• age-related macular degeneration (AMD);
  • apolipoprotein E (ApoE);
  • case-control study;
  • complement factor H (CFH);
  • genetic risk factor;
  • HTRA1;
  • LOC387715


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

Purpose:  Recent studies strongly support the role of genetic factors in the aetiology of age-related macular degeneration (AMD). We investigated the frequency of Tyr402His polymorphism of the complement factor H (CFH) gene, Ser69Ala polymorphism at LOC387715, rs11200638 polymorphism of the HTRA1 gene and different apolipoprotein E (ApoE) alleles in Hungarian patients with AMD in order to determine the disease risk conferred by these factors.

Methods:  In a case-control study, we performed clinical and molecular genetic examination of 105 AMD patients (48 patients in the early and 57 in the late subgroup) and 95 unrelated healthy controls. Detailed patient histories were recorded with the use of a questionnaire focusing on known risk factors for AMD.

Results:  In the early AMD subgroup, homozygous CFH, LOC387715 or HTRA1 polymorphisms conferred a 4.9-fold (95% confidence interval [CI] 1.7–14.2), 7.4-fold (95% CI 2.1–26.2) or 10.1-fold (95% CI 2.5–40.8) risk of disease, respectively. In the late AMD subgroup, carriers of two CFH, LOC387715 or HTRA1 risk alleles were at 10.7-fold (95% CI 3.7–31.0), 11.3-fold (95% CI 3.2–40.4) or 13.5-fold (95% CI 3.3–55.4) greater disease risk, respectively. Two CFH and one LOC387715 risk alleles in combination conferred a 15.0-fold (95% CI 3.2–71.0) increase in risk, whereas two LOC387715 risk alleles combined with one CFH risk allele was associated with a 14.0-fold (95% CI 2.1–95.1) increased risk for late AMD. ApoE alleles neither increased disease risk nor proved to be protective.

Conclusions:  The CFH, LOC387715 and HTRA1 polymorphisms are strongly associated with the development of AMD in the Hungarian population. The association is particularly pronounced when homozygous risk alleles are present and in the late stages of the disease.


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

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly population worldwide (Hageman et al. 2005). The prevalence of the disease increases with age, reaching as much as 15% in people aged > 80 years (Friedman et al. 2004). Early diagnosis and prevention are extremely important because present therapeutic options are largely limited to the late stages of the disease.

Early stages of AMD are characterized by retinal pigment abnormalities and the accumulation of small deposits called drusen under the macular area of the retina. Advanced forms of the disease are characterized by the development of geographic atrophy (GA) of the retinal pigment epithelium and/or subretinal neovascular membrane (SRNVM; exudative form of AMD) formation. Although the aetiology of the disease is still largely unclear, several risk factors have been linked to AMD, including advanced age, ethnicity, smoking, high cholesterol level, blue-light exposure, oxidative damage, hypertension, obesity and inflammation (Haines et al. 2005; Klein et al. 2005). Recently, important genetic factors have been recognized in the background of AMD. A single nucleotide polymorphism (SNP) in the 1q31 chromosome region encoding a Tyr402His (Y402H) variant in the complement factor H (CFH) gene proved to account for high disease risk in the White populations examined (Edwards et al. 2005; Haines et al. 2005; Klein et al. 2005). The association is particularly prominent between advanced stages of the disease and the homozygous form of the polymorphism. Odds ratios (ORs) range between 3.3 and 11.52 (Haines et al. 2005; Klein et al. 2005; Zareparsi et al. 2005; Kaur et al. 2006; Seitsonen et al. 2006; Sepp et al. 2006; Simonelli et al. 2006) for patients homozygous for the risk allele, between 2.1 and 2.82 for carriers of at least one risk allele (Edwards et al. 2005; Hageman et al. 2005; Magnusson et al. 2006) and between 1.4 and 4.6 for heterozygous patients (Haines et al. 2005; Klein et al. 2005; Souied et al. 2005; Zareparsi et al. 2005; Seitsonen et al. 2006; Sepp et al. 2006; Simonelli et al. 2006). Complement factor H regulates complement activity by inhibiting C3 convertase and its product C3b. Recent evidence shows that the Y402H polymorphism alters binding affinity of CFH to C-reactive protein (CRP) and necrotic cells, probably resulting in the modification of local complement activation and inflammation responses (Herbert et al. 2007; Sjoberg et al. 2007; Yu et al. 2007).

