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Macular Degeneration: Causes, Diagnosis and Treatment : Causes, Diagnosis and Treatment [1 ed.]
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Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved. Macular Degeneration: Causes, Diagnosis and Treatment : Causes, Diagnosis and Treatment, Nova Science Publishers, Incorporated, 2011.

EYE AND VISION RESEARCH DEVELOPMENTS

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MACULAR DEGENERATION: CAUSES, DIAGNOSIS AND TREATMENT

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Macular Degeneration: Causes, Diagnosis and Treatment : Causes, Diagnosis and Treatment, Nova Science Publishers, Incorporated, 2011.

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EYE AND VISION RESEARCH DEVELOPMENTS

MACULAR DEGENERATION: CAUSES, DIAGNOSIS AND TREATMENT

EDDIE J. CAMPBELL Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

AND

LESLIE M. MCMANN EDITORS

Nova Science Publishers, Inc. New York

Macular Degeneration: Causes, Diagnosis and Treatment : Causes, Diagnosis and Treatment, Nova Science Publishers, Incorporated, 2011.

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com

NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Macular degeneration : causes, diagnosis, and treatment / editors, Eddie J. Campbell and Leslie M. McMann. p. ; cm. Includes bibliographical references and index.

ISBN:  (eBook)

1. Retinal degeneration. 2. Age factors in disease. I. Campbell, Eddie J. II. McMann, Leslie M. [DNLM: 1. Macular Degeneration. 2. Age Factors. 3. Genetic Predisposition to Disease. 4. Risk Factors. WW 270] RE661.D3M325 2010 617.7'35--dc22 2010051738

Published by Nova Science Publishers, Inc. † New York

Macular Degeneration: Causes, Diagnosis and Treatment : Causes, Diagnosis and Treatment, Nova Science Publishers, Incorporated, 2011.

Contents Preface Chapter I

Chapter II

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Chapter III

vii Correlation of Phenotype and Genotype in Age-Related Macular Degeneration Henry E. Wiley and Chi-Chao Chan Role of Anti-VEGF Therapies in Neovascular AMD: What the Clinical Trials Tell Us Shripaad Y. Shukla, Daniel F. Kiernan and Jennifer I. Lim Surgical Management of Age-Related Macular Degeneration: Past, Present and Future Treatment Options Pradeep S. Prasad and Jean-Pierre Hubschman

Chapter IV

Hereditary and Age-Related Macular Degeneration Nancy Skoura, Josephine Hoh and Ju Liu

Chapter V

Emerging New Rationale for the Treatment of Pathogenic Retina Based on Molecular Genetic and Biochemical Studies Barkur S. Shastry

Chapter VI

The Role of Genes in the Pathogenesis of AMD Peter James Francis

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29

49 69

99 117

vi Chapter VII

Contents Ocular Blood Flow in Degenerative Myopia Galina Dimitrova and Satoshi Kato

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Index

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133 141

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Preface This new book presents topical research in the study of macular degeneration, including the correlation of phenotype and genotype in agerelated macular degeneration; role of anti-VEGF therapies in neovascular AMD; the biochemical and genetic studies that support the treatment of the pathogenic retina and ocular blood flow in degenerative myopia. Chapter I - Beginning with the recent discovery of risk-modifying polymorphisms associated with the complement factor H (CFH) gene, multiple genetic association studies have better defined the heritable determinants of susceptibility to age-related macular degeneration (AMD). With identification of variants in genes including CFH, complement component 2 (C2), complement component 3 (C3), complement factor B (CFB), complement factor I (CFI), and the age-related maculopathy susceptibility 2 (ARMS2) / high-temperature requirement A serine peptidase 1 (HTRA1) locus, which together account for the bulk of genetic contribution to AMD risk, considerable effort has been dedicated to discovering associations between key polymorphisms and specific aspects of the disease phenotype. The authors review the literature on correlation between phenotype and genotype in AMD, outline areas for future inquiry, and discuss the potential application of such knowledge to building predictive models of AMD risk and response to treatment. Chapter II - Choroidal neovascularization (CNV) as a component of neovascular age-related macular degeneration (NVAMD) remains a challenging clinical problem. It is known that vascular endothelial growth factor (VEGF) plays a critical role in the development of CNV. Because of this, a variety of anti-VEGF therapies have been developed to combat NVAMD. These agents represent a significant improvement in the therapeutic

