The relationship between vascular endothelial growth factor −2578C/A polymorphism and age-related macular degeneration

Age-related macular degeneration (AMD) is a complex, multifactorial disease in which genetic and environmental factors interact.¹ Three independent teams^2–4 simultaneously demonstrated a significant association between the Y402H polymorphism of the complement factor H (CFH) gene and exudative AMD in North American subjects; the contribution of the Y402H polymorphism of the CFH gene to exudative AMD susceptibility has also been demonstrated in the French population.⁵ Molecular dissection of AMD pathogenic processes has identified vascular endothelial growth factor (VEGF) as among the most important factors, which led to the development of VEGF inhibition therapies⁶ with anti-VEGF antibodies for neovascular AMD. Vascular endothelial growth factor is important for retinal health, because of its involvement in vascular development, in the survival of nascent retinal vessels, and in maintaining stable mature vessels.⁷ VEGF, a 45-kDa homodimeric glycoprotein,⁸ has initially drawn much attention as an important mediator of retinal ischemia-associated intraocular neovascularisation;⁹ VEGF is produced from many cell types within the eye, and studies have shown that VEGF levels are elevated in vitreous and aqueous fluids in the eyes of individuals with proliferative diabetic retinopathy.^10,11 The VEGF gene is located on chromosome 6, at location 6p21.3, and consists of 8 exons.¹² At least 30 single nucleotide polymorphisms (SNPs) in this gene have been described.¹³ Three common SNPs are of particular interest: –2578C/A (rs699947) and –1154 G/A, in the promoter region,¹⁴ and –634C/G (in the 5′ untranslated region), that are reportedly related to VEGF protein production.^15,16 In this study, we have aimed at analyzing whether the rs699947 C/A polymorphism of the VEGF gene is associated with exudative AMD in French patients.

During the last 3 years at the Center of Molecular Neurogenetics, we recruited a total number of 242 consecutive unrelated exudative AMD patients, all originating from Paris and the Parisian region in France. All have at least three generations of french ancestry, and North-Africans are excluded from the study. Criteria for inclusion were as follows: women and men aged 65 or older; unilateral or bilateral choroidal neovascularization; presence of any type of drusen (small, moderately large or large); no association with other retinal disease. Exudative AMD was diagnosed according to the guidelines of the international classification.¹⁷ Patients with exudative AMD underwent an ophthalmological examination, including visual acuity measurement and fundus examination. All the participants in the study provided informed consent according to the French legislation, in agreement with the Declaration of Helsinki for Research Involving Human Subjects. Firstly, all the patients were genotyped for the SNP rs1061170 (the Y402H polymorphism), located in exon 9 of the CFH gene, by PCR-directed sequencing using primer sequences and conditions previously published.¹⁸ The results showed that 68 (28.1%) out of the 242 exudative AMD patients belonged to the protective TT genotype. These 68 patients were selected for this study concerning the VEGF rs699947C/A polymorphism; their demographic characteristics are summarized as follows: 29 (42.6%) were males and the mean age was 74.1 ± 6.5 years. The patients were compared to 68 normal subjects (matched according to sex and age), originating from the same geographic Parisian region. All were also non-North-African Caucasians, with at least three generations of French ancestry. A proportion of only 30% (21) of them were homozygous for the protective TT Genotype at CFH Y402H.

The genotyping of the polymorphism rs699947C/A of the VEGF gene was carried out after amplification of the target DNA by PCR, followed by restriction fragment length polymorphism. DNA was amplified using 10ng of total genomic DNA, in a 25µL reaction volume containing 20mM Tris-HCl (pH 8.4), 2.5mM MgCl2, 50mM KCl, 0.2mM of each dNTP, 20 pmol of each primer (forward, 5′-GGATGGGGCTGACTAGGTAAG-3′; reverse, 5′-AGCCCCCTTTTCCTCCAAC-3′) and 1.5 U of Platinium Taq polymerase (Gibco BRL). PCR was carried out using a thermal cycler (Gene Amp ® PCR system 9700; Applied Biosystems). The reaction conditions were an initial denaturation step (94°C, 5 min), 30 rounds of PCR consisting of 94°C for 40 sec, 62°C for 1 min and 72°C for 40 sec, with a final extension time of 5 min at 72°C. The PCR product is a fragment of 325 bp. About 10µL of the amplified product were then digested for 3 hrs at 37°C with 5 units of BglII. The digested products were separated on 2.5% agarose gel in the presence of ethidium bromide. The uncut 325bp indicated homozygous C allele (the CC genotype); the homozygous A allele (the AA genotype) was represented by DNA bands with 202- and 123-bp fragments, and heterozygous genotype CA was displayed by a combination of 325-, 202- and 123-bp fragments.

