Analysis of interleukin-6 gene polymorphism and its serum levels in Indian age-related macular degeneration patients

• Introduction
• Methods
• Results
• Discussion

Abstract

Background: Age-related macular degeneration (AMD) is a complex condition involving multiple factors. The condition is associated with numerous inflammatory indicators, including cytokines. Single-nucleotide polymorphisms in cytokine genes can also modify gene expression, perhaps contributing to the development of the disease. The objective of the present study was to examine the correlation among IL-6 SNPs (rs1800795, rs1800796, and rs1800797) and the serum levels of IL-6 in AMD patients treated at the Regional Institute of Ophthalmology of Pt. B.D. Sharma PGIMS, Rohtak (Haryana), India.

Methods: This case-control study included 131 patients diagnosed with AMD using precise ophthalmic examinations, such as slit lamp examination, fundoscopy, and ocular coherence tomography. To provide a basis for comparison, we also enlisted 100 healthy individuals as controls. Serum IL-6 protein levels were measured in both patients and controls using an enzyme-linked immunosorbent assay kit (ELISA). Genotyping IL-6 SNPs was performed using the PCR and DNA Sanger sequencing technique.

Results: IL-6 serum levels were considerably elevated in individuals with AMD compared to the control group (p < 0.05). Statistically significant differences were seen in the genotype frequencies of rs1800795 (p = 0.027) and rs1800797 (p = 0.0011) among the AMD patients and the healthy controls. Furthermore, strong correlations were observed between rs1800795 and the likelihood of developing AMD based on the heterozygous (OR = 2.04; p = 0.025), dominant (OR = 1.80; p = 0.035), and over-dominant models (OR = 2.10; p = 0.0094). Additionally, there were notable associations between rs1800797 and vulnerability to AMD through heterozygous (OR = 3.21; p = 0.009), dominant (OR = 2.74; p = 0.004), and over-dominant (OR = 3.11; p = 0.002) models. The rs1800795, rs1800796, and rs1800797 haplotypes C-G-A and G-G-A were linked to an elevated risk of AMD (p = 0.005, p = 0.024. respectively).

Conclusions: Our findings indicated a significant elevation in IL-6 serum levels among the AMD patient group compared to the control group. The interleukin-6 gene polymorphisms rs1800795 and rs1800797 were linked to an elevated risk of AMD in our study population.

Age-related macular degeneration (AMD) is a multifactorial ailment defined by the gradual degradation of retinal cells. This condition results in irreversible visual loss in individuals aged 50 years and above [1,2]. Inflammation is a prevalent element in the development of many diseases, including AMD. It is often described as a complicated signaling cascade that is essential for recognizing and eliminating foreign chemicals as well as encouraging tissue recovery. However, continuous inflammation and excessive production of proinflammatory molecules can lead to chronic illnesses such as AMD, malignancies, type 2 diabetes mellitus, and neurologic disorders [3].

The age-related eye disease study (AREDS) identifies three main stages of AMD—early, intermediate, and late. People with early AMD usually have no signs of the disease and are identified by the presence of drusen, along with retinal pigment defects. Drusen is the accumulation of lipids, proteins, and collagen in the retinal pigment epithelium (RPE) as well as Bruch’s membrane [4]. In the intermediate stage, there is evidence of at least one large drusen, numerous small to medium-size drusen, or geographic atrophy (GA) that does not affect the macular region. There are two types of late-stage AMD: dry or atrophic AMD alongside GA affecting the RPE, and neovascular or exudative AMD that involves choroidal neovascularization (CNV) and detached units in the RPE. These conditions can lead to central vision impairment [5].

The exact causes of AMD remain unclear; hence, it is considered a multifactorial condition linked to genetic predisposition, environmental factors, disrupted lipid metabolism, immune responses leading to chronic inflammation, oxidative stress, and extracellular matrix dysfunction [6]. Progressive damage to the photoreceptors is seen in late-stage AMD due to abnormal RPE function and an imbalance in oxygen and nutrient flow between the outer retina and blood vessels, leading to increased permeability of the choriocapillaris [7]. Drusen formation, inflammation, and subsequent neurodegeneration in the macular region are characteristic features of the disease [8].

