July 2017
Volume 58, Issue 9
Open Access
Cornea  |   July 2017
TNF-α Genetic Predisposition and Higher Expression of Inflammatory Pathway Components in Keratoconus
Author Affiliations & Notes
  • Muneeza Arbab
    Translational Genomics Laboratory, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
  • Saira Tahir
    Translational Genomics Laboratory, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
  • Muhammad Khizar Niazi
    Armed Forces Institute of Ophthalmology, Rawalpindi, Pakistan
    Army Medical College, Rawalpindi, Pakistan
  • Mazhar Ishaq
    Armed Forces Institute of Ophthalmology, Rawalpindi, Pakistan
    Army Medical College, Rawalpindi, Pakistan
  • Alamdar Hussain
    Translational Genomics Laboratory, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
  • Pir Muhammad Siddique
    Shifa International Hospital, Islamabad, Pakistan
  • Sadia Saeed
    Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan
  • Wajid Ali Khan
    Al-Shifa Eye Trust Hospital, Rawalpindi, Pakistan
  • Raheel Qamar
    Translational Genomics Laboratory, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
    Pakistan Academy of Sciences, Islamabad, Pakistan
  • Azeem Mehmood Butt
    Translational Genomics Laboratory, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
  • Maleeha Azam
    Translational Genomics Laboratory, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
  • Correspondence: Maleeha Azam, Department of Biosciences, COMSATS Institute of Information Technology, Park Road, Tarlai Kalan, Islamabad-45600, Pakistan; malihazam@gmail.com
  • Azeem Mehmood Butt, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad 45550, Pakistan; azeem.butt@comsats.edu.pk
  • Footnotes
     RQ, AMB, and MA contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science July 2017, Vol.58, 3481-3487. doi:10.1167/iovs.16-21400
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      Muneeza Arbab, Saira Tahir, Muhammad Khizar Niazi, Mazhar Ishaq, Alamdar Hussain, Pir Muhammad Siddique, Sadia Saeed, Wajid Ali Khan, Raheel Qamar, Azeem Mehmood Butt, Maleeha Azam; TNF-α Genetic Predisposition and Higher Expression of Inflammatory Pathway Components in Keratoconus. Invest. Ophthalmol. Vis. Sci. 2017;58(9):3481-3487. doi: 10.1167/iovs.16-21400.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To date keratoconus (KC) pathogenesis is undefined; however, the involvement of inflammatory pathways in disease development is becoming apparent. In the present study, we investigated the role of a promoter region polymorphism rs1800629 (−308G>A) in the inflammatory pathway component TNF-α and its effects on the expression of TNF-α and downstream molecules tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), and interleukin 6 (IL-6) in KC development.

Methods: TNF-α promoter polymorphism rs1800629 (−308G>A), was genotyped in 257 sporadic KC patients and 253 healthy controls. Enzyme-linked immunosorbent assay (ELISA) was performed to assess for the −308G>A genotypes. Quantitative polymerase chain reaction (qPCR) was carried out to compare the mRNA expression of TNF-α, TNFR1, TNFR2, RELA, and IL6 in the corneal tissues of 20 KC patients and 20 donor controls.

Results: The −308G>A genotype GA was found to be significantly associated with KC development (dominant model [odds ratio (OR) = 6.67 (95% confidence interval [CI] = 4.28–10.42), P < 0.001]) and allele-A (OR = 4.30, 95%CI = 2.93–6.34, P < 0.001). TNF-α serum levels were significantly raised in patients with GA genotype (196.5 ± 69.5 pg/mL) compared to reference genotype GG (21.7 ± 8.2 pg/mL) (P < 0.0001). There was a significant overexpression of TNF-α (P = 0.002), TNFR2 (P = 0.0001), RELA (P = 0.0117), and IL6 (P = 0.0007) in the KC corneal tissues as compared to the control.

Conclusions: The GA genotype of the TNF-α −308G>A polymorphism is a significant genetic risk factor for the pathogenesis of KC. Moreover, this single nucleotide polymorphism (SNP) was observed to be associated with deregulated expression of downstream molecules, thus further reinforcing the role of the inflammatory pathway components in the development of KC.

