Association of TAS2R16 gene (rs860170, rs978739, rs1357949) polymorphisms and TAS2R16 serum levels in patients with age-related macular degeneration

ABSTRACT Background The aim of this study is to determine the association of TAS2R16 (rs860170, rs978739, rs1357949) gene polymorphisms and TAS2R16 serum levels in patients with the occurrence of age-related macular degeneration (AMD). Methods Subjects with early AMD, subjects with exudative AMD, and healthy controls participated in the study. DNA was isolated by salting out leukocytes from peripheral venous blood. Single nucleotide polymorphisms (SNPs) were analysed by RT-PCR. TAS2R16 levels were determined by enzyme-linked immunosorbent assay (ELISA) using the Abbexa Human Taste Receptor Type 2 Member 16 (TAS2R16) ELISA kit. Statistical data analysis was performed using “IBM SPSS Statistics 27.0” and SNPstats statistical data analysis programmes. Results The TAS2R16 rs860170 TT genotype is statistically significantly less frequent in the exudative AMD group than in the control group, whereas the TAS2R16 rs860170 C allele gene is statistically significantly more frequent in the exudative AMD group. Each C allele of TAS2R16 rs860170 is associated with a 2.8-fold increased probability of occurrence of exudative AMD. The C allele of TAS2R16 rs860170 is statistically significantly more frequent in men and women with exudative AMD than in the control group. The C allele of TAS2R16 rs860170 is associated with a 2.8-fold increased odds of occurrence of exudative AMD in women and a 2.9-fold increased odds of occurrence of exudative AMD in men. In TAS2R16 (rs860170, rs978739, and rs1357949), the T-T-A haplotype is associated with a 2.6-fold decreased likelihood of developing early AMD and the T-T-A haplotype is associated with a 3.2-fold decreased likelihood of developing early AMD in women. For TAS2R16 (rs860170, rs978739, and rs1357949), carriers of the T-T-G and T-T-A haplotypes are associated with a 2.2- and 3.2-fold decreased probability of exudative AMD, respectively. Individuals with the C-C-A haplotype are 9.2-fold more likely to develop exudative AMD. Specifically, the C-C-A haplotype is associated with a 9.3-fold increased likelihood of exudative AMD in men. In contrast, women with the T-T-A haplotype are 5.6-fold less likely to develop exudative AMD. Conclusion TAS2R16 plays an important role in the development of AMD.


Introduction
Age-related macular degeneration (AMD) is an acquired, chronic, and progressive neurodegenerative disease of the retina that results in significant or irreversible central visual impairment when the macula is affected (1,2).AMD is divided into initial and late forms, which in turn are subdivided into initial, exudative, and atrophic forms.The initial form is characterised by drusen formation between the retinal pigment epithelium and the basement membrane.In contrast, the exudative form is characterised by neovascularization that damages the retina and leads to vision loss.Although AMD is the third leading cause of blindness worldwide, it is a disease that primarily affects the elderly (3,4).As the disease progresses, the visible image deteriorates, recognition of objects, reading, writing, and driving become difficult, and the retinal damage that occurs can lead to vision loss.AMD is a multifactorial disease with a multifactorial aetiology, and the risk is determined by genetic and modifiable factors used to reduce vision loss (5,6).As more genes are identified in various studies, advances in genetics are an excellent tool to detect the progression of AMD.Increasing knowledge of genetics is leading to the discovery of new gene variants associated with the disease and their exploration.TAS2R16 is a protein-coding gene associated with taste receptor cells of the tongue and palate epithelium and the perception of bitterness (7,8).TAS2R16 is also associated with the development of nicotine dependence and the risk of alcohol dependence, and is thought to influence cancer, longevity, and the inflammatory process in the gut (9)(10)(11).In this study, we will investigate whether the TAS2R16 gene and its polymorphisms and TAS2R16 serum levels have an impact on the occurrence of AMD, as there is no evidence in the scientific literature to date of an association between our chosen gene and the disease.However, TAS2R16 has been identified in human neuronal tissue and is thought to suppress the inflammatory response by inhibiting the release of cytokines and other mediators (12).The expression of TAS2R receptors has been detected in human leukocytes, especially lymphocytes and mast cells, and their stimulation by TAS2R agonists leads to inhibition of the expression of IL −13, IL −4, IL − 5, tumour necrosis factor (TNF-α), IL −1β, and interferon (IFN-γ) (13,14).Innate immune cells (macrophages, dendritic cells, neutrophils, lymphocytes) are also involved in the pathogenesis of NV.Cytokines, including IL −1β, IL −6, IL −8, IL −10, IL −17, TGF-β, IFN-γ, TNF-α, etc, stimulate angiogenesis so that photoreceptor cells are progressively damaged in late AMD, while cytokines such as IL −4, IL −12, IFN-β inhibit angiogenesis (15).Therefore, the aim of our study is to determine the associations of TAS2R16 (rs860170, rs978739, rs1357949) gene polymorphisms and TAS2R16 serum levels in patients with the occurrence of early and exudative age-related macular degeneration.

