Analysis of Toll-Like Receptor 2 Polymorphism (rs5743704) in Saudi Patients with Primary Open-Angle Glaucoma

Abstract

Aims: To investigate whether the single-nucleotide polymorphism (SNP) rs5743704 in the toll-like receptor 2 gene is associated with primary open-angle glaucoma (POAG) or any of its clinical indices in a Saudi cohort. Method: Ninety-five unrelated POAG cases and 95 controls of Saudi origin were genotyped utilizing a TaqMan^® assay. The association between genotypes and various clinical indices important for POAG were investigated. Results: The genotypic and allelic frequencies among cases were not significantly different when compared to controls. The minor allele frequency was 0.021 in cases and 0.011 in controls. No significant association was seen with intraocular pressure and cup/disc ratio. However, carriers of the C/A genotype had higher number of anti-glaucoma medications compared to controls (p = 0.04). Conclusion: SNP rs5773704 is not associated with POAG in a Saudi population. Despite sample size limitation, the association of the minor allele A with higher number of anti-glaucoma medications suggests a possible indirect role for this SNP in predicting disease severity.

Introduction

Toll-like receptors (TLRs) are membrane-spanning proteins that play an important role in the regulation of innate immunity (Akira et al., 2006). Studies in the past have revealed strong alterations in the naturally occurring IgG autoantibody repertoires in glaucoma patients (Joachim et al., 2003; Grus et al., 2004; Joachim et al., 2005; Joachim et al., 2007), suggesting a possible role of autoimmunity in the neurodegenerative process of the disease (Rieck, 2013). TLR2 is one of the most characterized and investigated members of the TLR family. It predominantly interacts with bacterial lipopolysaccharide and heat shock proteins (HSPs), such as HSP60 and HSP70, which are potentially involved in glaucoma (Akira et al., 2001). TLR2 only recognizes endogenous HSPs (Miggin and O'Neill, 2006). TLR2 is expressed in microglia and astrocytes from glaucomatous human retinas (Luo et al., 2010). Single-nucleotide polymorphisms (SNPs) in TLR2 have been previously associated with susceptibility to sepsis (Schroder et al., 2003), asthma (Eder et al., 2004), and cancer (de Oliveira and Silva, 2012). Likewise, promoter polymorphism in TLR2 is shown to regulate transcriptional activity of TLR2 (Tahara et al., 2008). In DBA/2J mice, an inherited model of glaucoma, 11 TLRs were upregulated in optic nerve head at early stages, including TLR2, whose gene product can recognize endogenous HSPs (Howell et al., 2011), emphasizing that immune responses are mediated, at least in part, by TLRs. Any dysregulation in the signaling pathway, possibly as a result of SNP(s), may alter the ligand binding capacity of TLRs and hence disturb the pro- and anti-inflammatory cytokines (Ferwerda et al., 2008) and modulate the disease process.

The present study investigated the possible association of a functional TLR2 SNP (rs5743704) with primary open-angle glaucoma (POAG) and important clinical indices used to assess disease severity.

Materials and Methods

Study population

The study adhered to the tenets of the Declaration of Helsinki, and all participants signed an informed consent. The study was approved by the College of Medicine Ethics Committee (approval number No. 08-657) at King Saud University. The study population was recruited at King Abdulaziz University Hospital in Riyadh, Saudi Arabia.

Saudi POAG cases (n = 95) satisfied the following clinical criteria: (i) thinning or notching of disc or retinal nerve fiber layer defect; (ii) abnormalities in visual field (e.g., arcuate scotoma, nasal step, paracentral scotoma, generalized depression) in the absence of other causes or explanation; (iii) age greater than 40 years; and (iv) open anterior chamber angles, bilaterally on gonioscopy. The exclusion criteria included evidence of secondary glaucoma, for example, pigmentary glaucoma, uveitic, and pseudoexfoliation, and history of long-term steroid use or ocular trauma. An ethically matched healthy control group (n = 95) included individuals of age >20 years, normal intraocular pressure (IOP) (<21 mmHg without any medication), open angles on gonioscopy, normal optic disc, and free from glaucoma on examination.

