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Keywords:

  • age-related macular degeneration;
  • cardiovascular risk factors;
  • genetics

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Purpose:  To investigate the association between genetic cardiovascular risk factors and exudative age-related macular degeneration (AMD) in a White Austrian population.

Methods:  Seventy-five unrelated AMD patients and 75 unrelated healthy, sex- and age-matched control patients were genotyped for the following 19 single nucleotide polymorphisms (SNPs) in 14 different genes: blood coagulation factor V (FV) R506Q, factor II (prothrombin) G20210A and factor XIII (FXIII) V34L; 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C; plasminogen activator inhibitor 1 (PAI-1) 4G/5G; endothelial protein C receptor (EPCR) 4600 A>G (A3 haplotype), 4678 G>C (A1 haplotype); apolipoprotein B (ApoB) R3500Q; apolipoprotein E (ApoE) E2/E3/E4; β-fibrinogen −455 G>A; human platelet antigen 1 (HPA1) a/b; angiotensin-converting enzyme (ACE) I/D; endothelial nitric oxide synthase (eNOS) 786 T>C, 894 G>T; lymphotoxin alpha (LTA) 804 C>A and 9p21 rs10757278. Genotyping was carried out by polymerase chain reaction (PCR) followed by reverse hybridization (CVD StripAssays; ViennaLab Diagnostics, Vienna, Austria).

Results:  No statistically significant association could be observed between AMD and the investigated genetic risk factors for cardiovascular disease (CVD). All factors seem to be uniformly distributed in the two groups of AMD patients and healthy controls. Two variables –β-fibrinogen: −455 G>A (p = 0.0786) and apolipoprotein E4 (p = 0.0636) – were not as far from association as the others.

Conclusion:  Our data show that the 19 tested CVD risk markers do not play a significant role in AMD. β-Fibrinogen and apolipoprotein E4 should be examined in a larger cohort.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Age-related macular degeneration (AMD) is a progressive degenerative disorder of the central macular region of the retina. It is the leading cause of legal blindness in people aged over 55 years in developed countries (Ambatti 2003).

The aetiology of AMD is complex and its pathogenesis remains poorly understood. The prevalence of both AMD and cardiovascular disease (CVD) is strongly age-dependent. There are reports providing evidence of associations between cardiovascular risk factors and AMD. Cardiovascular risk factors like smoking, high blood pressure, serum cholesterol, intake of calcium channel blockers and increased body mass index revealed an association with AMD in different studies (Klein et al. 2007). The Blue Mountain study also showed some evidence of links between cardiovascular risk factors and AMD; in particular, a history of stroke or any cardiovascular event [such as myocardial infarction (MI) or angina] were stated by the authors to predict AMD (Tan et al. 2007).

In the present study we investigated a possible association between candidate gene markers for inherited risk for CVD and AMD by using 19 single nucleotide polymorphisms (SNPs) in 14 different genes:

  •  eNOS −786 T>C polymorphism causes a higher susceptibility to coronary heart disease (CHD) and 894 G>T (Glu298Asp) is related to an increased risk of premature MI (Cooke et al. 2007).
  •  The 804 C>A (Thr26Asn) exchange in the gene-encoding lymphotoxin alpha (LTA) on chromosome 6p21 has been associated with susceptibility to CHD and to large-vessel-associated ischaemic stroke (Topol et al. 2006).
  •  Studies of never-treated hypertensive patients and European and Chinese populations suggested that cardiovascular risk factors are associated with the D allele of the angiotensin-converting enzyme (ACE) gene (Muthumala et al. 2007).
  •  The human platelet antigen 1 (HPA1) a/b polymorphism is an important inherited risk factor for MI. The polymorphism is also a risk factor for coronary thrombosis, the progression of coronary artery disease, early-onset MI and stroke, particularly in smokers.
  •  The SNP rs10757278 is one of several clustered together in a region of chromosome 9 that has been linked to increased risk for CHD and potentially to diabetes (Helgadottir et al. 2008).
  •  Blood coagulation factor V (FV) Leiden (R506Q) results in the observed hypercoagulable state, which leads to an increased risk for venous thromboembolism (VTE) (Braun et al. 1996).
  •  It has been reported that the FV H1299R mutation increases the risk of venous thrombosis and CVD risk in carriers of the FV R506Q mutation (Lunghi et al. 1996).
  •  The G20210A mutation in the prothrombin gene had been identified as a candidate gene for venous thrombosis in families with a history of VTE (Varga & Moll 2004).
  •  5,10-Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with reduced enzyme activity and elevated plasma homocysteine levels in conjunction with folate deficiency. Homozygosity predisposes to arterial and venous thrombosis in the presence of additional risk factors. The MTHFR A1298C polymorphism is also associated with reduced MTHFR enzyme activity (Sofi et al. 2005).
  •  Blood coagulation factor XIII (FXIII) activation is known to be strongly influenced by a common polymorphism in the FXIII A-subunit gene (Val34Leu), which has been shown to be protective against MI, ischaemic stroke and deep vein thrombosis (Gemmati et al. 2007).
  •  The 4G allele of plasminogen activator inhibitor 1 (PAI-1) (Serpin E1) is considered to be a mild risk factor for venous thrombosis and MI (Lijnen 2005).
  •  Carriers of the endothelial protein C receptor (EPCR) A3 haplotype show higher soluble EPCR plasma levels and are predisposed to VTE and foetal loss (Medina et al. 2005).
  •  The Apo B R3500Q mutation is the major cause of familial hypercholesteraemia (Real et al. 2003).
  •  The Apo B R3500Q mutation is the major cause of familial hypercholesteraemia (Real et al. 2003).
  •  Apo E2 is associated with type III hyperlipoprotenaemia and with both increased and decreased risk for atherosclerosis. E4 has been associated to atherosclerosis and Alzheimer’s disease (Dedoussis 2007).
  •  The β-fibrinogen −455 G>A polymorphism is consistently associated with differences in fibrinogen levels and, therefore, should be associated with CHD and ischaemic stroke risk, if fibrinogen is a causal factor (Lip 1995).

