Abstract
Objective: Our study was aimed to analyze the relationship between retina-specific ATP-binding cassette, sub-family A, member 4 (ABCA4) gene polymorphisms and gene-environment interactions with age-related macular degeneration (AMD) susceptibility. Methods: 98 AMD patients and 110 healthy controls, matched in age and sex, were enrolled in this study. ABCA4 polymorphisms (2633C>A, 5646G>A and 6389T>A) were determined by direct sequencing. Differences of genotype and allele distributions were analyzed by χ2 test. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were adopted to represent the relative risk of AMD. Gene-environment interactions were analyzed using crossover analysis. Results: 2633C>A polymorphism had no obvious correlation with AMD risk. Genotype AA and allele A in 5646G>A polymorphism significantly increased the risk of AMD (OR=4.753, 95% CI=1.249-18.085; OR=1.944, 95% CI=1.209-3.126). 6389T>A polymorphism AA genotype had no significant correlation with AMD risk, but the A allele distinctly enhanced the AMD risk (OR=1.681, 95% CI=1.071-2.639). Afterwards, we analyzed the interactions between ABCA4 polymorphisms and smoking on AMD. Smoking had interactions with all of 2633C>A (CC+CA), 5646G>A and 6389T>A polymorphisms, and the interactions were significantly correlated with AMD. Conclusions: 2633C>A (CC+CA) genotype, 5646G>A and 6389T>A polymorphisms of ABCA4 gene and smoking are susceptible factors for AMD, and the interactions of ABCA4 polymorphisms with smoking increased the risk of AMD.
Keywords: ABCA4, polymorphisms, smoking, Interactions, AMD
Introduction
Age-related macular degeneration (AMD), a serious blinding disease caused by the damage in retinal pigment epithelium (RPE), is one of the most common reasons for the loss of vision in middle-aged and elderly people [1]. Statistics show that the morbidity of AMD, is about 1.6% among people aged 50-65, increases to 30% among people over 75 years old [2]. AMD can be divided into two kinds: dry and wet, and the latter is even more devastating to eyesight. AMD symptoms mainly manifest as non-infectious damage in macular area, lipid deposition, the formation of new blood vessels and pathological changes of RPE cells, Bruch membranes and choroid blood vessel beds. Early features of AMD are pigment abnormalities and the formation of glass film warts, and the late features are geographic atrophy and choroid neovascularization. Specific pathogenesis of AMD has not yet been recognized, but AMD is currently considered as a syndrome induced by environmental and genetic factors and/or their interactions. AMD is related to many factors such as age, inheritance, birth defect, smoking, dietary habit, chronic light damage, cardiovascular disease, malnutrition, iris pigment, farsightedness, body chronic inflammation and complement activation [3-5]. Numerous researches manifest that the heritability of AMD can be up to 71% and that genetic factors play important roles in the occurrence of AMD [6-8].
Using genome scan integration analysis, Fisher et al. discovered that the gene polymorphisms closely related to AMD susceptibility were on chromosomes 10q26, 1q, 2p and 3p [9]. It has been found that ATP-binding cassette, sub-family A, member 4 (ABCA4) gene has a certain relationship with the occurrence of AMD [10-12]. However, some other researches did not confirm ABCA4 gene polymorphisms were in relevance with AMD occurrence [13,14].
Thus it can be seen that the influences of ABCA4 gene mutations to AMD are not explicit. Therefore, in the present study we explored the correlation between ABCA4 gene 2633C>A, 5646G>A and 6389T>A polymorphisms with AMD susceptibility. Then we investigated the correlation between the interactions of ABCA4 polymorphisms and smoking with AMD susceptibility.
Materials and methods
Subjects of the study
This is a case-control study, all subjects were unrelated Chinese Han population. This study, complied with the provisions of Helsinki declaration, was approved by the ethics committee of General Hospital of People’s Liberation Army and obtained the written informed consent from all participants. Cases and controls were matched in age and gender.