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options for NVAMD. This chapter will discuss evidence for their efficacy based on completed clinical trials and also emerging studies using new therapies. Chapter III - Despite recent advances in the management of age-related macular degeneration (AMD), the disease continues to be a leading cause of blindness among the elderly with an increasing prevalence in industrialized nations. While intraocular pharmacotherapy in the form of vascular endothelial growth factor (VEGF) inhibition has vastly improved visual and functional outcomes in patients with the early to moderate stages of exudative AMD, the management of non-exudative and advanced exudative AMD continues to be a challenge with disappointing clinical results for both patients and physicians. Surgical therapies in the form of submacular surgery and macular translocation have had variable success but may be employed in patients with either very advanced AMD or in those with disease recalcitrant to VEGF inhibition, laser photocoagulation, photodynamic therapy or a combination thereof. The following chapter serves as a summary of the past, present and burgeoning surgical options for the management of AMD. Current therapeutic indications for surgery in AMD include advanced atrophic AMD and some forms of exudative macular degeneration, including large submacular hemorrhage and exudative AMD not responsive to traditional therapy. Surgical techniques to be reviewed in this chapter include submacular surgery, macular translocation, and submacular hemorrhage displacement. Novel surgical therapies and indications to be discussed include surgically-implantable pharmacologic devices, gene therapy, retinal pigment epithelium transplantation, and pars plana vitrectomy for vitreomacular traction. Chapter IV - Age-related macular degeneration (AMD) is the most common cause of blindness in people over 60. Based on the twin studies genetic contributions to AMD are as high as 75% among overall risks. Recent genetic association studies have pointed that the polymorphisms of several genes were associated with increased risks for AMD. The first part of this chapter will provide a systematic review on AMD associated genes suggested by various studies with the individual and combined relative risks in conferring this disease. Since these genetic advancements are still in infancy with regards to preventions and therapeutic targets, in the second part of the chapter the authors will discuss the approaches for further understanding the roles of the genetic associations. They will focus on the complement factor H (CFH) and the complement component 3/the complement factor B (C3/CFB) as they are by far the most firmly established risk factors associated with AMD. To investigate if there are other genetic variants in the immunological

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Preface

ix

pathways that may also be the risk factors predisposing individuals to AMD, one can resequence genes of the components of entire complement pathways and/or the MHC/HLA highly variable regions. The authors argue, from the epidemiological perspective, there is also a need of efforts in collecting plasma/serum and designing proteomic analyses in addition to genomic analysis. All together this chapter aims to pave the way for the following chapters that present mechanistic and functional insights from the disease associated genes to define pathogenesis of AMD. Chapter V - The retina (back of the eye) is a multicellular component of the eye and is responsible for phototransduction processes. In this enzymatic amplification, the light energy is converted into electrical stimuli that initiates neural response to light. Unfortunately, the same retina is also one of the most common sites for various disorders that result in blindness. Although the total number of genes involved in the normal structure and function of the eye are not known, application of molecular genetic and biochemical techniques to study the genetic eye diseases has enriched our understanding of several inherited disorders. As a result, the biochemical and genetic basis of some of the eye disorders are beginning to be understood. For instance, biochemical studies of many degenerative retinal disorders revealed that they involve misfolding, aggregation and retention of mutant proteins in the endoplasmic reticulum (ER) similar to many neurodegenerative diseases. This abnormal deposition of misfolded proteins may induce ER stress and proteasomal dysfunction that may ultimately lead to cell death. Because chaperones are shown to suppress aggregate formation of mutant proteins, at least in theory the first rationale is that if the expression of molecular chaperones can be increased by using inducible drugs, it may be possible to alleviate proteinmisfolding diseases. Molecular genetic studies on the other hand, have provided another rationale for the improved treatment. For instance, individual variations in drug response and adverse drug reactions are well known in eye care. Although substantial studies that link genetic variants to inter-individual difference in medicinal drug response have not been reported in the eye field, there are some small scale studies that seem to associate the drug response to the genotypes of patients in two major eye disorders namely age-related macular degeneration (AMD) and glaucoma. Therefore, the second rationale is that, if the genotype of the patients at a particular locus is known then it may be possible to increase the safety and efficacy of the medicinal drug. Taken together, these results suggest that it may be possible to optimize the eligibility criteria or perform the therapy in a customized manner and to produce a decreased amount of abnormal deposition of misfolded proteins to delay the

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degenerative processes. It is hoped that these new approaches may provide opportunities in the future to redefine and develop new treatment strategies that will one day treat, prevent or cure many blinding disorders. However, there are many challenging questions that need to be addressed and it remains to be established whether these are of any use in the clinic. In this short article an attempt has been made to highlight some of the biochemical and genetic studies that support these new rationale for an improved treatment. Chapter VI - Age-related macular degeneration (AMD), akin to other common age-related diseases, has a complex pathogenesis and arises from the interplay of genes, environmental factors and personal characteristics. The past decade has seen very significant strides towards identification of those precise genetic variants associated with disease. That genes encoding proteins of the (alternative) complement pathway (CFH, C2, CFB, C3, CFI) are major players in etiology came as a surprise to many but has already lead to the development of therapies entering human clinical trials. Other genes replicated in many populations ARMS2, APOE, variants near TIMP3 and genes involved in lipid metabolism have also been implicated in disease pathogenesis. The genes discovered to date can be estimated to account for approximately 50% of the genetic variance of AMD and have been discovered by candidate gene approaches, pathway analysis and latterly genome-wide association studies. Next Generation sequencing modalities and meta-analysis techniques are being employed with the aim of identifying the remaining rarer but perhaps, individually more significant sequence variations, linked to disease status. Complementary studies have also begun to utilize this genetic information to develop clinically-useful algorithms to predict AMD risk and evaluate pharmacogenetics. In this article, contemporary commentary is provided on rapidly progressing efforts to elucidate the genetic pathogenesis of AMD as the field stands at the end of the first decade of the 21st Century. Chapter VII - Degenerative myopia is among the leading causes of blindness in the working population. The circulatory irregularities observed in myopia are thought to be related to the chorioretinal and vascular pathology of degenerative myopia (thinning and atrophy of the choroid and retina; straightening of the retinal vessels, small calibre and scarce choroidal arteries; thinning and loss of the choriocapillaris, etc.). In experimental studies in chicks, the choroidal blood flow decreased as the axial length of the eye increased. The reduction of choroidal blood flow was not a cause but a consequence of ocular enlargement in experimental myopia. In clinical studies of high myopia, the choroidal and retinal blood flow were reported decreased

