|Molecular Vision 2007;
Received 6 August 2007 | Accepted 30 October 2007 | Published 26 November 2007
A tag-single nucleotide polymorphisms approach to the vascular endothelial growth factor-A gene in age-related macular degeneration
Andrea J Richardson, F.M.
Amirul Islam, Robyn H. Guymer, Melinda Cain, Paul N. Baird
Centre for Eye Research Australia, University of Melbourne, Australia
Correspondence to: Andrea Richardson, Centre for Eye Research Australia, 32 Gisborne Street, East Melbourne, Victoria, 3002, Australia; Phone: +61 3 9929 8621; FAX: +61 3 9662 9916; email: email@example.com
Purpose: Age-related macular degeneration (AMD) is the leading cause of poor vision in the developed world, and its pathogenesis remains unknown. The most devastating form of end stage disease is neovascular or wet AMD, where there is abnormal growth of new blood vessels under the retina. Vascular endothelial growth factor (VEGF) is thought to be a major player in the stimulus of this abnormal growth of blood vessels. We undertook a case-control association study to investigate the VEGF-A gene, a known angiogenic gene that has previously been associated with AMD.
Methods: We recruited 577 individuals with AMD (early, atrophic, and neovascular AMD) and 173 ethnically matched controls for our study. We employed a tag-single nucleotide polymorphisms (tSNP) approach to investigate this gene using a series of seven tSNPs that encompassed the coding region of the VEGF gene as well as its promoter. Alleles were determined by a MALDI-TOF based approach followed by statistical analysis.
Results: One SNP (rs3024997) showed evidence of departure from Hardy-Weinberg equilibrium in only the AMD cases. Therefore, it was retained for further analysis. All other SNPs in our study showed no departure from Hardy-Weinberg equilibrium. No association was found between any of the VEGF tSNPs analysed in our study and AMD nor any of its sub-types.
Conclusions: Using a tSNP approach, we found no evidence of an association of these SNPs within the VEGF-A gene being associated with either AMD or any of its subtypes in our population.
Age-related macular degeneration (AMD) is the leading cause of permanent blindness in developed countries, affecting approximately 50 million people worldwide . In Australia, AMD contributes to 50% of all blindness (presenting visual acuity <6/60) . A combination of genetic and environmental influences is thought to be responsible for AMD, and recent findings have implicated a number of immune response genes associated with this complex disease [3-6].
The end-stage lesions in AMD can manifest as choroidal neovascularization, where abnormal blood vessels grow from the choroid underneath the retina and leak, thus causing rapid deterioration in central vision. Alternatively, the end stage lesion can also present as geographic atrophy (GA), where there is a slow process of atrophy of the photoreceptors and retinal pigment epithelium. It is not well understood what determines progression of a patient with early AMD to one of these end stage lesions.
It is well established that the formation of blood vessels occurs by angiogenesis and vascular endothelial growth factor (VEGF) has been identified as a key molecule in promoting angiogenesis . VEGF can potentially induce vascular leakage and inflammation by triggering the increased production and permeability of capillary endothelial cells .
Until recently, treatment options for AMD have been severely limited. There are now several new treatments using either pegaptanib (Macugen) an oligonucleotide aptamer that targets only the VEGF165 isoform, or the monoclonal antibodies ranibizumab (Lucentis), and bevacizumab (Avastin), that target VEGF-A [8,9].
Two studies reported significant association to the VEGF gene. In the first study, association with VEGF and AMD was found in a case-control cohort with single nucleotide polymorphism (SNP) rs2010963, and in a family cohort with two intronic SNPs (rs833070 and rs3025030) . The second study, which involved 45 patients with neovascular disease, identified a haplotypic association in both the promoter and an intronic region of the VEGF gene .
While the VEGF gene is primarily associated with neovascular disease, a previous study  has found association in a family-based study using early, atrophic and neovascular patients combined. Hence, our study included all three sub-types of AMD to ensure a thorough investigation of this gene. In order to better characterize possible association of the VEGF gene with AMD, we undertook a tag-SNP (tSNP) approach to maximize coverage of the gene, using a large case-control study.
