Many pathways and processes involved in the progression of systemic disorders such as cancer, cardiovascular disease and autoimmune diseases are also thought to be involved in AMD. It is therefore likely that valuable insights into the pathophysiology of AMD may be gained by studying pathways common to AMD and other well-characterised diseases that may overlap in pathogenesis. In fact, cross-talk between disease fields has already proven beneficial for the treatment of AMD: anti-vascular endothelial growth factor (anti-VEGF) antibodies initially developed for the treatment of cancer are currently the most effective treatment for neovascular AMD .
The important role of vitamin D in diseases of complex aetiology, its anti-angiogenic properties and an association between vitamin D serum levels and early AMD led us initially to explore the association between vitamin D and neovascular AMD by examining vitamin D-associated epidemiological factors and genetic variants of vitamin D metabolism genes in an extremely discordant sibling pair cohort. Here, we showed a protective effect of UV exposure on the development of neo-vascular AMD. Further, we identified several CYP24A1 SNPs significantly associated with all subtypes of AMD in four diverse cohorts, both retrospectively and prospectively. Moreover, these significant associations were demonstrated in a meta-analysis of the four cohorts. To the best of our knowledge, this was the first study to demonstrate a protective effective of UV exposure on the development of AMD and a genetic link between vitamin D metabolism and AMD.
The role of sunlight in AMD pathophysiology remains controversial. Human exposure to natural sunlight may be both beneficial and detrimental. One benefit of cutaneous sunlight exposure is the production of vitamin D. Studies have also shown that vitamin D produced as a result of sunlight exposure reduces the risk of autoimmune diseases such as multiple sclerosis (MS) and inflammatory bowel disease (IBD) . By contrast, natural sunlight (ie UV exposure) has long been known to have harmful effects on the human body, including permanent DNA damage. For example, studies have linked sunlight exposure to skin cancer, for both melanomas [64, 65] and carcinomas . In dermatological malignancies, the most detrimental factor is UVB, but UVA may also play a role.
To distinguish between the effects of general sunlight exposure and specifically UV exposure, sunlight exposure was self-assessed by patients and UV exposure was quantified separately by examining UV radiation, as measured in irradiance of UVB (290 nm to 320 nm), UVA (320 nm to 400 nm) and UV index. In addition, we examined vitamin supplementation, as well as sun-related variables including iris colour, self-reported sun exposure and skin cancer history. We also measured 25(OH)D in sera as a biomarker for vitamin D levels. To the best of the authors' knowledge, this was the first examination of sunlight exposure in the context of established AMD risk genotypes and smoking.
Quantification of sunlight exposure is difficult, as it is an ongoing life-long process [67–79]. One of the first studies of ocular exposure to UV based on interview data and field measurements in Chesapeake Bay watermen demonstrated no association between UV exposure and AMD . Further analysis of this same dataset showed that there was in fact an association between AMD and ocular exposure, but to blue light specifically [76, 78]. Alternatively, various studies have tested the association of sunlight exposure and AMD by measuring various sun-related variables. For example, Khan et al. investigated the role of variables such as iris colour, place of residence and hair colour but found no association between AMD and these sun related factors . Cruickshanks et al. measured lifetime number of sunburns, use of hats and sunglasses and number of hours spent outdoors, and showed an association between leisure time spent outdoors and risk of early AMD . Our study demonstrated a protective effect of moderate levels of UV radiation exposure and AMD, independent of smoking history and CFH and HTRA1 genotype. While experimental studies have shown that UV light has damaging effects on the retina--primarily via oxidative damage and formation of reactive oxygen species--previous epidemiological studies have shown inconsistent results regarding an association between UV light and risk of AMD [67–79]. UV light is part of the sunlight spectrum, but only UVA (320-400 nm) and UVB (280-320 nm) reach the Earth's surface. UV light has traditionally been considered harmful because it induces DNA damage and causes oxidative damage to RPE cells [82–84]. Furthermore, UV light has been shown to have pro-angiogenic properties. UVA-irradiated RPE cells have been shown to have increased levels of prostaglandin-endoperoxide synthase 2 (also known as cyclooxygenase-2 [COX2]), which is implicated in choroidal neovascularisation . UVB-induced tumours express elevated levels of factors associated with angiogenesis, including VEGF, matrix metallopeptidase 2 (MMP2) and matrix metallopeptidase 3 (MMP3) .
Emerging evidence shows that solar radiation may have beneficial effects, however. Epidemiological studies have demonstrated that sunlight reduces the incidence and mortality of various cancers, including non-Hodgkin's lymphoma and melanoma [88, 89]. UV light is known to suppress the immune response and growing evidence points to the role of inflammation in AMD pathogenesis. Since aberrant inflammation is believed to be a contributing factor to AMD, the immunosuppressive effects of UV radiation may provide some protective effect.
We specifically examined the role of UV light and AMD in this study, although previous studies have mainly looked at ambient sunlight exposure. Previous studies have been inconsistent about the role of sunlight exposure and AMD [67–79, 91, 92]. Some studies suggested a positive association,[69, 76, 91, 92] while others found no correlation [71, 78, 93]. To our knowledge, our study was the first to show a protective influence of UV light on neovascular AMD. Our finding may contrast with previous studies as a result of using a larger number of neovascular cases (n = 133) than that used in most previous studies. Furthermore, previous studies measured ambient solar radiation, which includes light of various wavelengths (UV, infrared, visible), while our study distinguished the contribution of UV irradiance.