Keratoconus (KC) is an ectatic eye condition characterized by a central or paracentral corneal thinning and an abnormal outward protrusion of the cornea. These structural changes result in irregular astigmatism, myopia, decreased corneal transparency, and its refractive power, thus affecting vision.1,2 Progression of KC is one of the major causes of corneal transplants in young adults worldwide. The disease pathogenesis is still inconclusive; being a multifactorial disease, KC has been shown to be associated with both genetic and environmental factors. 
Previous investigations into disease pathogenesis have implicated the role of oxidative stress–induced inflammation in disease progression. Keratoconic tissues were found to have an imbalance in the expression of aldehyde dehydrogenase 3 (ALDH3), superoxide dismutase (SOD), and catalase, which are involved in the elimination of reactive oxygen species (ROS), reactive aldehydes, and nitric oxides (NO). Inability of the diseased cornea to remove these reactive species results in enhanced oxidative stress, which triggers inflammation and apoptosis of the affected cell.3 In addition, exogenous factors including UV radiation, allergens, and mechanical trauma also harm the corneal cells and stimulate the production of ROS.4 Recent studies on keratoconic tissues have demonstrated higher expression of proinflammatory marker tumor necrosis factor α (TNF-α), cytokine nuclear factor κβ (NF-κβ), interleukin 6 (IL-6), and anti-inflammatory marker transforming growth factor β (TGF-β)5,6; in addition, quantitative studies by Balasubramanian et al.,7,8 Sorkhabi et al.,9 Lema and Durán,10 and Jun et al.11 (IOVS 2009:ARVO E-Abstract 3533) have shown increased levels of IL-6 in keratoconic tears. Studies on corneal thinning disorders including KC have also reported aberration in the DNA repair mechanism and accumulation of immune cells and increased TNF-α.12,13 Pannebaker et al.,14 Lema and Durán,10 Sorkhabi et al.,9 Balasubramanian et al.,7 and Jun et al.11 found higher concentration of TNF-α in tears and corneal tissue of KC samples; in addition, Kim et al.15 also reported increased keratocyte apoptosis in KC corneas. These findings therefore have shifted the KC disease paradigm from noninflammatory to inflammatory corneal disorder. However, the exact inflammatory mechanism in disease manifestation is yet to be established. A TNF-α promoter region functional single nucleotide polymorphism (SNP: rs1800625; −308G>A) has been reported to have clinical significance in different diseases, because this SNP affects TNF-α transcription. In this SNP the presence of the A-allele increases the binding of transcription factor to the promoter region of TNF-α, thereby altering its expression.16,17 
Being a proinflammatory cytokine, TNF-α is produced by many cell types in response to inflammation, infection, and other environmental stresses. In inflammation, TNF-α interacts with its receptors, tumor necrosis factor receptor 1 (TNFR1) and tumor necrosis factor receptor 2 (TNFR2), thus initiating the activation of the caspases cascade and consequently two transcription factors, activation protein-1 (AP1) and NF-κβ.18 Activated NF-κβ therefore induces cytokines that trigger the immune response, which include activation of TNF-α itself, IL-1, IL-6, and IL-8, as well as adhesion molecules including vascular cell adhesion molecule 1 (VCAM-1) and intracellular cell adhesion molecule 1 (ICAM-1), which lead to the recruitment of leukocytes at the site of inflammation.19 
Genetically, the TNF-α −308G>A A-allele results in overexpression of TNF-α, which consequently affects its downstream molecules. To date no study has been conducted to validate the genetic role of TNF-α −308G>A SNP in the development of KC and its consequent effect on the expression of important components of the inflammatory pathway. We therefore investigated the genetic role of inflammatory pathway component TNF-α, and its consequent effect on the downstream molecules TNFR1, TNFR2, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), and IL-6 in KC manifestation. This was done by genotyping TNF-α −308G>A polymorphism and assessing its effect on TNF-α serum levels. In addition, we performed expression profiling of TNF-α and its downstream components in corneal tissue of KC patients and donor controls. 
Methods
Ethics Statement
The current study was approved by the Ethics Review Board of the Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, and conforms to the tenets of the Declaration of Helsinki. The study purpose was explained to all the participants and written consent was obtained prior to blood and tissue sample collection. The donor corneas were used as controls during the analysis. 
Study Design and Sample Collection
The study comprised genotyping TNF-α promoter sequence polymorphism rs1800629 (−308G>A) in a total of 510 individuals (257 KC patients and 253 healthy controls) and serum and mRNA expression profile determination of TNF-α and its downstream molecules TNFR1, TNFR2, RELA, and IL6 in corneal tissues in a total of 40 corneal tissues, including 20 keratoconic and 20 donor control corneas. The blood samples of sporadic KC cases were collected from Armed Forces Institute of Ophthalmology (AFIO), Rawalpindi, Pakistan, and Al-Shifa Eye Trust Hospital, while tissue samples were obtained from the AFIO. The mean age of the patients and that of the control (negative for any eye disease, inherited disorder, or major health problem) was 20.84 years and 21.38 years, respectively. The disease inclusion criteria in the present study were the inclusion of cases with KC as a prime disease or KC due to infection or excessive eye rubbing but with a positive family history; cases without a family history who developed KC secondary to infection or excessive rubbing, or cases with trauma to the eye or having other corneal pathologies, were excluded. The diagnosis of KC patients was carried out by corneal pachymetry having mean corneal thickness of 462.5 μm ± 8 (normal thickness is 554.9 μm ± 7.4) and topography with the keratometric reading (k-max) greater than 48 D (normal k-value is less than 45 D). 
Genotyping of TNF-α −308G>A
Genomic DNA was isolated from whole blood by a standard phenol–chloroform extraction method.20 Briefly, the protocol consisted of genotyping TNF-α rs1800629 by the polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method, using a pair of forward primer 5′-AGG CAA TAG GTT TTG AGG GCC AT-3′ and reverse primer 5′-GTA GTG GGC CCT GCA CCT TCT-3′ as described previously.21 PCR was performed in a total volume of 25 μL containing 40 to 50 ng genomic DNA, 0.2 mM deoxynucleotide triphosphates (dNTPs), 1.5 mM MgCl2, 0.2 μM each of reverse and forward primer, 1× Taq buffer, and 1 U Taq polymerase. The thermal cycling was carried out by an initial denaturation of the genomic DNA at 95°C for 7 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, primer annealing at 60°C for 30 seconds, and extension at 72°C for 45 seconds, followed by a final extension step at 72°C for 10 minutes. 
The PCR product (12 μL) was subjected to restriction enzyme digestion at 37°C overnight with 10 U NcoI restriction enzyme according to the manufacturer's instructions (Fermentas, Burlington, ON, Canada). The digested PCR products were resolved on 4% agarose gels, where the PCR products carrying the homozygous variant A-allele remained uncut (212 bp), while the one carrying the wild-type G-allele homozygously was digested into two fragments of 192 and 20 bp and the heterozygous genotpyes GA had three fragments (212, 192, and 20 bp) (Fig. 1). The RFLP genotyping was further validated by Sanger sequencing of 10% of the respective RFLP genotypes. 
Figure 1
 
