Related Macular Degeneration and Cutis Laxa

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Genetic studies of age-related macular degeneration

Baas, D.C.

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cover.indd 1 31-10-12 08:36 Genetic Studies of Age-related Macular Degeneration

Dominique C. Baas

Chapter 0.indd 1 23-10-12 19:24 The research described in this thesis was conducted at the Netherlands Institute for Neuroscience (NIN), an institute of the Royal Netherlands Academy of Arts and Sciences, Department of Clinical and Molecular Ophthalmogenetics, Amsterdam, The Netherlands. Publication of this thesis was financially supported by:

Academisch Medisch Centrum Amsterdam (AMC) Landelijke Stichting voor Blinden en Slechtzienden (LSBS) Nederlands Instituut voor Neurowetenschappen (NIN) Rotterdamse Stichting Blindenbelangen Stichting Blindenhulp Stichting Blinden-Penning Coverphoto Photo inside : beech tree bark with an eye design (www.shutterstock.com) Book design and layout: : birch tree bark with an eye design (www.shutterstock.com) Print: Wilma Witkamp Buijten & Schipperheijn ISBN: 978-90-9027159-0

Chapter 0.indd 2 23-10-12 19:24 Genetic Studies of Age-related Macular Degeneration

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op dinsdag 18 december 2012, te 12:00 uur

door

Dominique Chantal Baas

geboren te Naarden

Chapter 0.indd 3 23-10-12 19:24 PROMOTIECOMMISSIE:

Promotor: Co-promotor: Prof. dr. A.A.B. Bergen Overige leden: Dr. T.G.M.F. Gorgels

Prof. dr. F. Baas Prof. dr. C.B. Hoyng Prof. dr. M. Kamermans Prof. dr. C.C.W. Klaver Prof. dr. R.O. Schlingemann Pr of. dr. A.H. Zwinderman

Faculteit der Geneeskunde

Chapter 0.indd 4 23-10-12 19:24 Table of contents

⎢ General Introduction 7

Chapter 1 ⎢ The ERCC6 Gene and Age-related 27 Macular Degeneration Chapter 2 ⎢ Multicenter Cohort Association Study of 55 SLC2A1 Single Nucleotide Polymorphisms Chapter 3 and Age-related Macular Degeneration

⎢ The Complement Component 5 Gene and 97 Age-related Macular Degeneration Chapter 4 ⎢ The Serping1 Gene and Age-related 123 Macular Degeneration (Letter) Chapter 5 ⎢ Geographic Atrophy in Age-related 135 Macular Degeneration and TLR3 (Letter) Chapter 6 ⎢ The Dynamic Nature of Bruch’s Membrane 143

Chapter 7 ⎢ Reduced Secretion of Fibulin 5 in 189 Age-related Macular Degeneration and Chapter 8 Cutis Laxa

⎢ General Discussion 207

Chapter 9 ⎢ Summary 235

⎢ Nederlandse Samenvatting 241

⎢ List of Publications 247

⎢ Dankwoord 251

⎢ Curriculum Vitae 257

⎢ Color Figures 261

Chapter 0.indd 5 23-10-12 19:24 Chapter 0.indd 6 23-10-12 19:24 CHAPTER 1 General Introduction

Chapter 1.indd 7 23-10-12 19:25 Chapter 1 The Retina

The retina is the light sensitive neural tissue lining the inner surface of the eye. It is

as thin as 0.4 mm and finds its embryologic origin in the developing brain. Therefore it may be considered a part of the central nervous system. The retina can be divided in the neural retina, adjacent to the vitreous, and the retinal pigment epithelium Figure 1 (RPE), which is attached to the vascular layer (the choroid) by Bruch’s membrane

(BM)( ). The neural retina is composed of multiple layers and contains many sensitive cells in the retina. They transform light into electrical and neurochemical different cell types including the photoreceptor cells. Photoreceptors are the light-

signals that are transmitted through the optic nerve to the brain. The two main types of photoreceptors are cones and rods. Cones function in photopic conditions (bright light) and provide high spatial resolution and mediate color vision. Rods enable vision in scotopic conditions (dim light) and are used for contrast, brightness and motion perception. The retinal pigment epithelium and Bruch’s membrane

The RPE is a polarized monolayer of pigmented neuroepithelial cells, attached side (Figure 1 to the photoreceptors on the apical side, and to the BM and choroid on the basal ). RPE cells are post-mitotic cells, hexagonally shaped, densely packed and joined by tight junctions. The RPE constitutes the outer blood-retinal barrier and regulates the transport of ions, nutrients and waste products between retinal photoreceptors and choroid. The RPE performs several crucial functions in preservation of sight, such as phagocytosis and degradation of photoreceptor outer segments (POS), recycling of visual pigments, light absorption and regulation of the ion balance in the subretinal (between the neural retina and RPE) space [1]. BM is a five-layered sheath of connective tissue consisting of 1) the basement membrane of the RPE; 2) the inner collagenous layer; 3) the elastin layer; 4) the outer collagenous layer; 5) the basement membrane of the choriocapillaris. Together, the RPE and BM play an essential role as a barrier between the subretinal space and the choroid and in the selective transport of molecules between the neural retina and choroidal circulation [2]. The macula

Figure 1). The yellow The macula is an oval, yellow-colored area of approximately 5 mm in diameter located in the center of the posterior pole of the human eye ( appearance is due to a high concentration of carotenoids in this area. The macula is 8

Chapter 1.indd 8 23-10-12 19:25 G�� I��

concentration of cones. the most important part of the eye for high-resolution daylight vision due to the high The central area of the macular region is represented by the fovea centralis (1.85 mm in diameter), which has a central pit, the foveola (diameter 0.35 mm). Outside the fovea is the parafovea (radius 0.5 mm), and the perifovea (radius 1.5 mm). The highest density of cone photoreceptor cells is found in the 1 fovea. The relative highest density of rod photoreceptor cells in the retina is present in a circular perifoveal ring [3,4].

Figure 1. Normal Macular Anatomy (left) and Normal Macular Fundus Appearance (right).

Reprinted with permission from The Macular Degeneration Research, a program of the American Health Assistance Foundation © 2012. http://www.ahaf.org/macular. A color version of this figure can be found in the color figures section. A�e-related Macular De�eneration

Age-related macular degeneration (AMD) is a late-onset, degenerative and progressive disorder of the macula with a multifactorial etiology [5]. It is the leading cause of severe visual impairment in the elderly in the Western world. As the elderly population is rising in number, AMD is becoming an important public health issue [6]. In the early stages of the disease, AMD causes no or hardly any visual impairment; functions such as driving a car, reading and face recognition are lost. Individuals who however, in late or end stages it leads to irreversible loss of central vision. Important

have advanced stages of AMD experience a 60% reduction in the quality of life [7]. Persons suffering from low vision are prone to depression and social isolation [8,9].

Chapter 1.indd 9 23-10-12 19:25 Chapter 1 AMD classification

A universal and uniform description of the disease phenotype is essential in order to study the genetics of a complex disease, such as AMD. The diagnosis of AMD can be made by visual inspection of the retina (fundoscopy or fundus photography). An

international and uniform fundus grading system for AMD was established in 1995 by Bird and coworkers (The International Classification and Grading System for AMD)[10]. Since that time, other definitions, classifications and modifications have been proposed for grading AMD. The most commonly used classification is the one et al proposed by the Age-Related Eye Disease Study (AREDS) [11], which is comparable to the grading system described by Bird ., but uses slightly different criteria for

classification of early AMD and geographic atrophy (GA). Throughout this thesis, Figure 2 only studies using (a modification of) The International Classification and Grading System for AMD ( ) or a similar protocol, like AREDS, were considered. Yellow extracellular deposits between the basal membrane of the RPE and the inner collagenous layer of BM, called drusen, are the first hallmark of AMD [12-14]. Clinically, two main drusen types can be distinguished: hard and soft. Hard drusen are small deposits characterized by a light yellow color, round shape and distinct/ well defined borders. Soft drusen may vary in size and shape, but are usually flatter and larger than hard drusen. They are characterized by pale yellow color and et al indistinct/ill-defined borders. Hard drusen sometimes develop into soft drusen. A subtype of soft drusen is reticular drusen. Klein . defined reticular drusen as ill-defined networks of broad, interlacing ribbons [15]. They have been shown to be associated with a high risk of progression to late stages of AMD. distinct without The early stage of AMD (early AMD) is characterized by the presence of either soft

drusen with pigmentary irregularities or soft indistinct drusen (Figure 2; stages 2 and 3 pigmentary irregularities and soft indistinct drusen with pigmentary irregularities ). At this stage, blurred vision and abnormal dark adaptation may occur, but the visual loss is relatively mild. Later stages of the disease (late AMD) involve two forms: the dry form (geographic atrophy; GA), and the wet (exudative) form, choroidal neovascularization (CNV). Combinations of both Figure 2; stage 4 forms also exists (mixed AMD). GA is characterized by progressive atrophy of the RPE, choriocapillaris, and photoreceptors ( ) and patients start to lose central vision and experience visual scotomas (localized areas of decreased or missing vision) in or near the center of the visual field. GA is the most common end Figure 2; stage 4). stage and accounts for 80-90% of AMD cases. CNV is characterized by abnormal growth of choroidal blood vessels through BM into the retina ( These new blood vessels, that grow in between the RPE and BM (sub-RPE space)

Chapter 1.indd 10 23-10-12 19:25 G�� I��

or into the subretinal space, are very fragile and tend to leak or bleed. This leads to accumulation of fluid and exudates or even hemorrhage in the sub-RPE and subretinal spaces. Vessel growth into the subretinal space causes damage to the photoreceptors and eventually results in detachment of the neural retina or the RPE. Consequently, vision abnormalities or loss of vision ensues [16]. Although CNV 1 accounts for less than 15% of late AMD prevalence, it is responsible for more than 80%Stage of cases of severe visionDefinition loss or legal blindness resulting from AMD [3]. 0 - - No signs of AMD 1 Hard drusen (<63 µm) only - - Soft distinct drusen (≤63 µm) only 2 Pigmentary abnormalities only, no soft drusen (≤63 µm) - - Soft indistinct drusen (≤125 µm) or reticular drusen only 3 Soft distinct drusen (≤63 µm) with pigmentary abnormalities Soft indistinct drusen (≤125 µm) or reticular drusen 4 with pigmentary abnormalities Geographic atrophy or choroidal neovacularization

Figure 2. Fundus Photographs Illustrating The Signs of AMD Graded According to a Modification of The International Classification and Grading System for AMD. (A B C D E F). Stage 4 (choroidal

). Stage 0; ( ). Stage 1; ( ). Stage 2; ( ). Stage 3; ( ). Stage 4 (geographic atrophy); ( et al neovascularization). Definitions of the stages are given in the text. Fundus photographs reprinted with permission from Redmer van Leeuwen . [17]. A color version of this figure can be found in the color figures section.

Chapter 1.indd 11 23-10-12 19:25 Chapter 1 Prevalence of AMD

Globally, AMD may currently affect as many as 50 million people. Several population- based studies like the Beaver Dam Eye Study (BDES), The Blue Mountains Eye Study (BMES) and The Rotterdam Study [18-20] described a higher population prevalence of early AMD (7.2%-15.6%) in comparison with late AMD (1.6%-1.9%). A sharp increase can be seen after 75 years of age in the prevalence of late AMD, with percentages in age groups being: 0.2% (55-64 yrs), 0.9% (65-74 yrs), 4.6% (75-84 yrs) and 13.1% (>85 yrs) [21]. Given the overall aging of the population, it is estimated that by the year 2020 at least 80 million people will be affected by AMD [22]. However, the overall AMD prevalence is different across various ethnic populations. For example, the prevalence of AMD is relatively high in the Western (Caucasian) populations compared with Asian populations. Nonetheless, AMD is becoming a public health issue worldwide due to senescence, life-style factors and the quick shift in demographics [23,24]. Risk factors

AMD has a multifactorial etiology. Both environmental and genetic risk factors contribute to AMD pathogenesis [25]. Environmental risk factors

Multiple environmental factors have been described as potential risk factors for AMD, among which age and smoking are the most consistent [26]. Aging

age affects retinal health is unclear. It may relate to the accumulation of free radical Aging is the most important risk factor for AMD [21,27]. The mechanism by which

related oxidative damage in the RPE and BM over the years. Indeed, oxidative damage may be the main driving force limiting longevity according to the free radical theory of aging [28,29]. Smoking

Numerous case-control, population-based, and prospective studies robustly point at tobacco smoking as a strong risk factor for AMD susceptibility. Tobacco smoking increases the risk for AMD from 1.7 to 3.2-fold in ever smokers, and from 1.9 to 4.5- fold in current smokers [30-32]. The number of cigarettes smoked is directly linked with the risk of developing advanced AMD [32]. Indeed, cigarette smoking has

Chapter 1.indd 12 23-10-12 19:25 General Introduction

numerous pathobiologically relevant effects: it reduces the plasma concentration 1 of antioxidants and the choroidal blood flow, and increases platelet adhesiveness, hypoxia, and the release of free radicals [33].

Other, less consistent AMD risk factors include: Dietary antioxidants

The harmful effects of oxidative stress in the retina may be reduced by dietary antioxidants. High dietary intake of zinc, the carotenoids, lutein and zeaxanthin, and of vitamins C and E, is associated with a reduced risk of AMD [34-36]. Vitamin C is found in the retina and protects the eye from light damage [37]. Vitamin E is found in the membranes of POS and is a powerful antioxidant that helps to protect cell membranes against damage by free radicals [38]. Lutein and zeaxanthin (found mostly in green leafy vegetables) are the two major components of the macular pigment of the retina. These carotenoids also hold antioxidant properties and may decrease the risk of AMD by absorption of short-wavelength, potentially phototoxic,

light [39,40]. Female sex

Female gender may be an AMD risk factor in persons aged over 75 years, with a relative risk for CNV possibly twice as high in women compared to age-matched men [21]. However, this issue is controversial, since regression models adjusting for age as a continuous variable, rather than by decade, showed no statistically significant effect of gender on the risk of AMD [41,42]. Ethnicity

Several studies have shown that AMD occurs more frequently in Caucasians than in persons of other ethnic origins. AREDS, the National Health and Nutrition Examination Survey III (NHANES-III), the Baltimore Eye Study (BES) and a population-based study in Barbados found that rates of early AMD in black people et al were similar to those found in white people. In contrast, the rates of CNV were lower in black populations [27,43]. Klein . recently described AMD prevalence in 4 ethnic groups aged 45-85 years [44]. The prevalence in African-Americans was the lowest (2.4%) whereas the prevalence in Hispanics (4.6%), Chinese (4.6%) and Caucasians (5.4%) was higher.

Chapter 1.indd 13 23-10-12 19:25 Chapter 1 Cardiovascular factors

Cardiovascular disease factors may also play a role in the development of AMD. However, so far, results of different studies have been inconsistent. Hypertension was shown to be associated with AMD in some studies [45-49], while other studies found no association [50,51]. The same holds for body mass index and the presence et al of cardiovascular disease (stroke, myocardial infarction or angina). The association between high cholesterol levels and AMD is also not clear: Tomany . described in et al 2004 that elevated total serum cholesterol levels were associated with a higher risk of GA, but not CNV [52], while Tan . described in 2007 that an increased total/ high density lipoprotein (HDL) ratio predicted both CNV and GA [50]. Increased serum levels of HDL cholesterol may lower the risk of late AMD [47,52]. Some studies suggest a direct association between elevated serum and diet cholesterol concentrations and AMD [52,53]. Sunlight

Finally, sunlight exposure was suggested as a risk factor in the development of AMD [52]. Whether or not sunlight exposure is involved in AMD is not clear. The BDES implied that persons exposed to sun more than 5 hours a day (during their whole life) had a higher 10-year incidence of early AMD than those exposed less than two hours [54]. So far, other studies could not confirm this [45]. Genetic predisposition

In addition to environmental factors, genetic factors greatly influence the susceptibility to AMD. Genetic predisposition for AMD was first demonstrated already 30 years ago: Twin studies showed a clearly greater concordance of AMD among monozygotic twins than dizygotic twins [55]. Estimates of heritability ranged from 46% to 71%, the highest rates being for advanced AMD [56]. Familial aggregation studies also provided evidence that genetic factors may play a key role in AMD pathogenesis by showing a higher disease risk among the first-degree relatives of AMD probands [57]. Over the last years, enormous progress has been made in the dissection of the genetic susceptibility for AMD. Progress has primarily come from the outcome of genetic association studies, in combination with data from gene expression and functional studies. Association studies are based on a comparison of unrelated affected and unaffected individuals from a population. Apart from the (disease) trait(s) under investigation, these populations must be highly similar to avoid misinterpretations. Genetic association studies test whether a particular allele or mutation is present at

Chapter 1.indd 14 23-10-12 19:25 General Introduction

a higher frequency among affected as compared to unaffected individuals [58,59]. one (APOE, CFH ARMS2 HTRA1, LIPC and TIMP3). 1 Below, we describe the most important genetic susceptibility genes in AMD one by and other complement factors, / APOE APOE As early as 1998, Klaver and coworkers discovered the first susceptibility gene for AMD: apolipoprotein E ( ) [60]. is central to the metabolism of low density lipoprotein (LDL) cholesterol and triglycerides, and has been associated with increased risk of a number of age-related disorders [61]. The gene for APOE is et al APOE located on chromosome 19q13.2, displaying three major allelic variants (ε2, ε3, and ε4). Klaver . showed a statistically significant protective effect of the ε4 allele against AMD, whereas the ε2 allele may be associated with a slightly increased risk of AMD [60]. In 2005, a major susceptibility gene for AMD on chromosome 1q32 [62-64] was CFH) gene and independently found in three different cohorts: The finding of the association between the Y402H polymorphism in the complement factor H ( AMD was a major breakthrough in ophthalmic research. The complement system CFH gene, four other genes of the is an important component of the innate immunity and plays amongst others a role CFB), I (CFI C2 C3)) were found in chronic low-level inflammation. Besides the complement system (complement factors B ( ), 2 ( ), 3 ( to be consistently associated with AMD. This indicates that the immune system plays a major role in the etiology of AMD [65-68]. Together, these associations account for a large part of the genetic contribution to AMD susceptibility. Another major susceptibility locus for AMD has been mapped to the chromosome 10q26 region by several genome-wide linkage studies [69-71]. This locus contains ARMS2), two genes. As yet it is not clear which one of these (or both) genes is involved in AMD. LOC387715 HTRA1) The genes are: age-related maculopathy susceptibility 2 ( also known as et al ARMS2 gene was [72,73] and the high-temperature requirement factor A1 gene ( [74-76]. Rivera . concluded that the A69S SNP in exon1 of the the most likely susceptibility allele of AMD [73]. The ARMS2 protein was previously immunolocalized to the mitochondrial outer membrane in mammalian cells and to ARMS2 the cytosol [77-79]. Until now, the functional characterization of this gene has been HTRA1 gene encodes a incomplete, but variation in the gene may increase vulnerability for oxidative damage, possibly in association with smoking [72]. The heat shock serine protease which regulates the degradation of extracellular matrix (ECM) proteoglycans. Proteolytic cleavage of proteoglycans by HTRA1 enables HTRA1 access of additional degradative matrix enzymes, such as collagenases and matrix metalloproteinases, to their substrates [80]. is expressed in retina and RPE and was also found in drusen by immunohistochemical staining [74].

