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NR 4-6/2009
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Distribution of the
C-460T polymorphism of the vascular endothelial growth factor
gene in age-related macular degeneration
Rozkład genotypów
polimorfizmu C-460T genu naczyniowo-śródbłonkowego czynnika
wzrostu w zwyrodnieniu plamki związanym z wiekiem
Szaflik Jacek P.1, Błasiak
Janusz2, Krzyżanowska Anna1, Zaraś
Małgorzata1, Janik-Papis Katarzyna2,
Borucka Anna I.1, Woźniak Katarzyna2,
Szaflik Jerzy1
1 Ophthalmology Department, Medical University of
Warsaw, Warsaw, Poland
Head: Professor Jerzy Szaflik, MD, PhD
2 Department of Molecular Genetics, University of
Lodz, Lodz, Poland
Head: Professor Janusz Błasiak, PhD |
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| Summary: |
Purpose: To assess
the association between genotypes and alleles of the
C-460T polymorphism of the vascular endothelial growth
factor (VEGF) gene and the risk of wet form of
age-related macular degeneration (AMD).
Materials and methods: 100 patients with
clinically diagnosed wet form of AMD and 104 healthy
individuals were enrolled in this study. The patients
were diagnosed by optical coherence tomography,
fluorescein angiography and indocyanin green angiography.
The allele-specific polymerase chain reaction was used
to determine the genotypes of the C-460T polymorphism of
the VEGF gene. The odds ratios (ORs) and 95% confidence
intervals (CIs) were calculated using a logistic
regression model to assess the assoctiation betweeen
genotypes of the C-460T polymorphism and AMD occurrence.
Results: A difference was observed in the
genotype distributions between patients and controls. An
association (OR 3.04, 95% CI 1.65-5.60) was found
between wet form of AMD and the C/T genotype. On the
other hand, the T/T genotype displayed the protective
effect against the disease.
Conclusion: The C-460T polymorphism of the
endothelial growth factor can be considered as a
potential marker for the wet form of age-related macular
degeneration. |
| Słowa kluczowe: |
age related macular
degeneration, AMD, vascular endothelial growth factor,
VEGF, genetic polymorphism. |
| Key words: |
zwyrodnienie plamki
związane z wiekiem, AMD, naczyniowo-śródbłonkowy czynnik
wzrostu, VEGF, polimorfizm genetyczny. |
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Introduction
Age-related macular degeneration (AMD) is the overall leading
cause of visual loss and neovascularization seems to be
primarily involved in the fatal outcome of the wet form of this
disease (see 1 for review). The process of neovascularization is
underlain by several angiogenic factors, including vascular
endothelial growth factor, VEGF, which seems to be crucial for
this process. Recent studies have documented the
immunohistochemical localization of VEGF in surgically resected
choroidal neovascular membranes from AMD patients (2). These
findings suggest a role for VEGF in the progression of
AMD-related choroidal neovascularization, raising the
possibility that VEGF inhibition may have therapeutic value for
this condition. Accordingly, clinical trials with anti-VEGF
antibodies and other VEGF inhibitors are currently ongoing (3).
VEGF or VEGF-A, is a member of a family of related proteins
consisting of VEGF-A, VEGF-B, VEGFC, VEGF-D and Placenta Growth
Factor. Along with Platelet-derived Growth Factor A and B (PDGF-A
and PDGF-B) they form a protein superfamily. The VEGF-A pre-mRNA
is alternatively spliced and is translated to give 5 polypeptide
products of 121, 145, 165, 189 and 206 amino acids (4). VEGF-A
is crucial to embryonic development and disruption of even one
allele of the VEGF-A gene is lethal (5).
The expression of the VEGF gene is modulated by a variety of
effectors including cytokines, lipopolysaccharide (LPS),
hormones and hypoxia (6). Dysregulated VEGF expression is
implicated in a number of disease pathologies. Increased VEGF
expression resulting in inappropriate VEGF-induced angiogenesis
is linked with tumour growth and metastasis, rheumatoid
arthritis and diabetic retinopathy. Conversely, the ability to
produce VEGF in response to hypoxia is linked to the development
of collateral vessels and protection against myocardial disease.
In the eyes of healthy subjects, VEGF is present only in a
minimal degree in endothelial cells of retinal and choroidal
vessels, retinal pigment epithelium (RPE) and intravascular
leukocytes of choroid and retina (7). Because VEGF is important
for the process of neovascularization, which, in turn, is
essential for wet form of AMD, the expression of the VEGF gene
may be of great importance in the pathogenesis of this disease.
This expression can be modulated by the variability of the gene,
which can take a form of polymorphism. Therefore, it is
reasonable to search for the association between sequence
variability of the VEGF gene and wet form of AMD. In the present
work we checked the association between genotypes and alleles of
the C-460T polymorphism of the VEGF-A gene and the risk of the
occurrence of AMD in wet form.
Materials and methods
Patients
Blood samples were obtained from patients with wet form of AMD
(n = 100) and healthy individuals (n = 104).