It is widely recognized that inflammation plays an important role in the progression of AMD. The release of inflammatory mediators in the posterior segment of the eye can result in dysfunction of the RPE, thereby contributing to the progression of AMD. Research on AMD patients has explored single nucleotide polymorphisms (SNPs) in various inflammatory factors, such as toll-like receptor 3, complement factor H, CX3C chemokine receptor 1 (CX3CR1), interleukin 6 (IL-6), TNF-alpha, IL-17, 1L-18, interleukin-1 beta (IL-1β), and interleukin 8 (IL-8) [9–13]. Genetic polymorphisms within genes influence individual differences in cytokine production, making AMD a complex condition that is not easily classified as a single-gene disorder. As the gene responsible for AMD exhibits a high degree of variability and interference, it becomes rational to explore the genetic variants and evaluate the function of inflammatory mediators in AMD pathology [10].

IL-6 is a potent pro-inflammatory cytokine with multiple functions. It is involved in regulating inflammatory processes and immune responses and plays a role in an array of physiologic functions, such as hematopoiesis, metabolism, and tissue repair. IL-6 is synthesized by various cell types, such as immune system cells (macrophages and T cells), endothelial cells, fibroblasts, and RPE cells. Changes in the IL-6 gene can impact how the gene is transcribed and how the cytokine is released [14]. IL-6 plays an essential role in T-cell proliferation and differentiation, thereby influencing the inflammatory response. It also adds to the acute phase of inflammation by drawing neutrophils to the inflamed area, which in turn activates IL-6 receptors in nearby cells and the internal IL-6 signaling pathway. Transition from the acute phase to chronic inflammation involves the replacement of neutrophils with monocytes. Misregulation of IL-6 can lead to chronic inflammation and autoimmune disorders due to its pivotal role in the initiation and resolution of inflammation [15].

IL-6 induces the liver to produce acute-phase proteins, such as C-reactive proteins, serum amyloid A, and fibrinogen. It may also enhance endothelial permeability directly or indirectly through the stimulation of vascular endothelial growth factor (VEGF), resulting in increased vascular permeability and the onset of choroidal neovascularization (CNV) in AMD. Due to its high serum levels in AMD patients, IL-6 is considered a risk factor for CNV in AMD [16]. IL-6 gene polymorphisms have been linked to several ocular disorders, including AMD [11,16–23].

Over 50 SNPs and five common haplotypes have been discovered in the IL-6 gene; however, only three promoter region SNPs, −174G/C (rs1800795), −572G/C (rs1800796), and −597G/A (rs1800797), have been widely investigated in numerous immune-related disorders, including rheumatoid arthritis, multiple sclerosis, diabetes, allergic diseases, chronic periodontitis, systemic sclerosis, and cancer [24–28]. It has been reported that these promoter polymorphisms are important regulators of the IL-6 gene and downstream protein levels, both in vitro and in vivo [17], affecting its expression in numerous diseases. The −174G/C polymorphism, in particular, plays a crucial role in regulating IL-6 transcription and plasma levels, affecting disease susceptibility and inflammatory responses. The activity of the IL-6 promoter is also modulated by the other two polymorphic sites, which interact to control the impact of the −174G/C polymorphism. This intricate regulation underscores the significant role of these polymorphisms in IL-6-mediated pathophysiology [29].

Recent studies, particularly those focusing on specific populations, including the Indian population, have highlighted the potential involvement of IL-6 in susceptibility to ocular disorders [11,16,17,19–22,30–32]. These findings indicate that IL-6 is a promising candidate for AMD research.

Given these insights, our study focused on IL-6 gene SNPs rs1800795, rs1800796, and rs1800797 for evaluation as possible risk factors for the disease in our cohort of AMD patients. The current study aims to find and compare the serum levels of IL-6 in AMD patients and controls and to investigate the association of these levels with specific gene polymorphisms.

Symptoms used to diagnose AMD encompass the presence of drusen (small or large), subretinal CNV, hemorrhages, edema, and areas of geographic and RPE atrophy. Demographic features of the patients and controls are shown in Table 2. Sunlight exposure and gender differences were not statistically significant (p > 0.05) between the AMD and control groups. However, age differences and smoking status between the two groups were found to be statistically significant (p < 0.05).