Chapter 1.indd 15 23-10-12 19:25 Chapter 1 ARMS2 HTRA1 In summary, although progress has been made, the precise involvement of and/or in AMD remains to be elucidated. LIPC In 2010, a genome-wide association study (GWAS) showed an association between LIPC is involved in triglyceride late AMD cases and a variant in the hepatic lipase gene ( ) [81]. This finding LIPC was replicated in eight additional cohorts [81,82]. hydrolysis and it affects serum HDL cholesterol levels [83]. The T allele of the SNP rs493258 consistently increases HDL serum levels and decreases AMD risk across all study populations [81,82]. TIMP3 At the same time, Chen and co-workers identified yet another AMD susceptible locus , a 100 kb upstream of the metalloproteinase inhibitor 3 gene ( ). The protein encoded by this gene is part of a family of inhibitors of matrix metalloproteinases in TIMP3 group of peptidases involved in degradation of the ECM [82]. Interestingly, mutations were previously also found in patients with Sorsby fundus dystrophy, an ABCA1, ABCA4, ABCR, early onset retinal disorder strongly resembling AMD [84]. ACE, C5, CETP, CFHR1-5, CST3, CX3CR1, ELOVL4, ERCC6, FBLN5, HMCN1, LPL, LRP6, Yet other candidate genes previously implicated in AMD are: PON1, SERPING1, SOD1-2, SYN3, TLR3-4, VEGF and VLDLR

[85-87]. The individual contribution of these genes as a risk factor for AMD appears to be heterogeneous or non-consistent across populations and relatively minor. Aim and approach of this thesis

Altogether, the (major) AMD susceptibility genes described above as well as a number of environmental risk factors suggest an important role for lipid metabolism, inflammatory and oxidative stress pathways, and a role for the ECM in the etiology associated with genetic variants in CFH and ARMS2/HTRA1 of AMD. Notwithstanding the recent discovery of the major genetic risks factors , a substantial part of the heritability of AMD still awaits clarification. The aim of this thesis was to further enlighten the genetic background of AMD based on these formerly implicated molecular pathways. In order to do so, we selected candidate genes based on their previously reported association with AMD and involvement in these AMD molecular pathways (oxidative stress, inflammation and ECM). In addition, we based our selection on the outcomes of our SNP genotyping assays that were done with 1.536 SNPs chosen from AMD gene expression studies carried out by our research group.

Chapter 1.indd 16 23-10-12 19:25 General Introduction Microarray analysis and SNP genotyping assays

in vivo in vitro 1 A part of our candidate gene identification was based on gene expression analyses in vivo studies, macular RPE cells from healthy human (22k microarray, Agilent technologies) of [88] and RPE cells (Baas, unpublished data). For our donor eyes (aged 63-78 years) were isolated by laser dissection microscopy and used for microarray studies. Gene expression was analyzed and functional annotation et al was performed in order to gain insight into the functional properties of the human in vitro RPE, as described by Booij . [88]. For our studies, human ARPE-19 cells were cultured for 84 days with and without the addition of bovine photoreceptor outer segments (POS) to the culture medium. Gene expression patterns were determined on several time points: 56 days (T0; start POS addition), 70 days (T1; 2 weeks POS addition) and 84 days (T2; 1 month POS) post-plating. Next, we performed functional annotation analysis in et al order to gain insight into the functional properties of the human ARPE-19 cell line (Baas .; unpublished results). The above described microarray experiments showed amongst others a role for oxidative stress, ECM and inflammation pathways. Based on these results we selected a total of 45 AMD candidate genes and screened all known common genetic variants of these genes in the Amsterdam-Rotterdam-Netherlands (AMRO- NL) study population. All subjects were Caucasian and recruited from the Erasmus University Medical Centre, Rotterdam and the Netherlands Institute of Neuroscience, Amsterdam, the Netherlands. The analysis was carried out with the Illumina reaction. GoldenGate SNP genotyping assay, capable of multiplexing 1.536 SNPs in a single

Study populations for testing genetic association In this thesis, the results of the genetic association studies found in the initial

discovery cohorts were replicated in a study population from the University of Southampton, Southampton, United Kingdom and/or (a subset of) populations of the International Age-related Macular Degeneration Genetics Consortium. This consortium includes eight international cohorts (Columbia University, New York, USA; University of Iowa, Iowa City, USA; AREDS cohort, NEI/NIH, USA; Rotterdam Study, Erasmus University, Rotterdam, The Netherlands; The AMRO-NL study, The Netherlands Institute for Neurosciences (NIN), Amsterdam, The Netherlands; University Eye Clinic, Reykjavik, Iceland; University of Würzburg, Würzburg, Germany; and the Center for Eye Research Australia, University of Melbourne,

Chapter 1.indd 17 23-10-12 19:25 Chapter 1

Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Australia). All cohorts contained patients and controls of European descent. Outline of this thesis

In this thesis, we describe seven studies related to three pathobiological AMD mechanisms (oxidative stress, immunological aspects and ECM turnover): Part 1. Aspects of oxidative stress

Oxidative stress was previously implicated in AMD. Indeed, the retina, and especially the RPE is exposed to high levels of oxidative stress. In this respect, a recent ERCC6 manuscript of Tuo and co-workers (2006) is of interest. They reported that a SNP ERCC6 chapter 2, (rs3793784) in the promoter region of the gene was associated with AMD. is involved in repair of oxidative DNA damage. In we analyzed ERCC6 the potential association of rs3793784 with AMD in four study populations. In addition, to examine a possible functional relationship, we determined mRNA human donor eyes. In chapter 3, expression levels in healthy and early AMD affected human RPE, isolated from SLC2A1 SLC2A1 regulates the we tested the hypothesis whether genetic variation in the glucose transporter 1 ( ) is associated with AMD. bio-availability of glucose in the RPE, which, as we hypothesized, may influence oxidative stress mediated AMD pathology. Part 2. Aspects of the immune system

Complement activation appears to play a key role in the pathogenesis of AMD. The complement component 5 (C5) protein was previously localized in drusen. C5 plays a role as a chemoattractant regulating local inflammatory processes, and plays a chapter 4, crucial role in the propagation of the complement reaction toward the membrane C5 attack complex. In we describe an extensive association analysis to test Chapter 5 the hypothesis whether complement gene variants (independently) mediate SERPING1) in the AMD susceptibility. portrays the involvement of a variant (rs2511989) SERPING1 in intron 6 of serpin peptidase inhibitor, clade G, member 1 ( development of AMD. As a member of the classical complement pathway, is a plausible candidate gene. Two recent studies provided conflicting data about the association between this gene and AMD. To resolve this issue, we genotyped rs2511989, in seven large case-control studies involving individuals from European TLR3 descent. Yet another gene involved in the innate immune system is toll-like receptor 3 ( ). Also for genetic variants in this gene, conflicting association results have

Chapter 1.indd 18 23-10-12 19:25 General Introduction chapter 6, TLR3 been described. In we, and our collaborators from the International AMD 1 Genetics Consortium describe that the association between and (dry) AMD is non-consistent between populations. Part 3. Aspects of the extracellular matrix

Chapter 7 consists of Bruch’s membrane is a complex extracellular matrix located between the RPE and the endothelial cells of the choriocapillaris. a review of BM. The chapter starts out with an introduction about the normal structure and function of BM and the changes that occur with age. We subsequently describe the formation and composition of drusen and illustrate that abnormalities in the BM play a key (FBLN5) were role in the pathogenesis of AMD. Amongst others, fibulin proteins are widespread FBLN5 components of ECM. Previously, rare missense mutations in fibulin 5 chapter 8) and determined the functional effects of missense mutations on reported in association with AMD. For that reason, we investigated the role of FBLN5 in AMD (

fibulin 5 expression. We also attempted to correlate the genotype with the AMD phenotype. chapter 9

In the final chapter, , we discuss the main findings of the thesis in the light of the current literature, we discuss strengths and weaknesses of the studies performed, and suggest future directions.

Chapter 1.indd 19 23-10-12 19:25 Chapter 1 References

1. Strauss O (2005) The retinal pigment epithelium in visual function. Physiol Rev 85: 845- 881. 2. Odgen TE, Schachat AP, and Ryan SJ (1994) St Louis, Mosby-Year Book, Inc. 2nd ed., 5-94. 3. Jager RD, Mieler WF, Miller JW (2008) Age-related macular degeneration. N Engl J Med 358: 2606-2617. 4. Gass JD (1997) Stereoscopic Atlas of Macular Diseases: Diagnosis and treatment. St Louis, Mosby-Year Book, Inc. 4th ed., 1-17. 5. Hawkins BS, Bird A, Klein R, West SK (1999) Epidemiology of age-related macular degeneration. Mol Vis 5: 26. 6. Congdon N, O’Colmain B, Klaver CC, Klein R, Munoz B, et al. (2004) Causes and prevalence of visual impairment among adults in the United States. Arch Ophthalmol 122: 477-485. 7. Rovner BW, Casten RJ (2002) Activity loss and depression in age-related macular degeneration. Am J Geriatr Psychiatry 10: 305-310. 8. Langelaan M, de Boer MR, van Nispen RM, Wouters B, Moll AC, et al. (2007) Impact of visual impairment on quality of life: a comparison with quality of life in the general population and with other chronic conditions. Ophthalmic Epidemiol 14: 119-126. 9. Mitchell J, Bradley C (2006) Quality of life in age-related macular degeneration: a review of the literature. Health Qual Life Outcomes 4: 97. 10. Bird AC, Bressler NM, Bressler SB, Chisholm IH, Coscas G, et al. (1995) An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group. Surv Ophthalmol 39: 367-374. 11. (2001) The Age-Related Eye Disease Study system for classifying age-related macular degeneration from stereoscopic color fundus photographs: the Age-Related Eye Disease Study Report Number 6. Am J Ophthalmol 132: 668-681. 12. Feeney-Burns L, Hilderbrand ES, Eldridge S (1984) Aging human RPE: morphometric analysis of macular, equatorial, and peripheral cells. Invest Ophthalmol Vis Sci 25: 195-200. 13. Green WR (1999) Histopathology of age-related macular degeneration. Mol Vis 5: 27. 14. Sarks SH (1976) Ageing and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol 60: 324-341. 15. Klein R, Davis MD, Magli YL, Segal P, Klein BE, et al. (1991) The Wisconsin age-related maculopathy grading system. Ophthalmology 98: 1128-1134. 16. Gehrs KM, Anderson DH, Johnson LV, Hageman GS (2006) Age-related macular degeneration- emerging pathogenetic and therapeutic concepts. Ann Med 38: 450-471. 17. van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT (2003) The risk and natural course of age-related maculopathy: follow-up at 6 1/2 years in the Rotterdam study. Arch Ophthalmol 121: 519-526. 18. Vingerling JR, Dielemans I, Hofman A, Grobbee DE, Hijmering M, et al. (1995) The prevalence of age-related maculopathy in the Rotterdam Study. Ophthalmology 102: 205-210.

Chapter 1.indd 20 23-10-12 19:25 General Introduction

19. Mitchell P, Smith W, Attebo K, Wang JJ (1995) Prevalence of age-related maculopathy in Australia. The Blue Mountains Eye Study. Ophthalmology 102: 1450-1460. 1 20. Klein R, Klein BE, Linton KL (1992) Prevalence of age-related maculopathy. The Beaver Dam Eye Study. Ophthalmology 99: 933-943. 21. Smith W, Assink J, Klein R, Mitchell P, Klaver CC, et al. (2001) Risk factors for age-related macular degeneration: Pooled findings from three continents. Ophthalmology 108: 697- 704. 22. Clemons TE, Milton RC, Klein R, Seddon JM, Ferris FL, III (2005) Risk factors for the incidence of Advanced Age-Related Macular Degeneration in the Age-Related Eye Disease Study (AREDS) AREDS report no. 19. Ophthalmology 112: 533-539. 23. Gupta SK, Murthy GV, Morrison N, Price GM, Dherani M, et al. (2007) Prevalence of early and late age-related macular degeneration in a rural population in northern India: the INDEYE feasibility study. Invest Ophthalmol Vis Sci 48: 1007-1011. 24. Krishnaiah S, Das T, Nirmalan PK, Nutheti R, Shamanna BR, et al. (2005) Risk factors for age- related macular degeneration: findings from the Andhra Pradesh eye disease study in South India. Invest Ophthalmol Vis Sci 46: 4442-4449. 25. de Jong PT (2006) Age-related macular degeneration. N Engl J Med 355: 1474-1485. 26. Thornton J, Edwards R, Mitchell P, Harrison RA, Buchan I, et al. (2005) Smoking and age- related macular degeneration: a review of association. Eye (Lond) 19: 935-944. 27. Coleman HR, Chan CC, Ferris FL, III, Chew EY (2008) Age-related macular degeneration. Lancet 372: 1835-1845. 28. Harman D (1956) Aging: a theory based on free radical and radiation chemistry. J Gerontol 11: 298-300. 29. Stadtman ER (2001) Protein oxidation in aging and age-related diseases. Ann N Y Acad Sci 928: 22-38. 30. Smith W, Mitchell P, Leeder SR (1996) Smoking and age-related maculopathy. The Blue Mountains Eye Study. Arch Ophthalmol 114: 1518-1523. 31. Seddon JM, George S, Rosner B (2006) Cigarette smoking, fish consumption, omega-3 fatty acid intake, and associations with age-related macular degeneration: the US Twin Study of Age-Related Macular Degeneration. Arch Ophthalmol 124: 995-1001. 32. Delcourt C, Diaz JL, Ponton-Sanchez A, Papoz L (1998) Smoking and age-related macular degeneration. The POLA Study. Pathologies Oculaires Liees a l’Age. Arch Ophthalmol 116: 1031-1035. 33. Christen WG, Glynn RJ, Manson JE, Ajani UA, Buring JE (1996) A prospective study of cigarette smoking and risk of age-related macular degeneration in men. JAMA 276: 1147- 1151. 34. (2001) A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch Ophthalmol 119: 1417-1436.

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35. Ho L, van Leeuwen R, Witteman JC, van Duijn CM, Uitterlinden AG, et al. (2011) Reducing the genetic risk of age-related macular degeneration with dietary antioxidants, zinc, and omega-3 fatty acids: the Rotterdam study. Arch Ophthalmol 129: 758-766. 36. van Leeuwen R, Boekhoorn S, Vingerling JR, Witteman JC, Klaver CC, et al. (2005) Dietary intake of antioxidants and risk of age-related macular degeneration. JAMA 294: 3101-3107. 37. Blanks JC, Pickford MS, Organisciak DT (1992) Ascorbate treatment prevents accumulation of phagosomes in RPE in light damage. Invest Ophthalmol Vis Sci 33:2814-2821. 38. Packer L, Landvik S (1990) Vitamin E in biological systems. Adv Exp Med Biol 264: 93-103. 39. Krinsky NI, Deneke SM (1982) Interaction of oxygen and oxy-radicals with carotenoids. J Natl Cancer Inst 69: 205-210. 40. Packer L (1993) Antioxidant action of carotenoids in vitro and in vivo and protection against oxidation of human low-density lipoproteins. Ann N Y Acad Sci 691: 48-60. 41. Mitchell P, Wang JJ, Foran S, Smith W (2002) Five-year incidence of age-related maculopathy lesions: the Blue Mountains Eye Study. Ophthalmology 109: 1092-1097. 42. Wang JJ, Rochtchina E, Lee AJ, Chia EM, Smith W, et al. (2007) Ten-year incidence and progression of age-related maculopathy: the blue Mountains Eye Study. Ophthalmology 114: 92-98. 43. Schachat AP, Hyman L, Leske MC, Connell AM, Wu SY (1995) Features of age-related macular degeneration in a black population. The Barbados Eye Study Group. Arch Ophthalmol 113: 44. 728-735. Klein R, Klein BE, Knudtson MD, Wong TY, Cotch MF, et al. (2006) Prevalence of age-related macular degeneration in 4 racial/ethnic groups in the multi-ethnic study of atherosclerosis. Ophthalmology 113: 373-380. 45. (2000) Risk factors associated with age-related macular degeneration. A case-control study in the age-related eye disease study: Age-Related Eye Disease Study Report Number 3. Ophthalmology 107: 2224-2232. 46. Hogg RE, Woodside JV, Gilchrist SE, Graydon R, Fletcher AE, et al. (2008) Cardiovascular disease and hypertension are strong risk factors for choroidal neovascularization. Ophthalmology 115: 1046-1052. 47. Hyman L, Schachat AP, He Q, Leske MC (2000) Hypertension, cardiovascular disease, and age-related macular degeneration. Age-Related Macular Degeneration Risk Factors Study Group. Arch Ophthalmol 118: 351-358. 48. Klein R, Klein BE, Tomany SC, Cruickshanks KJ (2003) The association of cardiovascular Study. disease with the long-term incidence of age-related maculopathy: the Beaver Dam Eye 49. Ophthalmology 110: 1273-1280. van Leeuwen R, Ikram MK, Vingerling JR, Witteman JC, Hofman A, et al. (2003) Blood pressure, atherosclerosis, and the incidence of age-related maculopathy: the Rotterdam Study. Invest Ophthalmol Vis Sci 44: 3771-3777. 50. Tan JS, Mitchell P, Smith W, Wang JJ (2007) Cardiovascular risk factors and the long- term incidence of age-related macular degeneration: the Blue Mountains Eye Study. Ophthalmology 114: 1143-1150.

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51. Klein R, Klein BE, Franke T (1993) The relationship of cardiovascular disease and its risk factors to age-related maculopathy. The Beaver Dam Eye Study. Ophthalmology 100: 406- 1 414. 52. Tomany SC, Wang JJ, van LR, Klein R, Mitchell P, et al. (2004) Risk factors for incident age- related macular degeneration: pooled findings from 3 continents. Ophthalmology 111: 1280-1287. 53. Mares-Perlman JA, Brady WE, Klein R, VandenLangenberg GM, Klein BE, et al. (1995) Dietary fat and age-related maculopathy. Arch Ophthalmol 113: 743-748. 54. Klein R, Klein BE, Jensen SC, Cruickshanks KJ (2001) Sunlight and the 5-year incidence of early age-related maculopathy: the Beaver Dam Eye Study. Arch. Ophthalmol 119:246-250. 55. Hammond CJ, Webster AR, Snieder H, Bird AC, Gilbert CE, et al. (2002) Genetic influence on early age-related maculopathy: A twin study. Ophthalmology 109: 730-736. 56. Seddon JM, Cote J, Page WF, Aggen SH, Neale MC (2005) The US twin study of age-related macular degeneration: relative roles of genetic and environmental influences. Arch Ophthalmol 123: 321-327. 57. Seddon JM, Ajani UA, Mitchell BD (1997) Familial aggregation of age-related maculopathy. Am J Ophthalmol 123: 199-206. 58. Shennan SL (1997) Evolving methods in genetic epidemiology. IV Approaches to non- mendelian inheritance. Epidemiol Rev 19: 44-51. 59. Khoury MJ, Beaty TH, Cohen BH (1993) Fundamentals of genetic epidemiology. Oxford 1993. 60. Klaver CC, Kliffen M, van Duijn CM, Hofman A, Cruts M, et al. (1998) Genetic association of apolipoprotein E with age-related macular degeneration. Am J Hum Genet 63: 200-206. 61. Ang LS, Cruz RP, Hendel A, Granville DJ (2008) Apolipoprotein E, an important player in longevity and age-related diseases. Exp Gerontol 43: 615-622. 62. Edwards AO, Ritter R, III, Abel KJ, Manning A, Panhuysen C, et al. (2005) Complement factor H polymorphism and age-related macular degeneration. Science 308: 421-424. 63. Haines JL, Hauser MA, Schmidt S, Scott WK, Olson LM, et al. (2005) Complement factor H variant increases the risk of age-related macular degeneration. Science 308: 419-421. 64. Klein RJ, Zeiss C, Chew EY, Tsai JY, Sackler RS, et al. (2005) Complement factor H polymorphism in age-related macular degeneration. Science 308: 385-389. 65. Fagerness JA, Maller JB, Neale BM, Reynolds RC, Daly MJ, et al. (2009) Variation near complement factor I is associated with risk of advanced AMD. Eur J Hum Genet 17: 100-104. 66. Gold B, Merriam JE, Zernant J, Hancox LS, Taiber AJ, et al. (2006) Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet 38: 458-462. 67. Maller JB, Fagerness JA, Reynolds RC, Neale BM, Daly MJ, et al. (2007) Variation in complement factor 3 is associated with risk of age-related macular degeneration. Nat Genet 39: 1200-1201. 68. Yates JR, Sepp T, Matharu BK, Khan JC, Thurlby DA, et al. (2007) Complement C3 variant and the risk of age-related macular degeneration. N Engl J Med 357: 553-561.

