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Human Molecular Genetics Advance Access originally published online on August 29, 2006
Human Molecular Genetics 2006 15(19):2955-2961; doi:10.1093/hmg/ddl238
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

VEGF polymorphisms are associated with neovascular age-related macular degeneration

Amanda J. Churchill1,*, James G. Carter1, Helen C. Lovell1, Conor Ramsden1, Steven J. Turner1, Anna Yeung2, Julia Escardo3 and Denize Atan1

1 Molecular Ophthalmology, University of Bristol, Bristol, BS1 2LX, UK, 2 West Midlands Regional Genetics Laboratory, Birmingham Womans, Hospital NHS Trust, Birmingham, B15 2TG, UK and 3 Bristol Eye Hospital, Bristol, BS1 2LX, UK

* To whom correspondence should be addressed at:, Bristol Eye Hospital, Lower Maudlin Street, Bristol, BS1 2LX, UK. Tel: +44 117 9284949; Fax: +44 117 9251421; Email: a.j.churchill{at}bristol.ac.uk

Received June 12, 2006; Accepted August 15, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly. Linkage has been shown to the vascular endothelial growth factor (VEGF) gene and ocular levels of VEGF are raised in individuals with the neovascular form of disease. To examine the role of VEGF further, we conducted a case–control study where 45 individuals with neovascular AMD and 94 age-matched controls were genotyped for 14 single nucleotide polymorphisms (SNPs) in the VEGF promoter and gene. The single SNP +674 CC genotype was significantly associated with AMD (OR=2.40, 95%CI 1.09–5.26, P=0.027). Haplotype analysis of SNPs +674, +4618, +5092, +9162 and +9512 revealed that CTCCT and TCACC were associated with AMD (OR=15.77, 95% CI 1.91–130.24, P=0.0161 and OR=9.95, 95%CI 3.22–30.74, P=0.000053, respectively). The haplotype TCACT was associated with the control group (P=0.0001832). Furthermore, haplotype analysis of promoter SNPs revealed that possession of the –460T, –417T, –172C, –165C, –160C, –152G, –141A, –116A, +405C haplotype was strongly associated with AMD (OR=18.24, 95%CI 2.25–148.25, P=0.0074). This is the most extensive analysis of the VEGF gene in AMD, demonstrating a clear association with the exudative form of disease, thereby creating the possibility for predictive testing. Smoking, high fat intake and hypertension are negative environmental risk factors in AMD, whereas increased consumption of dietary antioxidants can have a protective effect. Identification of those at risk in the population would allow individual counselling with lifestyle advice to reduce the risks of blindness. (Genbank accession nos M63971 and AF437895).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Age-related macular degeneration (AMD) is a devastating condition causing painless loss of central vision in ~28% individuals over 75 years of age (1,2). This can be a gradual loss due to the atrophy of the retinal pigment epithelium or more sudden due to haemorrhage from a choroidal neovascular membrane.

Many therapies have been tried in order to slow the progression of visual loss in neovascular AMD with limited success, including teletherapy and high dietary intake of beta carotene, vitamins C, E and zinc (3,4). The most promising drugs to emerge from recent clinical trials inhibit the effects of vascular endothelial growth hormone, vascular endothelial growth factor (VEGF)-A, a major player in the control of angiogenesis (5,6). VEGF-A contains nine exons and is alternatively spliced to produce isoforms of differing lengths, 189, 165 and 121 amino acids. VEGF 165 is the most predominant isoform in the eye and is further spliced into an ‘a’ or ‘b’ isoform, the difference being the presence at the C-terminus of either exon 8 or exon 9, respectively (7). VEGF 165a has potent angiogenic properties, whereas VEGF165b has anti-angiogenic properties. What controls splicing is still largely unknown but the balance between these two isoforms is vital in maintaining vascular homeostasis. Previously, we have measured a relative increase in VEGF165a compared to VEGF165b in vitreous samples from diabetics with proliferative eye disease (7). Kliffen et al. have shown that total VEGF 165 is elevated in individuals with AMD although the individual isoforms have not been measured (8). Anti-VEGF therapies are emerging as the most successful treatment to date for neovascular AMD. Unfortunately these drugs do not discriminate between the angiogenic and anti-angiogenic isoforms, can only be administered once the diagnosis has been established and after visual loss has occurred (9). Multiple intravitreal injections are required and the financial costs are high.

