Association between Allergic Conjunctivitis and Cytokine Related Genetic Polymorphisms in a Pediatric Population

Research Article

Austin J Clin Ophthalmol. 2018; 5(1): 1086.

Association between Allergic Conjunctivitis and Cytokine Related Genetic Polymorphisms in a Pediatric Population

Demir S¹*, Kiliç R², Ates ö³, Benli I4, Alim S¹, Ersekerci TK5 and Günes A¹

¹Department of Ophthalmology, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey

²Ahi Evran University Faculty of Medicine, Department of Ophthalmology, Kirsehir, Turkey

³Department of Medical Biology, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey

4Department of Biochemistry, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey

5Darende State Hospital, Department of Ophthalmology, Malatya, Turkey

*Corresponding author: Selim Demir, Department of Ophthalmology, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey

Received: February 05, 2018; Accepted: March 02, 2018; Published: March 22, 2018


Purpose: Cytokines play important role in inflammatory and allergic diseases. Recent studies have demonstrated that IL-4 and IL-10 single nucleotide polymorphisms (SNPs) are associated with allergic diseases such as asthma or allergic rhinitis. Therefore, we aimed to evaluate the possible association between these SNPs and allergic conjunctivitis including seasonal allergic conjunctivitis (SAC) and vernal keratoconjunctivitis (VKC) in a pediatric population.

Methods: A case-control association study of 108 patients with allergic conjunctivitis (69 SAC and 39 VKC) and 95 controls were recruited. IL-4- 590C/T (rs2243250), IL-10-592C/A (rs1800872), IL-10-819C/T (rs1800871), and IL-10-1082G/A (rs1800896) SNPs were evaluated by real-time polymerase chain reaction-based DNA analysis. The frequency of alleles and distribution of genotypes were assessed by the chi-squared test.

Results: All studied polymorphisms satisfied Hardy-Weinberg equilibrium. Age and sex distributions were similar between the patients and control groups (p>0.05). There were no significant differences between patients and controls in the distributions of genotype and allele frequencies of the studied SNPs (p>0.05).

Conclusion: In this study, there was no association of the analyzed SNPs located in IL-4 and IL-10 with allergic conjunctivitis, suggesting that SNPs included in our study have no significant role in causing allergic conjunctivitis in the pediatric population.

Keywords: Allergic conjunctivitis; Interleukin 4; Interleukin 10; Single nucleotide polymorphism


Allergic conjunctivitis is a common and complex inflammatory condition of the eye. It has a broad-spectrum from acute mild form of seasonal/perennial allergic conjunctivitis (SAC/PAC) to chronic vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) [1]. The signs and symptoms of allergic conjunctivitis are influenced by many factors including genetics, environmental factors, ocular microbial flora, and immune regulation mechanisms [2]. The most important pathogenetic role of forming SAC and PAC are common allergens binding to immunoglobulin E (IgE) on mast cells and triggering type I hypersensitivity response. T helper (Th) 2-cell-mediated responses and eosinophil infiltration as well as IgEmediated hypersensitivity reactions are the leading pathogenetic way to forming VKC and AKC [3]. However, the causes leading to allergic conjunctivitis is not fully known.

Allergic conjunctivitis has a significant impairment of selfrated health status during an ocular allergy episode [4]. Although allergic conjunctivitis is seen at all ages, it occurs in childhood more often. Other allergic diseases such as allergic rhinitis, asthma, atopic dermatitis or food allergy which may affect an individual’s quality of life often co-exist with allergic conjunctivitis [5].

Cytokines play important role in recruitment and activation of leukocytes. Pro-inflammatory cytokines such as interleukin 4 (IL-4) and tumor necrosis factor-alpha (TNF-a) are over-expressed mainly during the active inflammatory phase of allergic conjunctivitis [6,7], while anti-inflammatory cytokines such as IL-10 and IL-12 may lower-expressed [8]. Previous studies have been also demonstrated that polymorphism of IL-4 [9,10] and IL-10 [8,11] are related with allergic diseases. Therefore, in this study, we aimed to evaluate possible association between allergic conjunctivitis and polymorphisms of IL-4 (590C/T), IL-10 (-592C/A), IL-10 (-819C/T), and IL-10 (-1082G/A) genes in a pediatric population.

