In our effort to identify EE-specific genetic susceptibility loci, we utilized a custom Illumina SNP genotyping chip to screen for variants within candidate genes involved in allergy and/or epithelial cell function. In order to account for multiple testing, the Bonferroni LD adjusted P-value required for statistical significance in this study was determined to be P = 3 × 10−4. As the coincidence of allergy is high (approximately 70%) in EE, we first chose to investigate the specificity of any potential associations of candidate gene SNPs by stratifying the discovery control cohort into two phenotypically defined groups based on a history of allergy (see Methods). When comparing EE to the allergic controls, we found that only TSLP harbored variants which reached statistical significance ( and , and Supplementary Table I). Indeed, seven SNPs in TSLP exhibited association with EE (4.11 × 10−5 ≤ P ≤ 3.29 × 10−3). However, when comparing EE to the non-allergic controls, no genes exhibited variants which reached statistical significance; the strongest observed TSLP SNP association only reached P = 7.35 × 10−3 in this analysis. When the two control groups were combined (allergic and non-allergic), the association strengthened, most likely owing to the increase in sample size of the control group. Interestingly, polymorphisms in IL4, a known allergy susceptibility gene, were associated with EE (best P = 1.33 × 10−3, OR = 1.91) using the non-allergic controls but not with the allergic controls (best P = 0.27) (see Supplementary Table I), emphasizing the importance of using phenotypic matched controls for identifying risk variants associated with primary disease. Moreover, these data highlight the specificity of the TSLP genetic association with EE.
TSLP SNPs are associated with EE as compared to allergic and/or non-allergic controls
Associations of TSLP SNPs with EE are independent of allergy phenotypes
We next aimed to replicate the observed genetic association with TSLP variants using a completely independent replication cohort of EE cases and controls (). Although the replication control cohort was composed of both allergic and non-allergic individuals, three TSLP SNPs were significantly associated with EE () in this independent analysis. In a meta-analysis of the associated TSLP variants from both the discovery and replication cohorts, the association of rs10062929 strengthened (combined P = 3.16 × 10−6) approximately 2-logs above the threshold of the Bonferroni adjustment. Moreover, the combined P-values for rs11466750 and rs11466749 were nominally significant (combined P = 8.48 × 10−4 and 1.86 × 10−3, respectively).
Replication of TSLP SNP association with EE in two independent case-control cohorts
The prevalence of EE in males is approximately 2.5-fold higher than in females. To determine whether TSLP variants contribute to this gender bias, we performed a gender-stratified analysis of EE cases and allergic controls from the discovery cohort and determined the SNPs with the greatest change in P-value in either the male or female cohorts (See Supplementary Table II). Of the nine TSLP SNPs, only rs10062929 and rs11466749 also associated in the female EE cohort (P = 0.016 and P = 0.048 in EE vs. female atopic controls, respectively), while the P-values of most variants in TSLP remained unchanged in the male-only analyses.
Interestingly, the TSLP receptor (TSLPR) is encoded on a pseudoautosomal region on Xp22.3 and Yp11.3, which further underscores the potential significance of finding SNPs in the TSLP/TSLPR pathway observed among males with EE17
. We were interested in testing the hypothesis that variants in the TSLPR may also associate with EE in a gender-specific manner. Therefore, we genotyped three coding SNPs recently validated from direct sequencing of the TSLPR gene (P.G., unpublished data): rs36139698, rs36177645, and rs36133495. Of these, the SNP rs36133495 (Ala to Val) was significantly associated with disease risk in a cohort of male EE patients and normal male controls (% Val/Val, 27% in male EE cases vs. 15% in male controls) with a P
= 0.039, OR = 2.05 ( and ). Conversely, rs36133495 was not significantly associated in a female case-control analysis (P
= 0.929, OR = 1.22). Taken together, these data present evidence that the TSLP signaling pathway may contribute to the male bias in EE.
A non-synonymous SNP in the TSLPR associates with EE in a gender-specific manner
A gender-specific association of a TSLPR SNP in male EE patients
In the lung and skin, TSLP is produced primarily by epithelial cells in response to Th2 cytokines or TLR3 agonists, subsequently targeting dendritic cells to develop Th2-polarizing activity including cytokine and chemokine production13, 18
; however, it has been observed that activated mast cells can also express TSLP to similarly drive Th2 responses12, 19, 20
. Indeed, we have recently observed increased expression of TSLP mRNA in the esophagus of EE patients compared with control individuals10
. Accordingly, we aimed to determine if primary esophageal epithelial cells could produce TSLP. Notably, exposure to the TLR3 ligand poly I:C (a dsRNA mimetic) robustly increased TSLP mRNA expression in as little as 3 hours post-treatment with either 10 or 100 µg/ml poly I:C (). These data suggest that the esophageal epithelium is a source of TSLP expression in EE.
TLR signaling stimulates TSLP expression in primary esophageal epithelial cells