Common ocular diseases, including uveitis, glaucoma, AMD, diabetic retinopathy, and cataract, can result in visual impairment and eventually lead to irreversible blindness [35], significantly diminishing quality of life. The development and progression of these conditions are heavily influenced by inflammation. While earlier research on inflammation in eye diseases has focused mainly on uveitis and dry eye disease, inflammation is now recognized as a factor in various other ophthalmic disorders. Identifying links between inflammatory biomarkers and ocular diseases could enable early diagnosis and timely intervention [22]. The objective of this study was to examine the potential relationship between IL-6 gene polymorphisms, serum levels, and AMD, a multifactorial condition with a complex cause.

Our study revealed that serum levels of IL-6 were significantly increased in AMD patients compared to controls, indicating an association between elevated IL-6 levels and AMD. Additionally, when analyzing the subtypes of AMD (dry and wet) separately against the controls, significant associations were found between IL-6 levels and AMD status for both types. These findings align with previous studies linking elevated IL-6 levels to AMD pathogenesis. For instance, Ambreen et al. (2015) reported significantly elevated serum IL-6 levels in patients compared to controls (p < 0.0001; Wilk’s λ = 0.064; F = 278.7), with a stronger association observed in individuals with dry AMD [11]. Čolak et al. (2017) observed that higher serum IL-6 levels were present exclusively in patients with neovascular AMD, while those with early AMD did not show increased levels (r = –0.999) [36]. Haas et al. (2015) observed significantly elevated serum IL-6 levels in patients with neovascular AMD (p = 0.037). The authors proposed that this could be partly linked to changes in visceral fat distribution in patients with AMD [37]. Klein and co-authors, on the other hand, published different results on the population of Beaver Dam, Wisconsin, which did not show an association between IL-6 serum levels and AMD (OR = 0.97; 95% CI = 0.80–1.17; p = 0.74) [38].

Our results demonstrated a significant difference in the genotype frequencies of IL-6rs1800795 and rs1800797 between AMD patients and controls, along with a significant difference in the allele frequency of IL-6rs1800797. For IL-6rs1800796, no statistically significant differences were observed in the distribution of allele or genotype frequencies between the two groups. Multiple clinical and observational studies have demonstrated a substantial association between IL-6 polymorphisms and an elevated risk of AMD [11,31]. However, findings from other studies, including genome-wide association studies (GWAS), have not consistently replicated these associations [22,39].

In our study, although differences in serum IL-6 levels were noted across genotypes, these differences did not reach statistical significance. Similar findings have been reported in other studies, where no significant association between IL-6 polymorphisms and serum IL-6 levels has been observed in various inflammatory disorders [40–43]. However, conflicting results have also been reported [11,17,22]. Our results suggest that although these SNPs are recognized for their regulatory effects on IL-6 gene expression in vitro and in vivo, their impact on serum IL-6 levels may vary depending on population characteristics and study design. The absence of a significant association may reflect the intricate interplay between genetic and environmental factors affecting IL-6 levels, rather than indicating that these SNPs lack biologic relevance [22,44]. Additionally, the deviation of rs1800797 from the Hardy–Weinberg equilibrium in the control group highlights the need to consider potential population stratification and other factors that may influence the genetic associations observed in our study.

External factors, such as smoking, may also contribute to the variability in IL-6 expression. Smoking is a well-established risk factor for AMD [31]. The significant elevation in serum IL-6 levels observed in smokers compared to non-smokers indicates that smoking may contribute to increased systemic inflammation in AMD patients [32].

Additionally, our haplotype analysis found that the C-G-A and G-G-A haplotypes were significantly associated with an increased risk of AMD. This finding is notable, as it adds a new dimension to the understanding of genetic risk factors for AMD, suggesting that specific haplotype combinations may be more informative than single SNP analyses.

Overall, our findings revealed that IL-6 polymorphisms, particularly rs1800795 and rs1800797, were linked with a higher possibility of developing AMD. Elevated serum IL-6 levels further underscore the potential role of inflammation in AMD pathogenesis. However, controversial results in the literature indicate that these associations may be influenced by population-specific factors and interactions with other genetic or environmental elements. Additional research is required to clarify these complex relationships and to better understand the mechanisms through which IL-6 and its polymorphisms contribute to AMD.