|Year : 2023 | Volume
| Issue : 1 | Page : 106-110
Investigating the association of IL12B and INFG Polymorphisms with the risk of pseudoexfoliation syndrome and glaucoma
Ghasem Fakhraie1, Jalaledin Ghanavi2, Kioomars Saliminejad3, Poopak Farnia2
1 Department of Ophthalmology, Glaucoma Service, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran
2 Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
|Date of Submission||20-Oct-2022|
|Date of Decision||12-Jan-2023|
|Date of Acceptance||17-Feb-2023|
|Date of Web Publication||14-Mar-2023|
Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University of Medical Sciences, Tehran
Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University of Medical Sciences, Tehran
Source of Support: None, Conflict of Interest: None
Background: Immune responses may be involved in the development of pseudoexfoliation (PEX), pseudoexfoliation glaucoma (PEXG), and primary open-angle glaucoma (POAG) pathogenesis. The aim of the present study was to evaluate the association of IL12B rs3212227 A/C and INFG rs1861494 T/C polymorphisms with the risk of PEX, PEXG, and POAG in an Iranian population. Methods: Totally, 55 POAG, 57 PEX, and 78 PEXG patient cases as well as 79 healthy controls were included in this study. Genotyping of the IL12B and INFG polymorphisms was performed by polymerase chain reaction and restriction fragment length polymorphism methods using TaqI and FauI restriction enzyme, respectively. Results: Results indicated that IL12B AC genotype was significantly higher in POAG (36.4%; P < 0.001; odds ratio [OR] = 4.0, 95% confidence interval [CI]: 1.7–10.0) and PEX patients (36.4%; P = 0.023; OR = 2.7, 95% CI: 1.1–6.9) compared to the control group (12.6%). The C allele could be considered a risk factor for POAG (P = 0.002; OR = 3.1, 95% CI: 3.1–6.8) and PEX (P < 0.001; OR = 3.4, 95% CI: 3.4–7.3). INFG TC genotype was significantly higher in PEX (38.6%; P = 0.007; OR = 2.8, 95% CI: 1.3–6.3) and PEXG patients (37.2%; P = 0.009; OR = 2.7, 95% CI: 1.1–6.9) compared to the control group (19.0%). The C allele seemed to be a risk factor for PEX (P = 0.002; OR = 2.8, 95% CI: 1.4–5.7) and PEXG (P = 0.009; OR = 2.4, 95% CI: 1.2–4.7). Conclusion: Overall, IL12B was associated with susceptibility to POAG and PEX, and IL12B C allele increased the risk of POAG and PEX. In addition, INFG was associated with susceptibility to PEX and PEXG, and the INFG C allele seemed to be a risk factor for PEX and PEXG.
Keywords: Glaucoma, IL12B, INFG, polymorphism
|How to cite this article:|
Fakhraie G, Ghanavi J, Saliminejad K, Farnia P. Investigating the association of IL12B and INFG Polymorphisms with the risk of pseudoexfoliation syndrome and glaucoma. Biomed Biotechnol Res J 2023;7:106-10
|How to cite this URL:|
Fakhraie G, Ghanavi J, Saliminejad K, Farnia P. Investigating the association of IL12B and INFG Polymorphisms with the risk of pseudoexfoliation syndrome and glaucoma. Biomed Biotechnol Res J [serial online] 2023 [cited 2023 Apr 1];7:106-10. Available from: https://www.bmbtrj.org/text.asp?2023/7/1/106/371689
| Introduction|| |
Glaucoma is the second cause of age-related blindness that affects roughly 3.5% of 40–80 years old people worldwide., It is a multifactorial disease and both genetic and environmental factors contribute to the etiology of glaucoma., This syndrome is the progressive loss of retinal ganglion cells associated with characteristic structural changes to the optic nerve and visual function, and is usually asymptomatic until the advanced visual field loss., Glaucoma is categorized as primary and secondary. Primary glaucoma occurs in the absence of identifiable causes and its most important risk factors are intraocular pressure (IOP), age, and family history; however, the biomolecular mechanisms are still poorly defined.
