Genes play an important role in age-related macular degeneration (AMD). In recent years, researchers have identified genetic variation that contributes to the development of AMD.
From the geneticist's point of view, AMD is a complex, late-onset trait in which multiple demographic, environmental, and genetic risk factors combine to cause disease. Genetic research is a powerful method for identifying the biologic pathways contributing to disease risk.
Genetic research has identified two major biologic pathways contributing to the development of AMD. The first pathway is a branch of the innate immune system called the alternative pathway of complement. The second pathway is a segment of DNA on chromosome 10 called age-related maculopathy susceptibility 2 (ARMS2). The biologic basis of this second pathway is not yet understood, but ARMS2 variants can increase the odds of developing AMD by up to sevenfold.
The first major genetic variants consistently associated with AMD were found in the complement factor H gene (CFH).1 Factor H is an inhibitor of activation of the alternative pathway of complement. Candidate gene studies in this pathway subsequently identified variation in the complement component 2, factor B, component 3, and factor I genes. Each of these genes encodes proteins important in our body's response to infection and damage.
Genetic variation in other pathways, including the classical complement pathway (SERPING1), danger-sensing pathways (TLR4, TLR3), lipid pathway (ApoE), immune response pathways (HLA, KIR), and the mitochondrial genome, have been reported. Most have not been adequately replicated, except for the ApoE2 variant protecting against AMD.
Research into the genetic roots of AMD in our laboratory has continued to focus on these and other important loci. A number of papers from us and our collaborators were presented at this year's Association for Research in Vision and Ophthalmology meeting and are summarized in this article.
COPY NUMBER VARIATION IN THE REGULATION OF COMPLEMENT ACTIVATION (RCA) REGION
The regulation of complement activation (RCA) region contains factor H and five related genes called CFH related 1 through 5 (Figure 1). The genes in the RCA region are sometimes deleted. A combined deletion of CFHR3 and CFHR1 occurs in about 15% of people and has been thought to protect against developing AMD. However, until recently an accurate method for determining the number of copies in individual subjects was not available.
Katherine Schmid-Kubista, MD, in my laboratory developed a multiplex ligation-dependent probe amplification (MLPA) assay that allows the determination of the exact number of copies of the CFHR3 and CFHR1 genes.2 The assay was used to genotype 252 subjects with and 249 without AMD to determine whether the number of copies of these genes altered the risk of developing AMD.
We found four combinations of copy number variations. Deletions of both CFHR3 and CFHR1 was identified in 14% of chromosomes, deletion of only CFHR3 was seen in 0.4%, deletion of only CFHR1 in 1.1%, and duplication of CFHR1 in 0.1%.
Deletion of both copies of CFHR3/CFHR1 was found to decrease the odds of having AMD eightfold. However, combined deletion of CFHR3/CFHR1 was always found on the same chromosome as a protective form of CFH. We were unable to demonstrate an independent effect of the combined deletion or a difference in subjects with or without the risk histidine amino acid at position 402 in factor H.
We concluded that deletion of CFHR3 and CFHR1 protected against the development of AMD at least in part because the deletion tagged protective forms of CFH. We have now developed an assay that determines the copy number of all genes in the RCA region and will be able to look at the impact of the common CFHR4 deletion (Figure 1) on AMD risk.
GENOME-WIDE ASSOCIATION STUDY
In order to identify novel genetic variation contributing to AMD, we performed a genome-wide association study.3 This study was led by Anand Swaroop, PhD, Gonçalo Abecasis, PhD, Dwight Stambolian, MD, and myself. We genotyped more than 370,000 single nucleotide polymorphisms (SNPs) in 2,136 unrelated white patients with AMD and 1,138 white control subjects using the HumanCNV370 Duo BeadChip (Illumina, San Diego, CA). Using resources from the HapMap consortium we predicted the genotypes at about 2.5 million additional common SNPs.
This study confirmed previously reported associations of known genetic variants with AMD, including SNPs at CFH, C2/BF, C3, and ARMS2. We also replicated the association between CFI and AMD for the first time. Two additional pathways contributing to AMD were also discovered, providing insight into the pathogenesis of AMD. These will be published later this year.
COMPLEMENT ACTIVATION IS INCREASED IN THE BLOOD OF AMD PATIENTS
We have become interested in how the genetic changes in complement genes alter the regulation of the alternative pathway of complement activation in human tissue and blood. Laura Hecker, PhD, of our laboratory presented a study of how these genetic variants altered complement protein levels and activation in blood from patients with and without AMD.4 These studies were performed in collaboration with the group of Martin Oppermann, PhD, in Germany.
We confirmed the increased complement activation in AMD patients compared with controls that was first reported by Dr. Oppermann's group at ARVO in 2008. We went on to show that complement activation in the blood of humans is under genetic control. Further, the genetic risks for AMD that are found in regions of complement proteins involved in regulation of complement activation in blood increased complement activation in all subjects. Genetic variants located in regions involved in tissue regulation of complement activation did not alter complement levels in blood.
We concluded that fluid phase inhibition of complement activation is decreased in subjects with AMD. This leads to increased activation of complement in the blood of AMD subjects. We believe that complement activation in the blood could mediate some of the effects of lifestyle choices such as diet and smoking and contribute to the development of AMD. This work is under revision and is expected to be published later this year.
CONCLUSIONS
Genetic risks account for about 80% of the risk of developing AMD. Modifiable lifestyle and environmental factors such as diet, exercise, obesity, and smoking also alter the risk of developing AMD. Most studies have observed that these are independent risks, illustrating the importance of educating your patients about the modifiable risks.
All of the genetic risks contribute to the formation of the maculopathy (inflammatory deposits in the outer retina such as drusen) that characterizes AMD. Although some reports have suggested that specific genetic variants contribute to complications of AMD (geographic atrophy, exudation), the majority of studies have not observed such effects.
With the exception of smoking, which appears to exert a major impact on progression to exudation, the known risks appear to increase the risk of having the maculopathy of AMD. The most important risk for developing the complications of AMD is the burden of the maculopathy, namely the drusen and pigment changes that you see in your patients.
While genetic research will continue to shed light on the mechanisms of disease, it is important that we counsel patients on the modifiable risk factors, such as smoking, exercise, and diet. Patients can reduce their risk for developing both the maculopathy and the complications of AMD by avoiding tobacco, exercising, and maintaining a healthy diet and body weight. The retina examination we perform is by far the single most important predictor of future vision loss. Thus, our examination and counseling are essential to the care of patients with AMD.
Albert O. Edwards, MD, PhD, is at the Mayo Clinic, in Rochester, MN, and at the Oregon Eye Associates and the Institute for Molecular Biology, University of Oregon, in Eugene, OR. Dr. Edwards has no commercial relationships relative to the information discussed in this article. He may be reached at 800-888-2020; or via e-mail at aoe102@gmail.com.
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