The distributions of genotypes and allele frequencies of VEGF rs699947C/A polymorphism are shown in Table 1. In both the controls and patients the genotypes were in Hardy-Weinberg equilibrium (controls: χ² = 4.28, 2 df, p = 0.06; patients: χ² = 1.26, 2 df, p = 0.58), but the proportions of the CA genotype are more elevated in patients (57.4%) than in controls (36.8%). The frequencies of A-allele were 0.39 and 0.52 in controls and patients, respectively. The chi-square test of independence showed that the A-allele was significantly associated with the risk of AMD (χ² = 6.4, 1 df, p = 0.01), having an OR of 1.7 (95% CI: 1.3–2.4). The percentages (numbers) of CC genotypes in controls and patients were 42.6% (29/68) and 19.1% (13/68), respectively. On the other hand, the percentages (numbers) of A-carrier genotypes in controls and patients were 57.4% (39/68) and 80.9% (55/68), respectively. The comparison of CC genotypes with A-carrier genotypes (that is CC versus CA+AA) revealed that the presence of one A-allele in the genotype was strongly associated with an increased risk of AMD (χ² = 12.5, 1 df, p = 0.001) and conferred an OR of 2.8 (95% CI: 1.6–4.8).

TABLE 1  Distributions of VEGF rs699947C/A genotypes and allele frequencies in controls and patients.

VEGF rs699947C/A polymorphism	Controls	Patients
Genotypes
CC	29 (42.6%)	13 (19.1%)
CA	25 (36.8%)	39 (57.4%)
AA	14 (20.6%)	16 (23.5% )
Allele frequencies
p [C]	0.61	0.48
q [A]	0.39	0.52

So, in this study we have investigated the association of the rs699947C/A polymorphism in the promoter region of the vascular endothelial growth factor gene with exudative age-related macular degeneration. Comparisons between French patients not having the at-risk allele for the Y402H polymorphism of the complement factor H gene and controls show that the A-allele of the rs699947C/A polymorphism is clearly associated with increased exudative AMD risk in our population; further, the analysis of genotypic frequencies revealed that subjects with at least one A-allele – that is, those with AA and CA genotypes – were 2.8 times more likely to suffer from exudative AMD than those with CC genotype.

Previous studies investigated the possible association between VEGF polymorphisms and AMD. In the first study,¹⁹ an association between VEGF and AMD was found in a case-control cohort with the single nucleotide polymorphism rs2010963 (identical to the G634C SNP), and in a family cohort with two intronic SNPs (rs833070 and rs3025030). The second study,²⁰ which involved 45 patients with neovascular disease, combined nine SNPs in the promoter and 5′ UTR region to form haplotypes. No association was found between G634C SNP and wet AMD, but an association (OR: 18.24; 95% CI: 2.25–148.25), although with large confidence limits, was found between one of the haplotypes and wet AMD. In addition, haplotype analyses of five intronic SNPs demonstrated two haplotypes associated with wet AMD and one other haplotype associated with the control group; but two other studies^21,22 failed to replicate associations. So, although some studies support a role for a genetic influence of VEGF in AMD, conclusive data cannot be drawn.

But in a more recently published study,²³ concerning 11 polymorphisms of the VEGF gene, a significant (p = 0.001) association was found between the rs833069 polymorphism (located in intron 2) of the VEGF gene and AMD; the authors noted an excess of the GG homozygotes for this marker. In this study the AMD risk is increased 1.7-fold per copy of the minor G allele in VEGF (OR: 1.68; 95% CI: 1.259–2.264); homozygous carriage of the G allele of rs833069 is associated with a >5-fold increased risk of AMD (OR: 5.28; 95% CI: 2.08–13.43) compared with homozygous carriage for the A allele.

The association of VEGF-rs699947A-allele and A-carrier genotypes with increased risk of exudative AMD we found in the present study is most likely due to the modification of the VEGF protein expression. Indeed the study published in 2004 reported that the A-allele was correlated with higher VEGF levels;¹⁵ however, in another study,¹⁶ the C-allele was associated with higher VEGF levels. Therefore further studies need to be carried out to establish the effect of the rs699947C/A polymorphism on protein level.

As our study proceeds, more data are likely to emerge in the future. Analyses of –1154 C/G polymorphism in the promoter region and –634C/G in the 5′ UTR of the VEGF gene are also in progress. The present pilot study is part of an ongoing effort to identify the genetic components, other than the Y402H polymorphism of the complement factor H gene, involved in the pathogenesis and progression of AMD. Further studies are needed to establish the findings reported here and to uncover the mechanisms through which these genetic variations contribute to an increased risk of exudative AMD.

ACKNOWLEDGMENTS

The authors thank Dr Philippe Bornet for medical assistance in this study. DNA extractions were realized by F. David and PCR experiments by C.T Lu. The authors have no financial or proprietary interest in any of the products, methods, or materials mentioned in this study.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Financial support: We thank the Novartis-Pharma firm for continuous support.