Genotyping rs5743704 of the TLR2 gene

rs5743704 (g.25511C>A) polymorphism in the TLR2 gene (NG_016229.1) was genotyped using the TaqMan^® SNP Genotyping assay ID: C__25607736_10 (Applied Biosystems, Inc., Foster City, CA) on the ABI 7500 real-time polymerase chain reaction (PCR) system (Applied Biosystems) as described previously (Abu-Amero et al., 2014). Each PCR reaction was performed in a 96-well plate in a total volume of 25 μL consisting of 1X TaqMan Genotyping Master Mix (Applied Biosystems), 1X SNP Genotyping Assay Mix, 20 ng DNA, and two no template (negative) controls. The cycling conditions for the real-time PCR on ABI 7500 included incubation at 95°C for 10 min, followed by 40 cycles of denaturation at 92°C for 15 s, and annealing/extension at 60°C for 1 min. The VIC^® and 6-carboxy-fluorescein (FAM) fluorescence levels of the PCR products were measured at 60°C for 1 min. Automated two-color allele discrimination software on ABI 7500 was used to identify both the genotypes of the TLR2 gene on a two-dimensional graph.

Statistical analysis

Data management, coding, and storage were done using Microsoft Excel 2010^® software (Microsoft Corporation; Redmond, WA). Data were analyzed using SPSS^® version 20.0 (IBM Inc., Chicago, IL) and StatsDirect^® statistical software, version 2.7.2 (StatsDirect Ltd., Cheshire, United Kingdom). The categorical variables were presented as frequencies and percentages, while continuous variables were presented as mean (± standard deviation, [SD]). Hardy-Weinberg Equilibrium (HWE) deviation was tested by Pearson's χ² test. Odds ratio was calculated and χ² test was used to detect any association between different characteristics and the genetic profiles (Fisher Exact test whenever indicated). Mann-Whitney U-test was used to investigate whether there was any significant difference between the normal homozygous (C/C) and the mutated heterozygous (C/A) genotypes. The confidence interval level was set to 95% and a p-value below 0.05 was considered statistically significant.

Results

Demographic characteristics

Both the recruited study groups showed an age interval of 20 years and above. As shown in Table 1, the mean age (SD) in the POAG cases was found to be 59.9 (13.3) years compared with 55.5 (11.9) years in the controls. In cases, 61 (64.2%) were male and 34 (35.8%) were female, whereas in controls, 70 (73.7%) were male and 25 (26.3%) were female. Both the groups were found to be similar in terms of age and gender (p = 0.129 and 0.158, respectively).

Table 1.

Comparison of Demographic Characteristics and Systemic Comorbidities Among Case and Control Groups

Characteristics	Case (n = 95) No. (%)	Control (n = 95) No. (%)	p
Mean age in years (SD)	59.9 (13.3)	55.5 (11.9)	0.129
Gender male	61 (64.2)	70 (73.7)	0.158
Female	34 (35.8)	25 (26.3)	—
Family history of glaucoma	13 (13.7)	3 (3.2)	0.009
Diabetes mellitus	55 (57.9)	43 (45.3)	0.072
Smoking	39 (41.1)	47 (49.5)	0.024
Hypertension	49 (51.6)	53 (55.8)	0.561
Coronary artery diseases	7 (7.4)	3 (3.2)	0.194
Hypercholesterolemia	16 (16.8)	8 (8.4)	0.081
Awareness of having glaucoma	17 (17.9)	0 (0.0)	<0.0001

SD, standard deviation.

Analysis of controls with cases in terms of clinical comorbidity with systemic diseases showed that both the groups were similar in terms of diabetes mellitus (p = 0.072), hypertension (p = 0.0561), coronary artery disease (p = 0.194), and hypercholesterolemia (p = 0.081). However, family history of glaucoma (p = 0.009), smoking (p = 0.024), and awareness of having glaucoma (p < 0.0001) were found to be statistically significant.

Genotype and allelic distribution

Table 2 shows the genotype and allelic frequency observed among cases and controls. The genotype frequencies did not deviate significantly from the HWE (p > 0.05). No mutant homozygous (A/A) genotype was detected in both the groups. The minor allele frequency was found to be 0.021 in cases compared with 0.011 in controls. Both the genotype and allelic distribution were found to be nonsignificant.

Table 2.

Genotype and Allele Frequency Distribution of SNP rs5743704 in Cases and Controls

Genotype	Case (n = 95) No. (%)	Control (n = 95) No. (%)	Odds ratio	95% Confidence intervals	p
C/C	91 (95.8)	93 (97.9)	—	—	—
C/A	4 (4.2)	2 (2.1)	2.04	[0.284-23.033]	0.683
A/A	0 (0.0)	0 (0.0)	—	—	—
C	186 (97.9)	188 (98.9)	2.02	[0.285-22.560]	0.685
A	4 (2.1)	2 (1.1)	—	—	—

SNP, single-nucleotide polymorphism.

