| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
2 and 4 Alleles of the Apolipoprotein Gene Are Associated with Age-Related Macular Degeneration
From the Centre for Eye Research Australia, University of Melbourne, East Melbourne, Victoria, Australia.
 |
Abstract |
|---|
 Top Abstract MethodsResultsDiscussionReferences |
|---|
METHODS. Three hundred twenty-two unrelated individuals with diagnosed AMD and 123 unrelated but ethnically matched control subjects were analyzed. All subjects completed a standard questionnaire and were given a fundus examination. A blood sample was collected for DNA extraction. The common allelic variants of apoE were screened through the use of polymerase chain reaction (PCR) and restriction enzyme digestion followed by statistical analysis.
RESULTS. Individuals with the 
3 4 genotype of apoE had an approximate halving of disease risk for late (end-stage) AMD (odds ratio [OR] 0.58, 95% confidence interval [CI] 0.34–0.98) relative to the 3 3 genotype at age of ascertainment. Stratification of late AMD into atrophic and neovascular disease revealed that the greatest protective effect for the 3 4 genotype was in individuals with atrophic disease (OR 0.35, 95% CI 0.13–0.92). Men with the 3 4 genotype also showed almost a threefold reduction in risk of disease in late AMD (OR 0.36, 95% CI 0.16–0.82). However, individuals with late AMD and the 2 3 genotype had a significantly earlier mean age of diagnosis of disease (3.4 years, P = 0.015) compared with those with the 3 3 genotype, and this was most evident in women (3.9 years, P = 0.011) and in individuals with neovascular disease (4.7 years, P = 0.003).
CONCLUSIONS. The alleles of apoE appear to have a role in the etiology of AMD, with the 4 allele being protective, or at the very least, delaying the age of diagnosis of disease, whereas the 2 allele appears to have a modifier effect by bringing forward the mean age of disease diagnosis.
The etiology of AMD is poorly understood, but it is most likely a complex disease in which environmental risk factors impact on an individual’s genetic background.4 5 Potential risk factors have been identified from epidemiologic studies. Of particular interest to this study are the risk factors associated with cardiovascular disease (CVD). These include smoking, which is strongly associated with a two- to fourfold increased risk of disease6 ; a high-fat diet, which is associated especially with neovascular disease7 8 9 ; high serum cholesterol; elevated blood pressure; atherosclerosis; and a history of CVD.10 11 12 13 The association of one or more of these risk factors in most epidemiologic studies on AMD raised the possibility that some pathogenic mechanisms may be shared between CVD and AMD.
The common allelic variants (2, 3, 4) of the apolipoprotein E (apoE) gene have been implicated in several diseases including CVD and 14 Alzheimer’s disease,15 as well as AMD. Indeed, to date it has been the most consistently associated gene with AMD. ApoE is involved in cholesterol transport throughout the body16 and facilitates the binding of lipoproteins to low-density lipoprotein (LDL) receptors, thereby maintaining the lipoprotein cholesterol available to the cell.16 ApoE is expressed in retinal tissue, in particular the retinal pigment epithelium (RPE) and Bruch’s membrane.17 Animal studies have also shown that induction of a hypercholesterolemic state after either a high-fat diet or apoE deficiency resulted in a significant thickening of Bruch’s membrane18 as well as accumulation of retinal debris.19
Several case–control studies have now been undertaken to examine the association of the common allelic variants (2, 3, 4) of the apoE gene with AMD. These studies have concentrated on white individuals from mainly European backgrounds,20 21 22 23 24 25 although one study has been undertaken in ethnic Chinese.26 Only three of the seven previous studies have shown a significant protective effect for the apoE 4 allele with late (end-stage) AMD (odds ratio [OR] 0.16, 95% confidence interval [CI] 0.05–0.4320 ; OR 0.43, 95% CI 0.21–0.8821 ; and a pooled study OR 0.54, 95% CI 0.41–0.70,24 ), whereas the protective nature of this allele was more modest in two other studies (OR 0.88, 95% CI 0.58–1.3522 ; and OR 0.4, 95% CI 0.1–1.223 ). In individuals of Chinese ethnicity, there was little evidence to suggest an involvement of the 4 allele of this gene in AMD (OR 0.83, 95% CI 0.23–2.53).26 In the studies in which association was identified with the 4 allele, the findings were inconsistent, with protection being found when both exudative or atrophic disease were pooled,21 24 or when only exudative disease was analyzed,20 or in individuals with familial disease who were younger than 70 years.22 In addition, these studies had a small number of individuals carrying the 4 allele, and that limited the investigators’ ability to perform disease subanalysis. Three of the studies also suggested that the 2 allele may increase the risk of AMD, although none of these reached statistical significance.21 23 24
As apoE is the one gene that has so far shown the most consistent replication in genetic association studies with AMD and, given the inconsistencies in the previous studies, it was imperative to investigate this association further. We performed a study of power sufficient to allow subclassification of AMD into different subsets to explore further any associations of the alleles of this gene and AMD.