Restriction fragment length polymorphism (RFLP) analysis of −308G>A (rs1800629) of TNF-α polymorphism. The Nco1 restriction enzyme–digested polymerase chain reaction (PCR) products were resolved using 4% agarose gel electrophoresis. Lanes 1, 3, 4, 5, and 6 represent homozygous GG genotype (192- and 20-bp fragment). The 20-bp fragment, being smaller in size, was not visible. Lane 7 represents heterozygous genotype GA (212-, 192-, and 20-bp fragments), while lane 2 represents homozygous AA genotype (undigested 212-bp fragment); lane 8 is of the marker ladder.
Figure 1
 
Restriction fragment length polymorphism (RFLP) analysis of −308G>A (rs1800629) of TNF-α polymorphism. The Nco1 restriction enzyme–digested polymerase chain reaction (PCR) products were resolved using 4% agarose gel electrophoresis. Lanes 1, 3, 4, 5, and 6 represent homozygous GG genotype (192- and 20-bp fragment). The 20-bp fragment, being smaller in size, was not visible. Lane 7 represents heterozygous genotype GA (212-, 192-, and 20-bp fragments), while lane 2 represents homozygous AA genotype (undigested 212-bp fragment); lane 8 is of the marker ladder.
Serum Concentrations of TNF-α −308G>A Genotypes
The TNF-α concentration in the serum samples of patients having heterozygous genotype GA and reference homozygous GG genotype was determined by using the Human TNF-α enzyme-linked immunosorbent assay (ELISA) Kit (BioLegend's LEGEND MAX, San Diego, CA, USA) per the manufacturer's instructions. 
mRNA Expression Profile
Corneal tissue from KC patients and deceased donors were collected in 2 mL cryovial (Thermo Scientific, Darmstadt, Germany) prefilled with RNAlater (Applied Biosystems, Carlsbad, CA, USA) and stored at −80°C till further use. Total RNA from the tissues was extracted using All-in-one purification kit (NORGEN; Biotek Corp., Thorold, Canada) following the manufacturer's instructions and quantified using a nanospectrophotometer (IMPLEN, München, Germany). The total RNA was then reverse transcribed using RevertAid First Strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) following the manufacturer's instructions. The undiluted cDNA products were stored at −20°C until further use. 
The primers for real-time quantitative polymerase chain reaction (qPCR) for expression analysis of TNF-α, TNFR1, TNFR2, RELA, and GAPDH were designed using an online primer designing tool (https://eu.idtdna.com/site; in the public domain; Integrated DNA Technologies, Coralville, IA; Table 1). The qPCR was performed using maxima SYBR Green/ROX qPCR kit (Thermo Scientific) per the manufacturer's instructions. The relative expression levels of TNF-α, TNFR1, TNFR2, RELA, and IL6 were computed using the 2ΔΔct method. GAPDH was used as an internal control for data normalization. 
Table 1
 
Primer Sequences of TNF-α, TNFR1, TNFR2, RELA, IL6, and GAPDH Gene
Table 1
 
Primer Sequences of TNF-α, TNFR1, TNFR2, RELA, IL6, and GAPDH Gene
Statistical Analysis
For the statistical analysis of the genotype data, χ2 test and logistic regression analysis under dominant as well as recessive models were performed where P ≤ 0.05 was taken as statistically significant, to which Yates' correction was applied to check the strength of P values and to correct type I error. 
For expression analysis, the difference between groups (cases and controls) according to the relative expression levels of TNF-α, TNFR1, TNFR2, RELA, and IL6, and age, sex, and disease severity was computed using the nonparametric Mann-Whitney U test. Statistical analysis was performed using SPSS software version 23 (SPSS, Inc., Chicago, IL, USA). P values were 2-sided, and a value equal to or less than 0.05 was considered to be statistically significant. 
Results
Genotyping
In the control group the SNP was in Hardy-Weinberg equilibrium (HWE: χ2 = 3.47, P = 0.06; Court lab - HW calculator.xls), while in the cases, the results were not consistent with HWE (χ2 = 22.02, P = 0.000003). A significant difference in the genotype distribution among the cases and controls was observed (χ2 = 87.9, P < 0.001), where the GA genotype frequency was higher in KC cases (52%) than controls (13.4%) and was found to be associated with the disease under the dominant model (DM) (odds ratio [OR] = 6.67, 95% confidence interval [95%CI] = 4.28–10.42, P < 0.001). In addition, allele-A was also found to be significantly higher in the KC (28%) cases when compared to healthy controls (8%) and showed disease association (χ2 = 66.47, P < 0.001; OR = 4.30, 95%CI = 2.93–6.34, P < 0.001; Table 2). 
Table 2
 
Genotype and Allele Frequency Distribution and Logistic Regression Analysis of TNF-α −308G>A Polymorphism in KC Cases and Controls
Table 2
 
Genotype and Allele Frequency Distribution and Logistic Regression Analysis of TNF-α −308G>A Polymorphism in KC Cases and Controls
Serum Concentration
The TNF-α serum levels were significantly higher in patients with disease-associated GA genotype (196.5 ± 69.5 pg/mL) compared to those having reference homozygous genotype GG (21.7 ± 8.2 pg/mL, P < 0.0001; Fig. 2). 
Figure 2
 