A similarly strong association has been confirmed between AMD and Ser69Ala polymorphism (rs10490924) of an unknown protein at LOC387715 (also designated ARMS2) in the 10q26 region (Jakobsdottir et al. 2005; Rivera et al. 2005; Ross et al. 2007; Shuler et al. 2007; Tanimoto et al. 2007). Possible interaction between the two SNPs was investigated by logistic regression, but no evidence of epistasis was found (Rivera et al. 2005). This suggests an additive model, with the two genes independently contributing to disease risk. Recently, a polymorphism in the promoter region of the HTRA serine peptidase 1 (HTRA1) gene (rs11200638) also located at 10q26 was found to confer high risk for AMD (Dewan et al. 2006; Yang et al. 2006; Cameron et al. 2007; Yoshida et al. 2007). LOC387715 and HTRA1 polymorphisms are located in close vicinity (rs10490924 and rs11200638 are in almost complete linkage disequilibrium) and it is still unclear which of the two plays a role in the pathomechanism of the disease and which serves only as a genetic marker. Apolipoprotein E (ApoE), a polymorphic gene with three common allelic variants on chromosome 19 (E2, E3 and E4) has also been connected to the pathogenesis of AMD. The E3 allele occurs most frequently in the White population. The E4 allele has been reported to decrease AMD risk, or at least to delay the occurrence of the disease (Baird et al. 2004). By contrast, the presence of the E2 allele is associated with younger age at diagnosis (Baird et al. 2004). It should be noted, however, that the association between ApoE and AMD is rather controversial. A recent Spanish study reported an increased risk for AMD as a result of the ApoE E4 allele, with an OR of 5.6 (Asensio-Sanchez et al. 2006), but a Norwegian group could not find any association between ApoE genotypes and AMD (Utheim et al. 2008). The possible modifying effect of ApoE genotypes in smoking-associated AMD has also been investigated. The highest disease risk for smokers was found in carriers of the E2 allele, with an OR of 4.6, whereas carriers of the E4 allele have a lower risk with an OR of 1.9, compared with non-smoking individuals (Schmidt et al. 2000).

The aim of our study was to elucidate whether these polymorphisms are associated with AMD in the Hungarian population and to evaluate disease risk with respect to different disease stages.

Materials and Methods

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


We performed a case-control study. In total, 105 patients with AMD and 95 unrelated ethnically matched healthy controls were identified in eastern Hungary at the Department of Ophthalmology, University of Debrecen and at the Department of Ophthalmology, Kenézy Gyula Hospital, Debrecen, between 2005 and 2007. The control group consisted of individuals attending the outpatient clinic for minor refractive disorders (myopia or hypermetropia < 3.0 D or presbyopia). To maximize the reliability of our study, we deliberately selected controls who were somewhat older than patients. The study was approved by the Institutional Ethics Committee of the University of Debrecen and the procedures strictly adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants. Detailed patient histories were recorded with the use of a questionnaire focusing on known or suspected non-genetic risk factors for AMD, such as cigarette smoking, exposure to blue light, medical history of acute myocardial infarction, ischaemic heart disease and deep venous thrombosis. Patients who had smoked at least 10 cigarettes daily for > 10 years were considered as smokers. Body mass index (BMI) was calculated on actual body height and weight values once it had been ascertained that no considerable change in body weight had occurred in the past 10 years. An average BMI was calculated in patients with significant loss or increase in body weight based on body weight values for the previous 10-year period.

Colour fundus photographs were taken in patients and controls. Fluorescein angiography (FA) was used to investigate patients with exudative AMD. Grading of the severity of the disease was based on the Age-Related Eye Disease Study (AREDS) grading system and the International Classification System (Bird et al. 1995; AREDS 2001; Davis et al. 2005; Ferris et al. 2005). For statistical analysis, patients were classified according to the more severely affected eye into two groups corresponding to early and advanced AMD. Cases with GA or exudative components accompanied by severe visual defects were classified as advanced AMD, whereas cases without any advanced features and with at least one druse > 125 μm or with a total area of intermediate drusen larger than 125 μm were considered to have early AMD. No signs of early AMD, such as abnormal pigmentation or large indistinct soft drusen, were observed in controls. Non-extensive small drusen were graded as controls. Colour fundus photographs and FAs were evaluated by two experienced ophthalmologists. Patients or controls with ocular diseases (e.g. diabetic retinopathy, pathologic myopia) that might interfere with the reliable evaluation of AMD were not included in the study.

Molecular genetic methods for the detection of the common ApoE alleles, CFH Y402H polymorphism, LOC387715 rs10490924 (A69S), HTRA1 rs11200638 polymorphisms

Genomic DNA was isolated from EDTA (ethylenediaminetetraacetic acid) or citrate anticoagulated blood using the QIAamp Blood Mini Kit (Qiagen GmbH, Hilden, Germany). For the detection of ApoE E2, E3 and E4 alleles, a previously published method (Hoffmann et al. 2001) was used. In the case of the other three mutations, novel polymerase chain reaction (PCR)-restriction digestion methods were developed. For the amplification of the CFH gene fragment spanning the Y402H polymorphic site, the oligonucleotide primers used by Haines et al. (2005) were used. The PCR reaction mix consisted of 10 pmol forward and reverse primer, 100 ng genomic DNA, 2.0 U Taq DNA polymerase and 200 μM/L of each dNTP in a PCR buffer containing 1.5 mM MgCl2. Amplification consisted of 40 cycles with the annealing temperature of 60 °C. Nla III restriction enzyme has three sites on the 587-bp wild-type PCR product, resulting in a 418-bp and additional smaller fragments. The T to C transition introduced a novel restriction site in the 418-bp product, resulting in a 333-bp fragment. The restriction products were run on a 3% agarose gel (NuSieve 3:1; Cambrex, East Rutherford, NJ, USA) and visualized using ethidium bromide. A representative electrophoretic pattern is shown in Fig. 1A.