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69. Weeks DE, Conley YP, Mah TS, Paul TO, Morse L, et al. (2000) A full genome scan for age- related maculopathy. Hum Mol Genet 9: 1329-1349. 70. Seddon JM, Santangelo SL, Book K, Chong S, Cote J (2003) A genomewide scan for age-related macular degeneration provides evidence for linkage to several chromosomal regions. Am J Hum Genet 73: 780-790. 71. Majewski J, Schultz DW, Weleber RG, Schain MB, Edwards AO, et al. (2003) Age-related macular degeneration--a genome scan in extended families. Am J Hum Genet 73: 540-550. 72. Schmidt S, Hauser MA, Scott WK, Postel EA, Agarwal A, et al. (2006) Cigarette smoking strongly modifies the association of LOC387715 and age-related macular degeneration. Am J Hum Genet 78: 852-864. 73. Rivera A, Fisher SA, Fritsche LG, Keilhauer CN, Lichtner P, et al. (2005) Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum Mol Genet 14: 3227-3236. 74. Yang Z, Camp NJ, Sun H, Tong Z, Gibbs D, et al. (2006) A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science 314: 992-993. 75. Jakobsdottir J, Conley YP, Weeks DE, Mah TS, Ferrell RE, et al. (2005) Susceptibility genes for age-related maculopathy on chromosome 10q26. Am J Hum Genet 77: 389-407. 76. Dewan A, Liu M, Hartman S, Zhang SS, Liu DT, et al. (2006) HTRA1 promoter polymorphism in wet age-related macular degeneration. Science 314: 989-992. 77. Fritsche LG, Loenhardt T, Janssen A, Fisher SA, Rivera A, et al. (2008) Age-related macular degeneration is associated with an unstable ARMS2 (LOC387715) mRNA. Nat Genet 40: 892-896. 78. Kanda A, Chen W, Othman M, Branham KE, Brooks M, et al. (2007) A variant of mitochondrial protein LOC387715/ARMS2, not HTRA1, is strongly associated with age-related macular degeneration. Proc Natl Acad Sci U S A 104: 16227-16232. 79. Wang G, Spencer KL, Court BL, Olson LM, Scott WK, et al. (2009) Localization of age-related macular degeneration-associated ARMS2 in cytosol, not mitochondria. Invest Ophthalmol Vis Sci 50: 3084-3090. 80. Grau S, Richards PJ, Kerr B, Hughes C, Caterson B, et al. (2006) The role of human HtrA1 in arthritic disease. J Biol Chem 281: 6124-6129. 81. Neale BM, Fagerness J, Reynolds R, Sobrin L, Parker M, et al. (2010) Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC). Proc Natl Acad Sci U S A 107: 7395-7400. 82. Chen W, Stambolian D, Edwards AO, Branham KE, Othman M, et al. (2010) Genetic variants near TIMP3 and high-density lipoprotein-associated loci influence susceptibility to age- related macular degeneration. Proc Natl Acad Sci U S A 107: 7401-7406. 83. Kathiresan S, Willer CJ, Peloso GM, Demissie S, Musunuru K, et al. (2009) Common variants at 30 loci contribute to polygenic dyslipidemia. Nat Genet 41: 56-65. 84. Weber BH, Vogt G, Pruett RC, Stöhr H, Felbor U (1994) Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby’s fundus dystrophy. Nat Genet 8: 352-6.

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85. Katta S, Kaur I, Chakrabarti S (2009) The molecular genetic basis of age-related macular degeneration: an overview. J Genet 88: 425-449. 1 86. Francis PJ, Klein ML (2011) Update on the role of genetics in the onset of age-related macular degeneration. Clin Ophthalmol 5: 1127-1133. 87. Deangelis MM, Silveira AC, Carr EA, Kim IK (2011) Genetics of age-related macular degeneration: current concepts, future directions. Semin Ophthalmol 26: 77-93. 88. Booij JC, van Soest S, Swagemakers SM, Essing AH, Verkerk AJ, et al. (2009) Functional annotation of the human retinal pigment epithelium transcriptome. BMC Genomics 10: 164.

Chapter 1.indd 25 23-10-12 19:25 Chapter 1.indd 26 23-10-12 19:25 CHAPTER 2 The ERCC6 Gene and Age-related Macular Degeneration

Dominique C. Baas, Dominiek D. Despriet, Theo G.M.F. Gorgels, Julie Bergeron- Sawitzke, André G. Uitterlinden, Albert Hofman, Cornelia M. van Duijn, Joanna E. Merriam, R. Theodore Smith, Gaetano R. Barile, Jacoline B. ten Brink, Johannes R. Vingerling, Caroline C.W. Klaver, Rando Allikmets, Michael Dean and Arthur A.B. Bergen

PLoS One 2010; 5(11):e13786

Chapter 2 20120725.indd 27 23-10-12 19:25 Chapter 2 Abstract

Background: Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the developed countries and is caused by both environmental and genetic factors. A recent study (Tuo et al., PNAS) reported an association between AMD and a single nucleotide polymorphism (SNP) (rs3793784) in the ERCC6 (NM_000124) gene. The risk allele also increased ERCC6 expression. ERCC6 is involved in DNA repair and mutations in ERCC6 cause Cockayne syndrome (CS). Amongst others, photosensitivity and pigmentary retinopathy are hallmarks of CS. Methodology/Principle Findings: Separate and combined data from three large AMD case-control studies and a prospective population-based study (The Rotterdam Study) were used to analyse the genetic association between ERCC6 and AMD (2682 AMD cases and 3152 controls). We also measured ERCC6 mRNA levels in retinal pigment epithelium (RPE) cells of healthy and early AMD affected human donor eyes. Rs3793784 conferred a small increase in risk for late AMD in the Dutch population (The Rotterdam and AMRO-NL study), but this was not replicated in two non-European studies (AREDS, Columbia University). In addition, the AMRO- ERCC6. Finally, we determined that ERCC6 expression in the human RPE did not depend on NL study revealed no significant association for 9 other variants spanning rs3793784 genotype, but, interestingly, on AMD status: Early AMD-affected donor eyes had a 50% lower ERCC6 expression than healthy donor eyes (P = 0.018). Conclusions/Significance: Our meta-analysis of four Caucasian cohorts does not replicate the reported association between SNPs in ERCC6 and AMD. Nevertheless, ERCC6 expression in the RPE suggest that ERCC6 may be functionally involved in AMD. Combining our data with those of the literature, we hypothesize our findings on that the AMD-related reduced transcriptional activity of ERCC6 may be caused by diverse, small and heterogeneous genetic and/or environmental determinants.

Chapter 2 20120725.indd 28 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration Introduction

Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the Western world. The prevalence of AMD rises sharply with age, affecting 4% of the population over the age of 60 and more than 10% of individuals older than 75 [1,2]. The early stages of AMD are characterized by drusen, which are focal depositions of waste material underneath the retinal pigment epithelium (RPE). 2 These early stages frequently progress over time into late AMD, which presents itself in two forms: geographic atrophy (dry AMD) and neovascular AMD (wet AMD). As a complex disease, AMD has environmental as well as genetic determinants. Environmental risk factors include age, smoking, hypertension and diet. Estimates of the genetic hereditability range up to 71% [3,4]. The strongest genetic associations with AMD were found with variants in the complement factor H (CFH) gene [5-8] and with SNPs in a chromosomal region (10q26) containing the HTRA1 and ARMS2 genes [9,10]. CFH is the main inhibitor of the alternative pathway of the complement system, and thus plays an important role in the control of the innate immune system. Recently, associations with AMD were also found for other genes of the complement cascade (complement factor B, C2, C3) [11-13], but, interestingly enough, not for C5, although it should be noted that only relatively common SNPs in C5 were screened [14]. Nonetheless, the accumulated evidence in the literature suggests a key role of the alternative complement pathway in AMD. Whether genetic variants in the HTRA1 or ARMS2 genes on 10q26, or both, are truly involved in AMD remains to be elucidated [15,16]. In terms of function, both genes could play a role in AMD: HTRA1 is a serine protease [9] which may affect the turnover of the ECM (i.e. Bruch’s membrane), while ARMS2 has been implicated in mitochondrial function and oxidative stress [17]. Indeed, the retina, and especially the RPE is exposed to high levels of oxidative stress [18]. The combination of high oxygen consumption, intense light exposure and the presence of photosensitizers such as lipofuscin, may lead locally to excessive oxidative damage, for example of the DNA. In this respect, the recent ERCC6/AMD manuscript of Tuo and coworkers (2006) is of interest. They reported that a single nucleotide polymorphism (SNP; rs3793784), in the promoter region of the ERCC6 DNA repair gene was associated with AMD. The SNP was associated with AMD susceptibility, both independently and through interaction with a SNP (rs380390) in CFH. In addition, they found that the ERCC6 SNP altered the expression level of the gene [19]. The ERCC6 gene is involved in DNA repair, and loss of function mutations in this gene cause Cockayne syndrome (CS) [20,21]. CS is an autosomal recessive progeroid

Chapter 2 20120725.indd 29 23-10-12 19:25 Chapter 2 disorder characterized by severely impaired physical and intellectual development. Among the many clinical features, photosensitivity and pigmentary retinopathy are

DNA lesions that are located in actively transcribed DNA and obstruct transcription hallmarks of CS [21]. Cells from CS patients are specifically defective in repair of [22,23]. Substrates for this transcription-coupled repair include UV or oxidative

stress induced DNA lesions [24-26]. DNA repair deficiency of UV-induced DNA lesions DNA lesions may be implicated in other CS symptoms, including the retinopathy, may explain photosensitivity in CS. DNA repair deficiency of oxidative stress induced and premature aging [27,28]. Targeted disruption of Ercc6 in the mouse resulted in a mouse model for CS [29,30], which showed spontaneous retinal degeneration characterized by a gradual photoreceptor loss [29]. Interestingly, the retina of the Ercc6 damage is involved in CS retinal pathology. -/- mouse is hypersensitive to X rays, which confirmed that oxidative DNA In order to further explore the role of oxidative DNA damage in AMD, the potential association of ERCC6 promoter SNP rs3793784: C>G (c.-6530C>G) with AMD was analyzed in the population-based Rotterdam study and, in parallel, in the Dutch AMRO-NL case-control study. Replication was performed in two large non-European AMD case-control (Columbia University and Age-Related Eye Disease Study (AREDS)) studies. We subsequently investigated the potential modifying effect of CFH Y402H, LOC387715 A69S and smoking. Next, nine other ERCC6 (tag) SNPs were genotyped in 375 cases and 199 controls from the AMRO-NL study population. Finally, to examine a possible functional relationship, we determined both rs3793784 dependent and normal ERCC6 mRNA expression levels in healthy and early AMD affected human RPE isolated from human donor eyes.

Results

Analysis of association for ERCC6 promoter polymorphism rs3793784 The potential association of ERCC6 c.-6530C>G (rs3793784) with AMD was analyzed in the population-based Rotterdam Study and, in parallel, in the Dutch AMRO-NL case-control study. In the Rotterdam Study, 427 of 6418 persons were diagnosed with early AMD and 78 persons with late AMD at entry of the study. The mean follow-up time was 7.85 years. In this period, 1039 persons remained without AMD (“controls”), while 509 progressed to early AMD and 93 to late AMD. In the AMRO-NL study, 331 unrelated AMD patients and 170 controls were genotyped for rs3793784. Of those, 84 persons were diagnosed with early AMD and 247 persons with late AMD at entry of the study. Genotype frequencies were in Hardy-Weinberg Equilibrium in both studies. The population frequency of ERCC6 G allele was 0.43.

Chapter 2 20120725.indd 30 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration ERCC6 c.-6530C>G genotype are presented in Appendix 1. Baseline characteristics stratified for the The risk of AMD for the ERCC6 c.6530C>G allele is summarized in Appendix 2. In ERCC6 and late

the Rotterdam Study, we found a significant association between was seen in the prevalent analyses. In contrast, analysis of the AMRO-NL study AMD in the incident analyses. Although not statistically significant, a similar trend ERCC6 2 risk alleles. To determine the risk for the general Dutch population, we combined showed no significantly increased disease OR for persons carrying potential data from the Rotterdam Study and the AMRO-NL study population. This yielded

rs3793784 (OR 1.39, 95%CI 1.02-1.89). Analyzing subtypes of late AMD separately a borderline significant increased risk of late AMD for persons homozygous for Appendix 2. We also analyzed the interaction of ERCC6 c.-6530C>G with three known prominent AMD (dry, wet or mixed) did not yield statistical significant results: risk factors in the largest combined population study available to us (the Dutch Rotterdam and AMRO-NL studies). As assessed by calculation of the synergy index ERCC6 variant and smoking was found

(SI) no significant interaction between the with CFH Y402H (SI 1.56, 95%CI 0.77-3.15) and LOC387715 A69S (SI 3.08, 95%CI (SI 3.33, 95%CI 0.39-28.66). Neither did we find a significant SI for the interaction 0.83-11.50). These results implied that these risk factors did not modify the relation of ERCC6 with AMD. Finally, rs3793784 was also screened in two non-European study populations (Columbia and AREDS). The results from the genotype analysis of these populations are shown in Table 1. Again, all the genotype frequencies followed HWE (data not

and AMD were found. Table 2 shows that combining all data from the four studies shown). However, in both populations, no significant associations between the SNP

did not result in statistically significant association. AMD association analysis of nine other ERCC6 variants We next selected nine other ERCC6 SNPs that span and tag the entire ERCC6 gene. The LD plot and the distinct haplotype blocks for the nine selected SNPs, as generated by Haploview [31], are presented in Figure 1a. The nine variants spanned two different haplotype blocks of the ERCC6 gene. Corresponding LD scores (D’ and R2) for each selected marker of the ERCC6 gene with a MAF>10% are also given (Figure 1b). The potential association of the SNPs with early and late AMD was analyzed in the AMRO- NL cohort. Genotype frequencies for all SNPs followed HWE (data not shown). None of the selected SNPs, including one in full LD with rs3793784 (rs2229760), showed Appendix 3).

a significant association with early or late AMD ( 31

Chapter 2 20120725.indd 31 23-10-12 19:25 Chapter 2 1 1 Late AMD Late N=668 227 (34.0) 0.42 N=365 No. (%) OR (95% CI) 149 (40.8) 169 (46.3) 0.89 (0.65-1.22) 47 (12.9) 0.82 (0.52-1.29) 0.36 1 Mixed AMD Mixed N=178 63 (35.4) 0.41 1 1 GA N=166 56 (33.7) 0.42 N=92 34 (37.0) 0.39 1 1 AREDS Columbia CNV N=324 108 (33.3) 0.42 N=273 No. (%) OR (95% CI) No. (%) OR (95% CI) 115 (42.1) 124 (45.4) 0.85 (0.60-1.19) 45 (48.9) 1.04 (0.63-1.71) 34 (12.5) 0.77 (0.47-1.36) 13 (14.1) 0.99 (0.49-2.03) 0.35 1 c.-6530C>G Genotypes in Two Non-European Study Populations. Study Non-European c.-6530C>G Genotypes in Two ERCC6 Early AMD Early N=253 83 (32.8) 0.4 1 1 0.41 0.36 No AMD (controls) All AMD cases No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) OR (95% CI) N=217 N=921 0.4 N=359No. (%) N=365 No. (%) OR (95% CI) 0.38 Table 1. Risk of Age-related Macular Degeneration for Macular Degeneration 1. Risk of Age-related Table allele. minor common allele, “a” indicates “A” allele MAF= minor frequency. atrophy; geographic GA= neovascularization; choroidal CNV= macular degeneration; AMD= age-related and late early and respectively group as reference group (with the control analysis with logistic regression estimated are ORs because of rounding. 100% not always Percentages variable). AMD as outcome rs3793784 Genotype (AA)Noncarrier 82 (37.8) 310 (33.6) Heterozygous (Aa)Heterozygous 98 (45.2) 462 (50.2) 1.25 (0.89-1.73) 136 (53.8) 1.37 (0.92-2.05) 161 (49.7) 1.25 (0.85-1.83) 80 (48.2) 1.19 (0.76-1.88) 85 (47.8) 1.13 (0.73-1.75) 326 (48.8) 1.20 (0.86-1.69) Homozygous (aa)Homozygous 37 (17.1) 149 (16.2) 1.07 (0.69-1.65) 34 (13.4) 0.91 (0.52-1.58) 55 (17.0) 1.13 (0.68-1.87) 30 (18.1) 1.19 (0.66-2.14) 30 (16.9) 1.05 (0.59-1.89) 115 (17.2) 1.13 (0.72-1.76) MAF(%) rs3793784 Genotype (AA)Noncarrier 135 (37.6) 149 (40.8) Heterozygous (Aa)Heterozygous 172 (47.9) 169 (46.3) 0.89 (0.65-1.22) Homozygous (aa)Homozygous 52 (14.5) 47 (12.9) 0.82 (0.52-1.29) MAF(%)

Chapter 2 20120725.indd 32 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration Table 2. Risk of Age-related Macular Degeneration for ERCC6 c.-6530C>G Genotypes in All Study Populations Combined (Rotterdam, Amsterdam, Columbia University and AREDS).

No AMD (controls) AMD cases

rs3793784 N=3143 N=2679

No. (%) No. (%) OR (95% CI) Genotype 2 Noncarrier (AA) 1057 (33.6) 898 (33.5) 1 Heterozygous (Aa) 1524 (48.5) 1316 (49.1) 0.89 (0.65-1.22) Homozygous (aa) 562 (17.9) 465 (17.4) 0.82 (0.52-1.29) MAF(%) 0.42 0.42 AMD= age-related macular degeneration; MAF= minor allele frequency. “A” indicates common allele, “a” minor allele. Percentages not always 100% because of rounding. ORs are estimated with logistic regression analysis (with the control group as reference group and respectively early and late AMD as outcome variable).