Primary open-angle glaucoma (POAG), an adult-onset disease, is the most common form of glaucoma. Studies have demonstrated a strong association between the development of POAG and a positive family history. Glaucoma is closely related to pseudoexfoliation (PEX) syndrome which is a well-recognized late-onset disease characterized by the accumulation of microscopic granular amyloid-like protein fibers. These deposits primarily accumulate in the eyes. PEX is the most commonly identifiable cause of secondary glaucoma, named pseudoexfoliative glaucoma (PEXG). PEXG presents with higher IOP and is typically more aggressive and less responsive to topical medications in comparison with POAG, and only 15% of PEX cases present with glaucoma. The etiology of PEX is not well characterized and it has been remained unclear why only a proportion of subjects with PEX develop glaucoma. The genetic background among individuals could explain to some extent the observed variability in phenotypes related to glaucoma and PEX and the corresponding different worldwide prevalence.
Genome-wide association studies have implicated several loci and genes involved in the pathogenesis of PEX, PEXG, and POAG.,,, Each of these studies has revealed sets of significantly associated genetic loci implicating biological pathways, which do not overlap with each other between several forms of glaucoma.
Although considerable biological insight from previous studies has been gained, further studies are still necessary to find out the implicated genetic variants in the development of glaucoma. Inflammation and immune reaction have a strong link with the pathology of glaucoma; it is proposed that cytokines may act as biomarkers to evaluate the severity of glaucoma and anti-inflammatory treatments are necessary for the therapy of glaucoma. In a recent study, probing the profiles of proinflammatory cytokines in aqueous humor of glaucoma patients have revealed that many inflammatory cytokines were highly elevated. In another study performed by Chua and coworkers, levels of 29 inflammatory cytokines were measured in the aqueous of glaucoma subjects and the results showed that patients with POAG had higher levels of IL12 (P = 0.011), IFNγ (P = 0.005), and CXCL9 (P = 0.047) compared to the controls.
In the present study, IL12 and INFG were proposed as possible genetic predisposing factors in glaucoma. Accordingly, the association of IL12B rs3212227 A/C in 3'untranslated region and intronic INFG rs1861494 T/C polymorphisms with the risk of PEX, PEXG, and POAG were studied in an Iranian population.
| Methods|| |
A total of 190 unrelated patients including 78 patients with PEX, 59 with PEXG, and 55 with POAG who had undergone detailed standardized ophthalmic examinations at the Department of Ophthalmology, Glaucoma Service, Farabi Eye Hospital (Tehran, Iran), as well as 79 healthy controls were recruited for the study participation. Patients and healthy controls were all of Iranian origin.
All subjects gave written informed consent and the study was approved by the Ethical Committees of the National Research Institute of Tuberculosis and Lung Disease (NRITLD) (IR.SBMU.NRITLD.REC.1401.128).
DNA extraction and genotyping
Five millilitres of peripheral blood samples were collected in tubes containing 200 μL of 0.5 M EDTA as the anticlotting factor and was subsequently stored at − 20°C before analysis. Genomic DNA was extracted from blood samples using the salting out method.
Genotyping of the IL12B rs3212227 A/C and INFG rs1861494 T/C polymorphisms was performed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Primer sequences related to each polymorphism are demonstrated in [Table 1].
|Table 1: Primer sequences and restriction fragment length polymorphism conditions for genotyping of IL12B and INFG polymorphisms|
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PCR reactions were carried out in a final volume of 25 μL containing: ×10 PCR buffer, 2 mM MgCl2 (Roche, Germany), 0.2 mM of each dNTPs (Sigma-Aldrich, United Kingdom), 10 pmol of the forward and reverse primers, 50 ng of template DNA, 1 uL of DMASO, and 1.25 U of Taq DNA polymerase (Sigma-Aldrich, United Kingdom) and sterile distilled water up to 25 μL was added.