Figure 1.  Molecular genetic detection of (A) complement factor H Y402H, (B) LOC387712 rs10490924 and (C) HTRA1 rs11200638 polymorphisms. Wild-type samples for the respective mutations are shown in lanes 4 (A), 2 (B) and 3 (C). Heterozygous genotypes are in lanes 2 (A), 3 (B) and 4 (C). Homozygous mutant genotypes are shown in lanes 3 (A), 4 (B) and 2 (C). The first lane is a 50-bp ladder molecular weight marker (MWM).

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For the amplification of the respective gene segments spanning rs10490924 and rs11200638 polymorphisms, the following oligonucleotides were used: rs10490924F (5′-TAC CCA GGA CCG ATG GTA AC-3′); rs10490924R (5′-GGG GTA AGG CCT GAT CAT CT-3′), and primers rs11200638F (5′-ATG CCA CCC ACA ACA ACT TT-3′), rs11200638R (5′-GGG GAA AGT TCC TGC AAA TC-3′). The PCR products were 316 bp and 322 bp long, respectively. Settings of the PCR reaction were essentially the same as above except for the annealing temperature (55 °C). Digestion with Sat I enzyme resulted in 191-bp, 71-bp and 54-bp restriction fragments in the case of wild-type (G) rs10490924 allele. The mutant T allele abolishes one restriction site and therefore 262-bp and 54-bp long fragments were detected on an agarose gel (Fig. 1B). Similarly, Xma III digestion resulted in 183-bp and 139-bp restriction products in the case of wild-type (G) rs11200638 allele; in the presence of the minor allele (A), the restriction site is lost, resulting in a 322-bp product (Fig. 1C).

For the verification of the mutation detection methods described above, randomly selected samples were sequenced with the same primers used for PCR amplification. No discrepancy was found.

The PCR-restriction fragment length polymorphism (RFLP) methods described above can be readily available for many laboratories and provide a simple, easy-to-use technique suitable for the analysis of these polymorphisms associated with AMD.

Statistical methods

Analysis of the Hardy–Weinberg equilibrium in both the control and patient groups was performed. The genotype frequency difference was tested using the chi-square test. Continuous variables were tested using Student’s t-test. Relative risks for AMD in persons with different genotypes and alleles were estimated by ORs computed by logistic regression. Linkage between the LOC387715 A69S and HTRA1 rs11200638 was analysed using a method proposed previously (Gaunt et al. 2007).

Potential confounders considered were age, gender, smoking, BMI and blue light exposure. We tested whether these factors were associated with the different genotypes in the control group and thus might confound the effects. If they were statistically significantly associated with a certain genotype, we adjusted for them in the analysis by logistic regression.

As smoking is a significant environmental risk factor for AMD, we tested the interaction between all polymorphisms and smoking in the logistic models by likelihood ratio test.


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

Characteristics of the study population

In total, 105 patients and 95 controls were enrolled in the present study. The mean ages of AMD patients and the healthy controls were 74.2 ± 10.0 years (mean ± standard deviation [SD]) and 78.1 ± 6.3 years, respectively (p = 0.001). Men accounted for 50.5% of control subjects and 54.1% of patients. Patients were divided into two subgroups according to the stage of AMD, which resulted in 48 patients in the early and 57 patients in the late-stage AMD subgroups. There was no significant difference between the ages of the early and late subgroups of patients (73.3 ± 11.5 years and 75.0 ± 8.6 years, respectively; p = 0.4). Smokers accounted for 37.5% of control subjects and 38.4% of AMD subjects.

Genotype and allele distribution of the polymorphisms analysed in the patient and control groups

The genotype distributions of the polymorphisms analysed are shown in Table 1. The control group showed no statistical difference from the Hardy–Weinberg equilibrium with respect to CFH genotypes, and the LOC387715 rs10490924 polymorphism was in Hardy–Weinberg equilibrium among the patients. In all but the ApoE gene the minor (risk) allele was overrepresented in the patients. The frequency of the CFH Y402H C allele was 0.538 and 0.332, that of the LOC387715 rs10490924 T allele was 0.478 and 0.295, and that of the HTRA1 rs11200638 A allele was 0.463 and 0.289 in patients and controls, respectively. It should be noted that the HTRA1 genetic data are based on the analysis of 103 patients. In the case of ApoE, the most common E3 allele was found in similar frequencies in patients and in controls (0.826 and 0.816, respectively), whereas the potential risk factor E2 allele was less frequent in patients than in controls (0.066 and 0.1, respectively). By contrast, the ApoE E4 allele was more frequent in the patient group (0.108 versus 0.084, respectively). The linkage disequilibrium between the HTRA1 rs11200638 and the LOC387715 rs10490924 polymorphisms was incomplete in six cases (three controls and three patients; r= 0.9382). In four of these cases, the homozygous T allele of LOC387715 was observed together with the heterozygous HTRA1 genotype; in the other two individuals, the heterozygous LOC387715 genotype was associated with HTRA1 wild genotype.

