Association between vascular endothelial growth factor +936 C/T gene polymorphism and age-related macular degeneration

Abstract

Objectives

The pathogenesis of age-related macular degeneration (AMD) remains unknown. Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and a target for inhibition therapy in wet AMD. This study investigated the association between the VEGF +936 C/T gene polymorphism and AMD, in a Chinese Han population.

Methods

Patients with AMD, and age- and sex-matched controls were enrolled. Restriction fragment length polymorphism was used to analyse the VEGF +936 polymorphism in the promoter and the 3′ untranslated region of the gene.

Results

The study included 200 AMD patients and 200 control subjects. There was a significantly higher prevalence of the TT genotype among AMD patients (9.0%) compared with controls (3.5%); the odds ratio for this genotype in AMD patients was 2.73 (95% confidence intervals 1.11, 6.68). There were no significant associations between any genotype and AMD subphenotypic categories (early, geographic atrophy, choroidal neovascularization).

Conclusions

The present study findings suggested that the VEGF +936 TT genotype was associated with AMD among Han Chinese patients.

Keywords

Introduction

Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in the developed world, in people aged ≥50 years.^1,2 The incidence of AMD is low in persons younger than 50 years of age (0.05%), but climbs to 11.8% in those over 80 years of age.³ Although current therapeutic options are largely limited to the late stages of the disease,⁴ advances in early diagnosis and major developments in treatment have substantially improved the prognosis for patients with AMD. Several risk factors for AMD (including increasing age,⁵ cigarette smoking,⁶ higher body mass index,^7,8 sun exposure,⁹ arterial hypertension¹⁰ and nutritional factors¹¹) have been identified. Moreover, a genetic component to AMD has been established, based on familial aggregation and linkage studies.^12–14

Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and a target for inhibition therapy in wet AMD;¹⁵ it has also been found to play a key role in choroidal neovascularization.¹⁶ Current treatments that inhibit VEGF activity are effective in improving vision in ∼40% of eyes with neovascular AMD.¹⁷ The human VEGF gene is located on chromosome 6p21.3 and is organized into eight exons and seven introns, with the coding region spanning ∼14 kb.^18–21 Several single-nucleotide polymorphisms (SNPs) in the VEGF gene affect its expression.^22–25 One of these, the +936 C/T polymorphism in the promoter and 3′ untranslated region (UTR) of the VEGF gene, affects VEGF plasma levels; carriers of the VEGF 936T allele have significantly reduced VEGF plasma levels.^26–28

Although some studies have evaluated the role of polymorphisms in the VEGF gene in AMD, relatively little about it remains known.^{15,26,29–37} The present study investigated the association between the VEGF +936 C/T gene polymorphism and AMD in a Chinese Han population.

Patients and methods

Study Population

Between January 2010 and January 2012, consecutive patients with AMD, and age- and sex-matched controls, attending the Department of Ophthalmology at the Chinese PLA General Hospital, Beijing, China, were enrolled in this population-based control study. The controls were randomly selected from healthy individuals (using a random-number table) who underwent routine physical examination during the same time period as the patients. All participants were of Han Chinese origin and came from the same geographic region, and all underwent complete ophthalmological examination (including visual acuity measurement, slit-lamp biomoscopic examination and dilated fundus examination). Patients with AMD underwent colour fundus photography, fluorescein angiography and optical coherence tomography.

Patients with AMD were diagnosed according to the International Classification System³⁸ and were subdivided into one of three phenotype categories: early AMD (stages 1a, 1b, 2a, 2b and 3); geographic atrophy (stage 4ga); exudative AMD (choroidal neovascularization; stage 4cnv). Patients with AMD had to have at least one soft drusen >0.125 µm in one eye to be eligible for study inclusion. Participants with a family history of AMD or ocular diseases that might simulate AMD or preclude its diagnosis (namely, previous laser photocoagulation, cryopexy, media opacity or inflammatory diseases) were excluded from the study.

The Institutional Review Board of the Chinese PLA General Hospital approved the study and written informed consent was obtained from all participants. All procedures adhered to the tenets of the Declaration of Helsinki.