ERCC6 mRNA expression in the human RPE ERCC6 gene expression, with the risk allele (G) ERCC6 SNP in DNA derived SNP rs3793784 reportedly influences from the retina of 23 well characterized healthy old and early AMD affected donor showing higher expression [19]. We first genotyped this eyes. We next determined ERCC6 mRNA expression in laser dissected RPE cells from the same eyes, since this cell type occupies a central position in AMD pathology. Late AMD was not considered for RPE expression studies because of the lack of suitable

genotype group. The results are presented in Figures 2a and 2b. Clearly, in the RPE, cell material. There were no significant differences in age distribution between each the risk allele (G) of rs3793784 did not lead to higher ERCC6 expression levels. A two way ANOVA was used to statistically test the effect of genotype and AMD status on ERCC6 rs3793784 genotype on ERCC6 expression (Figure 2a; P = 0.293). In addition, the expression. The ANOVA did not detect an independent significant effect for combined effect of genotype and AMD status on ERCC6 either (P = 0.531). However, the ANOVA did interaction expression was not significant AMD status on ERCC6 expression, independent of the rs3793784 genotype (Figure demonstrate a significant effect of 2b, P = 0.018). Interestingly, the expression of ERCC6 in healthy RPE was nearly twice as high as in early AMD affected RPE (Figure 2b). The “raw” data of the three housekeeping genes and the ERCC6 gene is presented in Appendix 4.

Chapter 2 20120725.indd 33 23-10-12 19:25 Chapter 2 A

Figure 1. Linkage Disequilibrium (LD) Display in Haploview of SNPs Encompassing the ERCC6 Gene. SNP selection was based on criteria like functional relevance, minor allele frequency (MAF)>10%, coverage of the main linkage disequilibrium (LD) blocks and tagging of the most common haplotypes. Tag SNPs were selected by use of Tagger, an option of Haploview (all SNPs were captured with a LD tagging criteria of r2>0.8). Figure 1 displays the 5 distinct haplotype blocks and all SNPs that were tested in the AMRO-NL study population (A). LD scores (D’ and R2) between markers genotyped. Note D’ above the diagonal and R2 scores below the diagonal (B).

Chapter 2 20120725.indd 34 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration

Figure 2. ERCC6 Expression Levels in Relation to Genotype and Disease Status. Mean ERCC6 expression level in human donor eyes in relation to rs3793784 genotype (C/C, N= 8; C/G, N= 11 and G/G, N= 4) (A). Mean ERCC6 expression level in human donor eyes in relation to “status”= (old) healthy or early AMD (N= 14 donor eyes with early AMD and 9 old healthy donor eyes) (B). Quantitative RT-PCR analysis, normalized to the geometric mean of three housekeeping genes (RPLP0, PPIA, and EEF1a1) [46,47]. Two way ANOVA was used to test the independent effect of status and genotype on the mean expression, as well as the interaction between these variables. Abbreviations: AMD= age-related macular degeneration.

Chapter 2 20120725.indd 35 23-10-12 19:25 Chapter 2 Discussion

We genotyped the ERCC6 promoter SNP rs3793784, which was previously associated with AMD, in the prospective, population based Rotterdam Study and, in parallel, in the Dutch AMRO-NL case control study. In addition, we screened two other large, non-European, study populations (Columbia University, NY and AREDS) of European-American descent. In total, this study consisted of 2682 AMD cases and 3152 ethnically matched controls.

Non-replication of the rs3793784 SNP association with AMD et al. (2006) [19], we

Contrary to the findings of the initial association study by Tuo and AMD in all study populations. failed to show a (consistent) statistically significant association between rs3793784

We only detected a significant risk in the incident analysis of the Rotterdam Study, selective mortality may be involved: persons with two copies of ERCC6 rs3793784 but not in the prevalent analysis. This prompted us to specifically analyze whether have a higher risk of AMD, and if these persons also die earlier, it could explain the discrepancy between prevalent and incident analyses. However, in the Rotterdam Study, the ERCC6 variant was not associated with increased mortality (not shown); in addition, there was no unequal age-related genotype distribution (not shown). Thus, the small difference between the incident and prevalent risks in the population based cohort probably occurred by chance due to relatively small patient sample sizes. In the AMRO-NL case-control study, association between the rs3793784 SNP and AMD was not found. The combined data of the two Dutch populations (the Rotterdam Study and AMRO-NL study) still showed a marginally increased risk of homozygous ERCC6 carriers for late AMD. Since this effect was largely caused by one sub-population only (incident cases and controls from the Rotterdam study), we chose to include two additional study populations. Replication of the association in two non-European study populations clearly failed: In both the Columbia University and AREDS data, no association was present between rs3793784 and AMD.

Tuo et al. [19] previously genotyped the ERCC6 SNP rs3793784 essentially in the Our negative association findings in the AREDS population are of particular interest: same this discrepancy be explained? One possible explanation is the large difference in AREDS cohort and did find a statistically significant association. How can the number of AREDS participants screened: Tuo et al. [19] genotyped 460 cases and 269 controls, whereas in our AREDS screen, 929 cases and 217 controls were genotyped. Further analysis showed that the MAF of rs3793784 in the cases are comparable in both studies (0.42/0.41). However, the MAF of rs3793784 in the

Chapter 2 20120725.indd 36 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration controls of both studies is remarkably different (AREDS Tuo: 0.35 and our AREDS study: 0.40). The Rotterdam Study, AMRO-NL study and the Columbia study yield MAFs of, respectively, 0.43, 0.42 and 0.38 in controls. Thus, we conclude that the

association with AMD, are most likely due to chance. In addition, in contrast with findings of Tuo and coworkers (MAF 0.35 in the controls), and the underlying the data of Tuo et al interaction between CFH Y402H or any of the other two major AMD risk factors: 2 . [19], we also did not find evidence for effect modification or smoking or the LOC387715 A69S SNP. Overall, the possible weak association in a single Dutch sub-population and the negative association in three large replication cohorts, imply that the ERCC6 C-6530>G polymorphism has a minor role, if any, in the pathogenesis of AMD. This conclusion is supported by a recent study by Chen and coworkers, who conducted a genome-wide association scan for AMD in 2157 cases and 1150 controls. Amongst other, they evaluated evidence for involvement of previously associated SNPs and found no association between the ERCC6 SNP rs3793784 and AMD [32]. Finally, it is of course possible that another genetic variant or a variant in linkage disequilibrium (LD) with ERCC6 c.-6530C>G, is a contributor to the potential AMD risk observed. In order to further elucidate the putative role of this gene in AMD we tested nine other common ERCC6 variants in the AMRO-NL study. One of these SNPs was in perfect LD with the previously associated SNP, rs2229760. None of the

strengthen the conclusion described above and suggest that common SNPs in the selected SNPs showed a significant association with early or late AMD. These results ERCC6 gene confer no or little risk for AMD.

ERCC6 expression in the human RPE is related to AMD status, not to rs3793784 genotype Tuo et al ERCC6 expression level: the risk allele (G) resulted . (2006) found that rs3793784, located in the untranslated 5’ flanking in 2-3 times higher ERCC6 expression than the C variant [19]. This is an intriguing region of the gene, influenced

retina more susceptible to AMD. We decided to examine ERCC6 mRNA expression finding since it suggests that higher expression of this DNA repair gene makes the in the human RPE, which occupies a central position in AMD pathology. In this cell

Instead, we found that ERCC6 reduced in type, we could not reproduce the paradoxical finding of Tuo and co-workers [19]. early AMD affected eyes, independent of rs3793784 genotype. RPE expression levels were significantly ERCC6 in the RPE of early AMD affected eyes, suggests that ERCC6 is involved in AMD. It is plausible that this involvement The significantly reduced expression of relates to oxidative stress and aging. Functional studies have implicated oxidative 37

Chapter 2 20120725.indd 37 23-10-12 19:25 Chapter 2 stress in the RPE in the aetiology of AMD [33,34], while epidemiological studies unanimously consider aging as the main risk for AMD [35]. At the same time, ERCC6 plays a role in repair of (oxidative) DNA damage. Loss of function mutations in ERCC6 cause the autosomal recessive disorder Cockayne syndrome, which includes many severe physical and neurologic features, with premature aging and retinopathy as hallmarks of the disease. These observations led to the hypothesis that (oxidative)

AMD [36]. This hypothesis was supported by a recent study, which compared DNA DNA damage and insufficient DNA repair contribute to aging pathology, including repair capacity in leukocytes of AMD patients and controls: Wozniak and co-workers found that AMD patients had higher levels of endogenous oxidative DNA damage and a lower repair capacity of DNA lesions [37]. We found that early AMD affected eyes had an almost 50% reduced expression of ERCC6 in the RPE. In CS patients both ERCC6 alleles carry loss of function mutations [20,21]. To our knowledge, 50% reduction in transcription levels in CS carriers has not been reported to lead to pathology. Yet, it should be noted, that clinical data on CS carriers are scarce and, most likely, they have not been screened for retinal (AMD) pathology. Interestingly, repair of the 8-oxoguanine DNA lesion was investigated in a CS family: It was found that cells from the heterozygous parents had intermediate levels of repair of this oxidative DNA lesion compared to their CS affected and unaffected siblings [38]. It is important in this respect that most retinal cells, including the RPE are post-mitotic cells and that they are exposed to extremely high levels of oxidative stress over the prolonged period of (a life-) time. It is therefore conceivable that in this particular organ, the repair of oxidative DNA damage may be even more demanding than in other tissues. Small but prolonged shortcomings in DNA repair may, eventually, at advanced age become manifest in more damage and cell death than in other tissues. In this way, reduced levels of ERCC6 in the RPE may well contribute to the development of AMD. The cause of the reduced ERCC6 transcription in RPE of early AMD affected eyes is not clear. However, given its implication in the multifactorial disorder AMD, and our inability to associate common ERCC6 SNPs with AMD, we hypothesize that the genetic involvement of ERCC6 in AMD, if any, may reside in (combination of) rare SNPs. Alternatively, the reduction in ERCC6 transcription levels in relation to AMD may also be due to other genetic factors or to environmental insults, such as prolonged smoking.

Chapter 2 20120725.indd 38 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration Conclusion

genetic variation in ERCC6 In summary, our study did not confirm a consistent relation between common between ERCC6 SNP rs3793784 and either smoking, CFH or LOC387715. Using RPE and AMD. We did not find a significant interaction

expression of ERCC6. from human donor eyes, we also could not confirm rs3793784 genotype dependent 2 ERCC6 in human RPE affected

We did find significantly lower expression levels of that ERCC6 may be functionally implicated in the aetiology of AMD. Combining our by early AMD, regardless of the rs3793784 genotype. The latter finding suggests data with those of the literature, we hypothesize that the AMD-related reduced transcriptional activity of ERCC6 expression may be caused by diverse, small and heterogeneous genetic and/or environmental determinants.

Materials and Methods

Ethics statement All studies were approved by the Ethics Committees of the Academic Medical Center Amsterdam, the Erasmus Medical Center Rotterdam, the Institutional Review Board of Columbia University and the Age-Related Eye Disease Study (AREDS) Access Committee. All studies followed the tenets of the Declaration of Helsinki. All participants provided signed informed consent for participation in the study, retrieval of medical records, and use of blood and DNA for AMD research.

Cases and controls Altogether, we utilized one prospective, population-based study and three case- control studies consisting of 2682 AMD cases and 3152 ethnically- and age-matched control subjects.

Study populations Genetic association of the ERCC6 SNP rs3793784 was analyzed in the Rotterdam Study and, in parallel, in the AMRO-NL case-control study population. Next, replication was carried out in two non-European study populations (Columbia University and AREDS). Finally, nine ERCC6 (tag) SNPs were genotyped in the AMRO- NL study population. In The Rotterdam Study all inhabitants of 55 years or older living in a suburb of Rotterdam, the Netherlands, were invited to participate [39,40]. The initial cohort consisted of 10,275 eligible individuals, of whom 7,983 (78%) participated.

Chapter 2 20120725.indd 39 23-10-12 19:25 Chapter 2 The ophthalmologic part of the study consisted of 9,774 eligible individuals, of whom 7,598 (78%) participated. Baseline examinations took place from 1990 to 1993; three follow-up examinations were performed in 1993-1994, 1997-1999,

and 2000-2005. Incident cases were defined as the absence of AMD in both eyes at The AMRO-NL study population consisted of 375 unrelated AMD patients and 199 baseline and its first appearance in at least 1 eye at follow-up. controls. All subjects were Caucasian and recruited from the Netherlands Institute of Neuroscience Amsterdam and Erasmus University Medical Centre Rotterdam (Amsterdam-Rotterdam-NL (AMRO-NL)), through newsletters, via patient organizations, and nursing home visits. Controls were aged 65 years and older, and were unaffected spouses or non-related acquaintances of cases, or individuals who attended the ophthalmology department for reasons other than retinal pathology. Cases and controls from the Columbia University study population, consisted of 368 unrelated individuals with AMD and 368 unrelated controls of European American descent, recruited at Columbia University (New York, NY) as previously described [6]. The AREDS study population was initially conceived as a long-term multicenter,

patients over the age of 55 years who self-reported to participating eye clinics prospective study of the clinical course of AMD and age-related cataract. Briefly, between 1992 and 1996 were considered for AREDS, and eligibility was based on AMD symptoms and retinal photographs. A total of 1,699 patients were selected and

and then randomized into intervention and non-intervention groups. In this sample enrolled in the clinical trial, all were put into one of five AMD severity categories, set, there were 929 AMD cases and 217 patients, free of AMD, acted as controls. The full details and results of the original AREDS study are reported elsewhere [41,42].

Diagnosis of AMD Study subjects from the Rotterdam, AMRO-NL and Columbia University study populations underwent ophthalmic examination and fundus photography covering macula after pupil dilatation at each visit (Topcon TRV- 50VT fundus camera, Topcon Optical Co, Tokyo, Japan). Signs of AMD in those a 35° field centered on the

study populations were graded according to (a modification of) the international classification and grading system for AMD [43]. Cases and controls from the AREDS study were classified as having or not having disease on the basis of the AREDS classification system [44]. Although AREDS uses slightly different criteria for four studies. Controls showed no, classification of early AMD and GA, the grading criteria were very similar for the or less than five small hard drusen, and no other 40

Chapter 2 20120725.indd 40 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration macular pathology. Early AMD cases had either soft distinct drusen with pigmentary irregularities or soft indistinct drusen with or without pigmentary irregularities. Late AMD cases presented with dry AMD, wet AMD, or a combination of both (mixed AMD). Genotyping 2 Standard DNA techniques were applied as described elsewhere [45]. Genotyping of the polymorphisms rs3793784 (ERCC6 c.-6530C>G), rs1061170 (CFH Y402H) and rs10490924 (LOC387715 A69S) were done with the Taqman assay (Applied Biosystems, Foster City, CA). Genotyping of the nine ERCC6 (tag) SNPs was performed using an Illumina GoldenGate assay on a BeadStation 500 GX (Illumina Inc., San Diego,CA, US).

ERCC6 Tag SNP selection for Illumina GoldenGate assay Nine SNPs were selected to span and tag the entire ERCC6 gene. SNP data were used from the Centre d’Étude du Polymorphisme Humain (CEPH) population (Utah residents with ancestry from northern and western Europe) by use of the International HapMAP Project. Available at: http://www.hapmap.org, NCBI build 36, dbSNP b126; Accessed January 7, 2009. SNP selection was based on criteria like functional relevance, minor allele frequency (MAF)>10%, coverage of the main linkage disequilibrium (LD) blocks and tagging of the most common haplotypes. Tag SNPs were selected by use of Tagger, an option of Haploview [31] (all SNPs were captured with a LD tagging criteria of r2>0.8).

Human donor eyes and ERCC6 expression Postmortem eyes Studies on human eye tissue were carried out in accordance with the Declaration of Helsinki on the use of human material for research. Donor eyes were obtained from the Corneabank Amsterdam. The method of selection of eyes for this study was essentially described elsewhere [18]. In summary, medical history of the donors revealed no pre-existing disorders, prolonged medication, or other prolonged

Histological analysis was performed on all donor eyes, e.g. by staining with the agonal states that could possibly influence RPE gene expression or mRNA quality. periodic acid Schiff staining protocol (of sections (10 mm long) of the macula) to identify and quantify drusen. Donor eyes were categorized into (1) “(old) healthy”, if the donor was older than 70 and histology revealed no drusen (on average 0-1 per

Chapter 2 20120725.indd 41 23-10-12 19:25 Chapter 2 10 macular cryo-sections), and (2) “early AMD”, if the donor was older than 70 and histology showed 30 or more drusen per 10 macular cryo-sections with a still intact RPE.

Rs3793784 genotyping Tuo et al. (2005) showed that ERCC6 rs3793784, located in the untranslated 5’ ERCC6 expression level: the G allele resulted in 2-3 times higher ERCC6 expression than the C variant [19]. Therefore, genotyping flanking region of the gene, influenced of this SNP was performed on all above described donor eyes. Genomic DNA was prepared from frozen bulbi by standard extraction protocol using phenol. PCR and direct sequence analysis was carried out as previously described [46] with the use of the ABI-310 automated sequencer (Applied Biosystems).

ERCC6 mRNA expression in the RPE of human donor eyes We measured ERCC6 mRNA expression in the RPE cells with real-time qPCR, in triplicate [47]. In summary, human donor eyes were snap-frozen in isopentane (Sigma-Aldrich, England) and stored at -800 macula were cut and RPE cells were dissected using a PALM laser dissection C. Cryosections of 20 μm from the microscope (P.A.L.M. Micro Laser Technologies AG). Total RNA was isolated with

Template cDNA for the real-time PCR was made by reverse transcription of 200 RNeasy mini (Qiagen) and amplified with the MessageAmp aRNA kit (Ambion). ng aRNA with Superscript III (Invitrogen). Real-time PCR reactions were carried out in a 20 ml volume using qPCR Core Kit Sybr Green I (Eurogentec) and the following primer set: 5’-5’AAATCTGTGCACTTTCCATAGAACTTC-3’ and 5’- Reverse:

TATTCTGGCTTGAGTTTCCAAATTC-3’. The levels of amplified product were detected (Applied Biosystems). Expression levels of ERCC6 were normalized using the geo- by real-time monitoring of SYBR Green I dye fluorescence in the ABI Prism 7300 mean of the expression of internal control genes RPLP0, PPIA, and EEF1a1 [47,48]. Over ten different combinations of primersets were tested to obtain, in our hands and under our experimental conditions, the optimum reference gene set for genes expressed in the RPE (and/or photoreceptors). Two way ANOVA was used to determine the independent effect of (AMD) status and rs3793784 genotype on the

was accepted at a P value of <0.05. mean expression as well as the interaction between these two variables. Significance

Chapter 2 20120725.indd 42 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration Statistical analysis Characteristics of the Dutch participants were compared among those affected and non-affected with analysis of covariance for continuous variables, and with logistic regression analysis for discrete variables adjusting for age and sex. Hardy-Weinberg Equilibrium (HWE) of the ERCC6, CFH and LOC387715 genotype distributions were tested using a Chi2 test. 2 In the Rotterdam Study, odds ratio’s for prevalent AMD were estimated with logistic regression analyses, and relative risks for incident AMD were estimated with Cox proportional hazards analyses. Logistic regression was used in the AMRO-NL and the replication studies of Columbia University and AREDS to calculate odds ratios

Inc) for risk of AMD with the major alleles as reference. For the Rotterdam Study and (ORs) and 95% confidence intervals (CI) in SPSS for windows (release 16.0; SPSS, the AMRO-NL study population, ORs were adjusted for age and sex. Interaction with ERCC6 c.-6530C>G on AMD was determined for smoking, CFH Y402H, and LOC387715 A69S contrasting late AMD with no AMD. Analyses were initially performed on separate data sets of the Rotterdam Study (prevalent AMD, incident AMD), the AMRO-NL case-control study (in parallel), and subsequently conducted on combined data from the Netherlands (of population-based and case-

for biological interaction was determined by calculating the synergy index (SI), control groups) and data of all population studies combined. Statistical significance which measures deviation from additivity of two risk factors [49,50]. Two way ANOVA analysis was performed in SPSS for windows (release 16.0; SPSS, Inc).