Box plot of the TNF-α (−308G>A) genotype serum analysis. The serum profiles of risk genotype GA (left red box) and reference GG genotype (right blue box) are shown. Asterisks indicate level of significant difference between analyzed groups where *** is highly significant.
Figure 2
 
Box plot of the TNF-α (−308G>A) genotype serum analysis. The serum profiles of risk genotype GA (left red box) and reference GG genotype (right blue box) are shown. Asterisks indicate level of significant difference between analyzed groups where *** is highly significant.
qPCR Analysis
Quantitative PCR revealed a significant upregulation of TNF-α (P = 0.002), TNFR2 (P = 0.0001), RELA (P = 0.012), and IL6 (P = 0.0007) in keratoconic tissues compared to the donor control corneas (Fig. 1). Though there was a slight difference in the expression of TNFR1 (mean = −8.6, median = −8.9) as compared to the controls (mean = −9.5, median = −9.7), this was not statistically significant (P = 0.239) (Fig. 3). 
Figure 3
 
The graphs represent the relative expression of TNF-α (A), TNFR1 (B), TNFR2 (C), RELA (D), and IL6 (E) gene. Values greater than zero represent significantly higher expression level, while less than zero represents low expression. Boxes represent mean, median, and standard deviation of the normalized gene (TNF-α, TNFR1, TNFR2, RELA, and IL6) expression (ΔCq) levels. N.S. indicates nonsignificant difference between keratoconic and control corneal tissues; asterisks indicate level of significant difference between analyzed groups where * = significant, ** and *** = highly significant.
Figure 3
 
