Conjunctival fibrosis and the innate barriers to Chlamydia trachomatis intracellular infection: a genome wide association study

Abstract

Chlamydia trachomatis causes both trachoma and sexually transmitted infections. These diseases have similar pathology and potentially similar genetic predisposing factors. We aimed to identify polymorphisms and pathways associated with pathological sequelae of ocular Chlamydia trachomatis infections in The Gambia. We report a discovery phase genome-wide association study (GWAS) of scarring trachoma (1090 cases, 1531 controls) that identified 27 SNPs with strong, but not genome-wide significant, association with disease (5 × 10(-6) > P > 5 × 10(-8)). The most strongly associated SNP (rs111513399, P = 5.38 × 10(-7)) fell within a gene (PREX2) with homology to factors known to facilitate chlamydial entry to the host cell. Pathway analysis of GWAS data was significantly enriched for mitotic cell cycle processes (P = 0.001), the immune response (P = 0.00001) and for multiple cell surface receptor signalling pathways. New analyses of published transcriptome data sets from Gambia, Tanzania and Ethiopia also revealed that the same cell cycle and immune response pathways were enriched at the transcriptional level in various disease states. Although unconfirmed, the data suggest that genetic associations with chlamydial scarring disease may be focussed on processes relating to the immune response, the host cell cycle and cell surface receptor signalling.

Figure 1. Results of GWAS Analysis using EMMAX.

(A) QQ plot: There is genome-wide deflation (λ = 0.982 SE = 1.74e⁻⁰⁵) of the test statistic when the phenotype is corrected for kinship only. (B) QQ plot: Phenotype correction for age, gender and pairwise kinship removed any deviation from the null expectation of the genome-wide test statistic (λ = 1.001, SE = 8.7e⁻⁷). (C) Manhattan plot showing index SNPs with P_EMMAX < 5 × 10⁻⁶.

Figure 2. Regional Plot of the most significant index SNP region (rs111513399, PREX2).

Window size 250 kb. LD with index SNP (R² value) is indicated by colour. LD structure was generated from the GWAS data after imputation. The most significant PREX2 region coincides with a common splice variation. Known transcript variants (A: NP_079146.2 and B: NP_079446.3) are indicated by horizontal red lines and exons are indicated by crosshatching verticals.

Figure 3. Summary of pathways analysis with ALIGATOR and PODA.

Blue circle shows root level Reactome hierarchy event terms and stable identifiers with at least one significant pathway under ALIGATOR. Red circle shows same for PODA analysis. Six branches contained significant pathways under both analyses. Ten branches contained no significant pathways in either analysis.

Figure 4. PREX2 is closely involved in processes surrounding TARP mediated Chlamydial entry.

Downstream signalling via RAC leads to changes in cell cycle control and actin skeleton rearrangements that facilitate infection. PREX2 can indirectly mediate downstream changes to cell cycle control and glucose homeostasis via RAC and Akt/p53.

Figure 5. Trachoma associated genes and pathways.

Potential roles for candidate genes (red) that were identified through this GWAS are indicated. Various significant cell surface receptors pathways including FGFR, GPCR, ILGFR1 and GLPR1 are linked to cell cycle control by PI3K/Akt/p53 signalling. Chlamydial elementary bodies are known to interact with this system via sos1 and vav2. Downstream signalling from these pathways can lead to actin remodelling (facilitating cell entry), cell cycle arrest and inhibition of apoptosis; all factors that facilitate parasitism. Glucose and sodium ion homeostasis resulting from p53/cell-cycle control may increase nutrient availability to the growing inclusion. Up-regulation of NF_KB, CTGF, MMP9 and TGFB are potential routes to fibrosis.