Acknowledgements

This work was in part supported by an unrestricted AMD research grant from Merck & Co, Inc, NJ, USA and grants from the Nederlandse Vereniging ter Voorkoming van Blindheid, The Dutch Macula Foundation, the LSBS, the Oogfonds Nederland, de Rotterdamse Vereniging Blindenbelangen (to A.A.B.) The generation and management of GWAS genotype data for the Rotterdam Study is

(nr. 175.010.2005.011, 911-03-012). This study is funded by the Research Institute supported by the Netherlands Organization of Scientific Research NWO Investments for Diseases in the Elderly (014-93-015; RIDE2), the Netherlands Genomics Initiative

060-810, and funding from the Erasmus Medical Center and Erasmus University, (NGI)/Netherlands Organization for Scientific Research (NWO) project nr. 050- Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry

Chapter 2 20120725.indd 43 23-10-12 19:25 Chapter 2 of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. The Columbia University study population is supported in part by the grants from the National Eye Institute EY13435 and EY017404; the Macula Vision Research Foundation; Kaplen Foundation; Wigdeon Point Charitable Foundation and an unrestricted grant to the Department of Ophthalmology, Columbia University, from Research to Prevent Blindness, Inc.

Chapter 2 20120725.indd 44 23-10-12 19:25 ERCC6

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repair and polymorphism of the hOGG1 gene in lymphocytes of AMD patients. J Biomed 37. Wozniak K, Szaflik JP, Zaras M, Sklodowska A, Janik-Papis K et al. (2009) DNA damage/ Biotechnol 2009: 827562.

in Cockayne syndrome group B cells. Nucleic Acids Res 27: 1365-1368. 38. Dianov G, Bischoff C, Sunesen M, Bohr VA (1999) Repair of 8-oxoguanine in DNA is deficient 39. Hofman A, Grobbee DE, de Jong PT, van den Ouweland FA (1991) Determinants of disease and disability in the elderly: the Rotterdam Elderly Study. Eur J Epidemiol 7: 403-422. 40. Hofman A, Breteler MM, van Duijn CM, Krestin GP, Pols HA et al. (2007) The Rotterdam Study: objectives and design update. Eur J Epidemiol 22: 819-829. 41. (2001) A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E and beta carotene for age-related cataract and vision loss: AREDS report no. 9. Arch Ophthalmol 119: 1439-1452. 42. Bergeron-Sawitzke J, Gold B, Olsh A, Schlotterbeck S, Lemon K et al. (2009) Multilocus analysis of age-related macular degeneration. Eur J Hum Genet 17: 1190-1199. 43. Bird AC, Bressler NM, Bressler SB, Chisholm IH, Coscas G et al. (1995) An international

strategy for ABCC6 in pseudoxanthoma elasticum. Genet Test 8: 292-300. 46. Hu X, Plomp A, Gorgels T, Brink JT, Loves W et al. (2004) Efficient molecular diagnostic 47. van Soest SS, de Wit GM, Essing AH, ten Brink JB, Kamphuis W et al. (2007) Comparison of human retinal pigment epithelium gene expression in macula and periphery highlights potential topographic differences in Bruch’s membrane. Mol Vis 13: 1608-1617.

Chapter 2 20120725.indd 47 23-10-12 19:25 Chapter 2 48. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034 49. Despriet DD, Klaver CC, Witteman JC, Bergen AA, Kardys I et al. (2006) Complement factor H polymorphism, complement activators, and risk of age-related macular degeneration. JAMA 296: 301-309.

Epidemiology 3: 452-456. 50. Hosmer DW, Lemeshow S (1992) Confidence interval estimation of interaction.

Chapter 2 20120725.indd 48 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration Supplementary appendix Appendix 1. Baseline Characteristics of the Study Populations. a. The Rotterdam Study ERCC6 c.-6530C>G ERCC6 c.-6530C>G ERCC6 c.-6530C>G Noncarrier (N=1872) Heterozygous (N=2790) Homozygous (N=1062) Age, mean (sd), y At baseline 68.87 (8.60) 68.63 (8.66) 68.92 (8.70) 2 At diagnose 73.05 (8.08) 72.83 (8.22) 73.10 (8.14) Women, No (%) 1071 (58.6) 1620 (58.1) 638 (60.1) Smoking status, No/Total (%) Never 617/1793 (34.4) 921/2757 (33.4) 377/1048 (36.0) Past 751/1793 (41.9) 1173/2757 (42.5) 459/1048 (43.8) Current 425/1793 (23.7) 663/2757 (24.0) 212/1048 (20.2) CFH Y402H Noncarrier 750/1789 (41.9) 1130/2727 (41.4) 416/1036 (40.2) Heterozygous 802/1789 (44.8) 1215/2727 (44.6) 473/1036 (45.7) Homozygous 237/1789 (13.2) 382/2727 (14.0) 147/1036 (14.2) LOC387715 A69S Noncarrier 1147/1805 (63.5) 1733/2771 (62.5) 666/1058 (62.9) Heterozygous 584/1805 (32.4) 928/2771 (33.5) 368/1058 (34.8) Homozygous 74/1805 (4.1) 110/2771 (4.0) 24/1058 (2.3)* * P<.05 compared to ERCC6 c.-6530 C>G noncarrier

b. The AMRO-NL study population ERCC6 c.-6530C>G ERCC6 c.-6530C>G ERCC6 c.-6530C>G Noncarrier (N=157) Heterozygous (N=257) Homozygous (N=87) Age, mean (sd), y 76.22 (7.17) 77.26 (7.20) 76.71 (8.79) Women, No (%) 90 (57.3) 150 (48.4) 44 (50.6) Smoking status, No/Total (%) Never 52/131 (39.7) 68/219 (31.1) 26/79 (32.9) Past 64/131 (48.9) 105/219 (47.9) 45/79 (57.0) Current 15/131 (11.5) 46/219 (21.0)* 8/79 (10.1) CFH Y402H, No/Total (%) Noncarrier 37/150 (24.7) 65/241 (27.0) 21/81 (25.9) Heterozygous 74/150 (49.3) 127/241 (52.7) 39/81 (48.1) Homozygous 39/150 (26.0) 49/241 (20.3) 21/81 (25.9) LOC387715 A69S, No/Total (%) Noncarrier 72/151 (47.7) 122/250 (48.8) 41/86 (47.7) Heterozygous 59/151 (39.1) 91/250 (36.4) 35/86 (40.7) Homozygous 20/151 (13.2) 37/250 (14.8) 10/86 (11.6) * P<.05 compared to ERCC6 c.-6530 C>G noncarrier

Chapter 2 20120725.indd 49 23-10-12 23:05 Chapter 2 a a a a OR OR HR OR 1 1 1 1 N (%) N (%) N (%) N (%) 3 (18.8) 6 (35.3) 10 (31.3) 20 (27.0) MIX (n=17) 0.59 MIX (n=74) 0,44 0.39 0.47 MIX (n=16) MIX (n=32) a a a a OR OR HR OR 1 1 1 1 N (%) N (%) N (%) N (%) 8 (20.0) 6 (20.7) 48 (28.7) 61 (26.6) CNV (n=29) 0.53 CNV (n=229) 0.53 0.46 0.47 CNV (n=40) CNV (n=167) a a a a OR HR OR 1 1 1 1 N(%) OR N (%) N (%) N (%) 10 (27.0) Separate subtype analysis for the late AMD cases the late for subtype analysis Separate 21 (56.8) 1.55 (0.73-3.29) 22 (55.0) 1.93 (0.86-4.33) 7 (43.8) 1.74 (0.45-6.72) GA (n=32) GA 10 (31.3) 15 (31.3) 35 (30.4) 6 (16.2) 1.12 (0.41-3.09) 10 (25.0) 2.36 (0.93-6.00) 6 (37.5) 3.67 (0.92-14.69) 0.45 Separate subtype analysis for the late AMD cases the late for subtype analysis Separate 25 (21.7) 1.27 (0.74-2.17) 49 (21.4) 1.43 (0.96-2.13) 16 (21.6) 1.45 (0.73-2.86) GA (n=115) GA 0.48 0.45 0.46 Separate subtype analysis for the late AMD cases the late for subtype analysis Separate AMD cases the late for subtype analysis Separate GA (n=37) GA GA (n=48) GA Genotypes. a a a a 1 OR OR HR OR 1.70(1.02-2.82) 1 1 1 1.92 (1.05-3.49) 1.39 (1.02-1.89) 21 (22.6) N (%) N (%) N (%) N (%) 22 (28.2) 73 (29.6) 116 (27.8) Late AMD (n=78) Late 0.51 Late AMD (n=418) Late 0.49 0.45 0.47 Late AMD (n=93) Late Late AMD (n=247) Late c.-6530C>G ERCC6 a a a a 1 1 1 1 OR OR HR OR 162 (31.8) 257 (50.5) 1.12 (0.92-1.37) 50 (53.8) 138 (32.3) 31 (36.9) 319 (33.0) 90 (17.7) 0.99 (0.77-1.29) 22 (23.7) N (%) 204 (47.8) 0.99 (0.78-1.26) 35 (44.9) 1.01 (0.57-1.77) 13 (40.6) 0.82 (0.35-1.91) 15 (51.7) 1.62 (0.62-4.22) 7 (41.2) 0.65 (0.20-2.08) 38 (45.2) 0.60 (0.32-1.09) 127 (51.4) 0.90 (0.56-1.44) 23 (47.9) 0.83 (0.36-1.90) 85 (50.9) 0.87 (0.52-1.45) 19 (59.4) 0.80 (0.32-2.03) 470 (48.6) 0.99 (0.84-1.17) 212 (50.7) 1.22 (0.95-1.57) 55 (47.8) 1.08 (0.69-1.68) 119 (52.0) 1.30 (0.94-1.80) 38 (51.4) 1.27 (0.73-2.22) 0.43 85 (19.9) 1.12 (0.83-1.50) 21 (26.9) 1.71 (0.90-3.25) 9 (28.1) 1.51 (0.60-3.84) 8 (27.6) 2.31 (0.79-6.78) 4 (23.5)15 (17.9) 1.14 (0.30-4.37) 0.93 (0.42-2.09) 47 (19.0) 1.37 (0.72-2.59) 10 (20.8) 1.33 (0.46-3.88) 34 (20.4) 1.47 (0.75-2.88) 3 (9.4) 0.42 (0.09-2.03) 178 (18.4) 1.00 (0.80-1.24) 90 (21.5) N (%) 0.44 0.41 0.43 Early AMD (n=509) AMD Early N (%) N (%) 1157 (31.9) 53 (31.2) 840 (32.7) No AMD (controls) (n=3629)No AMD (controls) (n=427) AMD Early N (%) 1795 (49.5) 92 (54.1) 1254 (48.9) 677 (18.7) 25 (14.7) 473 (18.4) No AMD (controls) (n=2567)No AMD (controls) (n=967) AMD Early N (%) 0.43 0.42 0.43 No AMD (controls) (n=170)No AMD (controls) (n=84) AMD Early N (%) Appendix 2. Risk of Age-related Macular Degeneration for Macular Degeneration 2. Risk of Age-related Appendix cases 1. Based on prevalent study a. The Rotterdam c.-6530C>G ERCC6 Noncarrier c.-6530C>G ERCC6 Noncarrier c.-6530C>G ERCC6 Noncarrier c.-6530C>G ERCC6 Noncarrier MAF= minor allele MIX= combination of GA+CNV; atrophy; geographic GA= neovascularization; CNV= choroidal macular degeneration; AMD= age-related Abbreviations: stage 0) in the genetic the risk of disease (AMD vs and represent risk of AMD, of the relative estimates are and HRs The ORs ratio. HR= hazard OR= odds ratio; frequency; heterozygous heterozygous heterozygous homozygous heterozygous MAF(%) homozygous homozygous homozygous 3. Based on prevalent and incident cases incident and prevalent 3. Based on population study the AMRO-NL and study the Rotterdam data from Pooled MAF(%) MAF(%) MAF(%) b. The AMRO-NL study population study The AMRO-NL b. cases incident 2. Based on study The Rotterdam

Chapter 2 20120725.indd 50 23-10-12 23:05 ERCC6

The Gene and Age-related Macular Degeneration Appendix 3. Odds Ratios (OR) and 95% Confidence Intervals (CI) of Early and Late Age-related Macular Degeneration Cases Versus Unrelated Controls of the Amsterdam Study Population for Single Nucleotide Polymorphisms in the ERCC6 Gene. No AMD (controls) Early AMD Late AMD rs12220258 N=193 N=93 N=276 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 132 (68.4) 67 (72.0) 1 198 (71.7) 1 2 Heterozygous (Aa) 55 (28.5) 25 (26.9) 0.92 (0.52-1.62) 71 (25.7) 0.87 (0.56-1.34) Homozygous (aa) 6 (3.1) 1 (1.1) 0.35 (0.04-3.02) 7 (2.5) 0.87 (0.27-2.80) MAF(%) 0.17 0.15 0.15

rs2228528 N=192 N=93 N=276 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 132 (68.8) 67 (72.0) 1 198 (71.7) 1 Heterozygous (Aa) 54 (28.1) 25 (26.9) 0.93 (0.53-1.65) 70 (25.4) 0.86 (0.56-1.33) Homozygous (aa) 6 (3.1) 1 (1.1) 0.35 (0.04-3.02) 7 (2.5) 0.86 (0.27-2.78) MAF(%) 0.17 0.15 0.15

rs2228529 N=188 N=93 N=275 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 117 (62.2) 54 (58.1) 1 183 (66.5) 1 Heterozygous (Aa) 62 (33.0) 33 (35.5) 1.19 (0.69-2.05) 81 (29.5) 1.19 (0.69-2.05) Homozygous (aa) 9 (4.8) 6 (6.5) 1.75 (0.58-5.32) 11 (4.0) 1.75 (0.58-5.32) MAF(%) 0.21 0.24 0.19

rs2229760 N=192 N=93 N=272 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 61 (31.8) 30 (32.3) 1 81 (29.8) 1 Heterozygous (Aa) 99 (51.6) 48 (51.6) 0.92 (0.52-1.62) 142 (52.2) 1.01 (0.65-1.57) Homozygous (aa) 32 (16.7) 15 (16.1) 0.87 (0.41-1.88) 49 (18.0) 1.11 (0.62-1.99) MAF(%) 0.42 0.42 0.44

rs2281793 N=193 N=92 N=275 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 57 (29.5) 26 (28.3) 1 76 (27.6) 1 Heterozygous (Aa) 99 (51.3) 49 (53.3) 1.03 (0.58-1.86) 139 (50.5) 0.96 (0.62-1.50) Homozygous (aa) 37 (19.2) 17 (18.5) 0.92 (0.44-1.95) 60 (21.8) 1.11 (0.63-1.94) MAF(%) 0.45 0.45 0.47

rs4253165 N=194 N=93 N=277 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 121 (62.4) 54 (58.1) 1 184 (66.4) 1 Heterozygous (Aa) 63 (32.5) 33 (35.5) 1.21 (0.71-2.08) 82 (29.6) 0.98 (0.64-1.49) Homozygous (aa) 10 (5.2) 6 (6.5) 1.56 (0.53-4.64) 11 (4.0) 0.76 (0.30-1.90) MAF(%) 0.21 0.24 0.19

Chapter 2 20120725.indd 51 23-10-12 19:25 Chapter 2 Appendix 3 (continued). No AMD (controls) Early AMD Late AMD rs4253211 N=193 N=93 N=277 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 157 (81.3) 79 (84.9) 1 224 (80.9) 1 Heterozygous (Aa) 32 (16.6) 14 (15.1) 0.92 (0.46-1.84) 49 (17.7) 1.11 (0.67-1.83) Homozygous (aa) 4 (2.1) 0 (0.0) sample size too small 4 (1.4) 0.89 (0.20-3.92) MAF(%) 0.1 0.08 0.1

rs4253231 N=190 N=93 N=274 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 159 (83.7) 80 (86.0) 1 226 (82.5) 1 Heterozygous (Aa) 29 (15.3) 10 (10.8) 0.71 (0.33-1.54) 47 (17.2) 1.08 (0.64-1.84) Homozygous (aa) 2 (1.1) 3 (3.2) 3.09 (0.49-19.31) 1 (0.4) 0.30 (0.02-3.86) MAF(%) 0.09 0.09 0.09

rs4838523 N=193 N=91 N=274 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 155 (80.3) 73 (80.2) 1 213 (77.7) 1 Heterozygous (Aa) 38 (19.7) 17 (18.7) 0.89 (0.47-1.69) 57 (20.8) 1.05 (0.65-1.69) Homozygous (aa) 0 (0.0) 1 (1.1) sample size too small 4 (1.5) sample size too small

MAF(%) 0.1 0.1 0.12 AMD= age- related macular degeneration; MAF= minor allele frequency. “A” indicates common allele, “a” minor allele. Percentages not always 100% because of rounding. ORs are estimated with logistic regression analysis (with the control group as reference group and respectively early and late AMD as outcome variable). Adjusted for age and sex.

Chapter 2 20120725.indd 52 23-10-12 19:25 ERCC6

The Gene and Age-related Macular Degeneration Appendix 4. RT-PCR Analysis of ERCC6 Gene Expression in RPE from Eye Donors With Early AMD or Non-affected Age-matched Donors. Raw values Normalized values

RPLP0 PPIA EEF1a1 ERCC6 ERCC6 Early AMD RPE donor eye nr 1 127141.98 29794.97 266778.14 7.86 3.59 2 2 271949.82 27580.88 192751,28 21.18 8.58 3 39560.72 15669.60 58290.81 13.11 18.17 4 43006.91 7820.69 90704.15 7.31 10.72 5 37111.56 13161.91 67649.00 6.40 9.14 6 55537.74 12115.62 59859.02 6.05 8.09 7 206256.63 30139.77 126710.89 16.03 13.81 8 112524.60 8799.35 63831.50 7.30 8.40 9 32683.64 40352.79 108173.00 37.78 33.14 10 53523.36 16339.55 27788.13 8.05 7.96 11 37891.35 25164.13 105822.06 10.07 12.73 12 14801.76 4733.79 34014.94 4.81 9.91 13 317584.49 137167.67 441477.67 51.08 16.51 14 179980.48 61265.23 314561.14 45.61 8.74 Average 109254 30722 139887 17 12 Matched old-healthy donor eye nr 1 43492.56 53352.97 168767.89 47.12 29.51 2 223618.64 2641.75 264641.71 25.29 21.51 3 386653.29 208455.65 614043.33 218.49 27.27 4 104698.48 13235.10 141945.83 29.70 23.40 5 48304.87 4874.64 27375.18 4.28 10.55 6 6938.85 2173.92 15751.27 3.97 29.34 7 11254.24 7131.95 40504.12 6.34 19.61 8 14144.77 1673.07 18509.53 3.44 20.77 9 64436.07 9024.81 87293.16 12.64 15.64 Average 100394 33618 153204 39 22 P-value early AMD vs. old-healthy 0.018 Both raw expression values for the three selected RPE housekeeping genes (RPLP0, PPIA, and EEF1a1) and ERCC6, as well as the resulting normalized expression values for ERCC6 are presented. AMD= age- related macular degeneration. Normalization= normalized to the geometric mean of three housekeeping genes (RPLP0, PPIA, and EEF1a1) [46,47]. For statistical details see materials and methods.