Methods
All subjects signed an agreement in accordance with the Declaration of Helsinki.The study was conducted in the Laboratory of Ophthalmology of the Institute of Neurosciences of the Lithuanian University of Health Sciences.A total of 324 subjects were studied, and two study groups were formed: the control group (n = 112) and the group of patients with AMD (n = 212).The patient groups were divided into two subgroups: Patients with early AMD (n = 100) and patients with exudative AMD (n = 112).
The control group consisted of individuals who had no ocular pathology at examination and agreed to participate in the study.
Inclusion criteria for healthy patients were as follows: (1) no ophthalmic eye diseases were detected in the detailed ophthalmic examination; (2) informed consent to participate; Exclusion criteria for healthy patients were as follows: (1) any ophthalmic diseases; (2) patients taking epilepsy and sedatives.
Exclusion criteria for patients with early and exudative AMD were: (1) related ocular diseases (high refractive error, cloudy cornea or lens opacity (nuclear, cortical, and posterior subcapsular cataract), excluding minor opacities, and patients with intraocular lenses, keratitis, acute or chronic uveitis, glaucoma, late age-related macular degeneration, optic nerve disease); (2) systemic diseases (diabetes mellitus, oncological diseases, systemic tissue disorders, chronic infectious diseases, conditions after organ or tissue transplantation); (3) colour fundus photography because of the opacity of the optical system of the eye or because of the quality of fundus photography; (4) congenital colour vision disorders were excluded by history; (5) patients taking epilepsy and sedatives.
Visual acuity (VA) was estimated from letter charts and reported in decimal notation.All patients were examined by slit-lamp biomicroscopy.Biomicroscopy was used to assess corneal and lens transparency.Intraocular pressure was measured at each examination.The patients' pupils were dilated with 1% tropicamide.After pupil dilation, funduscopy was performed with a direct monocular ophthalmoscope and slit lamp using a double aspheric lens of + 78 diopters.The results of the examination were recorded on standardised forms developed for this study.Colour fundus photographs were taken with a fundus camera at half wide angle (OPTON SBG, 30 degrees).Photographs were taken with the focus on the centre of the fovea.Optical coherence tomography was performed in all AMD patients (OCT), and fluorescein angiography was performed in patients with suspected late AMD after examination of OCT.For this study, we used the classification system for AMD formulated in the previous Age-Related Eye Disease Study (4): Early AMD consisted of multiple small drusen and multiple intermediate (63-124 μm diameter) drusen or abnormalities of the retinal pigment epithelium.Extensive intermediate drusen characterised early intermediate AMD and at least one large druse (≥125 μm diameter) or geographic atrophy that did not involve the centre of the fovea.

DNA extraction and genotyping
DNA extraction and analysis of TAS2R16 gene polymorphisms were performed at the Ophthalmology Laboratory of the Neuroscience Institute of the Lithuanian College of Health Sciences.DNA extraction was performed by the salting-out method from venous blood samples.Single nucleotide polymorphisms (SNPs) were determined using TaqMan® genotyping assays (Thermo Scientific, Pleasanton, CA, USA).Genotyping of TAS2R16 rs860170, rs978739, rs1357949 was performed by real-time PCR (RT-PCR) according to the manufacturer's recommendations using a Step One Plus RT-PCR system (Applied Biosystems, Foster City, CA, USA) and an allele discrimination program.The program analyzed each genotype based on the fluorescence intensity of the different detectors (VIC and FAM).