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in affected versus control eyes. Fluorescein angiography studies have described specific features of choroidal neovascularization (CNV) in degenerative myopia. Color Doppler imaging studies suggest that increased vascular resistivity in the choroidal circulation may be involved in the pathogenesis of myopic CNV. In this review, the authors summarize and discuss the results from the studies that investigated ocular blood flow in degenerative myopia.

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In: Macular Degeneration Editors: E. Campbell and L. McMann

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Chapter I

Correlation of Phenotype and Genotype in Age-Related Macular Degeneration Henry E. Wiley and Chi-Chao Chan National Eye Institute, National Institutes of Health, Bethesda, Maryland

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Abstract Beginning with the recent discovery of risk-modifying polymorphisms associated with the complement factor H (CFH) gene, multiple genetic association studies have better defined the heritable determinants of susceptibility to age-related macular degeneration (AMD). With identification of variants in genes including CFH, complement component 2 (C2), complement component 3 (C3), complement factor B (CFB), complement factor I (CFI), and the agerelated maculopathy susceptibility 2 (ARMS2) / high-temperature requirement A serine peptidase 1 (HTRA1) locus, which together account for the bulk of genetic contribution to AMD risk, considerable effort has been dedicated to discovering associations between key polymorphisms and specific aspects of the disease phenotype. We review the literature on correlation between phenotype and genotype in AMD, outline areas for future inquiry, and discuss the potential application of such knowledge to building predictive models of AMD risk and response to treatment.

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Age-related macular degeneration (AMD) is a neurodegenerative disease of the retina that represents the most common cause of irreversible visual impairment among people over age 50 in developed countries.[1] Risk is multifactorial, relating to age and various environmental, dietary, and genetic factors. Heterogeneity of disease phenotype and onset late in life have complicated identifying the determinants of disease, but advances in genetic epidemiology and technology have recently transformed our understanding of heritable risk factors and provided insights into AMD pathophysiology.

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Ocular Phenotype The clinical and histopathological hallmark of AMD is the druse. Drusen appear as discrete yellow or white concretions visible ophthalmoscopically external to the outer retina. They are characterized by their size (small, medium, or large), borders (hard or soft), distribution (central or peripheral), number, and associated pigment changes. Histopathologically, drusen represent collections of amorphous and heterogeneous material between the basal lamina of the retinal pigment epithelium (RPE) and the inner collagenous layer of Bruch‘s membrane.[2, 3] The appearance of a few small drusen is considered a normal consequence of aging, but a greater number of larger drusen in the macula increases risk for development of geographic atrophy (GA), choroidal neovascularization (CNV), and serous and hemorrhagic detachment of the neurosensory retina and RPE—the advanced stages of disease responsible for irreversible impairment of central vision.[4] AMD is clinically heterogeneous. Drusen and associated pigment changes are pleomorphic and can resemble deposits and pigmentary alterations seen in other eye diseases. Vision loss stems from two distinct advanced stages of disease. GA involving progressive degeneration of macular RPE and photoreceptors typically begins in the parafoveal region and advances gradually to involve the center of the retina. Neovascular or exudative AMD, consisting of invasion of the sub-RPE or subretinal space by aberrant neovascularization from the choroidal or retinal circulation, can be indolent or rapidly progressive, resulting in disciform scarring that sometimes obscures other features of the disease.

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Figure 1. Photomicrograph of drusen. Drusen (arrow) represent focal collections of a heterogeneous material between the basal lamina of the RPE and the inner collagenous layer of Bruch‘s membrane. The RPE overlying drusen is often attenuated (arrowhead).

Various grading schema have been developed to classify the stages of disease. The Age-Related Eye Disease Study (AREDS) classification is one such system, based on standardized color fundus photography and validated in prospective clinical trials as a valuable tool for stratifying patients according to risk of disease progression and vision loss.[5 ]The value of the AREDS classification system in the clinic rests in its straightforward application to disease risk stratification and identification of individuals who would benefit from effective preventive therapy based on the results of routine ophthalmological examination.

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Figure 2. AREDS classification of AMD. Participants in the Age-Related Eye Disease Study were assigned to 1 of 4 categories based on characteristics of the eye with more severe disease. Drusen were divided into small, medium (also termed intermediate), and large using cut-offs at 63 and 125 microns (125 microns defined as the width of retinal venules at the optic nerve head margin, and 63 microns defined as half this width) and were counted if within 2 disc diameters of the center of the macula. Macular pigmentary changes could be considered sufficient for diagnosis of Early AMD even in the absence of drusen, but care was taken in such participants to exclude other causes for maculopathy.