Medical history was obtained from all subjects. The patients
underwent ophthalmic examination including best corrected visual
acuity, intraocular pressure, slit-lamp examination, and fundus
examination using non-contact and contact fundus lenses with a
slit lamp.
Diagnosis of wet form AMD was confirmed by optical coherence
tomography (OCT), fluorescein angiography (FA), and in some
cases indocyanin green angiography (ICG). OCT evaluated retinal
thickness, the presence of subretinal fliud and intraretinal
oedema; angiography assessed the anatomical status of the
retinal vessels, the presence of choroidal neovascularisaton (CNV)
and leakage.
The OCT examinations were performed with Stratus OCT model 3000,
software version 4.0. The FA and ICG examinations were completed
with a Topcon TRC-50I IX fundus camera with the digital Image
Net image system (ver. 2.14; Topcon Co., Tokyo, Japan).
The Local Ethic Committee approved the study and each patient
gave a written consent.
Determination of the VEGF genotype
DNA was isolated from peripheral blood leukocytes by using
proteinase K digestion and phenol/chloroform extraction.
Genotypes of the C–460T polymorphism was determined by the
allele specific PCR. Two forward primers annealed to the
polymorphic site T: 5’-TGCGTGTGGGGTTGAGGGT-3’ or
C: 5’-TGCGTGTGGGGTTGAGGGC-3’ and reverse primer:
5’-CCCGCCGCAATGAAGGGGA-3’ were used. The PCR was carried out in
a MJ Research, INC thermal cycler, model PTC-100 (Waltham, MA,
USA), in a total volume of 25 µl, containing 50 ng genomic DNA,
10 pmol each primer (Sigma Proligo, Sigma-Aldrich, Mannheim,
Germany), 200 mM each dATP, dCTP, dGTP and dTTP (Boeringer,
Mannheim, Germany), 20 mM Tris-HCl (pH 8.4) 50 mM KCl, 2 mM
MgCl2 and 1 unit Taq polymerase. The thermal cycling conditions
were 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 45 s
at 61°C and 1 min at 72°C. PCR-amplified DNA was analyzed on a
3% agarose gel and visualized by ethidium bromide staining.
Typical results of genotype analysis are displayed in Fig. 1.
Statistical analysis
The significance of the differences of observed alleles and
genotypes between groups was tested using the chi‑ test-based
analyses. The ORs and 95% CIs were calculated using a logistic
regression model. The two-group comparisons of continuous data
with normal distribution (verified with Shapiro-Wilk’s test)
were performed with t-test, the remianing data were analyzed
with the non-parametric Mann-Whitney U test. ANOVA was used to
identify parameters that would make significant differences when
comparing more than two groups; Scheffe’s test was then used as
the post hoc multiple comparison test. Analyses were performed
using STATISTICA 6.0 software (Statsoft, Tulsa, OK, USA).
Results and discussion
All the patients and controls were divided into three genotypes
of the C–460T polymorphism of the VEGF-A gene promoter: C/C, C/T
and T/T (Table 1). There was a significant (χ2=45.12, p < 0.001)
difference in genotype distributions between patients and
controls. An intermediate association (odds ratio 3.04, 95%
confidence interval 1.65-5.60) was found between wet form of AMD
and the C/T genotype. On the other hand, the T/T genotype
displayed the protective effect against wet form of AMD (OR
0.30, 95% CI 0.15-0.59). In general, the C allele increases the
risk of AMD in wet form and the T allele has a protective
influence against this disease.
Commonly accepted genetic factors indicating occurrence/
progression of a disease are mutations, which can contribute to
the disease phenotype, especially if they occur with a
relatively high frequency, taking form of gene polymorphisms.
The progression of AMD from its dry to wet form is still unclear
and is a matter of discussion. This justify research on specific
indicators, which can be attributed to this disease. Furthermore,
the correlation of VEGF production, initiated by different
physiological stimuli, with a specific genotype may allow
identification of individuals who exhibit high or low levels of
VEGF production predisposing them to VEGF-mediated pathologies.
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Intense angiogenesis is a hallmark of the wet form of AMD and is
underlying by high activity of VEGF. This activity may be a
consequence of alternative splice of the VEGF gene, which can be
spliced to form the angiogenic (VEGFxxx) and potentially
anti-angiogenic (VEGFxxxb) families of isoforms (8,9). The
regulation of splicing originating from upstream regions of the
gene could result in alterations in the recruitment of splicing
factors to the RNA polymerase complex and control of the speed
of the polymerase reaction. It is suggested that some of the
upstream polymorphisms, e.g. G-460C could affect this process.
Recently, it was showed that in the eye of diabetic patients
VEGF splicing was switched from an antiangiogenic to a
pro-angiogenic environment and -460C allele might be critical
factor for the balance of VEGF isoforms (10). In our previous
work we showed that the C-460T polymorphism of the VEGF gene may
be associated with diabetic retinopathy when combined with
another polymorphism of the promoter of this gene, G-634C (11).