Several case–control studies have confirmed the association of VEGF single nucleotide polymorphisms (SNPs) with diseases as diverse as breast cancer, kidney disease, ankylosing spondylitis, alzheimer's disease, prostate cancer and idiopathic recurrent abortions (10–15). More recently VEGF SNPs have been used as biomarkers to predict individuals at risk of developing oral cancer (–460 SNP) and as a prognostic indicator in breast cancer survival (16,17). An association has been demonstrated between the C–634G aka C+405G polymorphism and increased risk of diabetic retinopathy in type 2 diabetics. The C allele correlated with elevated levels of serum VEGF in the Japenese population (18). Functional work showed differing haplotypes at –460/+405 that can modify both basal promoter activity (by 71% from wild-type sequence) and mean induction by phorbol ester (from 5-fold to 8.5-fold) (19). Furthermore, an association has been shown between the presence of the +405C allele and the –460TT/+405CC haplotype and severity of retinopathy of prematurity (20).

Although most previous studies have focused on SNPs in the promoter region, it is well known that introns within the VEGF gene contain transcription factor binding sites and that these areas may be important in regulating VEGF production and/or influence splicing. A recently published study on AMD has demonstrated linkage to the VEGF gene in both familial and case–control datasets (21). The five SNPs used in the study were chosen to span across the whole VEGF gene. While individuals recruited to this study had a variety of phenotypes, this is the most promising evidence to date that the VEGF gene is associated with exudative AMD.

Our study was designed to determine whether a single VEGF polymorphism or haplotype could be associated with the neovascular form of AMD. Identification of such would not only increase our understanding of the biology of the disease but might allow a prediction of those ‘at risk’ in the general population. We analysed eight SNPs within the promoter (between nucleotides –460 and –116), one SNP (+405) in the 5' untranslated region (where one might expect transcriptional control to be the most affected) and five intronic SNPs spanning the VEGF gene. Using the fact that linkage disequilibrium exists across the VEGF gene, each of the five SNPs we genotyped predicted the genotype of several other SNPs in the VEGF gene (a process known as ‘haplotype-tagging’) allowing us extended coverage of the VEGF gene. Figure 1 shows the position of the SNPs used in the study and the areas of linkage disequilibrium. Table 1 gives the alternative nomenclature for the intronic SNPs (to ease comparison with other studies) and the location of the associated ‘tagged SNPs’ that fall within the blocks of linkage disequilibrium.


Figure 2381
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  		Figure 1. Diagram showing the position of the 14 SNPs analysed in this study, the associated ‘tagged’ SNPs and the blocks of linkage disequilibrium across the VEGF gene. The position of the SNPs are shown by arrows above the gene and the blocks of linkage disequilibrium associated with each htSNP are shown as shaded boxes below the gene.

 


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  		Table 1. Location and different nomenclature of selected htSNPs and their associated ‘tagged’ SNPs. Five htSNPs were selected using SNP tagger from a database of 44, using the eight most common haplotypes (35 individuals at frequency >3%)

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Analysis of the TYRP1ex2 and TP53 observed genotypes shows the study population was in Hardy–Weinberg Equilibrium for all alleles investigated. No significant difference between control and disease group polymorphism distributions suggests that no selection bias was present (data not shown). Samples selected for sequencing produced genotypes in agreement with those observed via allele-specific PCR.

