GENETICS OF RETINA FEATURE STORY Recent Findings in Genetic Research in AMD BY ALBERT O. EDWARDS, MD, PHD enes play an important role in age-related Research in Vision and Ophthalmology meeting and are macular degeneration (AMD). In recent years, summarized in this article. researchers have identified genetic variation that contributes to the development of AMD. COPY NUMBER VARIATION GFrom the geneticist’s point of view, AMD is a complex, IN THE REGULATION OF COMPLEMENT late-onset trait in which multiple demographic, environ- ACTIVATION (RCA) REGION mental, and genetic risk factors combine to cause disease. The regulation of complement activation (RCA) Genetic research is a powerful method for identifying the region contains factor H and five related genes called biologic pathways contributing to disease risk. CFH related 1 through 5 (Figure 1). The genes in the Genetic research has identified two major biologic RCA region are sometimes deleted. A combined dele- pathways contributing to the development of AMD. The tion of CFHR3 and CFHR1 occurs in about 15% of peo- first pathway is a branch of the innate immune system ple and has been thought to protect against developing called the alternative pathway of complement. The sec- AMD. However, until recently an accurate method for ond pathway is a segment of DNA on chromosome 10 determining the number of copies in individual subjects called age-related maculopathy susceptibility 2 (ARMS2). was not available. The biologic basis of this second pathway is not yet Katherine Schmid-Kubista, MD, in my laboratory understood, but ARMS2 variants can increase the odds developed a multiplex ligation-dependent probe amplifi- of developing AMD by up to sevenfold. cation (MLPA) assay that allows the determination of the The first major genetic variants consistently associated exact number of copies of the CFHR3 and CFHR1 genes.2 with AMD were found in the complement factor H gene The assay was used to genotype 252 subjects with and (CFH).1 Factor H is an inhibitor of activation of the alterna- 249 without AMD to determine whether the number of tive pathway of complement. Candidate gene studies in copies of these genes altered the risk of developing AMD. this pathway subsequently identified variation in the com- We found four combinations of copy number varia- plement component 2, factor B, component 3, and factor I tions. Deletions of both CFHR3 and CFHR1 was identi- genes. Each of these genes encodes proteins important in fied in 14% of chromosomes, deletion of only CFHR3 was our body’s response to infection and damage. seen in 0.4%, deletion of only CFHR1 in 1.1%, and dupli- Genetic variation in other pathways, including the clas- cation of CFHR1 in 0.1%. sical complement pathway (SERPING1), danger-sensing Deletion of both copies of CFHR3/CFHR1 was found pathways (TLR4, TLR3), lipid pathway (ApoE), immune to decrease the odds of having AMD eightfold. response pathways (HLA, KIR), and the mitochondrial However, combined deletion of CFHR3/CFHR1 was genome, have been reported. Most have not been ade- always found on the same chromosome as a protective quately replicated, except for the ApoE2 variant protect- form of CFH. We were unable to demonstrate an inde- ing against AMD. pendent effect of the combined deletion or a difference Research into the genetic roots of AMD in our labora- in subjects with or without the risk histidine amino acid tory has continued to focus on these and other impor- at position 402 in factor H. tant loci. A number of papers from us and our collabora- We concluded that deletion of CFHR3 and CFHR1 pro- tors were presented at this year’s Association for tected against the development of AMD at least in part 78 IRETINA TODAYISEPTEMBER 2009 GENETICS OF RETINA FEATURE STORY Figure 1. Variation in the number of copies of genes within the regulation of complement activation (RCA) region.The location bar across the top shows the base pair location on chromosome 1. Note that the RCA region is located within band 31.3 of chro- mosome 1. CFH was the original gene, and the CFHR genes arose through duplication during evolution.The bottom panel shows copy number changes observed in a variety of genome-wide genetic studies. Note that deletions (blue) in the CFHR3/CFHR1 region are common, as are deletions of CFHR4.The image was downloaded from the Database of Genomic Variants at http://projects.tcag.ca. because the deletion tagged protective forms of CFH. We changes in complement genes alter the regulation of the have now developed an assay that determines the copy alternative pathway of complement activation in human number of all genes in the RCA region and will be able to tissue and blood. Laura Hecker, PhD, of our laboratory look at the impact of the common CFHR4 deletion presented a study of how these genetic variants altered (Figure 1) on AMD risk. complement protein levels and activation in blood from patients with and without AMD.4 These studies were GENOME-WIDE ASSOCIATION STUDY performed in collaboration with the group of Martin In order to identify novel genetic variation contributing Oppermann, PhD, in Germany. to AMD, we performed a genome-wide association study.3 We confirmed the increased complement activation in This study was led by Anand Swaroop, PhD, Gonçalo AMD patients compared with controls that was first Abecasis, PhD, Dwight Stambolian, MD, and myself. We reported by Dr. Oppermann’s group at ARVO in 2008. genotyped more than 370,000 single nucleotide polymor- We went on to show that complement activation in the phisms (SNPs) in 2,136 unrelated white patients with AMD blood of humans is under genetic control. Further, the and 1,138 white control subjects using the HumanCNV370 genetic risks for AMD that are found in regions of com- Duo BeadChip (Illumina, San Diego, CA). Using resources plement proteins involved in regulation of complement from the HapMap consortium we predicted the geno- activation in blood increased complement activation in types at about 2.5 million additional common SNPs. all subjects. Genetic variants located in regions involved This study confirmed previously reported associa- in tissue regulation of complement activation did not tions of known genetic variants with AMD, including alter complement levels in blood. SNPs at CFH, C2/BF, C3, and ARMS2. We also replicated We concluded that fluid phase inhibition of comple- the association between CFI and AMD for the first time. ment activation is decreased in subjects with AMD. Two additional pathways contributing to AMD were This leads to increased activation of complement in also discovered, providing insight into the pathogenesis the blood of AMD subjects. We believe that comple- of AMD. These will be published later this year. ment activation in the blood could mediate some of the effects of lifestyle choices such as diet and smoking COMPLEMENT ACTIVATION IS INCREASED and contribute to the development of AMD. This work IN THE BLOOD OF AMD PATIENTS is under revision and is expected to be published later We have become interested in how the genetic this year. SEPTEMBER 2009 IRETINA TODAYI 79 GENETICS OF RETINA FEATURE STORY CONCLUSIONS in Vision and Ophthalmology; May 5, 2009; Fort Lauderdale, FL. Genetic risks account for about 80% of the risk of developing AMD. Modifiable lifestyle and environmen- tal factors such as diet, exercise, obesity, and smoking also alter the risk of developing AMD. Most studies have observed that these are independent risks, illus- trating 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 com- plications 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 rela- tive to the information discussed in this article. He may be reached at 800-888-2020; or via e-mail at [email protected]. 1. Edwards AO, Ritter R 3rd, Abel KJ, Manning A, Panhuysen C, Farrer LA. Complement factor H polymorphism and age-related macular degeneration. Science. 2005;308(5720):421-424. 2. Schmid-Kubista KE, Tosakulwong N, Wu Y, et al. Contribution of copy number variation in the regulation of complement activation locus to development of age-related macular degeneration. Invest Ophthalmol Vis Sci. June 24, 2009. [Epub ahead of print] 3. Swaroop A, Chen W, Stambolian D, et al. Genome wide association study (GWAS) of age-related macular degeneration (AMD). Paper presented at: Annual Meeting of Associ- ation for Research in Vision and Ophthalmology; May 4, 2009; Fort Lauderdale, FL.