DNA Extraction and Genotyping

Genomic DNA was isolated from 3–5 ml ethylenediaminetetra-acetic acid (20 g/l) or 4% sodium citrate (citrate 136, sodium 408 mmol/l) anticoagulated venous blood, using the QIAamp® Blood Mini Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer’s instructions. Samples were stored at 4°C until analysis. PCR reactions were carried out in a 20-µl reaction volume containing 100 µg genomic DNA, 25 pmol/l each primer, 0.2 mmol/l deoxyuceotide triphosphate, 10 mmol/l Tris–HCl (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl₂, and 1 U of Taq polymerase (Takara Shuzo, Otsu, Japan). The following primers were used: forward 5′- AGGAAGAGGGACTCTGCGCAGAGC-3′ and reverse 5′- TAA ATG TATGTATGTGGGTGGGTGTGTCTACAGG-3′. The PCR cycle conditions consisted of an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 30 s at 94°C, 30 s at 62°C, 30 s at 72°C, and a final elongation step at 72°C for 10 min, using a GeneAmp®-PCR System 9700 thermal cycler (Applied Biosystems®, Foster City, CA, USA).

The VEGF +936 C/T polymorphism was genotyped using restriction fragment length polymorphism. Briefly, PCR products were digested overnight at 37°C with NlaIII (30 U of enzyme/µg of DNA) (New England Biolabs, Beverly, MA). Digested PCR products were then resolved on a 3% agarose gel and stained with ethidium bromide for visualization under UV light. The VEGF +936T allele was cut into two fragments of 122 and 86 base pairs, whereas the VEGF +936C allele remained uncut, with a length of 208 base pairs.

Statistical Analyses

All analyses were performed using Predictive Analytics Software version 18.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Comparison of continuous or categorical variables between the AMD group and controls were compared using a two-sided Student’s t-test or χ²-test, respectively. Comparison of VEGF +936 C/T genotype frequencies between AMD and controls was assessed by Pearson's χ²-test, and odds ratios (ORs) and 95% confidence intervals (CI) were calculated. The association between the AMD phenotype and genotype was examined using a logistic regression model, and the ORs and 95% CIs were calculated. Genotype distribution was tested for Hardy–Weinberg equilibrium using the χ²-test. A P-value <0.05 was considered to be statistically significant.

Results

Characteristics of the 200 patients with AMD and 200 control subjects are shown in Table 1. There were no statistically significant differences in mean age, sex distribution, smoking status, body mass index, hypertension, hyperlipidaemia, diabetes or cardiovascular disease between the two groups. Of the 200 patients with AMD, roughly half had choroidal neovascularization, with a further quarter each having early AMD or geographic atrophy.

Table 1.

Characteristics of patients with age-related macular degeneration (AMD), and age- and sex-matched healthy controls, in a study of vascular endothelial growth factor (VEGF) gene +936 C/T gene polymorphis.

Parameter	AMD patients^a n = 200	Controls^a n = 200
Age, years	70.6 ± 8.4	70.5 ± 8.3
Sex
Male	90 (45.0)	94 (47.0)
Female	110 (55.0)	106 (53.0)
Smoking status
Never	119 (59.5)	122 (61.0)
Ever	81 (40.5)	78 (39.0)
Body mass index, kg/m²	23.9 ± 0.8	23.8 ± 0.7
Hypertension	97 (48.5)	95 (47.5)
Hyperlipidaemia	41 (20.5)	38 (19.0)
Diabetes	17 (8.5)	18 (9.0)
Cardiovascular disease	28 (14.0)	27 (13.5)
AMD phenotypic categories
Early	52 (26.0)	N/A
Geographic atrophy	49 (24.5)	N/A
Choroidal neovascularization	99 (49.5)	N/A

Data presented as n (%) patients or mean ± SD.

No statistically significantly differences (P ≥ 0.05); two-sided Student’s t-test or χ²-test.

N/A, not applicable.

The prevalence of the VEGF +936 T/T genotype was 9.0% among patients with AMD compared with 3.5% among controls (OR: 2.73 [95% CI 1.11, 6.68]; Table 2). Genotype and allele frequencies were in Hardy–Weinberg equilibrium in both groups. There were no significant associations between genotypes and AMD phenotypic categories (Table 3).

Table 2.

Prevalence of the vascular endothelial growth factor (VEGF) +936 C/T gene polymorphism among patients with age-related macular degeneration (AMD), and age- and sex-matched control.