Table 1.   Genotype distribution and allele frequencies of the analysed mutations in patients and controls.
MutationDistribution in casesDistribution controls in = 95AlleleAllele frequency in casesAllele frequency in controls
Early cases (= 48)Late cases (= 57)All cases (= 105)
  1. * HTRA1 genotyping data are missing in two early phase patients.

  2. CFH = complement factor H.

E alleles3/4 + 4/4919111920191314E20.0660.100
2/3 + 2/24861110101718E30.8260.816
2/40024221 1E40.1080.084
LOC387715 rsl0490924GG1735132330294345LOC387715
TT1225142526254 4T0.4780.295
HTRA1* rsl1200638GG1735132330294345HTRA1*
AA1123122123223 3A0.4630.295
CFH Y402HTT1735122l2928394lCFH
CC1531234038367 7C0.5380.332

Odds ratios for the different polymorphisms

Carriers of two copies of the 1277C CFH allele (CC genotype) were at a 7.3-fold greater risk (95% confidence interval [CI] of the OR 2.8–18.7; p < 0.001), whereas carriers of at least one risk allele (homozygous mutant [CC] and heterozygous [TC] genotypes) were at a 1.8-fold greater risk (95% CI 1.0–3.3; p = 0.046) for AMD compared with wild-type (TT genotype) subjects in the study population. Compared with wild-type individuals, homozygous CC genotypes were at 4.9-fold (95% CI 1.7–14.2; p = 0.003) and 10.7-fold (95% CI 3.7–31.0; p < 0.001) greater risk for early and late AMD, respectively. The CFH genotype was associated statistically significantly (p = 0.005) with gender in controls. Adjustment for gender, however, did not change the result.

Homozygosity (TT genotype) for the LOC387715 polymorphism conferred a 9.1-fold (95% CI 2.9–28.8; p < 0.001) increased risk of AMD, in detail a 7.4-fold (95% CI 2.1–26.2; p = 0.002) increased risk for early and an 11.3-fold increased risk (95% CI 3.2–40.4; p < 0.001) for late AMD. When mutation carriers were combined (TT/GT genotypes), a 2.0-fold (95% CI 1.1–3.6, p = 0.02) increased risk for the disease was observed. None of the potential confounders were associated with the LOC337815 genotype in the control group; therefore we did not adjust for them in the analysis.

Individuals with two copies of the HTRA1 risk allele (AA genotype) carried an 11.6-fold (95% CI 3.2–42.3; p < 0.001) increased disease risk, whereas carriers of at least one risk allele (homozygous mutant [AA] and heterozygous [GA] genotypes) had a 2.2-fold (95% CI 1.2–4.0; p = 0.009) increased risk of the disease compared with subjects with no risk allele (GG genotype). Homozygosity for the risk allele caused a 10.1-fold (95% CI 2.5–40.8; p = 0.001) increase in disease risk for early AMD and a 13.5-fold (95% CI 3.3–55.4; p < 0.001) increase in risk for late AMD. None of the potential confounders were associated with the HTRA1 genotype in the control group and therefore we did not adjust for them in the analysis.

The risk for AMD was somewhat increased in persons with only one risk allele of LOC387715 or HTRA1, but the associations were not statistically significant.

Compared with double wild-type subjects, individuals carrying two CFH (CC genotype) and one LOC387715 risk alleles (GT genotype) had a 9.3-fold (95% CI 2.4–35.8; p = 0.001) greater risk for AMD, which was even more prominent (OR 15.0, 95% CI 3.2–71.0; p = 0.001) in the late AMD subgroup. However, this genotype combination was not significant when it was tested in the early AMD subgroup (OR 4.8, 95% CI 1.0–24.1; p = 0.057). Homozygosity for the LOC387715 gene polymorphism (TT genotype) in combination with one CFH risk allele (CT genotype) conferred a 10.7-fold elevated disease risk (95% CI 1.9–58.7; p = 0.007) compared with double wild-type individuals. The same combination was associated with an 8.0-fold (95% CI 1.2–54.7; p = 0.03) and a 14.0-fold higher disease risk (95% CI 2.1–95.1; p = 0.007) in the early and late AMD subgroups, respectively. Altogether six patients and none of the controls carried both polymorphisms in homozygous form. A summary of these genotype combinations and the risks represented by them is shown in Table 2. No significant difference in the frequencies of the ApoE alleles was observed between the control and patient groups. Neither of the alleles increased disease risk nor proved to be protective. None of the potential confounders were associated with the ApoE genotype in the control group and therefore we did not adjust for them in the analysis.

Table 2.   Selected genotype combinations and the represented risks for age-related macular degeneration.
Genotype combinationNumber (%) of individuals
ControlsEarly casesLate cases
  1. CFH = complement factor H.