Materials and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Study design

For the prospective single-blinded case–control study 75 unrelated patients with exudative AMD and 75 unrelated control patients aged 55 years and older were recruited. All participants were White, lived in the same geographical area of Austria and were seen at the local department of ophthalmology. Written informed consent was obtained prior to enrolment. The study was performed in accordance with the Austrian Gene Technology Act, the tenets of the Declaration of Helsinki and the guidelines of the local ethics committee.

All AMD patients enrolled in the present study were classified according to the Age-related Eye Disease Study system (geographic atrophy, predominantly classic choroidal neovascularization, minimal classic choroidal neovascularization, occult choroidal neovascularization and retinal angiomatous proliferation) (Age-related Eye Disease Study Research Group 2001). Exudative AMD was diagnosed by ophthalmoscopic fundus examination, optical coherence tomography and fluorescein/indocyanine angiography. Patients with hereditary diseases, polypoidal choroidal vasculopathy or secondary CNV caused by pathological myopia (> −2 dioptres, spherical equivalent), angioid streaks, inflammatory or infectious chorioretinal disease, trauma or diabetic retinopathy were excluded from the study.

The age- and sex-matched control group, comprising patients recruited to our department for cataract surgery, had a thorough eye examination with a detailed fundus examination. Exclusion criteria for controls were evidence of any stage of age-related maculopathy, macular haemorrhages of any cause or media opacities resulting in impaired visualization of the macula – cataract grade 3 and 4 of the Lens Opacities Classification System III (LOCS-system) (Chylack et al. 1993).

Genotyping

Genotyping was carried out in a random order by an experienced technician who was masked to the disease status of the samples. Venous blood (5 ml) was withdrawn from each patient and collected in an ethylenediamnetetraacetic acid (EDTA)-containing tube. Genomic DNA was isolated from whole blood using a commercial kit (QIA-AMP DNA blood mini kit; Qiagen, Vienna, Austria). Genotyping for 19 SNPs (listed in Table 1) was performed by polymerase chain reaction (PCR) and reverse hybridization to allele-specific oligonucleotides immobilized on membrane test strips (CVD StripAssays; ViennaLab Diagnostics, Vienna, Austria). The hybridization and detection procedure was carried out in a temperature-controlled test-strip processor (profiBlot-T48; Tecan, Männedorf, Switzerland).

Table 1.   p-values of the analysed genetic cardiovascular risk factors.
GenePolymorphismp-value
Endothelial nitric oxide synthase (eNOS; NOS3)−786 T>C0.9083
894 G>T (Glu298Asp)0.5455
Lymphotoxin alpha (LTA)804 C>A0.7031
Angiotensin-converting enzyme (ACE)I/D0.1302
Human platelet antigen 1 (HPA1; Gp IIIa)a/b (L33P)0.4929
Apolipoprotein B (Apo B)R3500Q0.9907
9p21 rs10757278A>G0.5406
Factor V LeidenG1691A; R506Q0.8001
Factor V R2 haplotypeH1299R0.7970
Prothrombin (factor II)G20210A0.1330
5,10-Methylenetetrahydrofolate reductase (MTHFR)A1298C0.9017
C677T0.2420
Factor XIII (FXIII)V34L0.2491
Plasminogen activator inhibitor 1 (PAI-1)4G/5G0.5644
Endothelial protein C receptor (EPCR)4600 A>G (A3)0.3463
β-Fibrinogen−455 G>A0.0786
Apolipoprotein E (ApoE)ApoE2/E3/E40.0636

Statistics

Statistical analysis was performed using sas 9.1 (SAS Institute Inc., Cary, NC, USA). All p-values < 0.05 were considered statistically significant, and no correction for multiplicity was needed. Variables were analysed by logistic regression. First univariate tests were performed, and for those variables that were statistically significant a multivariate regression was calculated.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

One hundred and fifty participants were included in the present study, in which 75 unrelated AMD patients were compared to 75 unrelated ‘healthy’ controls. Age and gender were similar in both groups. The AMD group consisted of 41 (54.6%) women and 34 (45.3%) men, and the mean age was 77.0 ± 7.3 years (range 56–92 years). The control group consisted of 37 (49.3%) women and 38 (50.6%) men, and the mean age was 76.5 ± 6.5 years (range 62–94 years).