Many ancillary tests have been employed to more completely characterize the disease phenotype, including auto-fluorescence fundus photography, fluorescein and indocyanine green angiography, optical coherence tomography (OCT), scanning laser ophthalmoscope microperimetry, and electrophysiologic testing. Fluorescein angiography (FA) has historically played an important role in sub-classifying CNV, while indocyanine green angiography has been useful in diagnosing polypoidal choroidal vasculopathy (PCV), which may represent a variant of AMD. There has been intense interest over the last decade in better characterizing the various stages of disease using OCT, which offers fast and non-invasive imaging of the macula at everincreasing resolution.

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Genetic Susceptibility

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Studies on twins and affected families have long suggested a heritable component to AMD risk. Genetic linkage studies suggested disease susceptibility loci on chromosomes 1q25-31 and 10q26.[6-9] But the greatest advances in our understanding of the genetics of AMD have occurred in the last five years. A series of reports in 2005, including the first successful genome-wide association study (GWAS), found a significant association between AMD and certain polymorphisms in the complement factor H (CFH) gene, which encodes an important regulator of the alternative pathway of the complement system.[10-13]

Figure 3. Gene regions with confirmed association to AMD susceptibility. Associations to AMD susceptibility for the gene regions above have been replicated in high-quality studies. Association signals for a number of other genes await confirmation.

Recent targeted association studies, which focus on a small number of polymorphisms in a small number of candidate regions of the genome, have identified a handful of additional genes important for AMD susceptibility. Spurred by evidence from linkage studies suggesting a locus of AMD risk on chromosome 10q26, and corroborated by findings of recent GWAS, [14, 15] testing of polymorphisms in the age-related maculopathy susceptibility 2 (ARMS2, formerly known as LOC387715) and high-temperature requirement A serine peptidase 1 (HTRA1) genes confirmed the importance of this

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region.[16-19] Guided by new appreciation of the role of the complement system in AMD, various groups have found association signals for polymorphisms in complement component 2 (C2), complement component 3 (C3), complement factor B (CFB), and complement factor I (CFI), [20-22] also recently confirmed by GWAS.[15] Taken together, the association signals from the above genes account for the majority of the heritable risk in AMD.[23] Variants in a handful of other genes have reported association to AMD. Some of these, such as the gene encoding apolipoprotein E (ApoE), have a plausible role in disease pathogenesis and have been implicated in multiple small studies.[24-27] Others await confirmatory evidence in more definitive investigations. Still others, like the toll-like receptor 3 (TLR3) gene, which encodes a cell-surface receptor involved in innate immune defense, have been studied with contradictory results, with replicative efforts failing to corroborate significant association.[28-30] Much work remains to be done in identifying the disease-causing variants at various susceptibility loci. For example, early reports suggested a key role for the CFH rs1061170 / Y402H missense variant, a single nucleotide polymorphism (SNP) encoding a switch in the protein amino acid sequence from tyrosine in the low-risk phenotype to histidine in the high-risk phenotype in populations of European descent.[10, 11, 13] But subsequent research has identified other polymorphisms more highly associated with AMD risk than the CFH Y402H variant [18, 31] and possible effect from adjacent partial CFH duplications, CFH-related genes (CFHR) 1-5.[32] Similarly, controversy persists about whether ARMS2, HTRA1, or both genes contain diseasecausing variants, because of the linkage disequilibrium among key polymorphisms.[23]

Genetic Association Studies Since publication of the first successful human GWAS in 2005, hundreds of studies have surveyed variants across the whole genome or across targeted regions of interest to identify genetic associations for a wide variety of diseases. Such approaches became feasible following sequencing of the human genome (the Human Genome Project) and the systematic cataloging of common SNPs and haplotypes (the International HapMap Project), and have been facilitated by the steadily falling costs of high-throughput DNA sequencing. The proliferation of such studies in the literature has provoked

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debate about establishing protocol design standards, peer-review guidelines, and requirements for replication of results in order to minimize the publication of spurious associations.[33] Genetic association studies for AMD use certain basic strategies to maximize the chance of discovering a true association. Cases and controls are typically chosen from an ethnically homogenous population, to minimize the possibility of false positives from population stratification. Selected cases exhibit prototypic phenotypic parameters of advanced disease (like GA or CNV in the presence of drusen) while those with confounding comorbid conditions (such as degenerative myopia, for example) are carefully excluded. Some studies use cases with a positive family history of AMD or even select from families with extensively documented pedigrees, in order to enrich for individuals with a documented heritable component of the disease. Controls are matched carefully for age, given its status as a powerful predictor of risk. Meticulous phenotyping lowers the number of cases necessary to discover a statistically significant association, and hinges on sound methods for standardized data collection and good caliber of fundus imaging. Though many studies use a cross-sectional design for its simplicity and economy, a longitudinal approach which follows cases and controls over time allows for more complete and accurate phenotyping, by allowing better differentiation of cases which present similarly at baseline but evolve differently with follow up. Great care must be taken in the analysis to appropriately adjust the threshold for statistical significance to account for multiple hypothesis testing (the testing of multiple phenotypic parameters for association to multiple genetic variants). Some genetic polymorphisms exhibit a high degree of linkage disequilibrium with one another, which can decrease the number of sites in a given locus that must be tested for significant association, but obfuscates identification of causal variants. Identification of haplotypes, consisting of combinations of alleles inherited together, can facilitate study of variation at a given locus in a population of interest. What constitutes an AMD ―case‖ varies among published case-control genetic association studies. In the first genome-wide association study on AMD, Klein et al. defined cases as eyes exhibiting at least one large druse (measuring > 125 microns in diameter) combined with evidence of sightthreatening AMD (GA or neovascular AMD), while controls had either no or few small drusen (measuring < 63 microns in diameter).[10] In one of the first targeted genetic association studies implicating susceptibility loci in CFH, Edwards et al. analyzed 400 AMD cases, consisting of 47% high-risk early AMD, defined as sufficient drusen in the macula to fill a circle 700 microns in