Promoter and 5'UTR SNP analysis
There were no statistically significant associations with the AMD or control group at any of the nine individual SNP loci. PHASE predicted 37 possible extended haplotypes for the promoter and 5'UTR. The haplotype –460T, –417T, –172C, –165C, –160C, –152G, –141A, –116A, +405C was significantly associated with AMD, being predicted in 8/45 cases (17.8%) versus 1/94 controls (1.1%) (OR=18.24, 95% CI 2.25–148.25, corrected P=0.007).

Haplotype-tagged SNP analysis
The CC genotype at +674 was significantly associated with the AMD group (OR=2.40, 95%CI 1.09–5.26, P=0.027) and presence of the T allele was significantly associated with the control group (OR=0.42, 95% CI 0.19–0.92, P=0.027) (Table 2). No significant associations were seen at the other four haplotype-tagged (ht) SNP loci.


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  		Table 2. Results of htSNP +674C>T genotype analysis. The +674CC genotype is associated with AMD and presence of the T allele is associated with controls

 
PHASE predicted 23 different haplotypes from the combined genotypes of the five htSNPs (+674, +4618, +5092, +9162, +9512). Three haplotypes were significant after correction for multiple analysis. The haplotypes CTCCT and TCACC were associated with AMD (OR=15.77, 95% CI 1.91–130.24, corrected P=0.0161 and OR=9.95, 95%CI 3.22–30.74, corrected P=0.000053, respectively), whereas the haplotype TCACT was associated with the control group (corrected P-value=0.0001832) (Table 3).


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  		Table 3. Combined htSNP haplotype analysis. Three out of 23 predicted haplotypes showed significant associations to a particular phenotype: two were associated with AMD and one was associated with the control group. Order of htSNPs in haplotype: +674, +4618, +5092, +9162, +9512

 
Combined haplotype analysis
When the promoter/5'UTR and htSNP genotypes were combined into a 14-SNP haplotype (representing 28 VEGF SNPs overall), PHASE predicted 109 haplotypes. No single haplotype was found to be significantly associated with either AMD or controls after correction for multiple analyses.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
There have been several studies illustrating the genetic element of some types of familial AMD with direct evidence of mutations in genes responsible for other macular dystrophies (22). The majority of AMD cases however do not fall into this category and association studies are providing a valuable insight into the pathophysiology of this complex condition (23–25). Recently, it has been found that the Y402H polymorphism in Complement Factor H is strongly associated with AMD (26–29). Furthermore, specific haplotypes in Factors B/C2 have been shown to be associated with AMD with one protective and two ‘at risk’ haplotypes (L9H and R32Q) emphasizing the importance of an inflammatory element in AMD (30).

Kliffen et al. (8) have demonstrated increased expression of VEGF in the retinal pigment epithelium and in the outer nuclear layer in post-mortem maculae obtained from individuals with AMD. VEGF has a strong role to play in the pathogenesis of neovascular AMD, and therapies that block the effects of VEGF have given promising early results with significant retention of central vision and reduction in macular swelling (31,32). VEGF production can be regulated by transcription factors and IGF-1, TNF, PDGF and transfection of epithelial cells with splice factors ASF2 and Srp40 all result in an upregulation of VEGF (33–35). It is not known where many of these transcription factors bind, but the promoter/5'UTR and intronic regions of VEGF are clear targets. Several studies have looked at SNPs in the VEGF promoter and 5'UTR and found associations to the severity of a variety of diseases where angiogenesis plays a pivotal role. The –460, –116 and +405 SNPs have been associated with the development or severity of diabetic retinopathy and increased levels of serum VEGF although there have been conflicting data for the +405 SNP (18,19,36–38). Awata et al. (18) found that the presence of the CC genotype was a significant risk factor for diabetic retinopathy in the Japanese population and that individuals with the CC genotype had higher serum VEGF levels. Haines et al. (21) showed an association to the +405C allele in a cohort of exudative AMD. Suganthalakshmi et al. (38) found that the CG genotype was associated with retinopathy in the Indian population. Watson et al. (36) found no association with the +405 genotype and retinopathy in a Caucasian population but showed highest levels of VEGF in individuals carrying the +405GG genotype. These conflicting results may be explained by the fact that it is not a single SNP that influences ocular angiogenesis but a combined haplotype that is important. We studied nine SNPs in the promoter and 5'UTR (C–460T, T–417C, C–172A, C–165T, C–160T, G–152A, A–141C, G–116A, G+405C) and found that the TTCCCGAAC haplotype is significantly associated with AMD (OR=18.24, 95% CI 2.25–148.25, corrected P=0.0074). Interestingly, we did not see an independent association with the +405 SNP but the +405C allele was present in the haplotype associated with neovascular AMD.