ELISA
TAS2R16 levels were determined by enzyme-linked immunosorbent assay (ELISA) using the Abbexa Human Taste Receptor Type 2 Member 16 (TAS2R16) ELISA kit.The standard solution (20 ng/ml) was prepared by mixing the TAS2R16 protein with 1 ml of 1X cell extraction buffer.The solution was kept at room temperature for 10 minutes and stirred again.7 tubes were numbered consecutively to generate a calibration curve for different concentrations.500 μl of 1X isolation buffer was added to each tube.500 μl of standard solution containing TAS2R16 protein was added to tube 1 and mixed.Then, 500 μl of the solution was added from tube 1 to tube 2, 500 μl from tube 3 to tube 3, and in that order to tube 6.No standard solution was added to tube 7, which remained without protein and was treated as a negative control.A plate with the required number of wells for the assay was prepared.To each well, 100 μl of the antibody cocktail and 100 μl of the serum samples and 100 μl of the standard solutions were added to the appropriate location on the plate.The standard solutions and samples were tested in duplicate.The plate was sealed with a foil and incubated in a plate shaker at 400 rpm for 2 hours at 37°C.At the end of incubation, the entire contents of the plate were discarded and 100 μl of the solution of detection reagent A was added, the plate was sealed, and incubated at 37°C for 1 hour.At the end of the incubation, the foil was removed and the contents of the plate were discarded.Each well was washed with 350 μl of 1X wash buffer, discarded, and this step was repeated three times.After the last wash, the plate was dried on a clean paper towel.Then, 100 μl of the solution of detection reagent B was added to each well and the plate was incubated again at 37°C for 1 hour.Then, 5 washes were performed again with wash buffer.Then, 90 μl of TMB substrate was added and incubated in a plate shaker at 400 rpm for 10-20 minutes at 37°C in a dark environment.To record the immunoenzymatic reaction in progress, we added 50 μl stop solution to each well and placed the plate in the plate shaker for 1 min to mix the solutions.Final reaction samples were obtained with TAS2R16 protein on the bottom of the wells.The absorbance was measured with a microplate reader at the required 450 nm, and the concentration was calculated from a calibration curve based on the standard solutions used.

Statistical analysis
Statistical analysis of the data was performed using IBM SPSS Statistics 27.0.Data are presented as absolute numbers (percentages) and median (IQR).The Mann-Whitney U test was used to detect differences between two independent groups.To compare the homogeneity of the genotype distribution of polymorphisms between AMD patients and controls, χ2 and Fisher's one-way and two-way criteria were used.Binary logistic regression analysis was used to estimate the odds ratio (OR) of AMD occurrence as a function of genetic inheritance patterns.The genetic models (codominant: heterozygotes vs wild-type homozygotes and homozygotes vs wild-type homozygotes; dominant: homozygotes with a rarer allele and heterozygotes vs wildtype homozygotes; recessive: homozygotes with a rarer allele vs wild-type homozygotes; recessive: homozygotes with a rarer allele vs wild-type homozygotes) were included in the analysis.Homozygotes with rarer allele vs wild-type homozygotes and heterozygotes; supradominant: heterozygotes vs wild-type homozygotes vs homozygotes with rarer allele; an additive model was used to model the effect of each rarer allele on the development of AMD).This analysis was performed with a 95% confidence interval (CI) for the group with AMD.The Akaike information criterion (AIC) was evaluated to select the best inheritance model, with the lowest value indicating the best-fitting model.After Bonferroni correction, differences were considered statistically significant when p < 0.05/3 (p < 0.017).