All individuals for our study were collected as part of our AMD inheritance study (AMDIS) and had an Anglo-Celtic ethnic background. Study participants with AMD were recruited through either outpatient clinics at the Royal Victorian Eye and Ear Hospital (RVEEH) or private ophthalmology practices in Melbourne. Control subjects were collected from the same community as part of the large population-based epidemiologic eye study, the Melbourne Visual Impairment Project (VIP) or through aged-care nursing homes. All participants undertook a standard risk factor and disease history questionnaire, which included questions on the age at diagnosis of AMD. The time of ascertainment was the time of enrollment into the AMDIS when a clinical examination was performed. A blood sample for DNA analysis along with a fundus photograph was obtained from each participant.
Written informed consent was obtained from all individuals, and ethics approval for the project was provided by the Human Research and Ethics Committee of the Royal Victorian Eye and Ear Hospital, Melbourne. The study was conducted in accordance with the Declaration of Helsinki and according to the National Health and Medical Research Council of Australia's statement on ethical conduct in research involving humans, revised in 1999.
Tag single nucleotide polymorphism selection
The selection of tSNPs for genotyping in the VEGF gene was undertaken through the use of the International HapMAP Project. The entire VEGF gene was covered, including 2 kb either side of the gene encompassing the promoter and 3' untranslated (UTR) regions. tSNPs were selected using the pairwise algorithm based on the CEU population (Utah residents with ancestry from northern and western Europe). The tSNPs selected (rs833061, rs25648, rs3024997, rs2146323, rs3025030, rs3025035, and rs10434) for our study were based on the full length isoform of the VEGF-A gene (see Figure 1 for SNP positions). A strong linkage disequilibrium (LD) tagging criteria of r2>0.8 was used and all SNPs had a minor allele frequency (MAF) of at least 0.1. Perfect LD is where D'=r2=1, where D is the difference in the measure of observed and expected frequencies of alleles in a haplotype and r2 is equal to D2 divided by the product of the allele frequencies at two loci. Complete LD indicates that one allele at a marker is always co-inherited with an allele at a second marker. The HapMAP data was sourced from NCBI Build 35.
Genotyping of tSNPs was performed as previously described  using the MassARRAY® platform (Sequenom, San Diego, CA) through the Australian Genome Research Facility, Brisbane, Australia. In brief, 2.5ng of genomic DNA was amplified by polymerase chain reaction (PCR) at each SNP site. An extension reaction (MassExtend; Sequenom, San Diego, CA) was then performed by adding DNA polymerase, dNTPs, and an extension primer, which allowed a 1-bp primer extension of either allelic variant at the polymorphic site. Deoxynucleotides incorporated at the polymorphic site were terminated with the incorporation of a di-deoxynucleotide, generating two allele-specific products of different masses. Samples were spotted onto a gene microarray (384 SpectroCHIP; Sequenom), and the chip was placed in the spectrometer and genotypes were simultaneously called in real time (SpectroTyper RT software; Sequenom).
The Hardy-Weinberg equilibrium (HWE) test was undertaken to check whether genotypes fell within a standard distribution, using the software program JLIN: a Java based linkage disequilibrium plotter . Participant characteristics in both control and groups were compared using the chi-square test or independent sample t test. The chi-square test was also employed to compare deviations of genotype frequencies in all participants and controls, case specifics from those expected under HWE, as well as genotype frequencies between total AMD cases and controls. AMD subtypes were also compared with controls by using the same statistical technique. All analyses were performed using the software SPSS (version 14.0; SPSS Inc, Chicago, IL).
A total of 577 AMD affected Caucasian individuals with a mean age of diagnosis of 73.3 years were recruited. As shown in Table 1, there was a significant excess of females in the AMD affected and control groups with a combined total of 65%. Of the affected individuals, 342 presented with neovascular disease, 101 presented with GA, and 50 presented with early stages of AMD (the presence of soft drusen >125 μm, with or without regions of hyperpigmentation). A further 173 control individuals with a mean age of 71.0 years were also recruited (Table 1).
