Predisposition to Childhood Otitis Media and Genetic Polymorphisms within the Toll-Like Receptor 4 (TLR4) Locus

Abstract

Background: Predisposition to childhood otitis media (OM) has a strong genetic component, with polymorphisms in innate immunity genes suspected to contribute to risk. Studies on several genes have been conducted, but most associations have failed to replicate in independent cohorts.

Methods: We investigated 53 gene polymorphisms in a Finnish cohort of 624 cases and 778 controls. A positive association signal was followed up in a tagging approach and tested in an independent Finnish cohort of 205 cases, in a British cohort of 1269 trios, as well as in two cohorts from the United States (US); one with 403 families and the other with 100 cases and 104 controls.

Results: In the initial Finnish cohort, the SNP rs5030717 in the TLR4 gene region showed significant association (OR 1.33, P = .003) to OM. Tagging SNP analysis of the gene found rs1329060 (OR 1.33, P = .002) and rs1329057 (OR 1.29, P = .003) also to be associated. In the more severe phenotype the association was stronger. This finding was supported by an independent Finnish case cohort, but the associations failed to replicate in the British and US cohorts. In studies on TLR4 signaling in 20 study subjects, the three-marker risk haplotype correlated with a decreased TNFα secretion in myeloid dendritic cells.

Conclusions: The TLR4 gene locus, regulating the innate immune response, influences the genetic predisposition to childhood OM in a subpopulation of patients. Environmental factors likely modulate the genetic components contributing to the risk of OM.

Fig 1. Comparison of the TLR4 variant rs13209060 association to otitis media.

The size of the circle has been scaled to show the relative sizes of each dataset (N = number of cases in case-control datasets or N = twice the number of probands in family-based analysis). Horizontal lines have been scaled to show the 95% confidence intervals (95% CI) of the odds ratios of the T-allele, the position of the circles on the x-axis shows the odds ratio for that dataset. For simplicity and to highlight the result with the strongest signal in the Finnish population, we only plotted results of the association of rs13209060-T with otitis media; additional data can be found in S6 Table.

Fig 2. Expression of TNFα in myeloid dendritic cells from children with recurrent episodes of acute otitis media or chronic otitis media with effusion.

The percentage of TNFα expressing cells in the unstimulated sample was subtracted from the percentage of the cells seen in the LPS stimulated sample. Children with the otitis media risk rs1329060-rs1329057-rs5030717 TCG TLR4 gene haplotype were compared to children with the protective CTA haplotype by Mann-Whitney U-test.

Fig 3. Relative mRNA expression of TNFα analyzed by RT-qPCR in peripheral blood mononuclear cells (PBMC) stimulated with LPS in subjects with protective rs1329060-rs1329057-rs5030717 CTA or risk TCG TLR4 haplotype.

The statistical difference between groups was assessed using the Mann-Whitney U-test. The expression level is shown as relative units calculated by the ddCT-method. Boxes indicate interquartile range (25%–75%) with the horizontal bar within the box indicating the median. Whiskers show minimum and maximum values, o = outlier.