Results
The study included 324 subjects divided into three groups: control group (n = 112), early AMD (n = 100), and exudative AMD (n = 112).After the groups were formed, genotyping of TAS2R16 rs860170, rs978739, and rs1357949 was performed.The cohort of patients with early AMD consisted of 100 individuals: 64 female (64%) and 36 male (36%).The mean age of the patients was 76 years.The cohort of exudative AMD consisted of 112 subjects: 74 females (66.1%) and 38 males (33.9%), mean age 74 years.The control group consisted of 112 subjects: 63 men (56.3%) and 49 women (43.8%).The median age of the control group was 74 years.There were no statistical differences between the groups with respect to age (early AMD vs. control group p = 0.230, exudative AMD vs. control group p = 0.601).However, statistically significant differences were found between the distribution of males and females within the groups (p < 0.001).For this reason, further analysis was modified by gender.Demographic data are presented in Table 1.

Influence of TAS2R16 rs860170, rs978739, rs1357949 on the occurrence of early-AMD
The genotype and allele distributions of TAS2R16 rs860170, rs978739, rs1357949 were evaluated, and no statistically significant differences were found between patients with early AMD and controls.The results are shown in Table S1 in the supplemental material.Binary logistic regression also did not yield statistically significant results (supplemental material Table S2).

Influence of TAS2R16 rs860170, rs978739, rs1357949 on the occurrence of early and exudative age-related macular degeneration in relation to gender
Analysis of the genotypes and alleles of the TAS2R16 gene rs860170, rs978739, and rs1357949 by sex revealed that the rs860170 genotypes (TT, CT, and CC) were statistically significantly different between men with exudative AMD and control groups: 21.1%, 63.2%, and 15.8% versus 34.9%, 65.1%, and 0%, respectively (p = 0.003).Analysis of genotype distribution (TT, CT and CC) in women with exudative AMD and control group also revealed statistically significant results: 23%, 55.4% and 21.6% vs. 38.8%,61.2% and 0%, respectively (p = 0.001).In addition, the C allele was found to be statistically significantly more prevalent in females in the exudative AMD group compared to the control group (49.3% vs. 30.6%,p = 0.004) (Table 4).
However, analysis of genotypes and alleles in the group of patients with early AMD did not reveal statistically significant differences compared to the control group (Supplementary Material, Table S3).Binary logistic regression analysis in men and women showed that each C allele of TAS2R16 rs860170 increased the odds of exudative AMD by 2.8-fold in women (OR = 2.786; 95% CI: 1.463-5.306;p = 0.002), and by 2.9-fold in men (OR = 2.917, 95% CI: 11.273-6.687,p = 0.011; p = 0.011) (Table 5).
In addition, binary logistic regression analyses were performed separately for men and women to assess the effect of TAS2R16 rs860170, rs978739, rs1357949 on the occurrence of early AMD, but no statistically significant results were found (Supplementary Material, Table S4).
We performed haplotype association analysis of TAS2R16 rs860170, rs978739, rs1357949 in patients with early and exudative AMD compared with a control group.The pairwise linkage disequilibrium between SNPs in AMD patients is shown in Table S5 and Table S6 in the supplemental material.

Serum TAS2R16 levels in AMD patients and control group subjects
An evaluation of serum TAS2R16 levels was performed in patients with early and exudative AMD and in control group subjects.However, serum TAS2R16 levels were not found to be statistically significantly different between patients with early AMD and the control group (0.881 (0.434) ng/ml vs. 0.886 (0.623) ng/ml, p = 0.930) (Figure 1).The same results were found when analysing serum TAS2R16 levels in patients with exudative AMD compared to the control group (1.137 (0.438) ng/ml vs. 0.886 (0.623) ng/ml, p = 0.792) (Figure 2).