98 diagnosed AMD patients in General Hospital of People’s Liberation Army were enrolled as cases. In case group, there were 67 males and 31 females with an average age of 70.3 ± 9.15. 110 healthy individuals (85 males and 25 females), with a median age of 68.9 ± 11.78, were selected from the physical examination center of the same hospital at the same period. Patients were confirmed through eye test, contact lens slit-lamp examination, OCT test, ophthalmofundoscopy, fundus photography and fundus fluorescence angiography inspection. Then the test was performed by ophthalmology doctors following standard inspection procedures. Clinical stages were classified according to the AMD classification standards published by Johanna et al. [15]. Controls were excluded, who had the possibilities of blood diseases, renal insufficiency, diabetes, benign or malignant tumors and other choroid retinal diseases. We collected the basic information of participates, including demographic data and smoking history. Smoking status in this article was divided into non-smoking and smoking (at least one cigarette a day for more than half a year).
DNA extraction
4 ml peripheral venous blood was collected from the subjects who had a 12 h fasting. The blood was put into anticoagulation tubes and preserved in refrigerator at -20°C. DNA was extracted with DNA extraction kit from Beijing Tiangen Biotech Co., Ltd and preserved at -20°C.
Polymerase chain reaction (PCR) amplification
PCR primers for polymorphisms referred literature [10] and synthesized by Shanghai GeneCore BioTechnologies Co., Ltd. Primer sequences were shown in Table 1. 50 µl PCR reaction system contained 4 µl of template DNA, each 1.5 µl of forward and reverse primers, 2 µl of dNTp and 1 µl of TaqDNA polymerase, 5 µl of 10 × buffer and the rest volume of sterile water. PCR reactions started with initial denaturation at 95°C for 6min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 45 s and extension at 70°C for 45 s, and the last final extension at 72°C for 5 min. PCR products were sequenced by Sangon Biotech (Shanghai, China).
Table 1.
Primer sequences of ABCA4 polymorphisms
| Site | Primer | Primer sequence |
|---|---|---|
| 2633C>A | Forward | 5’-AGATCTTATAGAACTGCGGTAAGG-3’ |
| Reverse | 5’-ATAGAGGGCCACCTCTGTGA-3’ | |
| 5646G>A | Forward | 5’-TTTGGCTCTTGCTCAGTTCC-3’ |
| Reverse | 5’-GGGCTCCTGAGGAAAGAAAT-3’ | |
| 6389T>A | Forward | 5’-CATCCCACAGGCAAGAGATT-3’ |
| Reverse | 5’-GCAGCAGGACTCTTCCAAGT-3’ |
Statistical analysis
Differences of genotype and allele frequencies between two groups were compared by χ2 test using PASW Statistics 18 software (statistical significance with P < 0.05). Hardy Weinberg equilibrium (HWE) were tested by PLINK1.07 software (P > 0.05 represented statistical significance). Odds ratios (ORs) and 95% confidence intervals (95% CIs) were adopted to represent the relative risk of AMD. Moreover, differences of features in case and control groups were detected by t test or χ2 test. Gene-environment interactions were analyzed by crossover analysis.
Results
Data analysis of subjects features
Table 2 listed the basic information of subjects. There were no obvious differences in age and sex between the two groups (P > 0.05). Smoking rate of the case group was higher than that of the control group and the difference had statistically significant (P < 0.01).
Table 2.
Features of subjects
| Feature | Case n = 98 (%) | Control n = 110 (%) | P |
|---|---|---|---|
| Gender | > 0.05 | ||
| Male | 67 (68.37) | 85 (77.27) | |
| Female | 31 (31.63) | 25 (22.73) | |
| Age (x ± s) | 70.3 ± 9.15 | 68.9 ± 11.78 | > 0.05 |
| Smoking status | < 0.01 | ||
| - | 33 (33.67) | 87 (79.09) | |
| + | 65 (66.33) | 23 (20.91) |
“-” stands for non-smokers; “+” stands for smokers. Smokers are defined as those people who consume at least one cigarette a day for more than six months.
HWE examination
Genotypes distributions of ABCA4 gene polymorphisms (2633C>A, 5646G>A and 6389T>A) in control group were satisfied HWE. The result showed that the distribution of the above polymorphisms had significant goodness of fit to the law (P > 0.05), suggesting the control group was in an equilibrium state and had good representativeness.