One SNP (rs3024997) showed evidence of departure from HWE but this was only apparent in cases and was therefore retained for further analysis. The other six SNPs showed no evidence of departure from HWE (Table 2). The relative positions of all SNPs used in our study are shown in Figure 1. The position of previously reported significant SNPs in relation to our tSNPs [10,11] are depicted in bold type (Figure 1).
The genotype distributions of the SNPs used in our study in total cases compared to controls, and also stratified by disease sub-type (neovascular, GA, or early AMD) are shown in Table 2. No evidence of association of any of the tSNPs was evident in either total AMD cases or stratified by disease sub-type (Table 2).
While gene expression studies have shown that the VEGF gene is involved in angiogenesis and in increased inflammation and vascular leakage [14,15] we were unable to demonstrate, using a tSNP approach, any apparent association between SNPs in the VEGF gene and AMD or with any AMD sub-type in our population.
tSNPs are SNPs in the genome that exist in high linkage disequilibrium with a number of other SNPs, that is, they can be used to identify other SNPs that are inherited together in a haplotype without the need for genotyping each SNP. The advantage of using a tSNP approach in establishing association of a gene with disease is that SNP tagging provides a useful tool for maximizing the coverage of SNPs based on LD. This obviates the need to genotype every SNP within a gene to identify possible associations with disease. Hence, using this approach we were able to capture the information from SNPs that had previously been associated with AMD but have never been subsequently verified, without having to re-analyse the same SNP.
The two previous case-control studies that undertook association studies in the VEGF gene examined individual SNPs in the VEGF gene and found association in both the 5' UTR  and in the intron 1 region  with neovascular AMD. These two previously associated SNPs were tagged in the same LD block by SNPs rs833061, rs25648, rs2146323 and rs3024997 in the current study (see Figure 1). Two other recent reports also appear to support our findings that there is unlikely to be any association of variants in the VEGF gene with AMD [16,17]. Our study extended these findings by using tSNPs that not only encompassed all previously described promoter SNPs but also the remainder of the coding region of the VEGF gene. A plausible explanation between the positive findings identified in previous studies may lie in the populations that were studied and the subtle allele frequency differences that may ensue. One study investigated a United States population in which the ancestry of the participants was unclear, whereas our study looked at patients who had a predominantly Anglo-Celtic background. One study that did investigate a United Kingdom population had a small sample of 45 people that may have influenced a false positive result as this was not verified in a second population.
A common feature of our study and previous studies is the lack of association of individual promoter SNPs and AMD in case-control analysis. It has previously been suggested that the strongest linkage occurs at the 5' end of the VEGF-A gene [10,11]. This reflected the hypothesis that SNPs from upstream regions of the VEGF gene affect splice alterations, which in turn could affect the regulation of angiogenesis . However, our findings do not support this conclusion.
The analysis of common diseases typically assumes that common variants are responsible for these diseases. However, if rare variants or mutations are responsible for AMD, such as polymorphisms with a MAF<0.1, then it is possible that these may be missed by using such a tSNP approach. We chose SNPs with a minimum allele frequency of 0.1, thereby capturing most of the SNPs in the HAPMAP database, with the hope that rare variants might also be in LD with the tSNPs.
In summary, our study has provided a thorough t-SNP based examination that encompassed the promoter region, the entire VEGF-A gene and its 3' UTR, using a large sized sample to investigate association. We found little evidence to support an association of the VEGF-A gene with AMD. Hence, we demonstrated inconsistent results with previous studies. However, recent evidence also indicates a lack of association with AMD and the VEGF gene, which supports our claim for a negative finding. The results from this study cannot exclude the possibility that rare variants in the VEGF gene are involved in disease causation. The next step might be to genetically investigate other VEGF family genes. Alternatively, it might also be useful to investigate SNPs in other angiogenic genes to identify whether associations exist in these genes and AMD.
This work was supported by the National Health and Medical Research Council of Australia (through a clinical fellowship to R.H.G.), J.A. COM Foundation, and the Ophthalmic Research Institute of Australia.
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