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diameter or drusen with more advanced features such as retinal pigment epithelial hyperplasia; 16% drusen-associated AMD complicated by pure GA; and 37% drusen-associated AMD complicated by exudation.[13] In a study reporting significant association of polymorphisms in C2 / CFB and C3 to advanced AMD, Francis et al. used slightly different definitions in each of three separate cohorts of AMD patients. In the first cohort, they defined cases as those manifesting GA or CNV. In a second cohort, containing individuals with a strong family history of AMD, they defined a category 2 consisting of mild to moderate drusen of any size, but 125 microns in minimum diameter) >393,744 square micrometers in area within 1500 microns of the fovea; and a category 4 consisting of GA or CNV. In a third cohort derived from the AREDS, they divided AMD cases into mild, intermediate, and advanced using the AREDS grading system.[34] Even small differences in AMD ―case‖ definition between studies may affect what associations are found significant.

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Phenotype-Genotype Correlation With establishment of many of the key genetic loci that distinguish those at high risk for AMD from those at low risk, there is great interest in refining how genetic influences impact distinct aspects of the clinical phenotype. The present literature on phenotype-genotype correlation in AMD is nascent, and a dismaying portion of the early data is conflicting. Contradictory results between studies no doubt stem in some cases from real differences in the populations and variables studied, and in other cases reflect disparities in methodology, quality of study design, or statistical power. We expect that continued application of genetic association analysis to data sets of sufficient size, detail, and quality should result in significant expansion and clarification of our knowledge in the near future. Much of the early work in this field seeks to refine the association between variants in key AMD susceptibility genes (predominantly CFH and ARMS2 / HTRA1) and various parameters of the AMD phenotype. It is logical to expect that gene variants predisposing to advanced AMD might be associated with other phenotypic indicators of severe disease, such as age at presentation, bilaterality of disease, worse visual acuity, or progression of disease. Andreoli et al. found that high-risk variants in ARMS2 / HTRA1 are associated with a younger age at presentation.[35] Shuler et al. reported that

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individuals homozygous for high-risk alleles at both CFH rs1061170 / Y402H and ARMS2 rs10490924 / A69S were significantly younger than those without homozygosity at both loci, [36] a finding later corroborated by Leveziel et al.[37] Chen et al. reported that the high-risk allele for HTRA1 rs11200638 was more frequently associated with bilateral than unilateral advanced AMD (GA or CNV).[38] Pai et al. analyzed a cohort followed longitudinally in the Blue Mountains Eye Study and found that those homozygous for the CFH Y402H high-risk allele (also called the C allele) were significantly more likely to manifest bilateral than unilateral soft drusen, but did not find an association between the homozygous high-risk genotype and bilaterality of GA or CNV.[39] Leveziel et al. prospectively evaluated a subset of 264 individuals drawn from a cohort of 1,216 AMD patients, including those homozygous for the low- or high-risk alleles for CFH Y402H and ARMS2 A69S (excluding heterozygotes at each locus to simplify analysis and avoid bias of a codominant effect). They found that those homozygous for high-risk alleles at both loci manifested a higher incidence of bilateral CNV and disciform scarring as well as lower visual acuity than those homozygous for low-risk alleles at both loci. Those homozygous for the high-risk allele at ARMS2 A69S only (homozygous for the low-risk allele at CFH Y402H) had lower visual acuity than those homozygous for the high-risk allele at CFH Y402H only (homozygous for the low-risk allele at ARMS2 A69S).[37] Seddon et al. studied a subset of the AREDS cohort followed longitudinally for progression from early or intermediate AMD to advanced AMD (GA, CNV, or AMD causing vision loss) and showed that the CFH Y402H and ARMS2 A69S highrisk alleles were each independently associated with such progression.[40] Multiple groups have published on the association between CFH variants and macular drusen in populations of European descent. Francis et al. studied various CFH polymorphisms and haplotypes in three independent populations with AMD, including a group of extended families each with multiple affected members, a group of sporadic cases of advanced AMD, and a subset of patients from the AREDS. Among other findings, they reported a progressively higher frequency of the CFH Y402H high-risk allele with increasing grade of AMD. Those manifesting intermediate AMD (large drusen and/or extensive medium drusen) showed a significantly higher frequency of the high-risk allele than those with early AMD (small and non-extensive medium drusen), and those with advanced AMD (foveal GA or CNV) exhibited a significantly higher frequency of the high-risk allele than those with intermediate AMD.[41] Magnusson et al. evaluated both an Icelandic and an American population of AMD patients and found a significant association