Amplification conditions started with an initial denaturation step of 5 min at 94°C, followed by 32 cycles of 20 s denaturation (94°C), 30 s of annealing (60°C), and 30 s of extension (72°C), and ended by a final extension for 10 min (72°C). All PCR products were subjected to electrophoresis on 2% agarose gel prepared in 1 × TAE, stained with ethidium bromide, and visualized by exposure to ultraviolet light.
Next, the PCR products of IL12B (243 bp) and INFG (366 bp) were digested overnight using TaqI and FauI restriction enzymes at 65°C and at 55°C, respectively. DNA fragments were subjected to 10% polyacrylamide gel electrophoresis and stained with silver nitrate. The expected DNA fragments after digestion by RFLP are shown in [Table 1].
Obtained data were analyzed using SPSS software, version 16.0 (SPSS, Chicago, IL., USA). Chi-square and Fisher's exact tests were performed to compare the genotype and allele frequencies between the groups. The Hardy–Weinberg equilibrium (HWE) was checked using the Chi-square. Two-tailed P < 0.05 were considered statistically significant.
| Results|| |
The analysis of descriptive variables showed that the distribution of age and gender was not significantly different between the controls and POAG, PEX, and PEXG subjects (P < 0.05).
Analysis of the HWE using the Chi-square revealed that the distribution of genotypes for IL12B and INFG polymorphisms did not deviate from the expected values by HWE in the control group (P = 0.382, and P = 0.351, respectively). In addition, the distribution of genotypes for IL12B and INFG in POAG patients (P = 0.625, and P = 0.603, respectively) and PEX patients (P = 0.081, and P = 0.467, respectively) were in HWE. On the other hand, genotype frequencies neither for IL12B (P = 0.004) nor for INFG (P = 0.035) were in the HWE in PEXG patients.
Distribution of the allele and genotype for IL12B and INFG polymorphisms in subjects is demonstrated in [Table 2] and [Table 3]. Results displayed that there was a significant difference in genotype distributions and allele frequencies of IL12B rs3212227 polymorphism between the POAG and PEX groups compared to the control group.
|Table 2: Allele and genotype distribution of IL12B rs3212227 A/C polymorphism among glaucoma patients and the healthy controls|
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|Table 3: Allele and genotype distribution of the INFG rs1861494 T/C polymorphism among glaucoma patients and the healthy controls|
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The IL12B AC genotype was significantly higher (P < 0.001; odds ratio [OR] = 4.0, 95% confidence interval [CI]: 1.7–10.0) in POAG patients (36.4%) in comparison with the control group (12.6%). In addition, the IL12B AC genotype was significantly higher (P = 0.023; OR = 2.7, 95% CI: 1.1–6.9) in PEX patients (36.4%) compared to the control group (12.6%). The IL12B C allele could be considered a risk factor for POAG (P = 0.002; OR = 3.1, 95% CI: 3.1–6.8) and PEX (P < 0.001; OR = 3.4, 95% CI: 3.4–7.3).
In addition, INFG rs1861494 polymorphism was associated with susceptibility to PEX and PEXG. The INFG TC genotype was significantly higher (P = 0.007; OR = 2.8, 95% CI: 1.3–6.3) in PEX patients (38.6%) in comparison with the control group (19.0%). The INFG TC genotype was significantly higher (P = 0.009; OR = 2.7, 95% CI: 1.1–6.9) in PEXG patients (37.2%) compared to the control group (19.0%). The INFG C allele seemed to be a risk factor for the PEX (P = 0.002; OR = 2.8, 95% CI: 1.4–5.7) and PEXG (P = 0.009; OR = 2.4, 95% CI: 1.2–4.7). In contrast, no significant association was found in genotype and allele frequencies of INFG rs1861494 polymorphism between the POAG patients and the control group.
| Discussion|| |
Glaucoma as one of the leading age-related causes of blindness is occurred due to the progressive destruction of retinal ganglion cells. At present, lowering IOP is the only available therapy for the glaucoma treatment; however, it may still lead to the blindness., Mechanistic studies have concentrated on immunological alterations in the pathogenesis of glaucoma. Cytokines are considered potential factors in the pathogenesis of glaucoma and may contribute to the regulation of death in retinal ganglion cells. Cytokines may stimulate the production of aqueous humor or prevent aqueous drainage in glaucoma.