Genotype	AMD patients n = 200	Controls n = 200	OR (95% CI)
CC	132 (66.0)	138 (69.0)	0.87 (0.57, 1.33)
CT	50 (25.0)	55 (27.5)	0.88 (0.56, 1.37)
TT	18 (9.0)	7 (3.5)	2.73 (1.11, 6.68)
C allele frequency	314 (78.5)	331 (82.8)	0.76 (0.54, 1.08)
T allele frequency	86 (21.5)	69 (17.3)	1.31 (0.92, 1.87)

Data presented as n (%) of patients or mean ± SD.

Pearson's χ²-test: OR, odds ratio; CI, confidence interval; calculated using logistic regression analysis.

Table 3.

Stratification analysis of vascular endothelial growth factor (VEGF) +936 C/T genotype frequency among patients with age-related macular degeneration (AMD.

Category	Cases		CC^a		CT^a		TT^a
Category	Cases	n (%)	OR (95% CI)	n (%)	OR (95% CI)	n (%)	OR (95% CI)
AMD genotype	200	132 (66.0)	1 (Reference)	50 (25.0)	1 (Reference)	18 (9.0)	1 (Reference)
AMD phenotype
Early	52	34 (65.4)	0.99 (0.61, 1.61)	12 (23.1)	0.92 (0.46, 1.86)	6 (11.5)	1.28 (0.49, 3.39)
Geographic atrophy	49	32 (65.3)	0.99 (0.60, 1.63)	13 (26.5)	1.06 (0.54, 2.11)	4 (8.2)	0.91 (0.29, 2.80)
Choroidal neovascularization	99	66 (66.7)	1.01 (0.69, 1.48)	25 (25.3)	1.01 (0.59, 1.73)	8 (8.1)	0.90 (0.38, 2.14)

OR, odds ratio; CI, confidence interval; calculated using logistic regression analysis.

No statistically significantly differences (P ≥ 0.05).

Discussion

The present study demonstrated that the VEGF +936 TT genotype was associated with AMD in Han Chinese patients. There were no significant associations between genotype and AMD phenotype.

The VEGF +936 C/T gene polymorphism is associated with many other diseases; for example, in Han Chinese populations, the VEGF +936 TT genotype has been associated with a higher risk of glioma³⁹ and with acute respiratory distress syndrome.⁴⁰ A meta-analysis found an association between the VEGF +936C allele and a decreased risk of oral cancer,⁴¹ which was in contrast to the findings of a case–control study that suggested that the VEGF +936C allele was associated with vascular invasion in oral squamous cell carcinoma.⁴² A further case–control study suggested that the VEGF +936 C/T polymorphism might be a genetic determinant for colon cancer in Koreans.⁴³ The VEGF +936T allele has been found to be associated with an increased risk of stage III–IV endometriosis in a Japanese population,⁴⁴ and a decreased risk of breast cancer.⁴⁵

There have been previous reports of genetic polymorphisms associated with AMD, including polymorphism of the heme oxygenase-1 and -2 genes,⁴⁶ mitochondrial DNA polymorphism A4917G,⁴⁷ complement factor H gene Y402H polymorphism⁴⁸ C3 R102G polymorphism⁴⁹ and high-temperature requirement A-1 LOC387715 gene A69S.¹⁴ Further associations have been found through meta-analyses, including the LOC387715/HTRA1 gene,⁵⁰ the human high-temperature requirement A-1 gene -512 G/A⁵¹ and rs11200638 in the promoter of HTRA1 gene.⁵²

A systematic review and meta-analysis of eight studies found strong evidence for an association between complement factor H and AMD, and indicated a multiplicative model (with each C allele increasing the odds of AMD by approximately 2.5-fold).⁵² A meta-analysis from 15 case–control studies (8905 participants) indicated strong protective effects against AMD for the variant alleles of four SNPs in the CFB/C2 gene (rs9332739, rs547154, rs4151667 and rs641153).⁵³ A meta-analysis of reported studies (n = 2600 participants) showed that the 32W variant of complement factor B was also associated with protection against AMD.⁵⁴

Limitations of the present study included the fact that it was a hospital-based, case–control study; thus, selection bias may have occurred and the participants may not have been representative of the general population. Only one SNP in the VEGF gene was examined, and the precise significance of the VEGF +936 C/T gene polymorphism in AMD remains unclear.

In conclusion, findings of this study indicate that the VEGF +936 TT genotype is found more frequently among Han Chinese patients with AMD than among control subjects. Further studies are needed to confirm this finding in larger groups of patients.

Footnotes

Declaration of Conflicting Interest

The authors declare that there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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