked DNA from the NAS, the Nurse's Health Study,9, 11 and the Physician's Health Study.9, 11 In total, 70 Caucasian cases with each diagnosis were gender matched to 140 Caucasian disease-free controls. There were also fewer T alleles in Caucasian asthmatics vs controls (OR 0.72, 95% CI 0.48–1.09), although this difference was not statistically significant (p=0.12). No other significant association between disease status and this SNP was noted.
While the study of the TLRs has focused on their role in innate immunity against infectious agents, they may also play a role in determining susceptibility to allergies, asthma, and autoimmune diseases.3 Altering early life balance from Th1 to Th2 lymphocyte predominance may enhance a child's risk for these diseases, the so-called ‘hygiene hypothesis.’12, 13 As activators of cytokine production in response to infection, TLRs may participate in early life Th1 immunophenotype development.14, 15 Genetic variation in these receptors may thus influence risk for either Th1 (infectious) or Th2 (atopic) disease. Additionally, two missense mutations of the TLR4 gene (Asp299Gly and Thr399Ile) have been associated with a decreased response to inhaled endotoxin in humans.16 In turn, endotoxin exposure confers protection on the development of allergy and asthma.17 While no prior studies have evaluated this effect in our gene of interest, TLR6, both TLR6 and TLR4 elicit similar cytokine responses upon exposure to bacteria,18 suggesting that alterations in TLR6 may result in a phenotype similar to that of TLR4. Interestingly, human mast cells express mRNA for TLR6.19 While also involved in host defense against bacterial infection, the mast cell plays a pivotal role in the pathogenesis of human asthma.20, 21
We tested the association of asthma with the only common (allelic frequency ⩾10% in each of our ethnic populations), non-synonymous TLR6 SNP (Ser249Pro). This nonconservative amino-acid change occurs in the extracellular domain of the TLR6 protein sequence in a region that is homologous and conserved in the mouse. Variation in Ser249Pro was significantly associated with protection from asthma in African Americans and trended towards protection from asthma in European Americans. Although the possibility exists that this association is a chance finding (no correction was made for multiple testing) or reflects population stratification within the African American cohort, the amino-acid change argues for biologic plausibility. While our African American sample size was small, it was powered at 80% to detect an association as extreme as the one reported and at 76% to detect an OR of at least 2.5 (equivalent to a protective OR of 0.4). This association is also consistent with an extension of the ‘hygiene hypothesis’ noted above. That is, while genetic variation in TLR6 may predispose an individual towards a greater risk of infection, that same variation may simultaneously encode for a protective effect towards asthma. The interethnic differences in odds ratios may reflect differences in environmental background, sample size, or epistatic interactions.
In conclusion, the resequencing of TLR6 in three ethnic populations has defined novel variants of potential relevance to immunologically mediated disease. While further work, including studies evaluating molecular characterization of TLR6 and replication of the association of this gene with the diagnosis of asthma are warranted, the association of a nonsynonymous SNP change with a protective effect from asthma is both intriguing and consistent with the known biology of the toll-like receptors.
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This work was supported by the Program for Genomic Applications, NIH R01: HL66795, Innate Immunity in Heart, Lung and Blood Disease, from the National Heart and Blood Institute, and by NIH 2T32: HL07427-21, The Clinical Epidemiology of Lung Diseases.
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Tantisira, K., Klimecki, W., Lazarus, R. et al. Toll-like receptor 6 gene (TLR6): single-nucleotide polymorphism frequencies and preliminary association with the diagnosis of asthma. Genes Immun 5, 343–346 (2004). https://doi.org/10.1038/sj.gene.6364096
- toll-like receptor 6
- innate immunity
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