Chapter 2 20120725.indd 53 23-10-12 19:25 Chapter 2 20120725.indd 54 23-10-12 19:25 CHAPTER 3 Multicenter Cohort Association Study of SLC2A1 Single Nucleotide Polymorphisms and Age-related Macular Degeneration

Dominique C. Baas, Lintje Ho, Michael W.T. Tanck, Lars G. Fritsche, Joanna E. Merriam, Ruben van het Slot, Bobby P.C. Koeleman, Theo G.M.F. Gorgels, Cornelia M. van Duijn, André G. Uitterlinden, Paulus T.V.M. de Jong, Albert Hofman, Jacoline B. ten Brink, Johannes R. Vingerling, Caroline C.W. Klaver, Michael Dean, Bernhard H. F. Weber, Rando Allikmets, Gregory S. Hageman and Arthur A.B. Bergen

Molecular Vision 2012; 18:657-674

Chapter 3 20120725.indd 55 23-10-12 19:26 Chapter 3 Abstract

Purpose: Age-related macular degeneration (AMD) is a major cause of blindness in the elderly and has a genetically complex background. This study examines the association between single nucleotide polymorphisms (SNPs) in the glucose transporter 1 (SLC2A1) and AMD. SLC2A1 regulates the bio-availability of glucose

mediated AMD pathology. in the retinal pigment epithelium (RPE), which might influence oxidative stress Methods: Twenty-two SNPs spanning the SLC2A1 gene were genotyped in 375 cases and 199 controls from an initial discovery cohort (the Amsterdam-Rotterdam- Netherlands study). Replication testing was performed in The Rotterdam Study (the Netherlands) and study populations from Würzburg (Germany), the Age Related Eye Disease Study (AREDS, United States), Columbia (United States) and Iowa (United States). Subsequently, a meta-analysis of SNP association was performed. Results SNPs (rs3754219, rs4660687 and rs841853) and AMD was found. Replication in : In the discovery cohort, significant genotypic association between three

five large independent (Caucasian) cohorts (4860 cases and 4004 controls) did not different for the controls and/or cases among the six individual populations. Meta- yield consistent results. The genotype frequencies for these SNPs were significantly

Conclusions: No overall association between these SLC2A1 SNPs and AMD was analysis revealed significant heterogeneity of effect between the studies. demonstrated. Since the genotype frequencies for the three SLC2A1 SNPs were

study corroborates previous evidence that population-dependent genetic risk significantly different for controls and/or cases between the six cohorts, this heterogeneity in AMD exists.

Chapter 3 20120725.indd 56 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration Introduction

Age-related macular degeneration (AMD) is the most common cause of severe visual impairment in Western countries, rendering the disease a major public health issue [1,2]. The prevalence of AMD increases strongly with age, affecting 4% of the population over the age of 60 and more than 10% of individuals older than 75 [2,3]. The early stages of the disease are characterized by drusen, focal depositions of extracellular material in Bruch’s membrane beneath the retinal pigment epithelium (RPE) [4,5]. Late stages of the disease include two forms: an atrophic (geographic atrophy (GA)) and an exudative form (choroidal neovascularization (CNV)). 3 AMD has a multifactorial etiology [6]. Age, smoking history, high body mass index,

The importance of genetic risk factors for AMD was highlighted in several recent hypertension, and hypercholesterolemia influence AMD predisposition [7]. studies. In addition to the complement factor H (CFH) gene, genetic association studies consistently implicated at least four complement genes (factors B (CFB) and I (CFI), components 2 (C2) and 3 (C3)) as well as one of two genes (ARMS2 and HTRA1) in the chromosomal region 10q26. Taken together, these data suggest that the complement system, oxidative stress, mitochondrial function and extracellular matrix turnover play a role in AMD [8-14]. The RPE is one of the key tissues involved in AMD and functions in several processes that are vital for the preservation of sight. The RPE layer constitutes the outer blood-

retina and the choroid [15]. Among other things, the RPE transports glucose to the retinal barrier and regulates transport of ions, fluid and metabolites between the photoreceptors. Glucose supply to the photoreceptors is essential since glucose is the preferred energy substrate for the metabolically highly active retina [16]. The sodium-independent glucose transporter SLC2A1 is the predominant glucose transporter in the retina [17,18]. SLC2A1 localizes to the apical and basolateral membranes of the RPE [19]. According to Beatty and colleagues, the retina is the ideal environment for generating free radicals and other reactive oxygen species (ROS) [20]. This may occur through similar transport mechanisms through the inner and outer blood retina barriers, and makes the retina an environment susceptible to oxidative damage. Fernandes et al. showed in 2011 that sustained oxidative stress can result in decreased glucose transport in retinal endothelial cells [21]. On the other hand, increased glucose transport and SLC2A1 expression are upregulated by hypoxia as shown by Takagi et al. [22]. Finally, increased serum glucose (hyperglycemia) might lead to impaired antioxidant protection [23] and increased ROS production [24]. In conclusion, DNA sequence variations or altered expression

Chapter 3 20120725.indd 57 23-10-12 19:26 Chapter 3 levels in SLC2A1 affect local oxidative stress. may influence glucose delivery to the retina and thereby profoundly Variants in SLC2A1 have been associated with diabetic retinopathy [25], type 2 (non- insulin-dependent) diabetes [26], diabetic nephropathy [27,28] and clear-cell renal cell carcinoma [29]. Finally, expression of SLC2A1 in retina and brain is altered in different pathophysiological conditions including hypoxia [22,30], Alzheimer’s disease [31] and epilepsy [32]. We hypothesized that genetic variants in SLC2A1

lead to changes in the glucose level in the RPE and neural retina, and alter the local could influence the glucose transport capabilities of this transporter. This would oxidative burden. Since multiple studies suggest that oxidative stress is implicated in AMD [20,33-35] we performed an extensive case-control association analysis, to test whether SLC2A1 gene variants are associated with this devastating disorder.

Methods

Study populations

of a total of 5235 AMD cases and 4203 ethnically- and age-matched control subjects. We employed five case-control studies and one prospective cohort study, consisting The studies were approved by the Ethics Committees of the Academic Medical Center Amsterdam, the Erasmus Medical Center Rotterdam, the University of Würzburg, the Age Related Eye Disease Study (AREDS) Access Committee and the Institutional Review Boards of Columbia University and the University of Iowa. All studies followed the tenets of the Declaration of Helsinki and all participants provided signed informed consent. The initial discovery sample, the Amsterdam-Rotterdam-Netherlands (AMRO-NL) study population, consisted of 375 unrelated individuals with AMD and 199 control individuals. Subjects were all Caucasian and recruited from the Erasmus University Medical Centre, Rotterdam and the Netherlands Institute for Neuroscience, Amsterdam, the Netherlands [36]. The second sample, the Rotterdam Study, is a prospective cohort study aimed at investigation of chronic diseases in older adults, as previously described [37]. The eligible population comprised all inhabitants aged 55 years or older of a middle- class suburb in Rotterdam, the Netherlands. The third sample, the Franconian AMD study (Würzburg, Germany), consisted of 612 cases and 794 age-matched control subjects, both cases and controls originating from the lower Franconian region of Bavaria, Germany [38]. The fourth sample, the AREDS (United States), included two parts: 1) a randomized

Chapter 3 20120725.indd 58 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration clinical trial to assess the effect of supplemental antioxidants on risk of AMD and cataract, which began in April 1992 and ended in November 2001; and 2) a longitudinal study of progression of AMD. The study participants were Caucasian, aged 55 to 80 years, and were recruited at 11 centers in the US from the clinic and general populations in those areas. The sample consisted of 936 AMD cases and 218 controls [39].

comprised 1104 unrelated individuals with AMD and 368 unrelated controls of The fifth sample, the Columbia University (United States) study population, European American descent, recruited at Colombia University [40]. The sixth sample, the Iowa University (United States) study population, comprised 3 1139 unrelated individuals with AMD and 403 unrelated controls of European American descent, recruited at the University of Iowa [41].

Diagnosis of AMD Subjects from all cohorts underwent ophthalmic examination and fundus photography as described [36-41]. Signs of AMD were graded according to a

except the AREDS [43] and the Franconian AMD study [38]. modification of the International Classification and Grading system for AMD [42]

SNP selection Twenty-two SNPs were selected that capture common variations in the SLC2A1 gene. SNP data were from the Centre d’Étude du Polymorphisme Humain (CEPH) population (Utah residents with ancestry from northern and western Europe) by use of the International HapMap Project. SNP selection was based on criteria like functional relevance, minor allele frequency (MAF)>10%, coverage of the main linkage disequilibrium (LD) blocks and tagging of the most common haplotypes. Tag SNPs were selected with Tagger, an option of Haploview [44] (all SNPs were captured with a LD tagging criteria of r2>0.8).

Genotyping Genomic DNA was isolated from peripheral leukocytes after venous puncture according to standard protocols. A total of 1536 SNPS, including 22 SLC2A1 SNPs, were genotyped in the AMRO-NL study population using an Illumina GoldenGate assay on a BeadStation 500 GX (Illumina Inc., San Diego,CA, US). This GoldenGate assay did not include our previous published complement factor 5 (C5) and ERCC6 versus AMD screenings. We currently screened all known common genetic variants,

Chapter 3 20120725.indd 59 23-10-12 19:26 Chapter 3 which met Illumina quality standards, in approximately 45 AMD candidate genes (DB and AB; data not shown). The Rotterdam Study was genotyped with the Illumina HumanHap 550K array (Illumina Inc., San Diego,CA, US). Quality control was performed using PLINK (version 1.01) [37]. The Franconian AMD study sample was genotyped with TaqMan SNP Genotyping assay for the SNPs rs4660687: A>C and rs3754219: A>C. Rs841853 was genotyped by polymerase chain reaction (forward primer: 5’-CCT CAG GGA ATA AAG CTA GTC TCC AG-3’; reverse primer: 5’-AGA CCA GCC AGA GGT TCC AAA-3’) followed by XbaI digestion and restriction fragment length analysis. The AREDS, Columbia and Iowa samples were also genotyped with TaqMan SNP Genotyping. All TaqMan assays were performed on ABI7300 Real-Time PCR systems (Applied Biosystems, Foster City, CA).

Statistical analysis To account for multiple comparisons, we determined, initially, three different

significance thresholds for the GoldenGate assay in the AMRO-NL study. The first of SNPs on the Illumina GoldenGate assay (n=1536; p= 3.25 x 10-5)). However, this was an overall Bonferroni corrected significance threshold per SNP (0.05/ total nr

(MAF) of the SNPs screened nor the co-occurrence of SNP variation in LD blocks into significance level takes neither the large difference in the minor allele frequency

account. Thus, next to this overall significance level, we also applied a gene-wise SLC2A1 is 0.05/ total nr of SLC2A1 significance threshold by dividing the nominal alpha by the number of SNPs per SNPs on the Illumina GoldenGate assay (n=22) (p= 2.27 x 10-3). Finally, as a positive gene. The gene-wise significance threshold for

complement factor 3 (C3) polymorphisms, well known in the literature to associate control to determine true statistical significance levels of our assay, we also included with AMD. We found an association with multiple C3 SNPs and AMD in our study, with p-values ranging from p=0.02 to p=0.0003 (not shown). The p-values of our initial SLC2A1 section). association findings ranged from p=0.02 to p=0.0006 (see the Results 2 test to test SNP distributions for conformity with Hardy–Weinberg equilibrium (HWE). We compared allele frequencies between In all studies, we employed the χ cases and controls using the Fisher’s exact test statistic. We estimated odds ratios

analysis and represent the risk of disease (AMD versus no AMD) in the genetic risk (ORs) and 95% confidence intervals (CIs) for the risk of AMD with logistic regression group divided by the risk of disease in the non-risk group (non-carriers). For the AMRO-NL and the Rotterdam Study, we adjusted all ORs for age and gender. To determine whether SLC2A1 SNPs were independent risk factors, we estimated their

Chapter 3 20120725.indd 60 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration effects in a model with an additional adjustment for known (genetic) risk factors: CFH Y402H, LOC387715 A69S, and smoking and their interactions with the SLC2A1 SNPs. All analyses were performed in SPSS for Windows software (release 16.0; IBM- SPSS, Inc., Chicago, IL). In the Rotterdam Study, association tests were performed using logistic regression in the PLINK software package (version 1.01) [45]. A meta- analysis was performed using Review Manager (RevMan) Version 5.0. assuming an additive genetic model. We tested heterogeneity between studies using Cochran’s I2 metric [46]. In the absence of heterogeneity (I2 Q statistic [46]. In addition, heterogeneity was quantified with the (the Mantel-Haenszel method) to calculate the pooled OR; otherwise, we used the statistic <25%), we used the fixed effects model 3 random effects model (the DerSimonian and Laird method). We compared genotype 2 test.

frequencies between the study populations with the Pearson χ Results

The AMRO-NL sample: associations of SLC2A1 SNPs with AMD. We initially genotyped 22 SNPs in 375 unrelated AMD patients and 199 controls of the AMRO-NL study population. Demographic characteristics of the AMRO-NL study population and all study populations used were essentially described previously [36,47]. SNP genotype frequencies were tested in controls and conformed to HWE, except for two SNPs (rs12407920, rs16830101; p=0.04) that were removed from further analysis. HWE-data, genotype distributions, and allelic p-values for the 22 SLC2A1 SNPs are presented in Table 1 allelic association with AMD: rs3754219 (p=0.0011), rs4660687 (p=0.0157) and . Three out of the 22 SNPs showed a significant rs841853 (p=0.0006). The reported initial associations in the AMRO-NL study did

(rs3754219 and rs841853) passed the gene-wise threshold as well as the positive not reach the overall Bonferroni significance threshold, but two out of three SNPs control (C3) association threshold. The risks of AMD for these three SLC2A1 SNPs, adjusted for age and gender, are presented in Table 2. When all AMD cases and

association with all three SNPs. SNP (rs3754219) showed a protective effect for controls of the AMRO-NL study were included in the analysis, we observed significant AMD (OR 0.44; 95%CI 0.26-0.74). The other SNPs (rs4660687, rs841853) showed a risk increasing effect with the highest ORs seen for homozygous carriers of the risk alleles of rs841853 (OR 2.24; 95%CI 1.13-4.41). We subsequently analyzed the

association only for rs4660687 (Table 2). For the late AMD subgroup, we observed a early and late AMD subgroups separately. For early AMD, we observed a significant

similar effect as seen for all AMD cases: a significantly increased risk for hetero- and 61

Chapter 3 20120725.indd 61 23-10-12 19:26 Chapter 3

2 HWE yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes Allelic p-value m.a.f aa AMD (cases) Aa number (%) AA 171 (47.2) 159 (43.9) 32 (8.8) 30.8 0.3652 232 (64.4) 117 (32.5) 11 (3.1) 19.3 0.5203 229 (63.3) 122 (33.7) 11 (3.0) 19.9 0.3419 293 (82.1) 64 (17.9) 0 (0.0) 9 0.0538 no 292 (82.0) 64 (18.0) 0 (0.0) 9 0.0509 no 118 (33.0) 184 (51.4) 56 (15.6) 41.3 0.0011 225 (62.2) 124 (34.3) 13 (3.6) 20.7 0.6935 219 (60.5) 129 (35.6) 14 (3.9) 21.7 0.8162 95 (34.1) 133 (47.7) 51 (18.3) 42.1 0.0157 224 (62.0) 124 (34.3) 13 (3.6) 20.8 0.6947 106 (29.4) 193 (53.6) 61 (16.9) 43.8 0.1193 108 (29.8) 193 (53.3) 61 (16.9) 43.5 0.1182 170 (47.2) 158 (43.9) 32 (8.9) 30.8 0.4003 231 (64.0) 119 (33.0) 11 (3.0) 19.5 0.5222 230 (63.5) 121 (33.4) 11 (3.0) 19.8 0.5767 232 (64.1) 119 (33.0) 11 (3.0) 19.5 0.4725 231 (64.0) 119 (33.0) 11 (3.0) 19.5 0.5222 150 (41.8) 168 (47.0) 41 (11.4) 34.9 0.0006 227 (63.4) 120 (33.5) 11 (3.1) 19.8 0.524 250 (69.3) 102 (28.3) 9 (2.5) 16.6 1 224 (62.0) 124 (34.3) 13 (3.6) 20.8 0.6947 224 (62.0) 124 (34.3) 13 (3.6) 20.8 0.6947 m.a.f No AMD (controls) Aa aa number (%) Single Nucleotide Polymorphisms in the AMRO-NL Study Study AMRO-NL in the Nucleotide Polymorphisms Single SLC2A1 and 22 Selected Macular Degeneration Age-related 1. Allelic Association Between Table Population. SNP ID AA test was used to test SNP distributions for conformity with HWE. conformity SNP distributions for test used to was test rs1105297 94 (51.4) 75 (41.0) 14 (7.7) 28.1 rs1770810 115 (62.8) 59 (32.2) 9 (4.9) 21 rs1770811 109 (59.9) 64 (33.7) 9 (4.9) 22.5 rs12407920 161 (89.9) 16 (8.9) 2 (1.1) 5.5 rs16830101 161 (89.9) 16 (8.9) 2 (1.1) 5.5 rs3754219 44 (24.6) 84 (46.9) 51 (28.5) 52 rs3754223 108 (59.7) 67 (37.0) 6 (3.3) 21.8 rs3768043 108 (59.0) 68 (37.2) 7 (3.8) 22.4 rs4660687 71 (47.3) 57 (38.0) 22 (14.7) 33.7 rs4660691 109 (59.6) 68 (37.2) 6 (3.3) 21.9 rs710221 64 (35.0) 96 (52.5) 23 (12.6) 38.8 rs710222 64 (35.2) 96 (52.7) 22 (12.1) 38.5 rs751210 94 (51.4) 75 (41.0) 14 (7.7) 28.1 rs841845 114 (62.3) 60 (32.8) 9 (4.9) 21.3 rs841848 114 (62.3) 60 (32.8) 9 (4.9) 21.3 rs841851 114 (62.3) 60 (32.8) 9 (4.9) 21.3 rs841852 114 (62.3) 60 (32.8) 9 (4.9) 21.3 rs841853 104 (58.1) 62 (34.6) 13 (7.3) 24.6 rs841856 113 (61.7) 61 (33.3) 9 (4.9) 21.6 rs841858 129 (70.5) 47 (25.7) 7 (3.8) 16.7 rs900836 109 (59.6) 68 (37.2) 6 (3.3) 21.9 rs900837 109 (59.6) 68 (37.2) 6 (3.3) 21.9 AMD= age-related macular degeneration; SNP= single nucleotide polymorphism; m.a.f= minor allele frequency; HWE= Hardy-Weinberg Equilibrium. “A” indicates common indicates “A” Equilibrium. HWE= Hardy-Weinberg allele m.a.f= minor frequency; nucleotide polymorphism; single SNP= macular degeneration; AMD= age-related allele, minor “a” allele. Genotype as frequencies a are given percentage of subjects genotyped. succesfully The allelic is p-value calculated with Fisher’s test. exact The χ