|
Methods |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
All patients were identified through either outpatient clinics at the RVEEH, private ophthalmology practices in Melbourne, or low-vision clinics. Late AMD was classified as either neovascular (wet) or atrophic (dry). Individuals who presented with both neovascular and atrophic forms of AMD were classified as having neovascular AMD. Early AMD was defined as the presence of soft drusen (>63 µm), with or without regions of hyperpigmentation. People with only hard drusen or with only pigmentary changes were not classified as having AMD, as it was imperative that classifications of individuals as positive for disease be beyond doubt. Cases were classified as familial AMD if two or more first-degree relatives were documented as having disease. Sporadic disease was classified as an individual having no known family history of AMD.
Control subjects were collected from the same community as part of the large population-based epidemiologic eye study, the Melbourne Visual Impairment Project (VIP),27 or through aged-care nursing homes. Individuals were selected as control subjects based on the presence of a normal fundus (<10 hard drusen classified as drusen <63 µm in size).
DNA Analysis
Genomic DNA was isolated from venous blood leukocytes using a standard phenol-chloroform extraction procedure.28 Polymerase chain reaction (PCR) amplification was undertaken with the primers AF(5'GCCTCCCCACTGTGCGA3'), AR(5'GGCCGAGCATGGCCTG3') to produce a 243-bp product encompassing amino acid position 112 (site A) and BF(5'ACCGAGGAGCTGCGGG3'), BR(5'CTCGCGGATGGCGCTGA3'), to produce a 150-bp product encompassing amino acid position 158 (site B). PCR conditions used in amplification were 94°C for 3 minutes, followed by 40 cycles of 94°C for 30 seconds, 61°C for 30 seconds, and 72°C for 30 seconds and a final extension cycle of 72°C for 10 minutes. Each of sites A and B contained a single nucleotide polymorphism (SNP), either being a C or T at site A or a C or T at site B, that resulted in either a cysteine (cys) or arginine (arg). Digestion of each PCR product was undertaken with the restriction enzymes AflIII for site A and HaeII for site B. Digested products were size separated on agarose gels. The combination of amino acids at sites A and B (cys/cys, cys/arg, or arg/arg) allowed the common allelic variants of either 2, 3, or 4 of the apoE gene to be scored. Dideoxynucleotide sequencing was undertaken on a representative sample to confirm the results of each restriction digestion profile.
Statistical Analysis
The Fisher exact test was used to calculate the probabilities for the tests of whether there was any (unadjusted) association between various kinds of AMD and different genotypes or alleles. Odds ratios adjusted for age and gender were obtained from logistic regressions, and t-tests were used to calculate the probabilities for the tests of whether there was any difference in the mean ages of cases and control subjects or the mean ages at diagnosis of the two different genotypes (SAS, ver. 8.02; SAS, Cary, NC). Analysis of AMD was undertaken by subclassification into early, late, neovascular, and atrophic, as well as familial and sporadic disease.
|
Results |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
2 = 2.29, df = 3, P = 0.515) or control subjects (2 = 5.42, df = 3, P = 0.143).
|
4 allele in late AMD (P = 0.037, Fisher exact test) than in control subjects. A significant result was also found when late disease was subgrouped into either neovascular or atrophic AMD (P = 0.091 and 0.018, respectively, for the apoE 4 allele; Fisher exact test). Of note, was the total absence of the 4 allele (0/14 individuals) in individuals who had atrophic disease with a family history of AMD (Table 2) . We did not detect any significant reduction in 4 allele frequency in individuals with early AMD when compared with control subjects (data not shown).
|
3 4 genotype constituted approximately 80% of all 4 alleles of apoE (Table 2) . We identified an approximate halving of disease risk in individuals with late AMD when the 3 4 genotype (OR 0.58, 95% CI 0.34–0.98) was compared with individuals with the 3 3 ancestral genotype adjusted for both age and gender (Table 3) . Classification of late disease into either neovascular or atrophic disease indicated that this protective effect was significant only in the atrophic disease group, showing an approximate threefold reduction in disease risk (OR 0.35, 95% CI 0.13–0.92; Table 3 ) when the 3 4 genotype was compared with the 3 3 genotype.
|
4 allele in men with late AMD (10.3%) than in control subjects (23.6%; P = 0.010, Fisher exact test). We identified an almost threefold reduction in late AMD risk for men with the 3 4 genotype (OR 0.36, 95% CI 0.16–0.82) in comparison with the 3 3 genotype. However, no significant association was found in women, although a protective trend was evident (OR 73, 95% CI 0.37–1.43) for the 3 4 genotype.