Although our gene expression studies on vitamin D metabolism genes in human donor eyes found no significant association as a function of age or disease status, differences in expression levels between the macular and extramacular regions were observed. Specifically, the expression of both VDR and CYP27A1 was detected in the RPE-choroid, and CYP27A1 and CYP27B1 were detected in the retina, thus suggesting that the vitamin D metabolic genes VDR and CYP27B1 may function differently between the maculae and extramaculae, whether diseased or non-diseased. Also, AMD may not be a localised disease but rather may manifest systemically, and this may explain why CYP24A1 was not detected in human retinal tissue regardless of disease status. The gene expression changes that we observed in retinal cell lines suggested that CYP24A1 activity may alter over time and this would support its role in a late-onset disease such as AMD.
There were several limitations to this study. First, there was the possibility of recall bias. There was no definitive method to confirm an individual's recall of personal sun exposure and thus data could have been prone to misclassification. We attempted to minimise this bias by using a quantitative measurement of UV exposure based on the geographical location where the participants had resided for the majority of their lifetime; however, assessment of UV radiation based on residential history represents potential rather than actual exposure and could be subject to error. Secondly, although the subjects were questioned about the amount of time they spent outdoors, no information was gathered about their use of protection against sunlight, such as hats and sunglasses, which shield against UV light. Further, the subjects' sera were collected at various times throughout the year, and thus serum levels of vitamin D measured in this study may not accurately have represented the true range among the patient population, since vitamin D levels are influenced by sunlight exposure, diet and age.
Although a previous study by Parekh et al. showed an inverse relationship between vitamin D levels and early AMD but not late AMD, this study was limited by a small number of patients with an advanced form of AMD (n = 10). A more recent study by Millen et al. was able to show a statistically significant association between serum 25(OH)D concentrations and early AMD in women younger than 75 years; specifically, that high serum 25(OH)D concentrations may be protective . In our study, we further investigated the association of serum levels of 25(OH)D, the circulating form of vitamin D, with neovascular AMD by analysing the serum levels of 50 extremely discordant sibling pairs (n = 100). Our results were similar to those found by Millen and colleagues: a trend towards higher levels of vitamin D in the serum of unaffected patients compared with neo-vascular patients, although the difference was not statistically significant. Since the ability to produce vitamin D is diminished with advanced age (ageing results in decreased amounts of cutaneous 7-dehydrocholesterol, and by 70 years of age vitamin D3 synthesis is reduced by approximately 75 per cent), it is not unexpected that serum vitamin D levels were low, given that the average age of our unaffected patients was 75.4 years. This could explain why the association was not significant. Additionally, since this was measured only once, the serum values reflected the vitamin D production/intake over a limited amount of time, thus suggesting enhanced random measurement error.
In addition to epidemiological data, the association of AMD with genetic variation in vitamin D pathway genes was further suggested by existing linkage data for genomic regions associated with AMD. The vitamin D pathway genes assayed in this study all lie within regions that have been associated with AMD -- specifically, 2q33 (CYP27A1), 12q13 (CYP27B1 and VDR), and 20q13 (CYP24A1).
The CYP24A1 SNPs significantly associated with neovascular AMD in the discovery cohort (either individually [rs6127118 and rs2769234] or as part of a haplotype [rs6068816, rs6127118, rs1570699, rs92760 and rs2762934]) were concentrated in the area between exon 6 and exon 12. This area was further examined and refined by direct sequencing and analysed in an extended sibling cohort. After controlling for smoking history, sex and age, we were able to show significant variation in CYP24A1 in all populations, both separately and, more importantly, in a meta-analysis. Variation within CYP24A1 was available in the dbGAP dataset http://www.ncbi.nlm.nih.gov/projects/gap/ but, when tested for association with AMD, no significance was observed. This lack of association could be explained by the small number of subjects in the dbGAP dataset, as compared with the current study (n = 500 and 2,528, respectively). Additionally, these data were based on a 100,000 SNP chip, and the CYP24A1 variation was imputed with low imputation quality, which is another limitation of the dbGAP dataset.
Moreover, pathway analysis showed that, in a hypothetical pathway, CYP24A1 was directly linked to another AMD-associated gene, HTRA1, for which the exact pathway is unknown. In a complex disease such as AMD, one would expect the combination of multiple effects, including the modest effect of CYP24A1, to contribute to disease causality. Although there was no statistical interaction seen between CYP24A1 and HTRA1, there may have been binding between the two that differed between those with and those without AMD. Further molecular studies should be performed to investigate this association.
An anti-angiogenic role for vitamin D has been well documented in the cancer literature [96–98]. Therefore, a role for vitamin D may be protective of AMD by its anti-angiogenic properties . For example, VEGF expression was downregulated after tumour cells were treated with vitamin D [25, 26]. Vitamin D may exert anti-inflammatory properties by enhancing T suppressor cell activity and down-regulating T helper cells, T cytotoxic cells and natural killer cells . At physiological concentrations, vitamin D has also been shown to protect cell proteins and membranes from oxidative damage . In addition to playing a role in cancer, the vitamin D pathway has been implicated in several autoimmune diseases. Several studies have found that low levels of vitamin D are associated with increased autoimmunity.
A role for vitamin D in AMD is not only plausible in terms of the known biological roles of vitamin D, but also because of the diminished ability to produce vitamin D with advanced age . The problem of vitamin D deficiency is not only prevalent among the elderly: as of 2005, approximately 40 per cent of men and 50 per cent of women aged over 18 from the USA were estimated to have inadequate levels of 25(OH)D .