Chapter 3 20120725.indd 62 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration 0.37 (0.21-0.66) 1.61 (1.01-2.57) 2.21 (1.42-3.39) 2.64 (1.28-5.46) 92 (46.7) Late AMD Late N=267 95 (35.6) 1 39.7 N=197 71 (36.0) 1 34 (17.3)40.6 1.55 (0.82-2.90) N=268 103 (38.4) 1 37.1 3 2.13 (1.15-3.93) 2.29 (1.04-5.01) 16 (17.6) 0.59 (0.28-1.27) 40 (15.0) 41 (50.0) 37 (40.7)7 (7.7) 1.44 (0.84-2.46) 131 (48.9) 1.32 (0.49-3.55) 34 (12.7) Early AMD Early N=91 No. (%)23 (25.3) OR (95%CI) 1 46.2 No. (%)N=82 OR (95%CI) No. (%)24 (29.3) OR (95%CI) 1 45.7 No. (%)N=91 OR (95%CI) No. (%)47 (51.6) OR (95%CI) 1 28 No. (%) OR (95%CI) 0.44 (0.26-0.74) 1.74 (1.13-2.70) 1.87 (1.27-2.77) 2.24 (1.13-4.41) 41.3 42.1 34.9 No. (%)44 (24.6) No. (%) 118 (33.0) OR (95%CI) 51.9 1 No. (%)71 (47.3) No. (%) 95 (34.1) OR (95%CI) 33.7 1 No. (%)104 (58.1) No. (%) 150 (41.8) OR (95%CI) 24.6 1 No AMDN=179 All AMD cases N=358 N=150 N=279 N=179 N=359 rs3754219 Genotype Noncarrier (AA) (Aa)Heterozygous (aa)Homozygous m.a.f(%) 84 (46.9) 51 (28.5) 184 (51.4)rs4660687 Genotype 56 (28.5) 0.82 (0.53-1.27)Noncarrier (AA) (Aa)Heterozygous (aa)Homozygous 52 (57.1)m.a.f(%) 57 (38.0) 1.32 (0.72-2.43) 22 (14.7) 133 (47.7) rs841853 132 (49.4)Genotype 51 (18.3) 0.73 (0.46-1.14) Noncarrier (AA) (Aa)Heterozygous 1.73 (0.96-3.12) (aa)Homozygous m.a.f(%) 62 (34.6) 17 (20.7) 13 (7.3) 168 (47.0) 41 (11.4) Table 2. Odds Ratios (OR) and 95% Confidence Intervals (CI) of AMD Cases vs. Controls of the AMRO-NL Study Population for Three SNPs in SLC2A1. SNPs for Three Population Study of the AMRO-NL Controls (CI) of AMD Cases vs. Intervals 2. Odds Ratios (OR) and 95% Confidence Table because of 100% add up to not always allele. minor Percentages common allele, “a” indicates “A” m.a.f= minor allele frequency. macular degeneration; AMD= age-related age and sex. for Adjusted logistic regression. by estimated were ORs rounding.

Chapter 3 20120725.indd 63 23-10-12 19:26 Chapter 3 homozygote carriers of rs841853 and for the heterozygote carriers of rs4660687 and a protective effect for homozygote carriers of rs3754219 (Table 2). Adjusting for three prominent AMD risk factors (CFH Y402H, LOC387715 A69S and smoking) did not modify the relation of any of the SLC2A1 SNPs with AMD (data not shown).

Independent replication studies have variable outcomes The three SNPs that showed allelic and genotypic association (rs3754219,

comparable ethnic (Caucasian) composition: The Rotterdam study, The Franconian rs4660687 and rs841853) were selected for replication in five study populations of AMD study, the AREDS, Columbia University and the University of Iowa. Figure 1a shows the linkage disequilibrium (LD) map in Haploview of the three SNPs and corresponding LD scores, and Figure 1b shows the LD map of all 22 SLC2A1 SNPs with minor allele frequency>10% screened in this study and illustrates the nine distinct haplotype blocks. The three associated SNPs are in 2 different blocks in the SLC2A1 gene (rs3754219 and rs841853 are in the same LD block). As can be seen, two SNPs (rs1770811 and rs841856) without allelic association are inside the LD block with rs3754219 and rs841853. Obviously, the observed (non-) associations

alleles. A possible explanation might also be differences in LD patterns (between the depend on the informativeness (MAF) of the specific SNPs and the distribution of SNPs in the same wild-type LD block) with an untyped causal variant.

Replication studies for SLC2A1 SNP rs3754219 The results of replicating the association between SNP rs3754219 and AMD (all cases or late AMD and controls only) are presented in Appendix 1. Genotype frequencies for rs3754219 followed the HWE in all study populations (data not shown). When all AMD cases were included, with rs3754219 in any of the replication cohorts. When we analyzed AMD subgroups we observed no significant association separately (Appendix 2 Study for the CNV cases (p=0.0554). In line with the results of the discovery ), we found a borderline significant effect in the Rotterdam cohort, we observed this possible protective effect for homozygous carriers of the risk allele. In contrast, we observed a risk increasing association effect between SLC2A1 rs3754219 and AMD (subtype) in the Columbia University study: This effect occurred in the homozygous carriers of the risk allele (OR 1.66; 95%CI 1.01-2.72) in the GA group (Appendix 2 replication cohorts. ). No significant associations were seen in the other

Chapter 3 20120725.indd 64 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration

Figure 1. Linkage Disequilibrium (LD) Display in Haploview of SLC2A1 SNPs. (A). SLC2A1 SNPs: rs3754219, rs4660687 and rs841853. Left: D’(D prime), Right: r² (r square). (B). All the 22 SNPs in the SLC2A1 gene with minor allele frequency>10% screened in this study and illustrating the 9 distinct haplotype blocks. LD scores: D’ (D prime) displays the raw D’ value, which is the normalized

covariance for a given marker pair and the correlation coefficient r². Darker shades represent stronger LD. Replication studies for SLC2A1 SNP rs4660687 The results of replicating the association between rs4660687 and AMD (all cases or late AMD and controls only) are presented in Appendix 1. Genotype frequencies followed the HWE in all study populations (data not shown). The association

Rotterdam Study (OR 1.39; 95%CI 1.12-1.72). Thus, both Dutch populations of rs4660687 with AMD, as found in the AMRO-NL study, was confirmed in the

Chapter 3 20120725.indd 65 23-10-12 19:26 Chapter 3

Appendix 3), showed a significant association for all AMD cases. However, in the other replication cohorts, no significant association was found. Upon subtype analysis ( the Rotterdam Study. Again, this effect was present in both Dutch populations. For we found a significant association between rs4660687 and the early AMD cases of this SNP, we also found a possible opposite effect: in the Franconian AMD study, we

increasing effect. This was seen for hetero- and homozygous carriers of the risk allele. observed a significant protective effect in the mixed AMD subgroup instead of a risk

AMD. In the three cohorts from the United States, we found no significant associations for

Replication studies for SLC2A1 SNP rs841853 The results of replicating the association between rs841853 SNP and AMD (all cases or late AMD and controls only) are presented in Appendix 1. Genotype frequencies followed the HWE in all study populations (data not shown). The association of

rs841853 with AMD, as found in the AMRO-NL study, was confirmed in the AREDS (OR 2.07; 95%CI 1.14-3.76) in all AMD cases. In the other replication cohorts, no sample: We observed a significant effect for homozygous carriers of the risk allele Appendix 4), we observed

significant association was found. Upon subtype analysis ( significant association between rs841853 and the CNV cases of the Rotterdam study. rs841853 and late AMD cases (Appendix 1). The effect was particularly profound Additionally, in the AREDS sample, we observed a significant association between for the CNV and GA subpopulations (Appendix 4). Finally, in the Columbia cohort,

effect was in the opposite direction: heterozygotes showed a protective association we found a significant association between rs841853 and the GA cases, but this instead of a risk increasing effect for GA. We also saw this protective effect for the late AMD cases of the Columbia cohort (Appendix 1 between rs841853 and AMD subtypes were seen in the two other study populations. ). No significant associations

Meta-analysis: heterogeneity of effect We performed a meta-analysis of the putative association between AMD and SLC2A1 SNPs rs3754219, rs4660687 and rs841853 across the independent study

between studies (with Cochran’s Q test and the I2 metric, Table 3 and Figures 2-4). For populations. We quantified the effect and assessed potential heterogeneity of effect

not only for all AMD cases combined (p=0.006; I2=70%), but also for CNV (p=0.02; rs3754219, significant heterogeneity of effect between studies was demonstrated, I2=65%) and late AMD cases (p=0.002; I2=73%). Furthermore moderate (not

significant) heterogeneity was seen for the GA and CNV + mixed cases, respectively. 66

Chapter 3 20120725.indd 66 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration No heterogeneity was found for the early and mixed AMD cases (Table 3). For the groups with I2 values >25%, we used the random effects model (the DerSimonian and Laird method) to calculate the pooled OR; otherwise, in absence of heterogeneity (I2

statistic <25%), we used the fixed effects model (the Mantel-Haenszel method). (all clinical subtypes of) AMD was found over all populations (Figure 2). Similarly, The summary OR indicated that no significant association between rs3754219 and I2=64%)

meta-analysis for rs4660687 showed significant heterogeneity (p=0.02; for all AMD cases. Moderate (but not significant) heterogeneity was seen for the or low heterogeneity was detected (Table 3 AMD subtypes with the exception of the GA, CNV and CNV+ mixed cases where no 3 (depending on the amount of heterogeneity), no association between rs4660687 ). Using a random or fixed effects model and (all clinical subtypes of) AMD was found over all populations (Figure 3). Also

cases combined (p=0.008; I2=68%) but also for CNV (p=0.0005; I2=80%) and late for rs841853, the between-study heterogeneity was significant; not only for all AMD AMD cases compared to the controls (p=0.002; I2

=74%). Moderate (not significant) for the early, GA and mixed cases (Table 3 heterogeneity was seen for CNV+ mixed cases and no or low heterogeneity was seen (depending on the amount of heterogeneity), the summary OR indicated that no ). Using a random or fixed effects model

of) AMD (Figure 4). significant associations were present between rs841853 and (all clinical subtypes

Genetic heterogeneity between study populations To explore the genetic contribution to the heterogeneity of effect between the populations, we assessed the differences in MAF and genotype frequencies between the study populations (Table 2 and Appendix 1). For rs3754219, we observed MAFs in the control populations ranging from 41% (Columbia University) to 52% (AMRO- NL). For the cases, we observed rs3754219 MAFs varying from 41% (AMRO-NL) to 46% (Franconian AMD study). For rs4660687, we observed MAFs in the control populations ranging from 34% (AMRO-NL) to 43% (AREDS). For the cases, the rs4660687 MAFs were nearly similar (41-43%) with the exception of the Franconian AMD study (MAF=37%). For rs841853, the MAF ranged from 25% (AMRO-NL) to 33% (The Franconian AMD study) in the control groups. For the cases, we observed rs841853 MAFs varying from 30% (Iowa) to 35% (AMRO-NL). Subsequently we compared the genotype frequencies of the controls between study populations for 2 test. For rs3754219 and rs841853, we found 2 overall p-values of respectively 0.027 and 0.024 all three SNPs using the Pearson χ (Table 4 significant differences with χ ). We found no significant differences in genotype frequencies between the 67

Chapter 3 20120725.indd 67 23-10-12 19:26 Chapter 3 control groups for rs4660687. Next, we compared the genotype frequencies for all populations’ cases. The rs3754219 genotype frequencies of all AMD cases combined

2 overall p-value=0.002) or AMD clinical subtypes of the six study populations did not show significant 2 differences. For rs4660687, we found a significant (χ difference in genotype frequencies of all AMD cases and the mixed cases (χ frequencies for the CNV subtype (p-value=0.024; Table 4). Detailed results of the overall p-value=0.049). For rs841853, we found significant differences in genotype individual analysis are presented in Appendix 5.

Table 3. Test for Heterogeneity of Effect Between Different Populations for SLC2A1 SNPs Rs3754219, Rs4660687 and Rs841853.

rs3754219 rs4660687 rs841853 Controls vs χ2 df p-value I2 χ2 df p-value I2 χ2 df p-value I2 All AMD cases combined 16.4 5 0.006 70% 14 5 0.02 64% 15.74 5 0.008 68% Early 4.2 5 0.52 0% 8.79 5 0.12 43% 4.27 5 0.51 0% GA 7.57 4 0.11 47% 1.96 4 0.74 0% 4.49 4 0.34 11% CNV 11.59 4 0.02 65% 2.84 4 0.59 0% 20.06 4 0.0005 80% MIXED 2.24 3 0.52 0% 4.92 3 0.18 39% 3.83 3 0.28 22% CNV + MIXED 5.83 4 0.21 31% 4.61 4 0.33 13% 5.73 4 0.22 30% Late (CNV+ GA+ MIXED) 18.77 5 0.002 73% 7.75 5 0.17 36% 19 5 0.002 74% Heterogeneity between studies was tested using Cochran’s Q statistic and the I-square (I2) statistic for inconsistency. Abbreviations: AMD= age-related macular degeneration; GA= geographic athrophy; CNV= χ2= Chi square; df= degrees of freedom.

choroidal neovascularization; Mixed= combination of GA+CNV;

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and Age-related Macular Degeneration

Figure 2. Meta-analysis of Rs3754219 for its Association With All AMD Cases (A), Early (B), GA (C), CNV (D), Mixed (E), CNV+Mixed (F) and Late AMD (G) Cases in Six Independent Study Populations. AMD= age-related macular degeneration; GA= geographic atrophy; CNV= choroidal

intervals (CI; horizontal lines) are presented for each study. Also shown is the black diamond of the summary neovascularization; Mixed cases= combination of GA + CNV. Odds ratios (ORs, squares) and 95% confidence I2 <25%) or the additive random effects model of DerSimonian and Laird when heterogeneity was present (I2 >25%). OR using either the additive fixed effects model of Mantel Haenszel when heterogeneity was absent ( Heterogeneity between studies was tested using Cochran’s Q statistic and the I-square statistic for inconsistency.

Chapter 3 20120725.indd 69 23-10-12 19:26 Chapter 3

Figure 3. Meta-analysis of Rs4660687 for its Association With All AMD Cases (A), Early (B), GA (C), CNV (D), Mixed (E), CNV+Mixed (F) and Late AMD (G) Cases in Six Independent Study Populations. AMD= age-related macular degeneration; GA= geographic atrophy; CNV= choroidal neovascularization; Mixed cases=

combination of GA+CNV. Odds ratios (ORs, squares) and 95% confidence intervals (CI; horizontal lines) are effects model of Mantel Haenszel when heterogeneity was absent (I2 <25%) or the additive random effects presented for each study. Also shown is the black diamond of the summary OR using either the additive fixed model of DerSimonian and Laird when heterogeneity was present (I2 >25%). Heterogeneity between studies was tested using Cochran’s Q statistic and the I-square statistic for inconsistency.

Chapter 3 20120725.indd 70 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration

Figure 4. Meta-analysis of Rs841853 for its Association With All AMD Cases (A), Early (B), GA (C), CNV (D), Mixed (E), CNV+Mixed (F) and Late AMD (G) Cases in Six Independent Study Populations. AMD= age-related macular degeneration; GA= geographic atrophy; CNV= choroidal neovascularization; Mixed cases= combination

of GA+CNV. Odds ratios (ORs, squares) and 95% confidence intervals (CI; horizontal lines) are presented for each Haenszel when heterogeneity was absent (I2 <25%) or the additive random effects model of DerSimonian and study. Also shown is the black diamond of the summary OR using either the additive fixed effects model of Mantel Laird when heterogeneity was present (I2 >25%). Heterogeneity between studies was tested using Cochran’s Q statistic and the I-square statistic for inconsistency.

Table 4. Comparison of Genotype Frequencies of the Controls and AMD (subgroup) Cases Between All Study Populations.

rs3754219 rs4660687 rs841853 χ2 df Overall p-value χ2 df Overall p-value χ2 df Overall p-value Controls 20.26 10 0.027 14.89 10 0.136 20.60 10 0.024 All AMD 8.49 10 0.581 28.30 10 0.002 8.37 10 0.593 Early 6.81 10 0.743 8.42 10 0.588 5.13 10 0.882 GA 6.05 8 0.641 4.21 8 0.838 7.90 8 0.443 CNV 10.97 8 0.204 12.53 8 0.129 17.60 8 0.024 CNV + mixed 2.46 6 0.873 10.75 6 0.097 3.90 6 0.691 Late AMD 8.72 10 0.559 16.73 10 0.081 17.74 10 0.059 Mixed AMD 5.63 8 0.689 15.59 8 0.049 6.99 8 0.538

Genotype frequencies were compared by using the Pearson χ2 test. Abbreviations: AMD= age-related macular χ2= Chi square; df= degrees of freedom. degeneration; GA= geographic athrophy; CNV= choroidal neovascularization; Mixed= combination of GA+CNV;

Chapter 3 20120725.indd 71 23-10-12 19:26 Chapter 3 Discussion

Three out of 22 SLC2A1 association with AMD in the AMRO-NL discovery cohort. Replication of these three SNPs tested showed a significant allelic and genotypic

inconsistent results. Meta-analysis revealed no overall association between SLC2A1 SNPs (rs3754219, rs4660687 and rs841853) in five independent cohorts yielded

and AMD. For all three SNPs, we observed significant heterogeneity of effect and high inconsistency across the study populations. Using a random or fixed effect model, we calculated pooled ORs, which were not significant for any of the SNPs in interpretation of genetic association studies is the lack of consistent reproducibility any of the studies. Such findings are not unique: one of the greatest challenges in the [48]. Hirschhorn and colleagues (2002) reviewed more than 600 positive associations between common gene variants and disease and found that the majority is not robust. Of the 166 presumed associations, which were studied three or more times, only six were consistently replicated [49]. Similarly, a meta-analysis of 301 published studies covering 25 different reported associations showed that less than half of the associations were consistent across the different study populations [50]. For AMD, at least two recent studies highlight the lack of consistent replication of association across large population studies: initial associations found between AMD and SNPs in the Toll Like Receptor 3 (TLR3) [51] and the Serpin Peptidase Inhibitor, Clade G, member 1 (SERPING1) genes [52], could not be replicated independently [47,53]. For SLC2A1 SNP rs841853 and diabetic nephropathy, a similar inconsistent association result across large study populations has been reported [54]. The SLC2A1 SNPs rs841853, rs3754219 and rs4660687 do not reside within known regulatory region(s) of the SLC2A1 gene (i.e., promoter, enhancers, and

out that these SNP apparently indeed do not affect gene expression. Obviously, the silencer elements). We entered these SNPs into the SNPExpress database to find possibility exist that one or more of the SNPs tag a true causal variant in or adjacent to the SLC2A1 gene which determines the possible genetic susceptibility to AMD. Interestingly, Tao and coworkers (1995) examined whether the association between the rs841853 SNP and non-insulin-dependent diabetes mellitus (NIDDM) could be due to other causal variants in LD with rs841853. Initially, such variant was not found [55]. However, after seven years, Ng et al. (2002) found a new putative causal variant in LD with rs841853 in an insulin-responsive enhancer element, potentially regulating SLC2A1 gene expression and/or function [56]. One of our study limitations is that we selected for common SLC2A1 SNPs, with MAFs>10%. Consequently, we could have missed both variants with MAFs<10%

72and outside the coding region that might influence the disease phenotype.