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between the CFH Y402H high-risk allele and soft drusen.[42] Droz et al. evaluated a Swiss population of 425 AMD patients and reported a significant association between the CFH Y402H high-risk allele and macular drusen, though they did not find any significant correlation to drusen area or pigmentary changes.[43] Munch et al. found in a Danish population-based study of 1007 individuals that small, hard drusen (even 20 or more per eye) were not associated with polymorphisms in CFH (Y402H variant), ARMS2 (A69S variant), HTRA1 (rs11200638), or CFB (rs641153 and rs4151667), but they did find an association between homozygosity for the CFH Y402H highrisk allele and macular drusen greater than 63 microns in diameter (medium and large drusen).[44] The weight of evidence suggests that there is an association between CFH variants and macular drusen, but there are studies that have found no such relationship.[45] Cuticular drusen, also known as basal laminar drusen, exhibit distinct clinical features and a different natural history that distinguish them from the typical drusen of AMD. Presenting as innumerable, small, round, uniform concretions external to the neurosensory retina, often arranged in clusters and better visualized by fluorescein angiography and auto-fluorescence photography than on clinical evaluation or by color fundus photography, basal laminar drusen can predispose to eventual development of typical large drusen, serous yellow exudative macular detachment, and occasionally choroidal neovascularization.[46, 47] Most recent evidence suggests that they are topographically, compositionally, and ultrastructurally similar to typical drusen of aging and AMD, and the reasons for their unique clinical characteristics are unknown.[48] A study by Grassi et al. reported significant association between the CFH Y402H high-risk allele and cuticular drusen. Interestingly, the high-risk allele was present in a significantly higher proportion of those with cuticular drusen than those with ―typical‖ AMD.[49] Boon et al. examined a small group of individuals with basal laminar drusen and found a lower frequency of the CFH Y402H high-risk allele (48%) than that reported by Grassi et al. (70%).[50] Several groups have noted an interesting association between CFH variants and peripheral retinal features including extra-macular drusen and reticular pigmentary changes. Shuler et al. studied a group of 956 AMD cases for association of the CFH Y402H high-risk allele with any of 34 different phenotypic parameters. Peripheral reticular pigmentary changes were significantly associated with the high-risk allele. Peripheral drusen were not, but the findings of other studies differ in this regard.[45] Droz et al. found significant association with peripheral drusen.[43] Seddon et al. found

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association between CFH variants Y402H and rs1410996 and both peripheral reticular pigment and peripheral drusen, adjusting for AMD grade. They did not find any such associations with variants in ARMS2, CFB, C2, or C3.[51] Munch et al. reported an association between the CFH Y402H high-risk allele and peripheral drusen, and did not find any such association to ARMS2 or CFB variants.[44] Such findings have spurred new interest in the peripheral retinal features of disease, which—though well-described in AMD—have not been included in traditional clinical grading schema. With good evidence to suggest that variants in CFH and ARMS2 / HTRA1 predispose to advanced AMD, a host of studies has examined whether these key polymorphisms predispose to CNV, GA, or show a relationship with more specific phenotypic features of severe disease. The balance of evidence suggests that CFH variants may predispose indiscriminately to GA or CNV.[36, 40, 42, 52] Whether ARMS2 variants preferentially predispose to CNV remains uncertain. Seddon et al. evaluated progression from early/intermediate AMD to advanced disease in a subset of the AREDS. As mentioned earlier, they found that CFH Y402H and ARMS2 A69S high-risk alleles were independently associated with progression to advanced AMD. They also found that the effect of the ARMS2 high-risk allele was stronger for neovascular disease (odds ratio 6.1; 95% CI, 3.3 – 11.2) than for GA (odds ratio 3.0, 95% CI, 1.4 – 6.5) relative to no progression for those homozygous for the high-risk alleles, albeit with overlapping confidence intervals. They found no such preferential effect for CFH Y402H.[40] Shuler et al. evaluated 775 AMD cases and found a significant association between the ARMS2 A69S high-risk allele and CNV (and related features such as subretinal hemorrhage and pigment epithelial detachment), but found no such effect for the CFH Y402H variant.[36] In a subsequent study they tested for association of the ARMS2 A69S high-risk allele with 16 distinct phenotypic parameters and concluded similarly that this variant represented an independent risk factor for development of CNV.[53] Maller et al., however, tested whether the ARMS2 A69S variant was associated with CNV versus GA in a large population of 1,238 individuals with advanced AMD, and could not find any statistically significant signal.[18] A few polymorphisms have been tested for association with specific features of neovascular AMD, such as CNV composition by fluorescein angiography (predominantly classic, minimally classic, or occult with no classic CNV) and lesion size, and findings have been somewhat inconsistent. Brantley et al. reported association between the CFH Y402H high-risk allele and predominantly classic CNV.[54] They were not able to replicate this

