Lack of Association Between Polymorphism rs4986791 in TLR4 and Primary Open-Angle Glaucoma in a Saudi Cohort

Introduction

Several studies in the past have reported alterations in the IgG autoantibody repertoires of patients with glaucoma strongly implicating a possible role of autoimmunity in the neurodegenerative processes leading to glaucoma (Wax et al., 1998; Joachim et al., 2003, 2007; Grus et al., 2004).

The toll-like receptors (TLRs) constitute a family of type 1 transmembrane proteins that consists of an intracellular TLR/interleukin-1 receptor homology domain, which is selectively activated by conserved pathogen-associated molecular patterns (Anderson, 2000), and play a crucial role in regulating both innate and adaptive immunity (Akira et al., 2001). The TLR4 is the most investigated member of the TLR family that functions as the signal-transducing receptor for lipopolysaccharides (LPSs) and is reported to play a crucial role in LPS-induced neuronal cell death in the central nervous system (Lehnardt et al., 2003). Moreover, mice lacking TLR4 show reduced neuronal apoptosis and decreased pathology in both retina and brain (Caso et al., 2007; Kilic et al., 2008). In addition, any dysregulation of TLR4 signaling pathway may be as a result of single nucleotide polymorphism(s) (SNPs) in the extracellular domain of TLR4, which might alter the ligand binding capacity and, hence, disturb the pro- and anti-inflammatory cytokines (Ferwerda et al., 2008).

SNPs in the TLR4 gene (OMIM 603030) located on chromosome 9q32-33 have been associated with the risk of normal-tension glaucoma (NTG) in the Japanese population (Shibuya et al., 2008), suggesting that TLR4 is associated with pathophysiology of glaucoma. The study investigated whether SNP rs4986791 (T399I) in exon 3 of the TLR4 gene contributed to primary open-angle glaucoma (POAG) in a Saudi cohort.

Materials and Methods

Results

Table 1 shows the demographic characteristics of the subjects included in this study. Analysis of controls with cases in terms of both demographic and clinical comorbidity with systemic diseases showed that both groups were similar in terms of age (p = 0.865), gender (p = 0.747), diabetes mellitus (p = 0.642), smoking (p = 0.404), hypertension (p = 0.062), coronary artery disease (p = 0.619), hypercholesterolemia (p = 0.077), and awareness to presence/absence of glaucoma (p = 0.248). However, “having a family history of glaucoma” was found to be significantly different (p = 0.014) between both the groups, which were almost fourfolds greater in cases (n = 11, 12.9%) than in controls (n = 3, 3.2%).

Table 2 shows the genotype and allele frequency distribution among our cases and controls. Although cases were 1.6 × (95% CI: 0.564-4.949) more likely to have heterozygous genotype, the OR did not reach the threshold of statistical significance (p = 0.340). The genotype frequencies did not deviate significantly from the HWE (p > 0.05). The minor allele frequency was 0.076 in cases and 0.042 in controls (OR = 1.9; 95% CI: 0.701-5.378; p = 0.183). Both the genotype and allele frequency among POAG cases and controls did not vary significantly.

With the exception of family history of glaucoma (p = 0.032), no significant association of genotypes was seen with age, gender, diabetes, hypertension, smoking, coronary artery disease, hypercholesterolemia, and other glaucoma-specific clinical indices such as intraocular pressure, cup/disc ratio, and number of antiglaucoma medications (Table 3).

Discussion

The innate immune system plays a fundamental role in the host defense system by triggering cellular signaling pathways to induce immune response genes, including inflammatory cytokines that secondarily activates the adaptive immune system (Schenten and Medzhitov, 2011). Failure of immune regulation of both proapoptotic and protective pathways may lead to the degeneration of the RGCs in glaucoma and highlight the role of immune involvement in the glaucoma pathogenesis. Targeting TLR signaling has implications in the infection control and downregulation of inflammation. The TLR4 SNP rs4986791 is one of the most studied among all TLR4 receptor SNPs and according to the SNPedia (http://www.snpedia.com/index.php/Rs4986791), it has been associated with diabetic neuropathy (Rudofsky et al., 2004), heart diseases (Zee et al., 2005), bacterial vaginosis (Genc et al., 2004), asthma (Yang et al., 2004), and dilated cardiomyopathy (Riad et al., 2012). These studies suggest that TLR4 variants may result in abnormal function, such as abnormal recognition of heat shock proteins or other ligands (Akira et al., 2001), and may contribute to the development of various diseases, including multifactorial disorders. Previous studies had shown that multiple SNPs in the TLR4 gene are associated with the risk of NTG in the Japanese population (Shibuya et al., 2008). The study suggested that the ligands and/or cytokines involved in the TLR4 signaling network may be risk factors for the development of NTG (Shibuya et al., 2008). Recently, Takano et al. (2012) not only confirmed the association of TLR4 polymorphisms with NTG in another Japanese cohort but also showed that they may play a role in the pathogenesis of POAG and pseudoexfoliation glaucoma. In this study, we investigated the possible association of SNP rs4986791 in a group of Saudi patients with POAG and controls from the same ethnicity.

Our study is largely negative when it comes to genotype and allele differences between both groups. However, despite sample size limitation, the distribution of different demographic and clinical characteristics of cases at presentation per different allele combination showed significant difference between mutant [T] allele and family history of glaucoma. Thus, it indicates that the presence of the “T” allele and family history of glaucoma may predispose people at higher risk of developing POAG plausibly by increased activation of pro-inflammatory response pathways. However, since the study is limited by the fact that it's performed in a specific ethnicity and studied in a relatively small number of patients, this link may need further evaluation in a larger cohort. Similar to our findings, the TLR4 polymorphisms were not found to be significantly associated with the risk of NTG in the South Korean population (Suh et al., 2011). In addition, in another study by Chen et al. (2012), TLR4 SNP rs7037117 was reported to be associated with late-onset POAG in Southern Chinese population, but not in the Northern Chinese, suggesting that the role of polymorphism(s) in TLR4 and glaucoma may be ethnic specific.

The TLR4 promotes transcription of genes involved in immune activation, including nuclear factor kappa B, and also mintage-activated protein kinase pathways (Seya et al., 2010). Upregulation of TLR2, TLR3, and TLR4 has been reported in human glaucoma donor eyes consistent with the findings of increased expression of HSPs (Luo et al., 2010). It is also plausible that TLR4 might affect the alterations of the aqueous humor dynamics and injury to glaucomatous retina in eyes with POAG. Several studies have investigated the functional consequences for the most frequent polymorphisms of TLR4: Asp299Gly (rs4986790) and TLR4 Thr399Ile (rs4986791) (Ferwerda et al., 2008; Hold et al., 2014). An initial study by Ferwerda et al. demonstrated that individuals with the Asp299Gly, but not the Thr399Ile polymorphism show a stronger pro-inflammatory tumor necrosis factor-α cytokine response following LPS stimulation compared to wild-type TLR4 individuals (Ferwerda et al., 2008). In another recent study, the authors reported that TLR4 Asp299Gly and Thr399Ile polymorphisms affect constitutive receptor activity leading to a dysregulated suboptimal immune response (Hold et al., 2014). The initial blunted responses observed to LPS challenge were followed by exaggerated immune responses suggesting that these variants may act in combination with other immune processes to influence the outcome during an infection. The conflicting observations in these studies indicate that the genetic background of a study population may also influence the response (Hold et al., 2014).

Conclusion

SNP rs4986791 in TLR4 is not associated with POAG in Saudi population. Future studies may explain the missing links between genetic mechanisms of innate immunity and adult-onset glaucoma.