Materials and Methods

Study population

A total of 203 pediatric subjects recruited from ophthalmology clinics of Gaziosmanpasa University Hospital, Sivas Numune Hospital, and Malatya Darende State Hospital were enrolled in this study. The study population consisted of 108 patients diagnosed with either SAC or VKC, and 95 control subjects. There were 69 (63.9%) patients with SAC and 39 (36.1%) patients with VKC. Age-matched healthy volunteers with non-allergic ocular complaints with refractive errors were selected randomly as a control group. The protocol was approved by the local ethics committee (13-KAEK-001) the study and written informed consent was obtained from each patient. The study was carried out in accordance with the Declaration of Helsinki.

Ocular examination

Subjects were asked about their symptoms with special attention to age at onset of symptoms, seasonal exacerbation, and personal and family history of allergic diseases, such as rhinitis, asthma, dermatitis, food allergies, and allergies caused by medication. The diagnosis of SAC was made based on patient history, and characteristic clinical signs and symptoms [12]. Diagnosis of VKC was made based on the three diagnostic criteria: (1) presence of conjunctival tarsal (cobblestone) and/or limbal papillae; (2) presence of eosinophils in the conjunctival scraping; and (3) persistent or recurrent symptoms of conjunctivitis [13]. The confirmation of VKC was made in the presence of at least two of the three findings. Exclusion criteria included giant papillary conjunctivitis and AKC. Children who have symptoms and/or findings of any systemic allergy were not included to this study, except for allergic rhinitis.

DNA isolation

Blood specimens were drawn into EDTA containing tubes, and genomic DNA samples were extracted from the peripheral leukocytes of the collected venous blood by High Pure PCR Template Preparation Kit (Roche Molecular Biochemical’s, Mannheim, Germany) according manufacturer’s instructions.


The procedure for genotyping the IL-4 (-590C/T) and IL-10 (-592C/A, -1082G/A, and -592C/A) polymorphisms were described previously [14,15]. Genomic DNA was isolated from peripheral leukocytes from EDTA anticoagulated blood using the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals). Melting curve analyzing was performed to genotype the subjects, with LC Fast Start Master Hybridization Probes buffer (Roche Diagnostics Inc), primers, and probes (Metabion international AG, Martinsried, Deutschland) and a Light Cycler 480 II Real-Time PCR System (Roche Diagnostics). The PCR was performed with 50ng DNA in a total volume of 10μl containing 1μl buffer, 4nM MgCl2, 0.5mM of each primer and 0.4mM of each hybridization probe according to the manufacturer’s instructions for 45 cycles of denaturation (95°C, 5s), annealing (58°C or 63°C, 10s) and extension (72°C, 20s). After amplification, a melting curve was generated by holding the reaction at 45°C for 30s and then heating slowly to 95°C with a ramp rate of 0.2°C/s. The fluorescence signal was plotted against temperature to give melting curves for each sample.

Statistical analysis

After the data obtained during the investigation had been coded, they were analyzed using SPSS 15.0 (SPSS Inc). Normality tests were applied to all measurement variables. Data were presented as mean ± SD. A P value of <0.05 was accepted as the level of statistical significance. The Mann–Whitney U test was used to compare the results of age and sex between the groups. The frequency of studied single nucleotide polymorphisms (SNPs) alleles and the distribution of genotypes were assessed by the chi-squared test. The ?2 test was used to evaluate the Hardy-Weinberg equilibrium for the distribution of the genotypes of the patients and the controls.


Of the 108 patients, 60 were males (55.5%) and 48 were females (44.5%). There were 52 males (54.7%) and 43 females (45.3%) in the control group. The mean age of patients and controls were 12.4±2.3 years and 12.9±2.4 years, respectively. There was no statistical difference between groups in age or sex (p=0.188 and p=0.907, respectively).

Detailed presentation of the distribution of the genotype and allele frequency of IL-4-590 C/T, IL-10-592C/A, IL-10-819C/T, and IL-10-1082G/A SNPs between the SAC or VKC patients and controls are shown in Table 1. The observed and expected frequencies of the polymorphism in both groups were within the Hardy–Weinberg equilibrium. There was no significant differences in the distributions of genotype and allele frequencies of the studied SNPs between patients and controls (p>0.05).

Citation: Demir S, Kiliç R, Ates ö, Benli I, Alim S, Ersekerci TK, et al. Association between Allergic Conjunctivitis and Cytokine Related Genetic Polymorphisms in a Pediatric Population. Austin J Clin Ophthalmol. 2018; 5(1): 1086.