It was previously suggested that presence of the –460C allele predicted the G allele at the +405 locus. Such linkage disequilibrium is common across the VEGF gene and forms the basis of htSNP design (36). The observed haplotype frequencies in 115 normal healthy individuals for the –460/+405 loci were calculated and it was found that while the –460C/+405G haplotype was the most common (observed frequency 0.522), linkage disquilibrium was not absolute. The –460T/+405C combination seen in our extended haplotype was observed with a frequency of 0.287. Unpublished data also confirms that the –460T, –417T, –172C, –165C, –160C, –152G, –141A, –116A, +405C haplotype is significantly associated with the presence of proliferative diabetic retinopathy making this a haplotype relevant to a wider range of angiogenic eye diseases.

Haines et al. (21) showed a positive linkage between SNPs spanning the VEGF gene and a large cohort of individuals with mixed forms of AMD. Although comparison of studies using different genetic populations can be unrewarding, our linkage disequilibrium data corroborates the Haines' study, showing strongest linkage across the first half of the VEGF gene (Table 4). We did not, however, find strong linkage across the tail end of the gene (comparison between SNPs 4 and 5). Two of the SNPs used in the Haines' study are genotyped here (HainesSNP 1=+405 and HainesSNP 3=+5092) and a further two SNPs are tagged to SNPs used in our study (HainesSNP 2 is tagged to +674 and HainesSNP 5 is tagged to +9162). Haines et al. showed an association between SNP 2 (tagged to our SNP +674) and exudative AMD in their familial group. This is particularly interesting in that we have demonstrated a strong association to SNP +674 in our exudative AMD cohort. We found that the CC genotype at +674 was significantly associated with the AMD group, whereas presence of the T allele was significantly associated with the control group. This data supports the hypothesis that the +674CC genotype is a potential risk factor for AMD, and that carriage of the T allele is potentially protective. The +674 SNP is located in Intron1, 1695 bp downstream from the start of Exon1. The Transplorer Program (BioBase, Wolfenbüttel, Germany) places this 85 bp upstream of a putative stress response element (SRE) binding site (TRANSFAC F$STRE_01) (39). SREs are often associated with transcription induction during stress periods such as hypoxia (40). Proximity of this SNP may alter the local environment, influence binding and increase VEGF production.


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  		Table 4. Linkage disequilibrium measurements for the five htSNPs. Data calculated using the normal control dataset. D' is given in the upper right half and r2 in the lower left half

 
Haplotype analysis of the five htSNPs (+674, +4618, +5092, +9162, +9512) revealed two haplotypes associated with AMD and one haplotype associated with the control group (Table 3). Closer inspection of the two htSNP haplotypes associated with AMD (CTCCT and TCACC) reveals that the presence of the C or T allele at +674 loses its individual significance. This variation may be due to the mixing of ‘at risk’ and ‘protective’ haplotypes in any individual since PHASE data requires genotype input for each individual. For example, a paternal haplotype inclusive of the +674C allele may have been carried with a +674T maternal haplotype, thus reducing the ‘at risk’ nature of this particular locus. Furthermore, the haplotype at any given locus is likely to be influenced by the haplotypes present at the other combined loci. It is clear, however, that possession of either of the CTCCT or TCACC haplotypes is a significant risk factor in AMD (ORs=15.77 and 9.95, respectively). TCACT appears to offer relative protection from AMD in that only 6.7% AMD cases compared to 48.9% controls were predicted to carry this htSNP haplotype.

