PEER REVIEWED

Genetic Biomarkers for AMD

FAREED ALI, MD, FRCS(C)

Age-related macular degeneration (AMD), in addition to being one of the most common diseases encountered by the retinal physician, is also one of the most rapidly evolving in terms of our level of understanding of its pathogenesis. AMD was initially a disease that was described and understood strictly based on its clinical appearance and with no therapeutic options available. However, over the last several years, there have been rapid gains in the understanding of all aspects of the disease on the molecular, cellular and genetic levels. This increased level of understanding has provided the opportunity for the development of new therapeutic and diagnostic options for AMD.

EARLY EMPIRICAL STUDIES

The importance of dietary factors in the development of age-related macular degeneration has long been recognized, and, in fact, dietary supplementation is still the only effective therapy for dry AMD that has been proved in a formal clinical trial.^1-3 Empirical observations and epidemiological studies also provided evidence that the development and progression of AMD was related to advanced age, smoking history, racial factors, hypertension, atherosclerosis, sunlight exposure, and other factors.^4-10

The groundwork for the idea that AMD is a heritable disease derived from early epidemiological studies that examined possible inheritance patterns in an attempt to determine the risks of developing AMD.^11-12 The fact that AMD risk was highly associated with a family history of the disease strongly suggested a genetic component to the disease.

PATHOGENESIS OF CLINICALLY OBSERVED LESIONS

Concurrently, great advances were being made in the understanding of the molecular and cellular processes underlying the development of age-related macular degeneration. Primary attention was given to identifying the pathogenesis and composition of some of the key morphological lesions associated with age-related macular degeneration, such as drusen, basal deposits, and thickening of Bruch's membrane.

Early breakthroughs included the identification of the important role played by inflammatory processes in the development of drusen and abnormalities at the Bruch's/retinal pigment epithelium (RPE) interface.^13-14 The hypothesis was that abnormal activation of the complement cascade pathway at the level of the RPEchoroid interface — specifically, the formation of membrane attack complexes — exerted damage on cellular structures in the choroid and the retina. This, in turn, led to the formation of the key retinal lesions observed clinically in age-related macular degeneration.

Another important pathological process identified early on was cellular apoptosis, which was felt to be an important cause of retinal pigment epithelium damage that was clinically observed in age-related macular degeneration.^15-16 The biological stressors that triggered apoptosis were believed to include ischemia, ultraviolet/bluelight exposure, abnormal lipoprotein accumulation, and oxidative stress. These biological stressors were also felt to be related to earlier identified clinical risk factors for AMD development and progression. In fact, oxidative damage and lipoprotein accumulation were directly implicated in drusen formation and in the initiation of the aforementioned abnormal inflammatory processes important in the development of age-related macular degeneration.^17-18

MOLECULAR GENETICS AND BIOLOGICAL MARKERS

Importantly, a confirmed genetic abnormality with a very strong and significant association with an increased risk of developing AMD was described.^19-22 This involved DNA variants, or single nucleotide polymorphisms (SNPs), of the complement factor H (CFH) gene. CFH's key role in the immune system is to regulate the complement cascade pathway. The fact that the earliest identified genetic risk factor involved the complement pathway of the immune system supported the earlier work that postulated a role for immune/inflammatory processes in the development of AMD. The increased understanding of the molecular genetic basis of AMD, in turn, led to the refinement of the cellular and biological theories regarding AMD development and progression.

For example, the CFH gene was specifically identified as having a key involvement in the development of soft drusen and advanced forms of AMD.²³ In addition, variations involving the complement component 3 (C3), complement factor B (CFB), and complement factor H–related 1 and 3 (CFHR1/R3) genes, key components of the complement pathway, were shown to be significant risk factors for AMD.^24,25 Further studies of the importance of inflammation in AMD also helped to refine the early theories involving oxidative damage and retinal lipoproteins and their role in initiating the abnormal inflammatory responses that are related to the identified genetic variations.^26-27

A potentially important biological marker for AMD development and progression may be related to a group of molecules known as heat shock proteins (HSPs). RPE cell loss through the process of apoptosis is now generally accepted to be a key pathophysiological feature of AMD.²⁸ RPE cells are susceptible to this process because they are postmitotic cells that are exposed for a long period of time to numerous cellular stress factors. These stress factors include many factors that, again, are implicated in the development and progression of AMD, such as ischemia, bluelight exposure, oxidative stress, and free-radical damage.^29-30 Such stresses at the cellular level induce apoptosis, but they also increase the cellular production of HSP, which helps to protect the cell from the stress factors and therefore reduces the risk of apoptosis.