Genotypes of all 14 genes were determined successfully in all participants. However, for the vast majority of the investigated markers no statistical significance for an increased risk between patient and control group could be seen (Table 1).

Only two CVD markers were not as far from association as the others: β-fibrinogen [(−455 G>A), p = 0.0786] and apolipoprotein E [(ApoE4), p = 0.0636] (Figs 1 and 2).

image

Figure 1.  Genotype distribution of β-fibrinogen −455 G>A in age-related macular degeneration patients (n = 75) and healthy controls (n = 75). X-axis: patients, controls; Y-axis: incidence. Normal = 0, heterozygous = 1, homozygous = 2 for the β-fibrinogen −455 G>A.

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image

Figure 2.  Genotype distribution of apolipoprotein E (ApoE) in age-related macular degeneration patients (n = 75) and in healthy controls (n = 75). X-axis: patients, controls; Y-axis: incidence. ApoE: E2/E2 = 0; E2/E3 = 1; E2/E4 = 2; E3/E3 = 3; E3/E4 = 4, E4/E4 = 5.

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Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

The prevalence of both AMD and CVD is strongly age-dependent. Several reports provide some evidence of associations between cardiovascular risk factors and AMD. Factors such as hypertension, previous vascular events, obesity and diabetes have been significantly associated with early and advanced AMD in several studies (Klein et al. 2007; Tan et al. 2007). Friedman hypothesized that hypertension and atherosclerosis increase the risk of AMD through the reduction of blood flow through the choroidal vasculature and lipid deposition in Bruch’s membrane, with a reduction of permeability leading to the upregulation of vascular endothelial growth factor (Friedman 2000). Inflammation has been postulated to be important in the pathogenesis of atherosclerosis and in AMD – like the C-reactive protein, which has been associated with AMD (Vine et al. 2005). Single nucleotide polymorphisms in complement factors are the most important potential risk factors (Seitsonen et al. 2008). Until now no comparable study analysing different genetic markers associated with cardiovascular diseases has been conducted in AMD patients.

In the eye, ApoE immunoreactivity has been localized to Müller cells, Bruch’s membrane, the retinal pigment epithelium (RPE), basal deposits and drusen. ApoE mRNAs are synthesized by Müller cells in the neural retina and by the RPE.

The association of ApoE to AMD is described inconsistently. Utheim et al. (2008) found no association between AMD and ApoE polymorphism in a population of 96 individuals aged 50–75 years. Fritsche et al. have defined an extended haplotype block encompassing the entire ApoE gene locus, including known coding as well as cis-regulatory promoter variants. Two extended ApoE haplotypes that are common in the general population were found to be significantly associated with AMD, namely G-G-G-G-epsilon2 and T-G-A-G-epsilon4. Fritsche et al. also found one extended epsilon3-haplotype, G-G-G-A-epsilon3, to be protective in the homozygous state. So they hypothesized that both the relative rate of ApoE isoform expression in conjunction with established functional differences of the respective isoforms may be crucial in mediating AMD pathology (Fritsche et al. 2009). In our study we did not look for the determination of extended haplotype combinations, including the functional promoter SNP rs405509, so this may explain our results. Our data show no association of the E4 allele in AMD patients compared to healthy patients (p = 0.0636).

The fibrinogen level is predictive of CHD risk. Meta-analyses reported a relative risk of 1.8 [95% confidence interval (CI) 1.6–2.0]. However, existing atherosclerosis may increase fibrinogen levels, and thus reverse causation will lead fibrinogen to predict future CHD events.

The most common polymorphism that has been studied in this regard is the −455G>A polymorphism in the promoter region of the β-fibrinogen gene. This polymorphism is consistently associated with differences in fibrinogen levels and, therefore, should be associated with CHD and ischaemic stroke risk if fibrinogen is a causal factor (Lip 1995). Up to now, no data have existed on the β-fibrinogen −455 G>A mutation and AMD patients.

Fibrinogen has been identified in drusen and Bruch’s membrane, and prior epidemiological evidence from cross-sectional studies supports a relationship between fibrinogen and AMD. The possible risk of AMD could be through reductions in choroidal blood flow, direct effects on the vascular wall, or other effects of chronic inflammation (Grunwald et al. 1998). Our data show no association between the mutation β-fibrinogen −455 G>A and AMD patients compared to control patients (p = 0.0786).

In conclusion, our data show no major association between the tested genetic cardiovascular risk factors and exudative AMD except that two variables –β-fibrinogen: −455 G>A (p = 0.0786) and apolipoprotein E4 (p = 0.0636) – were not as far from association as the others.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References