When the 2 allele was considered in this study, there was a slight but not significant increase in the frequency of this allele in late AMD (9.9%) compared with control subjects (6.9%). There was no significant increased risk of disease with the 2 3 genotype in late AMD (OR 1.69, 95% CI 0.79–3.61), although there was a trend for an increased risk of late AMD. In addition a threefold increased risk of atrophic disease was found when comparing those with familial disease with control subjects. However, this finding was of borderline significance when compared with those with the 3 3 genotype (OR 3.58, 95% CI 0.98–13.1; Table 3 ).
We investigated whether the 2 3 and the 3 4 genotypes had any effect on the mean age of diagnosis of disease compared with the reference 3 3 genotype. In individuals with the 2 3 genotype, a significantly younger age of diagnosis was detected (3.4 years, P = 0.015) in late cases (Table 4) . This finding was most evident in women (3.9 years, P = 0.011) and in individuals with neovascular disease (4.7 years, P = 0.003). When we considered the 3 4 genotype, we did not observe a significant difference in age of diagnosis for late cases.
|
|
Discussion |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
4 allele has been reported in two population-based studies,20 21 as well as in a pooled case–control study,24 yet not in other studies. In our study we were able to replicate the finding of a significant protective effect for the 4 allele of apoE and late AMD. We were also able to subclassify late AMD into either atrophic or neovascular disease and show a significant protective effect for the 4 allele in the atrophic group.
In the two previous studies that found a similar protective effect for the 4 allele and late AMD, either all AMD cases were analyzed together21 or only neovascular disease was considered.20 The pooled AMD study24 classified disease based on disease type and reported a protective effect in both atrophic (OR = 0.40) and neovascular disease (OR = 0.61), similar to our findings of OR = 0.35 and OR = 0.65, respectively. When we subclassified individuals into those who had atrophic disease as well as disease that was familial in origin, we noted a total absence of the 4 allele of apoE in these individuals—an observation that has not been reported before. Although the number of individuals in this group was small (n = 14), the finding adds further support to the notion that the 4 allele provides a greater protective effect in individuals with atrophic disease than in those with neovascular disease.
The protective effect that we observed for the apoE 4 allele in familial index cases has also been reported in familial individuals under the age of 70 years.22 However, in another study, no such protective effect of this allele was reported in familial cases.25 The apoE 4 allele frequency was 8.7% in our atrophic AMD compared with frequencies of 8.9%22 and 12.4%25 in the previous studies, whereas in our control subjects, it was 17.9% compared with 12.4%25 and 14.6%22 in these studies. The variation in 4 allele frequency in the three studies presumably reflects ethnic differences between the populations. It is known that the 4 allele of apoE exhibits a population gradient across Western Europe, with the frequency of this allele varying from 20% in Finland and Sweden to 8% in Italy.29 Matching of cases and control subjects based on ethnic origin is therefore critical in any association study. In the present study, we sought to minimize problems of ethnic mismatching by including only those cases and control subjects where individuals could report the ancestry of all four of their grandparents back to the United Kingdom or Ireland.
We noted a significantly lower frequency of the apoE 4 allele in men with late AMD compared with control subjects. This was estimated to lead to an almost threefold reduction in disease risk in men with the 3 4 genotype compared with men with the 3 3 genotype. This finding differed to the previous pooled study of AMD,24 in which no significant association of the apoE 4 allele was detected with gender. The 4 allele of apoE has been reported to be associated with an increased risk of coronary heart disease in men but not in women.30 Further investigation of this observation would be useful in determining how the 4 allele may influence risk of AMD, depending on gender.
We confirmed the consistent trend of an increased risk of end-stage AMD when the 2 allele of apoE was present. We were also able to demonstrate the novel finding of an earlier age of diagnosis of disease of approximately 3.4 years in individuals in whom an 2 3 genotype was present. This finding has not been documented before in AMD and further study are needed to replicate it.
Association studies designed to dissect the components of a complex disease such as AMD are difficult, and we took several steps to improve the design and minimize potential pitfalls. These included the selection of ethnically matched cases and control subjects; the examination of all individuals in the study, including control subjects; and the identification of a sample size large enough to provide the power to undertake the association study.
In conclusion, we were able to demonstrate a protective association for the 4 allele in late AMD and an apparent earlier age of diagnosis of disease in individuals with an 2 3 genotype compared with 3 3. We were also able to show that the protective effect of 4 was greatest in men and in individuals with atrophic disease. However, it still remains to be determined how the alleles of the apoE gene influence the outcome of AMD and why the association of alleles with disease is the opposite of that in CVD and Alzheimer’s disease, in which the 4 allele is a risk factor for disease and the 2 allele is protective. Once the pathogenic mechanisms of AMD are better understood, it is hoped that these enigmas can be resolved.
|
Footnotes |
|---|
Submitted for publication October 10, 2003; revised December 2, 2003, January 20, 2004; accepted January 22, 2004.