Genotype effect on systemic comorbidities and specific glaucoma indices

Table 3 shows the comparison of demographic characteristics, systemic comorbidities, and specific clinical indices among cases according to genotypes. Statistically significant differences were observed for diabetes mellitus (p < 0.0001), hypertension (p = 0.0002), coronary artery disease (p < 0.0001), hypercholesterolemia (p < 0.0001), and awareness of having glaucoma (p < 0.0001). The analysis of glaucoma-specific _indices at presentation with genotypes revealed that with the exception of number of antiglaucoma medications used (p = 0.040), both intraocular pressure and the cup/disc ratio did not show any significant correlation (p = 0.513 and 0.525, respectively).

Table 3.

Comparison of Demographic, Systemic Comorbidities, and Glaucoma-Specific Indices Among Cases According to Genotypes

Characteristics	C/C (n = 91)	C/A (n = 4)	p
Mean age in years (SD)	61.1 (12.3)	57 (22.3)	0.055
Gender male	59 (64.8)	2 (50)	0.054
Female	32 (35.2)	2 (50)	—
Family history of glaucoma	12 (13.2)	1 (25)	0.059
Diabetes mellitus	54 (59.3)	1 (25)	<0.0001
Smoking	37 (40.7)	2 (50)	0.254
Hypertension	48 (52.7)	1 (25)	0.0002
Coronary artery diseases	5 (5.5)	2 (50)	<0.0001
Hypercholesterolemia	14 (15.4)	2 (50)	<0.0001
Awareness of having glaucoma	17 (18.7)	0 (0)	<0.0001
Intraocular pressure	35.4 (7.2)	34.5 (11.0)	0.513
Cup/disc ratio	0.71 (0.19)	0.66 (0.16)	0.525
Number of antiglaucoma medications	2.91 (0.6)	2.3 (0.5)	0.040

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 activate 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 glaucoma pathogenesis. Targeting TLR signaling has implications in infection control and downregulation of inflammation. TLR2 polymorphism (rs5743704) is the second most commonly studied polymorphism in the TLR family. rs5743704 was previously associated with pancreatitis occurrence and severity (Matas-Cobos et al., 2015) (Matas-Cobos et al., 2015), idiopathic recurrent vaginal candidiasis (Rosentul et al., 2014), cancer risk (Kutikhin, 2011), and hepatocellular carcinoma in chronic hepatitis (Nischalke et al., 2012). A possible association of TLR2 SNPs was investigated in the Japanese normal-tension glaucoma patients with negative outcome (Nakamura et al., 2009). However, to the best of our knowledge, SNP rs5743704 has never been investigated in relation to POAG.

Our study is largely negative when it comes to genotype and allele differences between groups and its direct association with POAG. However, analysis of possible association between genotypes and various clinical indices important for POAG showed an association between the C/A heterozygous genotype and number of antiglaucoma medications. The number of antiglaucoma medications is usually an indirect indicator of glaucoma severity. Thus, it is possible that this SNP may potentially serve as a marker for glaucoma severity. A markedly reduced frequency of rs5773704 A observed in a Saudi cohort suggests that a large sample size with high power of study is required to detect a significant relative risk.

TLR promotes transcription of genes involved in immune activation, including nuclear factor Kappa B and mintage-activated protein kinase pathways [seya (Seya et al., 2010). Upregulation of TLR2, in addition to TLR3, and TLR4, has been reported in human glaucoma donor eyes consistent with the findings of increased expression of HSPs. (Luo et al., 2010). Thus, it is also plausible that TLR2 might affect the alterations of the aqueous humor dynamics and injury to glaucomatous retina in eyes with POAG.

Conclusion

The study provides no direct association of TLR2 SNP rs5773704 with POAG, but does show an association with number of antiglaucoma medications, suggesting a possible role in predicting disease severity. However, it has to be stated that this study is limited by the fact that it is performed in a specific ethnicity and evaluated in a relatively small number of patients. If this association is proven in various ethnic groups and a large cohort, then this SNP could serve as a marker for glaucoma severity.

Footnotes

Acknowledgment

The authors would like to thank the Glaucoma Research Chair at the Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia, for funding this study.

Author Disclosure Statement

No competing financial interests exist.

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