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finding in a subsequent study of 86 patients receiving bevacizumab for CNV, but found in this latter study that the CFH Y402H low-risk allele and the ARMS2 A69H high-risk allele were both associated with larger CNV lesions.[55] Wegsheider et al. and Goverdhan et al. reported association between the CFH Y402H high-risk allele and classic CNV, [56, 57] while Andreoli et al. and Seitsonen et al. did not.[35, 58] While other groups have not replicated the finding by Brantley et al. that the CFH Y402H variant is associated with larger CNV, [58, 59] there is some evidence to corroborate a possible relationship between the ARMS2 A69S high-risk allele and greater lesion size.[35, 60] Retinal angiomatous proliferation (RAP), a form of exudative AMD in which neovascularization resides at least partly within the neurosensory retina, sometimes with anastomosis between retinal and choroidal vessels, has been studied by a few groups for association to variants in CFH and ARMS2. Though, like other forms of advanced AMD, it appears to be associated with variants in both genes when compared to controls, [61] there has not been any consistent evidence to link this subtype of neovascular AMD to a particular genotype.[56, 62] Variants in a few other genes have been studied for association to CNV. Francis et al. evaluated three separate populations totaling 3,137 individuals and found that variants in C2 / CFB and C3 were independently associated with progression from early/intermediate to advanced AMD, but without preferential evolution to CNV versus GA.[34] Fang et al. tested 515 patients with CNV and 253 controls for association to variants in the vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor 2 (VEGFR2) genes using a haplotype-tagging SNP approach to cover the coding sequences plus two kilobases of noncoding sequence on either side of both genes. They did not find a significant relationship to any of the tested SNPs.[63] Immonen et al. studied a smaller population with and without CNV and found no association between three tested VEGF SNPs and CNV, lesion composition, or lesion size.[64] While variants in CHF, ARMS2, C2 / CFB, and C3 have all been associated with progression to advanced AMD, none of these has been found to predispose to GA instead of CNV. Yang et al. reported that a variant in tolllike receptor 3 (TLR3) was associated with GA and not CNV, [28] but this finding was not replicated in a subsequent report by Edwards et al and Cho et al.[29, 30] Scholl et al. looked at whether variants in CFH, C3, or ARMS2 might exert an effect on growth rate of GA, but did not find any such evidence in a cohort of 99 individuals with bilateral GA followed longitudinally with auto-fluorescence imaging.[65] Klein et al. evaluated for association between

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variants in CFH, ARMS2, C2, C3, APOE, and TLR3 and growth rate or progression of GA among 114 individuals from the AREDS followed longitudinally with color fundus photography. The only nominally significant association was between growth rate of GA and the ARMS2 A69S high-risk variant, though this was not corroborated by association with other measures of progression, such as evolution from extrafoveal GA to foveal GA, or from unilateral GA to bilateral GA.[66] Polypoidal choroidal vasculopathy (PCV) is a distinct clinical entity involving abnormal dilations of choroidal blood vessels predisposing to serous and hemorrhagic pigment epithelial detachment, subretinal exudation and hemorrhage, and CNV, with a higher incidence in Asian populations. It shares certain features with neovascular AMD, but controversy persists about how closely the two are related. Discovery of gene variants imparting susceptibility to AMD has afforded the opportunity to explore whether PCV shares the same genetic vulnerabilities. Kondo et al. reported significant association of two variants in ARMS2 / HTRA1 (rs10490924 / A69S and rs11200638) with both PCV and typical AMD compared to controls in a Japanese population.[67] They subsequently discovered association between 7 of 12 tested CFH variants and PCV.[68] Gotoh et al. found similar frequency of the CFH Y402H and the HTRA1 rs11200638 SNPs in a Japanese population of 116 individuals with typical neovascular AMD and 204 individuals with PCV.[60] Lee et al. found association between PCV and variants in CFH (rs3753394 and rs800292) and ARMS2 / HTRA1 (rs10490924 / A69S and rs11200638) in a Chinese population of 72 individuals with PCV and 93 normal controls. However, they did not find association to the CFH Y402H variant or to polymorphisms tested in C2 / CFB.[69] Finally, Lima et al. recently found that variants in CFH (rs547154 and rs1061170 / Y402H), ARMS2 (rs10490924 / A69S), and CFB / C2 (rs1410996) associated with advanced AMD were also significantly associated with PCV in a population of 368 individuals with advanced AMD, 55 patients with PCV, and 368 controls.[70] The evidence to date suggests that AMD and PCV share similar genetic susceptibilities at multiple loci, though the key variants and allele frequencies may differ between Asian populations manifesting a greater incidence of PCV and European populations with predominantly typical neovascular AMD. There is great interest in elucidating how genotype influences treatment of AMD. Some of the early work explores possible associations between various gene variants and response to photodynamic therapy (PDT), seldom used now as monotherapy of typical exudative AMD since publication of pivotal trials proving the efficacy of the anti-VEGF agent, ranibizumab.[71, 72] Chowers et