Disclosure: P.N. Baird, None; E. Guida, None; D.T. Chu, None; H.T.V. Vu, None; R.H. Guymer, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Paul N. Baird, Centre for Eye Research Australia, 32 Gisborne Street, East Melbourne, Victoria 3002, Australia; pnb{at}unimelb.edu.au.
|
References |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Swaroop, K. E. Branham, W. Chen, and G. Abecasis Genetic susceptibility to age-related macular degeneration: a paradigm for dissecting complex disease traits Hum. Mol. Genet., October 15, 2007; 16(R2): R174 - R182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Liew, A. Shankar, J. J. Wang, R. Klein, M. S. Bray, D. J. Couper, A. R. Sharrett, and T. Y. Wong Apolipoprotein E Gene Polymorphisms and Retinal Vascular Signs: The Atherosclerosis Risk in Communities (ARIC) Study Arch Ophthalmol, June 1, 2007; 125(6): 813 - 818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Tikellis, C. Sun, M. B. Gorin, R. Klein, B. E. K. Klein, E. K. M. Larsen, D. S. Siscovick, L. D. Hubbard, and T. Y. Wong Apolipoprotein E Gene and Age-Related Maculopathy in Older Individuals: The Cardiovascular Health Study Arch Ophthalmol, January 1, 2007; 125(1): 68 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hye, S. Lynham, M. Thambisetty, M. Causevic, J. Campbell, H. L. Byers, C. Hooper, F. Rijsdijk, S. J. Tabrizi, S. Banner, et al. Proteome-based plasma biomarkers for Alzheimer's disease. Brain, November 1, 2006; 129(Pt 11): 3042 - 3050. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. P. Conley, J. Jakobsdottir, T. Mah, D. E. Weeks, R. Klein, L. Kuller, R. E. Ferrell, and M. B. Gorin CFH, ELOVL4, PLEKHA1 and LOC387715 genes and susceptibility to age-related maculopathy: AREDS and CHS cohorts and meta-analyses Hum. Mol. Genet., November 1, 2006; 15(21): 3206 - 3218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Thakkinstian, S. Bowe, M. McEvoy, W. Smith, and J. Attia Association between Apolipoprotein E Polymorphisms and Age-related Macular Degeneration: A HuGE Review and Meta-Analysis Am. J. Epidemiol., November 1, 2006; 164(9): 813 - 822. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. T.V.M. de Jong Age-Related Macular Degeneration N. Engl. J. Med., October 5, 2006; 355(14): 1474 - 1485. [Full Text] [PDF]
|
|
|
|
 |
|
 P. N. Baird, F. M. A. Islam, A. J. Richardson, M. Cain, N. Hunt, and R. Guymer Analysis of the Y402H Variant of the Complement Factor H Gene in Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4194 - 4198. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Kaur, A. Hussain, N. Hussain, T. Das, A. Pathangay, A. Mathai, A. Hussain, R. Nutheti, P. K. Nirmalan, and S. Chakrabarti 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., September 1, 2006; 47(9): 3729 - 3735. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Jun, B. E. K. Klein, R. Klein, K. Fox, C. Millard, J. Capriotti, K. Russo, K. E. Lee, R. C. Elston, and S. K. Iyengar Genome-Wide Analyses Demonstrate Novel Loci That Predispose to Drusen Formation Invest. Ophthalmol. Vis. Sci., September 1, 2005; 46(9): 3081 - 3088. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Malek, L. V. Johnson, B. E. Mace, P. Saloupis, D. E. Schmechel, D. W. Rickman, C. A. Toth, P. M. Sullivan, and C. Bowes Rickman Apolipoprotein E allele-dependent pathogenesis: A model for age-related retinal degeneration PNAS, August 16, 2005; 102(33): 11900 - 11905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Fisher, G. R. Abecasis, B. M. Yashar, S. Zareparsi, A. Swaroop, S. K. Iyengar, B. E.K. Klein, R. Klein, K. E. Lee, J. Majewski, et al. Meta-analysis of genome scans of age-related macular degeneration Hum. Mol. Genet., August 1, 2005; 14(15): 2257 - 2264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zareparsi, M. Buraczynska, K. E.H. Branham, S. Shah, D. Eng, M. Li, H. Pawar, B. M. Yashar, S. E. Moroi, P. R. Lichter, et al. Toll-like receptor 4 variant D299G is associated with susceptibility to age-related macular degeneration Hum. Mol. Genet., June 1, 2005; 14(11): 1449 - 1455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Keller, M. Small, and S. J. Fliesler Enzyme blockade: a nonradioactive method to determine the absolute rate of cholesterol synthesis in the brain J. Lipid Res., October 1, 2004; 45(10): 1952 - 1957. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