CFH Y402H TT genotype LOC387715 rsl0490924 GG genotype (double wild-type)16 (16.8%)5 (10.4%)4 (7.0%)
CFH Y402H CC genotype LOC387715 rsl0490924 GT genotype4 (4.2%)6 (12.5%)15 (26.3%)
CFH Y402H CT genotype LOC387715 rsl0490924 TT genotype2 (2.1%)5 (10.4%)7 (12.3%)
Odds ratios for the genotype combinations tested against double wild-type individuals
CFH Y402H CC genotype LOC387715 rsl0490924 GT genotypeEarly cases OR 4.8 (95% CI 1.0–24.1; p = 0.057) Late cases OR 15.0 (95% CI 3.2–71.0; p = 0.001) All cases OR 9.3 (95% CI 2.4–35.8; p = 0.001)
CFH Y402H CT genotype LOC387715 rsl0490924 TT genotypeEarly cases OR 8.0 (95% CI 1.2–54.7; p = 0.03) Late cases OR 14.0 (95% CI 2.1–95.1; p = 0.007) All cases OR 10.7 (95% CI 1.9–58.7; p = 0.007)

Smoking did not modify the effect of the polymorphisms studied; p-values of the likelihood ratio tests for interaction were 0.9, 0.4, 0.8 and 0.8 for CFH, LOC387715, HTRA1 and ApoE genotype, respectively. No significant association of the examined non-genetic factors and disease risk could be demonstrated. The distribution of the analysed genetic risk factors is summarized in Table 1 and ORs associated with them are shown in Table 3.

Table 3.   Crude odds ratios conferred by the analysed genetic factors in the study population with respect to different stages of age-related macular degeneration.
 Odds ratio (95% CI)
All casesEarly casesLate cases
  1. CFH = complement factor H.

CFH Y402H CC versus TT7.3 (2.8–18.7)4.9 (1.7–14.2)10.7 (3.7–31.0)
p < 0.001p = 0.003p < 0.001
CFH Y402H CC + TC versus TT1.8 (1.0–3.3)1.3 (0.6–2.6)2.6 (0.2–5.6)
p = 0.046p = 0.515p = 0.013
CFH Y402H TC versus TT1.0 (0.5–2.0)0.7 (0.3–1.7)1.5 (0.6–3.3)
p = 0.898p = 0.480p = 0.366
LOC387715 rs10490924 TT versus GG9.1 (2.9–28.8)7.4 (2.1–26.2)11.3 (3.2–40.4)
p < 0.001p = 0.002p < 0.001
LOC387715 rs10490924 TT + GT versus GG2.0 (1.1–3.6)1.4 (0.7–3.0)2.7 (0.3–5.6)
p = 0.02p = 0.314p = 0.009
LOC387715 rs10490924 GT versus GG1.4 (0.8–2.6)1.0 (0.4–2.1)2.0 (0.9–4.3)
p = 0.282p = 0.913p = 0.083
HTRA1 rs11200638 AA versus GG11.6 (3.2–42.3)10.1 (2.5–40.8)13.5 (3.3–55.4)
p < 0.001p = 0.001 p < 0.001
HTRA1 rs11200638 AA + GA versus GG2.2 (1.2–4.0)1.6 (0.8–3.4)2.9 (1.4–6.1)
p = 0.009p = 0.196p = 0.004
HTRA1 rs11200638 GA versus GG1.6 (0.9–3.0)1.1 (0.5–2.4)2.2 (1.1–4.8)
p = 0.126p = 0.831 p = 0.037


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

Our knowledge of the major genetic contributors involved in the pathogenesis of AMD has increased substantially in the last few years. However, the prevalences of these genetic risk factors show marked differences in different populations, which makes it necessary to establish specific allele frequencies for each. Therefore, we decided to analyse the ApoE, CFH, LOC387715/HTRA1 representative alleles in a clinically well characterized Hungarian AMD patient group with corresponding controls. A novel molecular genetic protocol for the detection of CFH, LOC387715 and HTRA1 polymorphisms is described in detail and can be readily used by any other molecular biological laboratory.

The risk attributed to the possession of the ApoE E2 allele could not be verified in our patients. Contrary to previous studies, the E2 allele was found to be less frequent in the patient than in the control group (allele frequencies were 0.066 and 0.1, respectively). Allele frequencies of the E3 allele did not show a statistically significant difference between the patient and control groups (0.826 in patients and 0.816 in controls), whereas the ApoE E4 allele was more frequent in the patient group (0.108) than in the controls (0.084). We could not detect a statistically significant association between ApoE alleles and AMD. Most previous studies on the association between ApoE and AMD report a higher frequency of ApoE E2 in patients than in controls. However, there are several exceptions. Reports from European (Asensio-Sanchez et al. 2006; Utheim et al. 2008) and Japanese (Gotoh et al. 2004) populations showed higher ApoE E2 frequencies in controls than in patients. Other than in a single study (Asensio-Sanchez et al. 2006), the frequency of ApoE E4 was higher in the control than in the patient group. Similarly to this recent report on a Spanish population, our study, surprisingly, demonstrated higher ApoE E4 and lower ApoE E2 frequency in patients than controls. The most plausible explanation for this discrepancy is that the number of individuals in our groups did not reach the level necessary to detect the minor risk attributed to ApoE. However, it is possible that there are geographic differences in the frequency of the ApoE alleles, which might result in different risks attributed by the ApoE alleles in populations of different ethnic origins.