The IL12 family members, including IL12, IL23, IL27, and IL35 are the only heterodimeric cytokines with unique and distinctive characteristics. IL12 is a proinflammatory/prostimulatory cytokine produced by activated macrophages, dendritic cells, and B cells in response to the pathogens and plays a key role in the development of T helper Type 1 cells.,, It has been shown that IL12 induces the production of IFNγ by T cells and natural killer cells.,,
In the present study, the association of two polymorphisms in two cytokine genes including IL12B rs3212227 A/C and INFG rs1861494 T/C was evaluated in patients with POAG, PEX, and PEXG and the control group. In genetic association studies, disease-free control groups from outbred populations should follow the HWE. In this regard, the evaluation of the HWE in our control group was in HWE for IL12B rs3212227 and INFG rs1861494 polymorphisms (P > 0.05).
Results of the present study indicated that IL12B rs3212227 polymorphism was associated with the increased risk of POAG and PEX, and INFG rs1861494 polymorphism was associated with susceptibly to PEX and PEXG in the Iranian population (P < 0.05). The IL12B AC genotype was significantly higher in POAG (36.4%) and PEX groups (36.4%) compared to the control group (12.6%) with ORs of 4.0 and 2.7, respectively. The IL12B C allele could be considered as a risk factor for the development of POAG (OR = 3.1) and PEX (OR = 3.4) in the Iranian population. Regarding the INFG polymorphism, the TC genotype was significantly higher in PEX (38.6%) and PEXG patients (37.2%) compared to the healthy controls (19.0%) with ORs of 2.8 and 2.7, respectively. The C allele seemed to be a risk factor for PEX (OR = 2.8) and PEXG (OR = 2.4).
In this study, the C allele was determined as the minor allele for IL12B rs3212227 and INFG rs1861494 polymorphisms with the frequencies of 0.08 and 0.10 in the control group, respectively. A comparison of these results revealed that they were lower than the global minor allele frequency (MAF) in dbSNP (https://www.ncbi.nlm.nih.gov) for IL12B rs3212227 (MAF = 0.40) INFG rs1861494 (MAF = 0.23) polymorphisms. This could be explained by the small sample size in the control group. The global MAF for each polymorphism is reported from genotyping of 2500 worldwide individuals by 1000 Genome Project.
To the best of our knowledge, the association of IL12B and INFG polymorphisms with glaucoma has not been previously studied. A few studies have evaluated the association of IL1 and IL6 gene polymorphisms with glaucoma. Results of a recent meta-analysis indicated that there was no significant association between the three polymorphisms of IL1 including IL1B rs16944, IL1A rs1800587, and IL1B rs1143634 with POAG. In another study, it was revealed that the IL6-174 G > C (rs1800795) polymorphism was not a risk factor for POAG.
Human and animal studies have indicated that inflammatory responses might contribute to the etiology of glaucoma. Inflammatory cytokines including IL6, TGFβ1, TGFβ2, IL6, IL8, IL10, IL12, α-serum amyloid A, IFNγ, and CXL9 have altered expression levels in the aqueous humor of eyes in glaucoma patients compared to the controls., Cytokine imbalance may modulate the immune microenvironment of eyes in glaucoma patients and hence influence optic neuropathy.
| Conclusions|| |
Our findings suggested that IL12B rs3212227 C/T polymorphism was associated with susceptibility to POAG and PEX. Moreover, the IL12B C allele increased the risk of POAG and PEX. Furthermore, INFG rs1861494 A/C was associated with susceptibility to PEX and PEXG, and the INFG C allele seemed to be a risk factor for PEX and PEXG in an Iranian population. These findings should be further investigated in other populations.
Limitation of study
There was no limitation in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]