A key HSP that has been recognized to play an important role in AMD is αB-crystallin.^31-32 It has been demonstrated that αB-crystallin production in RPE cells can be increased when the cells are subjected to heat shock and oxidative stress-induced injury. However, it has also been discovered that the αB-crystallin levels are higher in RPE cells in the macula compared to cells in more peripheral retinal areas. Also, the higher levels of αB-crystallin can often be found in cells adjacent to areas of drusen.

These findings support the notion that the macular area is subjected to higher levels of overall cellular stress, which in turn explains why the typical findings of AMD, such as drusen, RPE clumping, and geographic atrophy, tend to occur in the macular area. In addition, these data suggest that αB-crystallin is a key biomarker for AMD, and it may be useful in the future for aiding in early diagnosis of AMD and predicting the risk of AMD progression. It may even play a future role in AMD therapy. Indeed, it has been shown that zinc plays a key role in the cellular protective functions of αB-crystallin, which explains the beneficial effects observed for zinc intake related to AMD onset and progression.³³

NEW THERAPEUTIC AND DIAGNOSTIC OPTIONS

The identification of the important roles played by cell apoptosis, oxidative damage, and abnormal inflammatory activation helps to explain the much earlier findings of the beneficial effects of nutrient supplements, such as betacarotene and lutein, on AMD development and progression. These nutrients help protect the retina from the damaging effects of oxidative stress and abnormal activation of inflammatory pathways. They also help to explain the mechanisms of action of earlier identified AMD risk factors, such as smoking and sunlight exposure. However, this refined and enhanced understanding of AMD pathogenesis also presented opportunities for new therapeutic options for AMD. A number of new pharmacological therapies for dry AMD, all of which have beneficial effects on complementmediated inflammation or oxidative retinal damage, are currently in the early stages of development.^34-36

One of the most intriguing developments related to the latest theories of AMD pathogenesis is the prospect of genetic testing to identify and quantify an individual's risk for developing AMD. These include noninvasive salivasample testing based on analysis of the aforementioned SNP of the complement factor H and complement C3 genes, as well as SNPs of other key genes associated with AMD.^37-38 Such tests can be extremely accurate at identifying the presence of the specific SNPs in question. However, a potential limitation of the utility of such a test is the recognized multifactorial nature of AMD risk factors and the fact that genetics alone cannot account entirely for an individual's risk of developing AMD.

There also exist the general concerns surrounding any genetic test for medical conditions regarding the induced stress on patients who are told they are at a high risk for developing a chronic and incurable disease. Furthermore, there is the possibility that the accuracy of genetic tests for AMD is overestimated and that these tests may result in a high rate of false positive results.³⁹ This again brings up the ethical issue of the negative impact a false positive genetic test result would have on an individual.

CONCLUSION

It can be argued that the only utility of genetic testing for AMD is in identifying those patients who require close ophthalmic follow-up, in which case one can simply follow those patients who have a certain number of AMD risk factors identified on a simple questionnaire. However, this argument ignores the practical limitations that exist for widespread screening and monitoring, due to issues such as a limited supply of doctors and travel difficulties for some patients. An accurate, simple, and accessible genetic test for AMD could enhance the optimal use of limited medical resources and improve patient compliance with recommended follow-up regimens. Furthermore, as new therapies for early stages of AMD become available, early intervention, which may be improved by accurate genetic testing, could become essential. Finally, the possibility exists that genetic testing may identify biomarkers that can predict response to future AMD therapies. RP

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