Correlation analysis of ABCA4 genotypes and alleles with AMD
Genotypes distributions of ABCA4 gene 2633C>A, 5646G>A and 6389T>A polymorphisms were shown in Table 3. In this study, ABCA4 gene 2633C>A polymorphism had no significant correlation with AMD risk. AA genotype and A allele in 5646G>A polymorphism were obviously higher in case group than that in control group (P < 0.05). The results indicated that AA was the pathogenic genotype of AMD (OR=4.753, 95% CI=1.249-18.085), and the A allele was the susceptible allele of AMD (OR=1.944, 95% CI=1.209-3.126). Meanwhile, A allele of 6389T>A polymorphism was more frequent in case group (P < 0.05), demonstrating the close relationship between A allele and AMD (OR=1.681, 95% CI=1.071-2.639). Although the AA genotype in 6389T>A polymorphism was higher in case group, the difference in case and control groups had no statistical significance (P > 0.05).
Table 3.
Genotypes and alleles distributions of ABCA4 polymorphisms
| Genotype/Allele | Case n = 98 (%) | Control n = 110 (%) | χ2 (P) | OR (95% CI) |
|---|---|---|---|---|
| 2633C>A | ||||
| CC | 64 (65.31) | 84 (76.36) | - | 1 |
| CA | 30 (30.61) | 23 (20.91) | 2.798 (0.094) | 1.712 (0.909-3.224) |
| AA | 4 (4.08) | 3 (2.73) | 0.524 (0.469) | 1.750 (0.378-8.097) |
| C | 158 (80.61) | 191 (86.82) | - | 1 |
| A | 38 (19.39) | 29 (13.18) | 2.955 (0.086) | 1.584 (0.935-2.684) |
| 5646G>A | ||||
| GG | 54 (55.10) | 77 (70.00) | - | 1 |
| GA | 34 (34.70) | 30 (27.27) | 2.460 (0.017) | 1.616 (0.885-2.950) |
| AA | 10 (10.20) | 3 (2.73) | 6.105 (0.013) | 4.753 (1.249-18.085) |
| G | 142 (72.45) | 184 (83.64) | - | 1 |
| A | 54 (27.55) | 36 (16.36) | 7.652 (0.006) | 1.944 (1.209-3.126) |
| 6389T>A | ||||
| TT | 53 (54.08) | 73 (66.36) | - | 1 |
| TA | 32 (32.65) | 30 (27.27) | 1.530 (0.216) | 1.469 (0.798-2.706) |
| AA | 13 (13.27) | 7 (6.36) | 3.666 (0.056) | 2.558 (0.956-6.847) |
| T | 138 (70.41) | 176 (80.00) | - | 1 |
| A | 58 (29.59) | 44 (20.00) | 5.153 (0.023) | 1.681 (1.071-2.639) |
Influences of the interactions of 2633C>A, 5646G>A and 6389T>A with smoking on AMD
Tables 4, 5 and 6 exhibited the influences of the interactions of 2633C>A, 5646G>A and 6389T>A polymorphisms with smoking on AMD, respectively. For 2633C>A polymorphism CC+CA genotypes carriers, the risk of occurrence of AMD was 8.974 times higher in smokers than in non-smokers. AA genotype smokers had 8.250 times AMD risk than CC+CA genotypes non-smokers. The results illustrated the smoking increased the susceptibility of 2633C>A polymorphism for AMD. As for 5646G>A polymorphism, smoking significantly increased the AMD risk (P < 0.05) both in GG+AA genotypes and AA genotype (OR=6.439, 95% CI=3.405-12.178; OR=20.606, 95% CI=2.480-171.220). In 6389T>A polymorphism, TT+TA and AA genotypes smokers had significantly high AMD risk than non-smokers, respectively (OR=6.467, 95% CI=3.377-12.386; OR=12.813, 95% CI=2.660-61.720).
Table 4.
Influences of the interactions of 2633C>A polymorphism with smoking on AMD
| 2633C>A genotype | Smoking | Cases | Controls | OR | 95% CI | P |
|---|---|---|---|---|---|---|
| CC+CA | No | 32 | 88 | 1.000 | - | - |
| CC+CA | Yes | 62 | 19 | 8.974 | 4.666-17.260 | 0.000 |
| AA | No | 1 | 2 | 1.375 | 0.121-15.686 | 0.797 |
| AA | Yes | 3 | 1 | 8.250 | 0.828-82.209 | 0.035 |
Table 5.