The graphs represent the relative expression of TNF-α (A), TNFR1 (B), TNFR2 (C), RELA (D), and IL6 (E) gene. Values greater than zero represent significantly higher expression level, while less than zero represents low expression. Boxes represent mean, median, and standard deviation of the normalized gene (TNF-α, TNFR1, TNFR2, RELA, and IL6) expression (ΔCq) levels. N.S. indicates nonsignificant difference between keratoconic and control corneal tissues; asterisks indicate level of significant difference between analyzed groups where * = significant, ** and *** = highly significant.
Discussion
In the present study we investigated the role of inflammatory pathway components in the development of KC and a found genetic association of TNF-α promoter region SNP −308G>A, with resultant elevated TNF-α serum levels in carriers of GA genotype, and also observed upregulation of TNF-α, TNFR2, RELA, and IL6 in keratoconic tissues, thus strengthening the role of autoinflammation in disease pathogenesis. 
TNF-α is a proinflammatory cytokine involved in systemic inflammation, apoptosis, and activation of other immune cells,22 and has a potential role in corneal wound healing. Imanishi et al.23 have reported TNF-α involvement in the regulation of corneal cells and in the maintenance of corneal transparency. In the present study, the frequencies of risk allele-A and the genotype GA were significantly higher in KC patients as compared to controls and were found to be associated with the disease. These results were consistent with the finding of Aoki et al.,24 Li et al.,25 and Zhang et al.,26 who also found −308 G>A risk allele-A to be associated with other inflammatory diseases like asthma, Graves' disease, chronic obstructive pulmonary disease, and psoriasis.27 We could not compare our results with other studies of KC as the current report is the first one to genotype this SNP in KC cases and also to investigate its effect on the expression of the gene. Polymorphisms in the noncoding regulatory regions including the promoter region of the genes have been reported to alter the gene expression levels, and these functional SNPs therefore represent an important but relatively unexplored class of genetic variations.28 Therefore, in this study, the predictive effect of −308 promoter SNP present upstream of the transcription start site with G-allele known as TNFA1 and A-allele as TNFA2 was analyzed by performing serum concentration comparison of the GA genotype patients with those having the GG genotypes. In addition, mRNA expression analysis of the KC corneas was done, which revealed elevated levels of TNF-α in the serum of GA genotype carriers and also in the keratoconic tissues. These results were found to be consistent with the findings of Gheita et al.,29 Abraham and Kroeger,17 Laddha et al.,30 Kroeger et al.,16 and Elahi et al.,31 who also observed elevated serum levels and mRNA expression of TNF-α in carriers of TNFA2 resulting in imbalance of inflammatory cytokines, leading to susceptibility to inflammatory diseases. The higher mRNA expression of TNF-α in keratoconic tissues in the present study was found consistent with the results of Shetty et al.32 and Cheung et al.,33 who also found upregulation of TNF-α at the mRNA level in epithelial and stromal cells of keratoconic cornea. In contrast to the current findings, Jun et al.11 have reported significantly decreased levels of TNF-α in the tears of KC patients. However, in none of these studies was the −308G>A SNP role investigated. 
TNF-α triggers its downstream molecules including two of its transmembrane receptors, TNFR1 and TNFR2; an ocular tissue–specific transcription factor RELA (NF-κβ); and other proinflammatory cytokines, IL-6, IL-1B, IL-11 and TNF-α itself. In the case of TNF-α binding to TNFR1, which contains a death domain, NF-κβ is activated, which stimulates the inflammatory response and in parallel, apoptosis of the cell. However, binding of TNF-α to TNFR2, which lacks the death domain, leads to the activation of NF-κβ, which triggers the inflammatory response.34 In the current study, in the KC corneal tissues, higher expression levels of TNFR2 were observed while TNFR1 expression levels