In summary, the C-460T polymorphism of the endothelial growth
factor can be considered as a potential marker for the wet form
of age-related macular degeneration.
Acknowledgement
This work was supported by the grant of Ministry of Science and
Higher Education number 3C68.
References:
1. Montezuma SR, Sobrin L, Seddon JM.: Review of genetics in age
related macular degeneration. Semin Ophthalmol 2007, 22,
229-240.
2. Lopez PF, Sippy BD, Lambert HM, Thach AB, Hinton DR:
Transdifferentiated retinal pigment epithelial cells are
immunoreactive for vascular endothelial growth factor in
surgically excised age-related macular degeneration-related
choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1996,
37, 855-868.
3. Andreoli CM, Miller JW: Anti-vascular endothelial growth
factor therapy for ocular neovascular disease. Curr Opin
Ophthalmol 2007, 18, 502-508.
4. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW:
The vascular endothelial growth factor family: Identification of
a fourth molecular species and characterisation of alternative
splicing of RNA. Mol Endocrinol 1991, 5, 1806-1814.
5. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS,
Powell-Braxton L, Hillan KJ, Moore MW: Heterozygous embryonic
lethality induced by targeted inactivation of the VEGF gene.
Nature 1996, 380, 439-442.
6. Watson CJ, Webb NJA, Bottomley MJ, Brenchley PEC:
Identification of polymorphisms within the vascular endothelial
growth factor (VEGF) gene: correlation with variation in VEGF
protein production. Cytokine 2000, 12, 1232-1235.
7. Lutty GA, McLeod DS, Merges C, Diggs A, Plouét J:
Localization of vascular endothelial growth factor in human
retina and choroid. Arch Ophthalmology 1996, 114, 971-977.
8. Bates DO, Cui TG, Doughty JM: VEGF165b, an inhibitory splice
variant of vascular endothelial growth factor, is down-regulated
in renal cell carcinoma. Cancer Res 2002, 62, 4123-4131.
9. Suganthalakshmi B, Anand R, Kim R, Mahalakshmi R,
Karthikprakash S, Namperumalsamy P, Sundaresan P: Association of
VEGF and eNOS gene polymorphisms in type 2 diabetic retinopathy.
Mol Vis 2006, 12, 336-341.
10. Perrin RM, Konopatskaya O, Qiu Y, Harper S, Bates DO,
Churchill AJ: Diabetic retinopathy is associated with a switch
in splicing from anti- to pro-angiogenic isoforms of vascular
endothelial growth factor. Diabetologia 2005, 48, 2422-2427.
11. Szaflik JP, Wysocki T, Kowalski M, Majsterek I, Borucka AI,
Blasiak J, Szaflik J: An association between vascular
endothelial growth factor gene promoter polymorphisms and
diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol. 2008,
246, 39-43.
The study was originally received: 16.05.2008. (1046)/
Praca wpłynęła do Redakcji 16.05.2008. (1046)
Accepted for publication: 20.04.2009/
Zakwalifikowano do druku 20.04.2009 r.
Fig. 1. Genotypes of the C/ T polymorphism at
the – 460 bp VEGF-A promoter region determined by the allele
specific PCR-detection (ASO-PCR) and analysed by 3% agarose gel
electrophoresis, stained with ethidium bromide and viewed under
UV light. Lane M displays molecular weight marker; the lanes
show the results of amplification with primer specific either to
the C allele or the T allele.
Ryc. 1. Genotypy polimorfizmu C/T w pozycji -460 obszaru
promotorowego genu VEGF-1 określone metodą allelo-specyficznej
PCR i analizowane przez elektroforezę w 3% żelu agarozowym,
barwienie bromkiem etydyny i naświetlanie UV. Ścieżka M zawiera
marker mas cząsteczkowych, pozostałe ścieżki – wyniki
amplifikacji ze starterami specyficznymi do allelu C lub T.
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Genotype |
AMD (n = 100) |
Controls (n = 104) |
OR (95% CI) |
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or Allele
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Number
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Frequency |
Number |
Frequency |
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C/C
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8
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0.08
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11
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0.11
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0.74 (0.28-1.91)
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C/T
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78
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0.78
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56
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0.54
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3.04 (1.65-5.60)
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T/T
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14
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0.14
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37
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0.35
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0.30 (0.15-
0.59)
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C
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94
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0.47
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78
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0.38
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1.48 (1.00-2.19)
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T
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106
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0.53
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130
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0.62
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0.67 (0.46-1.00)
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Tab. I. The distribution of
genotypes, frequency of alleles of the
C-460T polymorphism of the VEGF-1 gene and odds ratio (OR) in
wet form of age related macular degeneration (AMD) patients and
controls without AMD.
Tab. I. Rozkład genotypów i częstość alleli polimorfizmu C-460T
genu VEGF-1 oraz analiza ilorazu szans (OR) dla chorych z mokrą
postacią zwyrodnienia plamki związanego wiekiem (AMD) i osób bez
AMD (kontrola).
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