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al. found no association between the CFH Y402H, ARMS2 A69S, and HTRA1 rs11200638 variants and response to PDT in an Israeli population with neovascular AMD.[59, 73] Brantley et al. found similarly that there was no association between the ARMS2 A69S variant and response to PDT, but they did find a significantly worse final visual acuity among individuals homozygous for the CFH Y402H low-risk allele.[74] Feng et al., on the other hand, found no association between the CFH Y402H variant and final visual acuity among 273 AMD cases treated with PDT, but tested nine tagging SNPs in the C-reactive protein (CRP) gene and found a significant association at two sites, rs2808635 and rs876538.[75] Immonen et al. found an intriguing association between the VEGF rs699947 and rs2146323 variants and effects of PDT, in the absence of any relationship between the tested variants and size or composition of CNV.[64] Tsuchihashi et al. found an association between certain CFH and ARMS2 / HTRA1 variants and treatment response to PDT in 110 Japanese individuals with exudative AMD, but did not adjust analysis for angiographic CNV subtype, confounding whether these variants were associated with disease phenotype or with response to treatment.[76] Others have started to explore how genotype may affect response to intravitreal injection of anti-VEGF agents such as ranibizumab and bevacizumab. Brantley et al. reported that in a population of 86 patients treated for neovascular AMD with serial intravitreal bevacizumab injection until quiescence, those homozygous for the CFH Y402H high-risk allele had significantly worse final visual acuity, adjusting for age, pre-treatment visual acuity, and lesion size. They found no association between treatment response and ARMS2 A69S genotype.[55] Lee et al. reported that among a population of 156 patients with neovascular AMD followed for at least nine months on serial ranibizumab treatment, those homozygous for the CFH Y402H high-risk allele required an average of one additional injection, but this finding only reached statistical significance in a recurrent event analysis.[77] Subsets of the AREDS cohort have been evaluated for pharmacogenetic effects between antioxidant / zinc supplementation and select gene variants. Klein et al. genotyped 876 AREDS participants with at least intermediate AMD at baseline for CFH Y402H and ARMS2 A69S. They found a significant interaction between the CFH Y402H variant and treatment. Among those homozygous for the CFH Y402H low-risk allele, 34% of those taking placebo progressed to advanced AMD, whereas only 11% of those taking antioxidants plus zinc progressed (reduction of 68%); among those homozygous for the high-risk allele, 44% of those taking placebo progressed, versus 39% of those taking antioxidants plus zinc (reduction of only 11%)

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(p=0.03). That is, those homozygous for the CFH Y402H high-risk allele were significantly less likely to benefit from AREDS antioxidant plus zinc supplementation. A similarly significant treatment interaction was noted between CFH Y402H genotype and treatment with zinc versus no zinc. No significant interactions were seen for the ARMS2 A69S variant.[78] Seddon et al. tested for treatment interaction between these same two gene variants and antioxidant and zinc supplementation in a larger AREDS cohort of 1,466 individuals and found no effect.[79] However, this study included patients with early AMD (AREDS category 2), for whom AREDS supplements did not show benefit in lowering risk of AMD progression, and this may have diminished the statistical power to detect the association reported by Klein et al. for AREDS patients with greater burden of disease at baseline (AREDS categories 3 and 4). In a separate report, Seddon et al. evaluated an AREDS subset of 1,446 individuals with early and intermediate AMD (AREDS categories 2 and 3) at baseline for progression to unilateral or bilateral advanced AMD. They found a statistically significant pharmacogenetic interaction which, like the findings of Klein et al., suggested that individuals homozygous for the CFH Y402H high-risk allele benefit less from AREDS antioxidant plus zinc supplementation than those heterozygous or homozygous for the low-risk allele. In their predictive model for incidence of advanced AMD, which combined demographic features, ocular phenotype data, information about environmental risk factors, and genotype (six variants characterized among five genes), the treatment interaction only increased the C statistic in their model by less than 1%, and had a similarly minor effect on sensitivity and specificity.[80] Finally, Francis et al. reported significant associations between variants in C2 / CFB, and C3 and advanced AMD in three separate cohorts, but did not find interaction with treatment in an AREDS subset of 876 individuals.[34]

The Future The field of genetic epidemiology is growing rapidly. In a recent review article on the HapMap and genome-wide association studies, Manolio and Collins indicate that between 2005 and 2008, over 150 risk loci were identified in over 60 common diseases and traits.[81] Genotyping platforms have become so readily available that it can be argued that the chief concern is no longer how to fund genetic association research, but instead how to ensure quality among the many reports submitted for publication.[33]

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Targeted and genome-wide association studies have already provided a good starting point in the search for disease-associated variants and in the ultimate quest for so-called ―causal‖ variants responsible for aspects of AMD pathogenesis. But recent fine-mapping studies around CFH have been instructive about the challenges and limits of association analysis as a means of identifying causal variants.[23] In 2005 multiple groups reported on the association between AMD and the CFH rs1061170 Y402H missense variant.[10, 11, 13] Substitution of the nucleotide T for the risk-imparting C at position 1,277 in exon 9 of the CFH gene results in a change at amino acid position 402 in the protein, with tyrosine in the low-risk polypeptide replaced by histidine in the high-risk variant. Functional consequence of this variant was suggested by the position of this amino acid substitution, which occurs in a region of CFH that binds heparin and C-reactive protein (CRP), and by speculation that a histidine-for-tyrosine substitution might alter the binding properties of the region.[82] Subsequent studies have reported multiple CFH variants more highly-associated with AMD than CFH Y402H, however, and many of these do not seem to alter the CFH protein, suggesting that the function of these polymorphisms may be regulatory.[31] In any case, fine mapping studies of the CFH region have revealed a situation more complex than originally thought, and the causal variants remain elusive. Comprehensive characterization of variation at loci of interest will clearly be important for understanding the association signals detected in preliminary studies. The role of rare genetic variants (those with minor allele frequencies of