did not show statistically significant difference compared to controls. Also, the higher levels of TNFR2 in the presence of increased concentrations of TNF-α can lead to activation of signaling pathways beyond the physiological levels, thus resulting in inflammation. The results were found consistent with the findings of Mohan et al.,35 Wan et al.,36 and Nakazawa et al.,37 who found upregulation of TNFR2 in the human corneal fibroblast, retinal ganglion cells in a mouse model of glaucoma, and retina of an oxidative-stress–induced retinopathy mouse model. In contrast to the current findings, Bryant-Hudson et al.,38 Shivanna et al.,39 and Tezel et al.40 have reported significantly lower expression levels of TNFR2 and higher expression levels of TNFR1 in mouse cornea infected with herpes simplex virus 1, cultured bovine corneal endothelial cells, and retina of normal and glaucomatous eyes. In the present study we found significantly higher levels of RELA in the corneal tissues of KC; these results were consistent with other findings, where an upregulation of NF-κβ was observed in human corneal fibroblasts, keratoconic and normal donor human corneas, and retina of retinal degenerative mice.5,35 This higher expression of NF-κβ in KC tissue compared to control tissues shows chronic and constitutive activation that further results in the constitutively higher expression of the other proinflammatory cytokines including IL6, IL1B, and IL11; this chronic activation may lead to chronic inflammation and thus the resultant pathology.41 Among these proinflammatory cytokines, IL-6 promoter region contains a putative binding site for NF-κβ, which explains the importance of NF-κβ for the activation of IL-6.42 The IL-6 higher expression in the current study was consistent with Jun et al.,11 Becker et al.,43 and Balasubramanian,7 who also observed IL6 upregulation in human KC cornea and tear fluid of KC patients. This further supports the role of the inflammatory pathway in KC development, as IL6 is among those proinflammatory cytokines that are primarily produced at sites of chronic and acute inflammation and induces an inflammatory response through its receptor.44 
None of the previous studies on KC have analyzed these major inflammatory pathway molecules to explore the genetic role of TNF-α and its downstream molecules specifically in KC corneal tissues. Our findings have shown higher expression levels of TNF-α not only in serum but also in tissues; moreover, increased expression of TNFR2, RELA, and IL6 also in Pakistani KC corneal tissues supports the idea that chronic inflammation may play a key role in KC development. To our knowledge this is the first study to report the predictive effect of TNF-α −308G>A promotor region polymorphism on the expression of TNF-α itself and its downstream signaling molecules including TNFR1, TNFR2, RELA, and IL6 in the corneal tissue. Our data therefore show that upregulation of TNF-α may lead to chronic activation and/or expression of downstream signaling pathway components involving TNFR2, RELA, and IL-6, hence leading to inflammatory pathway–induced disease manifestation. 
In conclusion, the positive genetic association of the TNF-α promoter region SNP −308G>A with KC in the current study has delineated the functional variant of an inflammatory pathway component for predisposition of this complex condition. Moreover, elevated serum TNF-α levels and mRNA expression upregulation of TNF-α, TNFR2, RELA, and IL6 in the keratoconic tissues suggest a strong correlation of TNF-α–induced higher expression of inflammatory pathway molecules in disease manifestation, therefore implicating KC as an inflammatory condition. 
Acknowledgments
The authors thank all participants for their cooperation in the study. 
Supported by the COMSATS Institute of Information Technology Islamabad Core Grant to RQ and Higher Education Commission Pakistan Grant No. 3738 to MA and RQ. 
Disclosure: M. Arbab, None; S. Tahir, None; M.K. Niazi, None; M. Ishaq, None; A. Hussain, None; P.M. Siddique, None; S. Saeed, None; W.A. Khan, None; R. Qamar, None; A.M. Butt, None; M. Azam, None 
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Figure 1
 