The biological role of CFH, a member of the innate immune system, is to regulate complement activity by inhibiting C3 convertase, which is an essential enzyme of the alternative complement pathway. Complement factor H also inactivates C3b, the product of C3 convertase, and thereby protects host arterial walls from excessive complement activity. This effect is augmented if CFH binds either to heparin or CRP. The tyrosine-histidine polymorphism, which is located in the region responsible for the binding of these molecules, may decrease the protective activity of CFH, thereby causing vessel injury, the consequence of which may be the neovascularization detected in late cases of AMD (Haines et al. 2005; Klein et al. 2005; Simonelli et al. 2006). Therefore, not only the risk for AMD but also for its progression may be associated with CFH genotypes. However, no clear association between the CFH Y402H polymorphism and exudative AMD lesion characteristics (angiographic subtype, lesion and SRNVM size) could be demonstrated in a very recent paper (Seitsonen et al. 2008).

Haines et al. (2005) found that the OR for AMD in homozygous patients increased from 3.33 to 5.57 when taking only neovascular AMD into consideration, which suggests that this polymorphism has a stronger association with exudative AMD than with non-exudative AMD. By contrast, Magnusson et al. (2006) found that carrying at least one risk allele contributed equally to the development of GA and neovascular AMD (ORs were 2.27 for patients with GA and 2.32 for patients with exudative AMD in the Icelandic cohort). The same study reported that the CFH variant also conferred similar risk for the formation of soft drusen, with an OR of 2.52 for carriers of at least one risk allele. Seddon et al. (2006) demonstrated in a large White study population that homozygous risk alleles for Y402H polymorphism were associated with a 7.4-fold elevation of risk for the development of advanced AMD and contributed equally to the development of GA and neovascular AMD (ORs 7.9 and 7.5, respectively). Similarly, Sepp et al. (2006) reported equal contributions of the homozygous at risk CFH allele in both forms of advanced AMD (ORs 6.0 and 5.1, respectively). Moreover, homozygous CFH risk alleles confer similar risks of progression from early and intermediate AMD to both advanced forms (ORs 2.6 for GA and 2.9 for CNV) (Seddon et al. 2007). These findings led to the suggestion that the known CFH risk allele participates in a common pathological pathway; however additional genetic and environmental factors may contribute to the development of the two different clinical forms of end-stage AMD (Magnusson et al. 2006).

The identification of the real underlying factor at the chromosome region 10q26 is still under extensive investigation and the results to date are inconclusive. Little is known about the biological role of the hypothetical gene LOC387715, but the predicted coded protein is highly conserved in humans and in chimpanzees (Kanda et al. 2007). By RT-PCR, Kanda et al. (2007) has suggested that LOC387715 is expressed in a placental cell line and weakly in other cell types as well, including the retina. Its subcellular localization has been shown to be in the mitochondrial outer membrane (Kanda et al. 2007). Risks for AMD caused by LOC387715 rs10490924 SNP were 8.21 for patients homozygous for the risk allele and 2.69 for heterozygous individuals in a German study performed recently (Rivera et al. 2005). All previous studies analysing both the LOC387715 and the HTRA1 variants have shown that the two polymorphisms are linked and therefore the ORs gained from these studies are very similar. The previously detected impact of rs11200638 on the expression of HTRA1 (Dewan et al. 2006; Yang et al. 2006) could not be confirmed (Kanda et al. 2007). Our results closely fit the previously available data on the risk of AMD resulting from LOC387715/HTRA1. Although we found strong associations with both early and late AMD, our data indicate that CFH Y402H and LOC387715/HTRA1 polymorphisms are associated with higher risk for advanced versus early AMD and suggest that these proteins and their polymorphisms have a more profound effect on the pathogenesis of advanced AMD compared with the early stages of the disease. Our data therefore raise the possibility that these polymorphisms might be involved not only in the development, but also in the progression of the disease, as shown by Seddon et al. (2007).

According to our results, 3% of the individuals analysed did not show complete linkage disequilibrium between the HTRA1 and LOC387715 polymorphisms, a proportion similar to that reported in very recently published data (Hughes et al. 2007). In addition to the established role of CFH in the pathogenesis of AMD, identifying the genuine underlying genetic risk factor for AMD in this part of the genome (i.e. HTRA1 or LOC387715) may be of great importance, especially in these discrepant cases as specific therapy may eventually be based on genetic data from patients and thus avoid inadequate or inefficient therapy.

Our finding that none of the control subjects carried two mutations (i.e. CFH and LOC387715/HTRA1) in homozygous form but that six individuals in the patient group (5.7% of all patients) carried double homozygous mutations suggests that carrying two homozygous risk alleles represents a particularly high disease risk.

Probably because of our moderate sample size, we were unable to demonstrate the modifying effect of smoking in relation to the LOC387715 rs10490924 polymorphism suggested by Schmidt et al. (2006). It should be noted that the subgrouping of smokers in the previous study made a more detailed analysis possible. Like ours, a recent study analysing the closely linked HTRA1 variant (Mori et al. 2007) failed to demonstrate any correlation; however, this study was performed not on a White, but on a Japanese cohort. Heterozygotes for LOC387715, HTRA1 had a somewhat higher risk for the development of AMD in our study (Table 3). However, none of the estimates were statistically significant because of the limited power of our study. Almost 600 participants would have been required to achieve an 80% power to detect a relative risk of 1.6.