It is not possible to state categorically that any of the four genotypes/haplotypes (+674 SNP, promoter haplotype or the two htSNP haplotypes) puts one at a greater risk of developing neovascular AMD without performing predictive studies over many years, but there is a strong association with the disease state. Potentially, a simple test may be developed to predict the susceptibility to AMD, for instance, determining whether an individual carried the +674CC genotype. Such a test might form the basis of lifestyle advice offered to young, healthy but susceptible, individuals. Studies on environmental risk factors often produce conflicting evidence but smoking and hypertension have been most consistently cited as negative risk factors in the development of AMD and some dietary supplements, in particular, anti-oxidants appear to offer some protection against AMD (41–43). Functional studies would allow further investigation into the role of the –460T, –417T, –172C, –165C, –160C, –152G, –141A, –116A, +405C promoter haplotype in VEGF production and further our understanding of what appears to be a haplotype associated with other neovascular retinal disease. This is the most comprehensive SNP analysis to date in AMD and has taken previous studies one step further in trying to understand the genetic basis of neovascular AMD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Patient selection
Cases (n=45) were recruited from the photodynamic therapy clinic at the Bristol Eye Hospital. All cases were over 50 years of age and had AMD, secondary to choroidal neovascular membranes demonstrated by fundus fluorescein angiography. Co-existing ocular pathology, such as high myopia, that might account for the neovascular membrane was excluded. Patient ages ranged from 51 to 94 years. Sex distribution was 37.8% male and 62.2% female with mean ages of 72.8 and 76.1 years, respectively. Controls (n=94) were age-matched to within 5 years of cases and recruited from spouses and healthy volunteers accompanying patients. A health questionnaire was completed to exclude those with a history of asthma, diabetes, cancer, psoriasis, rheumatoid arthritis and systemic lupus erythematosis. All controls underwent visual acuity testing, anterior segment and fundus examination. Sex distribution of control subjects was 35.1% male and 64.9% female, with mean ages of 72.6 and 73.2 years, respectively (age range: 55–89 years). All study participants were Caucasians of Northern European origin. Ethics approval for the study was obtained from the United Bristol Healthcare Trust and protocols conformed to the tenets of the Declaration of Helsinki. A venous blood sample was obtained from each participant after informed written consent.

DNA preparation
DNA was extracted by rapid salting technique and quantified by spectrophotometry (SpectraMax Plus, Molecular Devices, Wokingham, UK) (44).

Selection bias
To ensure there was no selection bias between the sample populations, each DNA sample was examined for Hardy–Weinberg Equilibrium using two independent polymorphic markers (in TYRP1ex2 and TP53PIN3) unrelated to AMD, as described in Powell et al. (45–47).