Discussion
The single nucleotide polymorphisms rs860170, rs978739, and rs1357949 of the TAS2R16 gene were studied in subjects with early AMD, exudative AMD, and healthy subjects.To our knowledge, there are no similar studies investigating TAS2R16 polymorphisms in AMD.Also, there are no data in the scientific literature analysing the direct association between AMD pathogenesis and selected TAS2R16 polymorphisms.TAS2R16 rs860170 has been found to be associated with longevity (16).It is well known that age is the main risk factor for the development of AMD (17).Malovini and co-authors reported that TAS2R16 rs978739 had a statistically significant association with longevity (p = 0.001) (18).Specifically, the frequency of the homozygous AA genotype gradually increased from 35% in subjects aged 20-70 years to 55% in centenarians, and haplotype analysis of polymorphisms showed that the TAS2R16 rs860170 and rs978739 polymorphisms correlated with longevity (OR = 0.74; 95% CI: 0.56-0.98;p = 0.033) (19).In our study, the TT genotype of TAS2R16 rs860170 is statistically significantly less frequent in patients with exudative AMD than in controls (22.3% vs. 36.6%,p = 0.019).According to the researchers, it is the TAS2R16 TT genotype that is associated with longevity in southern Italy, and the distribution of its genotypes (CC and TT) is statistically significant (p = 0.05) (20).In our study, a distribution analysis of all genotypes in men and women with exudative AMD revealed a statistically significant distribution of genotypes between men with exudative AMD and controls (p = 0.003) and between women with exudative AMD and controls (p = .001).Given the effective role of the immune response in longevity (21,22), researchers have investigated whether TAS2R16 is also involved in the prevention of skin ageing (23).TAS2R16 is known to be present in human skin tissues (24).Chung et al. found that increased expression of TAS2R16 prevents skin ageing (p < 0.01; p < 0.05), and it is suggested that TAS2R16 May be used to treat skin ageing and other skin lesions in the future (23).It is suggested that TAS2R16 May increase the tolerability of salicylic acid and other similar agents used as analgesics, anti-inflammatory and antipyretic agents (25).TAS2R16 rs860170 is known to be associated with food preference, and the perception of the bitterness of salicylic acid is associated with the A allele of TAS2R16 rs860170 (p = 0.001) (25,26).Inflammation and foods that modulate the release of anti-inflammatory mediators can be considered a crucial mechanism in human evolution and pathology (chronic inflammation) (27,28).Overexpression of TAS2R16 has been shown to improve wound healing by constricting the scratch wound (p < 0.05) (23).In contrast, Zhou and co-authors suggest that TAS2R16 expression is associated with anti-inflammatory effects by inhibiting the release of cytokines and other mediators (12).The development of NV is a major feature of exudative AMD associated with the release of cytokines (29), and the interaction between proinflammatory and antiinflammatory cytokines ultimately leads to a chronic inflammatory response cheng (30,31).In our study, we found that in men, each C allele of TAS2R16 rs860170 was associated with a 2.9-fold higher risk of exudative AMD (p = 0.011), while in women this risk was 2.8-fold higher (p = 0.002).
In 2017, a study by Barontini and co-authors on the association between TAS2R16 polymorphisms and colorectal cancer, which included the rs860170, rs978739, and rs1357949 polymorphisms we studied, showed that the TAS2R16 gene has no significant effect on colorectal cancer susceptibility but may be important for the development of rectal cancer (11).It is known that early AMD is associated with a fivefold increased risk of dying from lung cancer (OR = 5.28; 95% CI: 1.52-18.40)(32), and the C allele of TAS2R16 rs1525489 is associated with an increased risk of colorectal cancer (p = 0.047 and p = 0.051, respectively) (11).Although this TAS2R16 polymorphism was not examined in our study, the C allele of TAS2R16 rs860170 was associated with a 2.8-fold increased risk of exudative AMD (OR = 2.810; 95% CI: 1.716-4.600;p < 0.001), as it was statistically significantly more frequent in the group of male patients with exudative AMD compared to the control group (47.4% vs. p = 0.036) and also in the group of female patients with exudative AMD (49.3% vs. 30.6%,p = 0.004).Although the studies do not directly link the rs860170, rs978739 and rs1357949 polymorphisms of the TAS2R16 gene to the occurrence of AMD, an analysis of the scientific literature shows that