Restriction fragment length polymorphism (RFLP) analysis of −308G>A (rs1800629) of TNF-α polymorphism. The Nco1 restriction enzyme–digested polymerase chain reaction (PCR) products were resolved using 4% agarose gel electrophoresis. Lanes 1, 3, 4, 5, and 6 represent homozygous GG genotype (192- and 20-bp fragment). The 20-bp fragment, being smaller in size, was not visible. Lane 7 represents heterozygous genotype GA (212-, 192-, and 20-bp fragments), while lane 2 represents homozygous AA genotype (undigested 212-bp fragment); lane 8 is of the marker ladder.
Figure 1
 
Restriction fragment length polymorphism (RFLP) analysis of −308G>A (rs1800629) of TNF-α polymorphism. The Nco1 restriction enzyme–digested polymerase chain reaction (PCR) products were resolved using 4% agarose gel electrophoresis. Lanes 1, 3, 4, 5, and 6 represent homozygous GG genotype (192- and 20-bp fragment). The 20-bp fragment, being smaller in size, was not visible. Lane 7 represents heterozygous genotype GA (212-, 192-, and 20-bp fragments), while lane 2 represents homozygous AA genotype (undigested 212-bp fragment); lane 8 is of the marker ladder.
Figure 2
 
Box plot of the TNF-α (−308G>A) genotype serum analysis. The serum profiles of risk genotype GA (left red box) and reference GG genotype (right blue box) are shown. Asterisks indicate level of significant difference between analyzed groups where *** is highly significant.
Figure 2
 
Box plot of the TNF-α (−308G>A) genotype serum analysis. The serum profiles of risk genotype GA (left red box) and reference GG genotype (right blue box) are shown. Asterisks indicate level of significant difference between analyzed groups where *** is highly significant.
Figure 3
 
The graphs represent the relative expression of TNF-α (A), TNFR1 (B), TNFR2 (C), RELA (D), and IL6 (E) gene. Values greater than zero represent significantly higher expression level, while less than zero represents low expression. Boxes represent mean, median, and standard deviation of the normalized gene (TNF-α, TNFR1, TNFR2, RELA, and IL6) expression (ΔCq) levels. N.S. indicates nonsignificant difference between keratoconic and control corneal tissues; asterisks indicate level of significant difference between analyzed groups where * = significant, ** and *** = highly significant.
Figure 3
 
The graphs represent the relative expression of TNF-α (A), TNFR1 (B), TNFR2 (C), RELA (D), and IL6 (E) gene. Values greater than zero represent significantly higher expression level, while less than zero represents low expression. Boxes represent mean, median, and standard deviation of the normalized gene (TNF-α, TNFR1, TNFR2, RELA, and IL6) expression (ΔCq) levels. N.S. indicates nonsignificant difference between keratoconic and control corneal tissues; asterisks indicate level of significant difference between analyzed groups where * = significant, ** and *** = highly significant.
Table 1
 
Primer Sequences of TNF-α, TNFR1, TNFR2, RELA, IL6, and GAPDH Gene
Table 1
 
Primer Sequences of TNF-α, TNFR1, TNFR2, RELA, IL6, and GAPDH Gene
Table 2
 
Genotype and Allele Frequency Distribution and Logistic Regression Analysis of TNF-α −308G>A Polymorphism in KC Cases and Controls
Table 2
 
Genotype and Allele Frequency Distribution and Logistic Regression Analysis of TNF-α −308G>A Polymorphism in KC Cases and Controls
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