Promoter SNPs
Nine SNPs were selected from a PubMed search on association studies of polymorphisms within the VEGF promoter and 5'UTR region: C–460T, T–417C, C–172A, C–165T, C–160T, G–152A, A–141C, G–116A, G+405C. All SNPs are numbered from the transcription start (Genbank accession no. M63971) (48). PCR primers were designed to incorporate the eight promoter SNPs in one fragment (Forward: 5'GGTGAGTGAGTGTGTGCG-3', Reverse: 5'CCGCTACCAGCCGACTTT-3'). Each DNA sample was amplified by hot-start PCR [30 µl reaction mix; 1xPCR Buffer II (ABI), 2 mM> Mg2+, 200 µM dNTP (GeneAmp, ABI), 1 µM Primer, 0.1% Triton X-100 (Sigma, St. Louis, MO, USA), 6% DMSO (Sigma), 1unit Taq (AmpliTaq Gold, ABI), 1.5 µl DNA] with a DYADTM DNA Engine Peltier Thermal Cycler (MJ Research). PCR conditions were: 5 min hot-start denaturation at 94°C; add Taq; 3 min at 94°C; 35 cycles at 94°C for 30 seconds, 62.9°C for 1 min, 72°C for 1 min; final extension period of 5 min at 72°C. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Crawley, UK), and sequenced [Genetic Research Instrumentation Ltd (GRI), Braintree, UK]. Sequence data were checked visually using Chromas v1.45 (Technelysium Pty Ltd, Queensland, Australia) and genotypes determined using Sequencher v4.01 software (GeneCodes, Ann Arbor, MI, USA), and VEGF data from GenBank (#AL136131) as a reference sequence. Genotyping for the +405 polymorphism (i.e. rs2010963) was conducted by PCR-RFLP analysis using the restriction enzyme BsmFI as previously described with minor modifications (18). We found that a 3 h digestion time gave better overall resolution of the products on a 2.5% agarose gel (G allele=196 bp and 274 bp, C allele=470 bp). A subset of samples were sequenced to verify the PCR–RFLP analysis.

Haplotype-tagged SNP selection
SNPs were selected from data obtained from the UW–FHCRC Variation Discovery Resource (SeattleSNPs) Database, a collaborative online resource of SNP data, developed by the University of Washington and the Fred Hutchinson Cancer Research Centre (49). Using the SeattleSNP haplotype data (haplotype frequency >3%), five htSNPs were selected (+674, +4618, +5092, +9162, +9512) using the SNPtagger Program, as SNPs representative of 19 different polymorphic sites (Table 1) (50). Genotype data were obtained using allele-specific PCR techniques (Table 5). Control primers were used to confirm true negative results using a 20 µl reaction mix [1xPCR buffer, 1.5–2 mM Mg2+, 200 µM dNTP (GeneAmp, ABI), 1 µM primer, 0.5 µM control primer, 1unit Taq [either AmpliTaq Gold (ABI) or NovaTaq (Novagen, Merk Biosciences, Beeston, UK) polymerases, 2 µl DNA] in a DYADTM DNA Engine Peltier Thermal Cycler (MJ Research). Cycling conditions were 5 min hot-start denaturation at 95°C; add Taq; 5 min at 95°C; 35 cycles of 95°C for 45 seconds, at 60–67.2°C for 45 seconds, 72°C for 1 minute; final extension period of 5 min at 72°C. Samples were separated using Gel Electrophoresis (1.5% w/v Agarose Gel, 0.5xTBE), and visualized under UV-light using Labworks v4.0.0.8 software (Ultra-Violet Products Ltd, Cambridge, UK). To ensure accuracy of allele-specific results, a randomized selection of sample PCRs were sequenced. Resultant data were collated, samples genotyped and frequencies analysed.


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  		Table 5. Primer sequences and amplimer size for VEGF polymorphism genotyping. SNPs are numbered from GenBank AF437895

 
Haplotype and statistical analysis
Individual haplotypes and their estimated population frequencies were inferred using the PHASE program, v2.1, with all parameters set at the default values (51,52). Statistical analysis was carried out on computer using SPSS© v11.5.0 (SPSS UK Ltd, Woking, UK), Epi Info 6 v6.04d software (Centers for Disease Control and Prevention, USA) and SISA (53). Pearson's {chi}2 Test and/or Fisher's exact test were used to compare patient and control groups for possible associations between SNP genotype/allele frequency and disease state. Statistical significance was assumed at P<0.05. Odds ratios were also calculated, adjusted by Haldane's correction where necessary. Because of the multiple analysis conducted, the Bonferroni correction was applied to produce corrected P-values.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the National Eye Research Centre, UK and The NHS United Bristol Hospital Trust Medical Research Committee.

Conflict of Interest statement. None of the authors have any conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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