TAS2R16 and its polymorphisms are indirectly associated with the disease, especially from the aspect of longevity.Because the polymorphisms we studied are specifically associated with longevity, age is the most important risk factor for AMD.The TAS2R genes have been widely linked to individual differences in taste perception, food preferences, diet, immune responses, and pathophysiological mechanisms.Could they influence ageing by regulating food intake processing and metabolism?In 2012, Campa et al. investigated the possible links between longevity and common genetic variations in the three TAS2R clusters on chromosomes 5, 7, and 12 in a population of 941 individuals aged 20 to 106 years from southern Italy (19).They found that five polymorphisms were associated with longevity: three in the TAS2R16 gene (rs6466849, rs860170 and rs978739), one in the TAS2R4 gene (rs2233998) and two in the TAS2R5 gene (rs2227264), but only rs978739 reached statistical significance.Melis et al. investigated the differences in genotype distribution and haplotype frequency of the TAS2R38 gene in a group of 94 centenarians and near-centenarians from Sardinia, Italy, and compared them with two control cohorts aged 18-35 years and 36-85 years (33).Malovi haplotype analysis of TAS2R16 gene polymorphisms showed that (rs1357949-rs6466849-rs860170-rs978739: T-A-A-G) was associated with longevity (GS = 0.74; 95% CI 0.56-0.98;p = 0.033) (18,19).In comparison, statistical analysis of exudative AMD showed that individuals with the rs860170, rs978739, rs1357949 haplotype C-C-A were associated with a 9.2-fold increased odds of exudative AMD (OR = 9.17; 95% CI: 1.88-44.81;p = 0.007).Functional TAS2R16 variants in African American families were correlated with alcohol consumption, and the TAS2R16 allele associated with a lower risk of alcohol dependence was also associated with a lower mean Maxdrinks score in African American families (9).All 4 classes of G proteins are involved in the regulation of inflammation leading to activation or inactivation of inflammatory cells.The diversity of ligand binding and transmembrane signaling by GPCRs are primarily responsible for mediating complex inflammatory (and anti-inflammatory) responses (34).Race and agerelated decrease in GPR143 signaling as observed in the prevalence of age-related macular degeneration (AMD) in Caucasians compared to other races (35).In 2019, researcher M. Shimazawa found that gene expression of Gpr35 was significantly increased in the RPE choroidal complexes of both mice administered NaIO3 and patients with nonexudative AMD compared with controls (36).Each of the six changes was found once in individual AMD patients and was considered a variant that could impair protein function and potentially cause retinal pathology (37).G protein-coupled receptors (GPCRs) have also been found to be particularly good targets for the application of systems pharmacology because they activate different signal transduction pathways that can culminate in a common response, which is very important in the search for potential treatments for various diseases (38).The human family of TAS2Rs consists of at least 25 GPCRs that vary widely in sequence and have approximately 30-70% amino acid homology (39).TAS2R16 is one of the best studied TAS2Rs, but its structure has not yet been solved.Evolutionary and mutational approaches are useful to determine the structure-function relationship of GPCRs, including TAS2Rs, in terms of ligand binding and receptor activation as long as the proteins function (40).
The strength of this study was that we analyzed for the first time TAS2R16 gene polymorphisms (rs860170, rs978739, rs1357949) and serum TAS2R16 levels in patients with agerelated macular degeneration.Future studies should include more patients with AMD, and TAS2R16 gene polymorphisms (rs860170, rs978739, rs1357949) and TAS2R16 serum levels in patients with age-related macular degeneration should be analyzed in more patients.

Figure 1 .
Figure 1.Serum TAS2R16 levels in early-AMD and control groups.*Mann-Whitney U test was used.

Figure 2 .
Figure 2. Serum TAS2R16 levels in early-AMD and control groups.*Student T test was used.

Table 2 .
Distribution of TAS2R16 rs860170, rs978739, rs1357949 genotypes and alleles in patients with exudative AMD and healthy controls.

Table 4 .
TAS2R16 rs860170, rs978739, rs1357949 genotype and allele distributions in patients with exudative AMD and in healthy controls, by gender.

Table 5 .
TAS2R16 rs860170, rs978739, rs1357949 Binary logistic regression analysis in patients with exudative AMD and controls by gender.