Systems biology-based analysis implicates a novel role for vitamin D metabolism in the pathogenesis of age-related macular degeneration

Abstract

Vitamin D has been shown to have anti-angiogenic properties and to play a protective role in several types of cancer, including breast, prostate and cutaneous melanoma. Similarly, vitamin D levels have been shown to be protective for risk of a number of conditions, including cardiovascular disease and chronic kidney disease, as well as numerous autoimmune disorders such as multiple sclerosis, inflammatory bowel diseases and type 1 diabetes mellitus. A study performed by Parekh et al. was the first to suggest a role for vitamin D in age-related macular degeneration (AMD) and showed a correlation between reduced serum vitamin D levels and risk for early AMD. Based on this study and the protective role of vitamin D in diseases with similar pathophysiology to AMD, we examined the role of vitamin D in a family-based cohort of 481 sibling pairs. Using extremely phenotypically discordant sibling pairs, initially we evaluated the association of neovascular AMD and vitamin D/sunlight-related epidemiological factors. After controlling for established AMD risk factors, including polymorphisms of the genes encoding complement factor H (CFH) and age-related maculopathy susceptibility 2/HtrA serine peptidase (ARMS2/HTRA1), and smoking history, we found that ultraviolet irradiance was protective for the development of neovascular AMD (p = 0.001). Although evaluation of serum vitamin D levels (25-hydroxyvitamin D [25(OH)D]) was higher in unaffected individuals than in their affected siblings, this finding did not reach statistical significance.

Based on the relationship between ultraviolet irradiance and vitamin D production, we employed a candidate gene approach for evaluating common variation in key vitamin D pathway genes (the genes encoding the vitamin D receptor [VDR]; cytochrome P450, family 27, subfamily B, polypeptide 1 [CYP27B1]; cytochrome P450, family 24, subfamily A, polypeptide 1 [CYP24A1]; and CYP27A1) in this same family-based cohort. Initial findings were then validated and replicated in the extended family cohort, an unrelated case-control cohort from central Greece and a prospective nested case-control population from the Nurse's Health Study and Health Professionals Follow-Up Studies, which included patients with all subtypes of AMD for a total of 2,528 individuals. Single point variants in CYP24A1 (the gene encoding the catabolising enzyme of the vitamin D pathway) were demonstrated to influence AMD risk after controlling for smoking history, sex and age in all populations, both separately and, more importantly, in a meta-analysis. This is the first report demonstrating a genetic association between vitamin D metabolism and AMD risk. These findings were also supplemented with expression data from human donor eyes and human retinal cell lines. These data not only extend previous biological studies in the AMD field, but further emphasise common antecedents between several disorders with an inflammatory/immunogenic component such as cardiovascular disease, cancer and AMD.

specifically, 2q33[48] (CYP27A1), 12q13[36] (CYP27B1 and VDR), and 20q13[36] (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 [27]. 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 [28]. At physiological concentrations, vitamin D has also been shown to protect cell proteins and membranes from oxidative damage [30]. 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 [99]. 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 [100].

Conclusion

In summary, using multiple lines of evidence, this study demonstrated an association between the vitamin D pathway and AMD. Specifically, there is a protective association between UV radiation and neovascular AMD which is independent of smoking and CFH and HTRA1 polymorphisms. Additionally, we identified an association between genetic variation in the major vitamin D catabolic gene, CYP24A1, and risk for all forms of AMD and, specifically, the neovascular form of AMD. Further experiments are needed to elucidate the role of vitamin D and UV light in the pathogenesis of AMD. Ongoing large-scale trials of supplemental vitamin D may provide a unique opportunity to examine the effects of vitamin D on AMD in a randomised trial setting. Such studies may have important implications for the prevention and/or treatment of neovascular AMD by the regulation of modifiable lifestyle factors that influence levels of vitamin D.

Declarations

Acknowledgements

We thank the patients and their siblings for their participation in this study. We also thank Anne Marie Lane, Amanda Harring and Trevor Arneberg for their help on this project and Fei Ji, for critical and helpful discussions on statistical analysis.

This work was supported by grants from the Lincy Foundation; the Massachusetts Lions; Friends of the Massachusetts Eye and Ear Infirmary; Genetics of Age-related Macular Degeneration Fund; MEEI; the Edward N. & Della L. Thome Memorial Foundation; an unrestricted grant from Research to Prevent Blindness, Inc. to the Department of Ophthalmology and Visual Sciences, University of Utah; Hope for Vision and the National Institutes of Health [EY014458, EY14104, EY017362, EY017404].

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