In conclusion, polymorphisms in CFH and LOC387715/HTRA1 genes represent substantial risk for the development of AMD, particularly for the advanced form in Hungary, whereas ApoE alleles do not. The risk is more pronounced in the later disease stages and when the polymorphisms are in homozygous form.


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

The first two authors (GL and AF) contributed equally to work. This work was supported by a Mecenatura Grant for the University of Debrecen, Medical and Health Science Centre, by the Hungarian National Research Fund (K68616 and F60643). IB was supported by a Bolyai János Fellowship from the Hungarian Academy of Sciences. This work was presented at the Association for Research in Vision and Ophthalmology Annual Meeting, 27 April to 1 May, 2008, Fort Launderdale, FL, USA. The contribution of Erika Dzsudzsak is gratefully appreciated.


  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • AREDS (The Age-Related Eye Disease Study Research Group) (2001): The Age-Related Eye Disease Study system for classifying age-related macular degeneration from stereoscopic color fundus photographs: the Age-Related Eye Disease Study Report Number 6. Am J Ophthalmol 132: 668681.
  • Asensio-Sanchez VM, Rodriguez-Martin T, Gala-Molina I & Rodriguez-Fernandez I (2006): Age-related macular degeneration: its association with the epsilon4 allele of the apolipoprotein E gene. Arch Soc Esp Oftalmol 81: 912.
  • Baird PN, Guida E, Chu DT, Vu HT & Guymer RH (2004): The epsilon2 and epsilon4 alleles of the apolipoprotein gene are associated with age-related macular degeneration. Invest Ophthalmol Vis Sci 45: 13111315.
  • Bird AC, Bressler NM, Bressler SB et al. (1995): An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group. Surv Ophthalmol 39: 367374.
  • Cameron DJ, Yang Z, Gibbs D et al. (2007): HTRA1 variant confers similar risks to geographic atrophy and neovascular age-related macular degeneration. Cell Cycle 6: 11221125.
  • Davis MD, Gangnon RE, Lee LY et al. (2005): The Age-Related Eye Disease Study severity scale for age-related macular degeneration: AREDS Report No. 17. Arch Ophthalmol 123: 14841498.
  • Dewan A, Liu M, Hartman S et al. (2006): HTRA1 promoter polymorphism in wet age-related macular degeneration. Science 314: 989992.
  • Edwards AO, Ritter R III, Abel KJ, Manning A, Panhuysen C & Farrer LA (2005): Complement factor H polymorphism and age-related macular degeneration. Science 308: 421424.
  • Ferris FL, Davis MD, Clemons TE et al. (2005): A simplified severity scale for age-related macular degeneration: AREDS Report No. 18. Arch Ophthalmol 123: 15701574.
  • Friedman DS, O’Colmain BJ, Munoz B et al. (2004): Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol 122: 564572.
  • Gaunt TR, Rodriguez S & Day IN (2007): Cubic exact solutions for the estimation of pairwise haplotype frequencies: implications for linkage disequilibrium analyses and a web tool ‘CubeX’. BMC Bioinformatics 8: 428.
  • Gotoh N, Kuroiwa S, Kikuchi T, Arai J, Arai S, Yoshida N & Yoshimura N (2004): Apolipoprotein E polymorphisms in Japanese patients with polypoidal choroidal vasculopathy and exudative age-related macular degeneration. Am J Ophthalmol 138: 567573.
  • Hageman GS, Anderson DH, Johnson LV et al. (2005): A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci U S A 102: 72277232.
  • Haines JL, Hauser MA, Schmidt S et al. (2005): Complement factor H variant increases the risk of age-related macular degeneration. Science 308: 419421.
  • Herbert AP, Deakin JA, Schmidt CQ et al. (2007): Structure shows that a glycosaminoglycan and protein recognition site in factor H is perturbed by age-related macular degeneration-linked single nucleotide polymorphism. J Biol Chem 282: 1896018968.
  • Hoffmann MM, Scharnagl H, Koster W, Winkler K, Wieland H & Marz W (2001): Apolipoprotein E1 Baden (Arg(180)–>Cys). A new apolipoprotein E variant associated with hypertriglyceridaemia. Clin Chim Acta 303: 4148.
  • Hughes AE, Orr N, Patterson C, Esfandiary H, Hogg R, McConnell V, Silvestri G & Chakravarthy U (2007): Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking. PLoS Med 4: 355.
  • Jakobsdottir J, Conley YP, Weeks DE, Mah TS, Ferrell RE & Gorin MB (2005): Susceptibility genes for age-related maculopathy on chromosome 10q26. Am J Hum Genet 77: 389407.
  • Kanda A, Chen W, Othman M et al. (2007): A variant of mitochondrial protein LOC387715/ARMS2, not HTRA1, is strongly associated with age-related macular degeneration. Proc Natl Acad Sci U S A 104: 1622716232.