Influences of the interactions between 5646G>A polymorphism and smoking on AMD
| 5646G>A genotype | Smoking | Cases | Controls | OR | 95% CI | P |
|---|---|---|---|---|---|---|
| GG+GA | No | 33 | 85 | 1.000 | - | - |
| GG+GA | Yes | 55 | 22 | 6.439 | 3.405-12.178 | 0.000 |
| AA | No | 2 | 2 | 2.575 | 0.348-19.047 | 0.338 |
| AA | Yes | 8 | 1 | 20.606 | 2.480-171.220 | 0.000 |
Table 6.
Effects of the interactions between 6389T>A polymorphism and smoking on AMD
| 6389T>A genotype | Smoking | Cases | Controls | OR | 95% CI | P |
|---|---|---|---|---|---|---|
| TT+TA | No | 32 | 82 | 1.000 | - | - |
| TT+TA | Yes | 53 | 21 | 6.467 | 3.377-12.386 | 0.000 |
| AA | No | 3 | 5 | 1.538 | 0.347-6.812 | 0.569 |
| AA | Yes | 10 | 2 | 12.813 | 2.660-61.720 | 0.000 |
Discussion
AMD, an eye disease seriously damaging eyesight, is characterized by degenerative changes and neovascularization in retinal macular area. AMD has replaced cataract to be the first inducement of blindness in the elderly. Data from the world health organization point out that there are about 30 million AMD patients all over the world and a half million of them lose their eyesight every year. In recent years, a variety of studies confirmed that gene polymorphisms and environment have great influence on the occurrence and development of AMD [16-18], and genes like ABCA4 have been proved to be related with the onset of AMD [7,8,10].
ABCA4 gene was first found on chromosome1p13-p21 by Allikmets et al. [19]. In the subsequent decades, many researches investigate the ABCA4 gene. It contains 50 exons, encodes proteins of 2273 amino acids and participates in transportation of essential molecules in and out of photoreceptor cells. ABCA4 mutations result in the occurrence of a series of retinal diseases such as AMD and retinitis pigmentosa [14,20]. Thus, in current case-control study, we performed a correlation analysis of three newly discovered polymorphisms [10] of ABCA4 with AMD. We found that there was no significant correlation between 2633C>A polymorphism and AMD risk. In 5646G>A polymorphism, AA genotype and A allele increased the risk of AMD about 4.753 and 1.944 times, respectively. 6389T>A polymorphism A allele was significantly increased the AMD risk, but the AA genotype had no obvious correlation with AMD risk. These results demonstrated that 5646G>A and 6389T>A polymorphisms were risk factors for AMD. But all of the evidence was insufficient to understand the etiology of AMD.
As we all know, AMD was influenced by complex factors, including genetic and environmental factors. Among environmental risk factors for AMD, smoking is the most important one. A study by Mitchell et al. showed that smoking was in relevance with the abnormal increase in RPE [21]. According to epidemiologic study, smoking correlated with geographic atrophy, choroidal thickness and increased occurrence of AMD [22-25]. Larger amount smoking significantly increase the incidence of AMD, the OR is 1.55 [23]. Hence, to slow the progression of AMD, one must quit smoking [26]. Nowadays, the influences of the interactions between smoking and genetic factors on AMD have become the hotspot in research field. Base on the 3 ABCA4 polymorphisms (2633C>A, 5646G>A and 6389T>A), we explored the gene-environment interactions on AMD. For 2633C>A polymorphism only, it had no obvious correlation with AMD. After interaction analysis, we found that, smokers with 2633C>A polymorphism CC+CA genotype might have increased risk of AMD, and the OR was 8.974. The result implied that the positive interactions might exist between the polymorphism with smoking in AMD onset. Interactions of smoking with 5646G>A polymorphism GG+GA and AA genotypes significantly increased the AMD risk. Comparison with single polymorphism, interactions of polymorphism and smoking increased the risk of AMD from 6.105 to 20.606. 6389T>A polymorphism had the same trend on AMD. Overall, interactions between ABCA4 gene mutations and smoking obviously increased the risk of AMD.
AMD was resulted from the interplay between environmental and genetic factors. This study proved the close correlation of ABCA4 gene 2633C>A, 5646G>A and 6389T>A polymorphisms as well as smoking with AMD. With the development of biological sciences and gene technology, a near future will see a new understanding of pathogenesis of AMD, and offer effective prevention and treatment methods for AMD. Based on the interactions of AMD with environmental factors, susceptible individuals can be prevented the occurrence of AMD through smoking cessation, healthy dietary and lifestyle.
Disclosure of conflict of interest
None.
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