  • Kaur I, Hussain A, Hussain N et al. (2006): Analysis of CFH, TLR4, and ApoE polymorphism in India suggests the Tyr402His variant of CFH to be a global marker for age-related macular degeneration. Invest Ophthalmol Vis Sci 47: 37293735.
  • Klein RJ, Zeiss C, Chew EY et al. (2005): Complement factor H polymorphism in age-related macular degeneration. Science 308: 385389.
  • Magnusson KP, Duan S, Sigurdsson H et al. (2006): CFH Y402H confers similar risk of soft drusen and both forms of advanced AMD. PLoS Med 3: 5.
  • Mori K, Horie-Inoue K, Kohda M et al. (2007): Association of the HTRA1 gene variant with age-related macular degeneration in the Japanese population. J Hum Genet 52: 636641.
  • Rivera A, Fisher SA, Fritsche LG, Keilhauer CN, Lichtner P, Meitinger T & Weber BH (2005): Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum Mol Genet 14: 32273236.
  • Ross RJ, Bojanowski CM, Wang JJ et al. (2007): The LOC387715 polymorphism and age-related macular degeneration: replication in three case-control samples. Invest Ophthalmol Vis Sci 48: 11281132.
  • Schmidt S, Saunders AM, De La Paz MA et al. (2000): Association of the apolipoprotein E gene with age-related macular degeneration: possible effect modification by family history, age, and gender. Mol Vis 6: 287293.
  • Schmidt S, Hauser MA, Scott WK et al. (2006): Cigarette smoking strongly modifies the association of LOC387715 and age-related macular degeneration. Am J Hum Genet 78: 852864.
  • Seddon JM, George S, Rosner B & Klein ML (2006): CFH gene variant, Y402H, and smoking, body mass index, environmental associations with advanced age-related macular degeneration. Hum Hered 61: 157165.
  • Seddon JM, Francis PJ, George S, Schultz DW, Rosner B & Klein ML (2007): Association of CFH Y402H and LOC387715 A69S with progression of age-related macular degeneration. JAMA 297: 17931800.
  • Seitsonen S, Lemmela S, Holopainen J et al. (2006): Analysis of variants in the complement factor H, the elongation of very long chain fatty acids-like 4 and the hemicentin 1 genes of age-related macular degeneration in the Finnish population. Mol Vis 12: 796801.
  • Seitsonen S, Jarvela I, Meri S, Tommila P, Ranta P & Immonen I (2008): Complement factor H Y402H polymorphism and characteristics of exudative age-related macular degeneration lesions. Acta Ophthalmol 86: 390394.
  • Sepp T, Khan JC, Thurlby DA, Shahid H, Clayton DG, Moore AT, Bird AC & Yates JR (2006): Complement factor H variant Y402H is a major risk determinant for geographic atrophy and choroidal neovascularization in smokers and non-smokers. Invest Ophthalmol Vis Sci 47: 536540.
  • Shuler RK Jr, Hauser MA et al. (2007): Neovascular age-related macular degeneration and its association with LOC387715 and complement factor H polymorphism. Arch Ophthalmol 125: 6367.
  • Simonelli F, Frisso G, Testa F et al. (2006): Polymorphism p.402Y>H in the complement factor H protein is a risk factor for age-related macular degeneration in an Italian population. Br J Ophthalmol 90: 11421145.
  • Sjoberg AP, Trouw LA, Clark SJ, Sjolander J, Heinegard D, Sim RB, Day AJ & Blom AM (2007): The factor H variant associated with age-related macular degeneration (His-384) and the non-disease-associated form bind differentially to C-reactive protein, fibromodulin, DNA, and necrotic cells. J Biol Chem 282: 1089410900.
  • Souied EH, Leveziel N, Richard F, Dragon-Durey MA, Coscas G, Soubrane G, Benlian P & Fremeaux-Bacchi V (2005): Y402H complement factor H polymorphism associated with exudative age-related macular degeneration in the French population. Mol Vis 11: 11351140.
  • Tanimoto S, Tamura H, Ue T, Yamane K, Maruyama H, Kawakami H & Kiuchi Y (2007): A polymorphism of LOC387715 gene is associated with age-related macular degeneration in the Japanese population. Neurosci Lett 414: 7174.
  • Utheim OA, Ritland JS, Utheim TP, Espeseth T, Lydersen S, Rootwelt H, Semb SO & Elsas T (2008): Apolipoprotein E genotype and risk for development of cataract and age-related macular degeneration. Acta Ophthalmol 86: 401403.
  • Yang Z, Camp NJ, Sun H et al. (2006): A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science 314: 992993.
  • Yoshida T, DeWan A, Zhang H et al. (2007): HTRA1 promoter polymorphism predisposes Japanese to age-related macular degeneration. Mol Vis 13: 545548.
  • Yu J, Wiita P, Kawaguchi R, Honda J, Jorgensen A, Zhang K, Fischetti VA & Sun H (2007): Biochemical analysis of a common human polymorphism associated with age-related macular degeneration. Biochemistry 46: 84518461.
  • Zareparsi S, Branham KE, Li M et al. (2005): Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration. Am J Hum Genet 77: 149153.