Chapter 3 20120725.indd 72 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration In addition, because our initial association values found in the AMRO-NL study

there was, initially, a somewhat increased possibility of false-positive association(s). passed only two out of three significance levels used (see Material and Methods), Furthermore, as we and others have discussed extensively elsewhere [36,57], inconsistent replication in genetic-association studies can be caused by numerous factors, including chance, variation in study design, phenotypic AMD (grading) differences and genotype errors [58]. Indeed, the effect of these factors is stronger

have played a role in our study [36]. In addition, genetic heterogeneity between in small cohorts, such as defined by clinical subtypes of AMD. These factors may 3 SLC2A1 SNP 4660687 in the populations may significantly affect the outcome of association studies [57]. It may early cases of the Dutch populations (AMRO-NL and the Rotterdam Study cohorts), be not coincidental that we find positive replication for where the allele and genotype distributions are more or less similar, but not in other populations. Interestingly, we found opposite associations for the three SLC2A1 SNPs (rs3754219, rs4660687 and rs841853) in two highly comparable populations. et al. in two different ethnic populations [59]. The source of this opposite effect in our studies is not clear, A similar “flip-flop” association was previously found by Lin and remains to be elucidated. To assess the contribution of “the genetic factor” in detail we compared the genotype frequencies of all study populations in relation to 2 test showed that genotype frequencies for SLC2A1 the heterogeneity of effect. The Pearson χ rs3754219 and rs841853 were significantly different for the controls of genotype distributions for the cases. For several SLC2A1 SNPs, including rs841853, the six individual populations. For rs4660687 we observed significant differences in genuine genetic differences between populations exist [27,28,60-63]. The fact SCL2A1 SNPs in highly comparable (Caucasian) populations, strongly suggests that the genetic contribution that we found significant genetic heterogeneity for three to the heterogeneity of effect observed in our study is substantial.

three SLC2A1 SNPs (rs3754219, rs4660687, rs841853) and AMD. The heterogeneous In summary, in this study we found no consistent significant association between SLC2A1 SNPs provide further evidence for population- dependent genetic risk heterogeneity in AMD [47,51-53]. Further studies in other, findings on these three

substantiate the potential role of SLC2A1 in AMD. ethnically similar and diverse populations are needed to falsify, confirm or

Chapter 3 20120725.indd 73 23-10-12 19:26 Chapter 3 Acknowledgements

and Dohme and by the Algemene Nederlandse Vereniging ter Voorkoming van This study was in part financed by an unrestricted research grant from Merck Sharpe Blindheid (both to A.A.B.). GWAS genotype data for the Rotterdam Study is supported

(nr.175.010.2005.011, 911-03-012). This study is funded by the Research Institute by the Netherlands Organization for Scientific Research (NWO) Investments for Diseases in the Elderly (014-93-015; RIDE2), the Netherlands Genomics Initiative (NGI)/ NWO project nr. 050-060-810, the EMC and Erasmus University, Netherlands Organization for Health Research and Development (ZonMw), the Ministries of Education, Culture and Science, and Health, Welfare and Sports, the European Commission, and the Municipality of Rotterdam. The Franconian AMD study would like to thank Claudia N. Keilhauer (Department of Ophthalmology, University of Würzburg, Germany) for recruiting individuals with AMD and the control subjects. Kerstin Meier (Institute of Human Genetics, University of Regensburg, Germany) for technical assistance. AREDS was supported (in part) by the Intramural Research Program of the National Cancer Institute, National Institutes of Health. The Columbia University study is supported in part by the grants from the National Eye Institute EY13435 and EY017404; the Macula Vision Research Foundation; Kaplen Foundation; Wigdeon Point Charitable Foundation and an unrestricted grant to the Department of Ophthalmology, Columbia University, from Research to Prevent Blindness. G.S.H. is supported by an unrestricted NIHR24 grant to the Department of Ophthalmology & Visual Sciences, University of Utah and a Senior Scientist Award (GSH) from Research to Prevent Blindness, inc. Baltimore, MD, USA. The University of Iowa would like to thank Chris Pappas for technical assistance.

Chapter 3 20120725.indd 74 23-10-12 19:26 SLC2A1

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and Age-related Macular Degeneration Supplementary appendix Appendix 1. Risk of AMD for SLC2A1 SNPs Rs3754219, Rs4660687 and Rs841853 in Five Replication Populations.

rs3754219 rs4660687 All AMD No AMD All AMD cases Late AMD No AMD cases

The Rotterdam study

N= 2221 N=1069 N=175 N=2221 N=1069 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) No. (%) Genotype Noncarrier (AA) 711 (32.0) 335 (31.3) 1 56 (32.0) 1 772 (34.8) 340 (31.8) Heterozygous (Aa) 1109 (49.9) 531 (49.7) 1.02 (0.86 -1.21) 89 (50.8) 1.04 (0.73-1.49) 1091 (49.1) 517 (48.4) 3 Homozygous (aa) 401 (18.0) 203 (19.0) 1.05 (0.84-1.30) 30 (17.1) 0.92 (0.57-1.47) 358 (16.1) 212 (19.8) m.a.f(%) 43 43.8 42.6 40.7 42.9 The Franconian AMD study N=607 N=794 N=708 N=608 N=793 No(%) No(%) OR (95% CI) No(%) OR (95% CI) No(%) No(%) Genotype Noncarrier (AA) 194 (32.0) 238 (30.0) 1 212 (29.9) 1 212 (34.9) 307 (38.7) Heterozygous (Aa) 298 (49.1) 380 (47.9) 1.04 (0.82-1.33) 338 (47.7) 1.04 (0.81-1.33) 296 (48.7) 379 (47.8) Homozygous (aa) 115 (18.9) 176 (22.2) 1.25 (0.92-1.69) 158 (22.3) 1.26 (0.92-1.71) 100 (16.4) 107 (13.5) m.a.f(%) 43.5 46.1 46.2 40.8 37.4 AREDS N=212 N=917 N=667 N=206 N=901 No(%) No(%) OR (95% CI) No(%) OR (95% CI) No(%) No(%) Genotype Noncarrier (AA) 56 (26.4) 289 (31.5) 1 205 (30.7) 1 73 (35.4) 330 (36.6) Heterozygous (Aa) 108 (50.9) 437 (47.7) 0.78 (0.55-1.12) 324 (48.6) 0.82 (0.57-1.18) 90 (43.7) 400 (44.4) Homozygous (aa) 48 (22.6) 191 (20.8) 0.77 (0.50-1.18) 138 (20.7) 0.79 (0.50-1.22) 43 (20.9) 171 (19.0) m.a.f(%) 48.1 44.6 45,0 42.7 41.1 Columbia University N=365 N=1091 N=730 N=363 N= 1089 No(%) No(%) OR (95% CI) No(%) OR (95% CI) No(%) No(%) Genotype Noncarrier (AA) 121 (33.2) 352 (32.3) 1 226 (31.0) 1 133 (36.6) 360 (33.1) Heterozygous (Aa) 187 (51.2) 524 (48.0) 0.96 (0.74-1.26) 351 (48.1) 1.00 (0.76-1.33) 174 (47.9) 548 (50.3) Homozygous (aa) 57 (15.6) 215 (19.7) 1.30 (0.91-1.85) 153 (21.0) 1.44 (0.99-2.09) 56 (15.4) 181 (16.6) m.a.f(%) 41.2 43.7 45.0 39.4 41.8 The University of Iowa N=383 N=1091 N=768 N=379 N=1058 No(%) No(%) OR (95% CI) No(%) OR (95% CI) No(%) No(%) Genotype Noncarrier (AA) 110 (28.7) 338 (31.0) 1 238 (31.0) 1 127 (33.5) 338 (31.9) Heterozygous (Aa) 194 (50.6) 532 (48.8) 0.89 (0.68-1.17) 376 (48.9) 0.89 (0.67-1.19) 191 (50.4) 542 (51.2) Homozygous (aa) 79 (20.6) 221 (20.2) 0.91 (0.65-1.27) 154 (20.0) 0.90 (0.63-1.28) 61 (16.1) 178 (16.8) m.a.f(%) 45.9 44.6 44.5 41.3 42.4

polymorphism; m.a.f= minor allele frequency. “A” indicates common allele, “a” minor allele. Percentages not AMD= age- related macular degeneration; CI= confidence interval; OR= odds ratio; SNP= single nucleotide always add up to 100% because of rounding. ORs were estimated by logistic regression. Adjustment for age and gender only in the Rotterdam Study. 79

Chapter 3 20120725.indd 79 23-10-12 19:26 Chapter 3 Appendix 1 (continued).

rs4660687 rs841853

All AMD cases Late AMD No AMD All AMD cases Late AMD

The Rotterdam Study

N=175 N=2217 N=1069 N=175 OR (95% CI) No. (%) OR (95% CI) No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI)

1 61 (34.9) 1 1027 (46.3) 493 (46.1) 1 76 (43.4) 1 1.11 (0.94-1.32) 81 (46.3) 0.99(0.70-1.42) 961 (43.3) 459 (42.9) 1.00 (0.86-1.18) 82 (46.9) 1.21 (0.87-1.70) 1.39 (1.12-1.72) 33 (18.9) 1.21(0.77-1.91) 229 (10.3) 117 (10.9) 1.11 (0.86-1.43) 17 (9.7) 1.11 (0.63-1.95) 42.0 32 32.4 33.1 The Franconian AMD study N=707 N=591 N=780 N=697 OR (95% CI) No(%) OR (95% CI) No(%) No(%) OR (95% CI) No(%) OR (95% CI)

1 273 (38.6) 1 265 (44.8) 374 (47.9) 1 333 (47.8) 1 0.88 (0.70-1.11) 338 (47.8) 0.89 (0.70-1.12) 260 (44.0) 326 (41.8) 0.89 (0.71-1.11) 290 (41.6) 0.89 (0.70-1.12) 0.74 (0.53-1.02) 96 (13.6) 0.75 (0.53-1.04) 66 (11.2) 80 (10.3) 0.86 (0.60-1.23) 74 (6.4) 0.89 (0.62-1.29) 30.7 33.2 31.2 31.2 AREDS N=655 N=215 N=924 N=671 OR (95% CI) No(%) OR (95% CI) No(%) No(%) OR (95% CI) No(%) OR (95% CI)

1 239 (36.5) 1 111 (51.6) 429 (46.4) 1 312 (46.5) 1 0.98 (0.70-1.38) 291 (44.4) 0.99 (0.69-1.41) 90 (41.9) 383 (41.4) 1.10 (0.81-1.50) 274 (40.8) 1.08 (0.79-1.49) 0.88 (0.58-1.34) 125 (19.1) 0.89 (0.58-1.37) 14 (6.5) 112 (12.1) 2.07 (1.14-3.76) 85 (12.7) 2.16 (1.18-3.96) 41,3 27,4 32,8 33,1 Columbia University N=727 N=355 N=1083 N=721 OR (95% CI) No(%) OR (95% CI) No(%) OR (95% CI) No(%) OR (95% CI)

1 241 (33.1) 1 158 (44.5) 519 (47.9) 1 357 (49.5) 1 1.16 (0.89-1.51) 367 (50.5) 1.16 (0.88-1.54) 170 (47.9) 451 (41.6) 0.81 (0.63-1.04) 292 (40.5) 0.76 (0.58-0.99) 1.19 (0.83-1.71) 119 (16.4) 1.17 (0.80-1.72) 27 (7.6) 113 (10.4) 1.27 (0.81-2.01) 72 (10.0) 1.18 (0.73-1.91) 41.6 31.5 31.2 30.2 The University of Iowa N=746 N=130 N=316 N=215 OR (95% CI) No(%) OR (95% CI) No(%) No(%) OR (95% CI) No(%) OR (95% CI)

1 243 (32.5) 1 67 (51.1) 153 (48.4) 1 100 (46.5) 1 1.07 (0.82-1.39) 384 (51.4) 1.05 (0.80-1.38) 49 (37.4) 136 (43.0) 1.21 (0.79-1.88) 100 (46.5) 1.37 (0.86-2.17) 1.10 (0.77-1.56) 119 (16.0) 1.02 (0.70-1.48) 14 (10.7) 27 (8.5) 0.84 (0.42-1.71) 15 (7.0) 0.72 (0.33-1.58) 41.7 29.6 30.1 30.2

Chapter 3 20120725.indd 80 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration Appendix 2. Risk of AMD (Subgroup) Cases for SLC2A1 SNP Rs3754219 in Five Replication Populations. No AMD Early AMD GA The Rotterdam study N= 2221 N= 894 N=72 rs3754219 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 711 (32.0) 279 (31.2) 1 18 (25.0) 1 Heterozygous (Aa) 1109 (49.9) 442 (49.4) 1.02 (0.85-1.22) 38 (52.8) 1.38 (0.78-2.46) Homozygous (aa) 401 (18.0) 173 (19.4) 1.07 (0.86-1.35) 16 (22.2) 1.56 (0.78-3.12) m.a.f(%) 43 44.1 48.6 The Franconian AMD study N=607 N=86 N=162 rs3754219 No(%) No(%) OR (95% CI) No(%) OR (95% CI) 3 Genotype Noncarrier (AA) 194 (32.0) 26 (30.2) 1 42 (25.9) 1 Heterozygous (Aa) 298 (49.1) 42 (48.8) 1.05 (0.62-1.77) 82 (50.6) 1.27 (0.84-1.92) Homozygous (aa) 115 (18.9) 18 (20.9) 1.17 (0.61-2.22) 38 (23.5) 1.53 (0.93-2.51) m.a.f(%) 43.5 45.3 48.8 AREDS N=212 N=250 N=167 rs3754219 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 56 (26.4) 84 (33.6) 1 54 (32.3) 1 Heterozygous (Aa) 108 (50.9) 113 (45.2) 0.70 (0.45-1.07) 73 (43.7) 0.70 (0.43-1.13) Homozygous (aa) 48 (22.6) 53 (21.2) 0.74 (0.44-1.23) 40 (23.9) 0.86 (0.49-1.52) m.a.f(%) 48.1 43.8 45.8 Columbia University N=365 N=361 N=208 rs3754219 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 121 (33.2) 126 (34.9) 1 59 (28.4) 1 Heterozygous (Aa) 187 (51.2) 173 (47.9) 0.89 (0.64-1.23) 103 (49.5) 1.13 (0.76-1.67) Homozygous (aa) 57 (15.6) 62 (17.2) 1.04 (0.67-1.62) 46 (22.1) 1.66 (1.01-2.72) m.a.f(%) 41.2 41.1 46.9 The University of Iowa N=383 N=323 N=79 rs3754219 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 110 (28.7) 100 (30.9) 1 30 (37.9) 1 Heterozygous (Aa) 194 (50.6) 156 (48.3) 0,88 (0.63-1.25) 34 (43.0) 0.64 (0.37-1.11) Homozygous (aa) 79 (20.6) 67 (20.7) 0.93 (0.61-1.42) 15 (19.0) 0.70 (0.35-1.38) m.a.f(%) 45.9 44.9 40.5

polymorphism; m.a.f= minor allele frequency; GA= geographic athrophy; CNV= choroidal neovascularization; AMD= age- related macular degeneration; CI= confidence interval; OR= odds ratio; SNP= single nucleotide Percentages not always 100% because of rounding. ORs are estimated with logistic regression analysis. Mixed AMD= combination of GA + CNV; N.P= not performed. “A” indicates common allele, “a” minor allele. Adjustment for age and gender only in the Rotterdam study.

Chapter 3 20120725.indd 81 23-10-12 19:26 Chapter 3 Appendix 2 (continued). CNV CNV + Mixed cases Mixed AMD The Rotterdam study N=64 N=103 N=39 No. (%) OR (95% CI) No. (%) OR (95%/CI) No. (%) OR (95% CI)

25 (39.1) 1 38 (36.9) 1 13 (33.3) 1 33 (51.6) 0.87 (0.51-1.48) 51 (49.5) 0.86 (0.56-1.32) 18 (46.2) 0.93 (0.45-1.92) 6 (9.3) 0.41 (0.17-1.02) 14 (13.6) 0.65 (0.35-1.22) 8 (20.5) 1.04 (0.42-2.56) 35.1 38.3 43.6 The Franconian AMD study N=420 N=546 N=126 No(%) OR (95% CI) No(%) OR (95% CI) No(%) OR (95% CI)

135 (32.1) 1 170 (31.1) 1 35 (28.8) 1 192 (45.7) 0.93 (0.70-1.23) 256 (46.9) 0.98 (0.75-1.28) 64 (50.8) 1.19 (0.76-1.87) 93 (22.1) 1.16 (0.82-1.65) 120 (22.0) 1.19 (0.86-1.65) 27 (21.4) 1.30 (0.75-2.26) 45 45.4 46.8 AREDS N=322 N=500 N=178 No(%) OR (95% CI) No(%) OR (95% CI) No. (%) OR (95% CI)

98 (30.4) 1 151 (30.2) 1 53 (29.8) 1 160 (49.7) 0.85 (0.56-1.27) 251 (50.2) 0.86 (0.59-1.26) 91 (51.1) 0.89 (0.56-1.42) 64 (19.9) 0.76 (0.46-1.25) 98 (19.6) 0.76 (0.48-1.20) 34 (19.1) 0.75 (0.42-1.33) 44.7 44.7 44.7 Columbia University N=522 No(%) OR (95% CI)

N.P. 167 (32.0) 1 N.P. 248 (47.5) 0.96 (0.71-1.30) 107 (20.5) 1.36 (0.91-2.02) 44.3 The University of Iowa N=649 N=689 N=40 No(%) OR (95% CI) No(%) OR (95% CI) No. (%) OR (95% CI)

197 (30.3) 1 208 (30.2) 1 11 (27.5) 1 318 (49.0) 0.92 (0.68-1.23) 342 (49.6) 0.93 (0.70-1.25) 24 (60.0) 1.24 (0.58-2.62) 134 (20.6) 0.95 (0.66-1.36) 139 (20.2) 0.93 (0.65-1.33) 5 (12.5) 0.63 (0.21-1.89) 45.1 45.0 40.0

polymorphism; m.a.f = minor allele frequency; GA= geographic athrophy; CNV= choroidal neovascularization; AMD= age- related macular degeneration; CI= confidence interval; OR= odds ratio; SNP= single nucleotide Percentages not always 100% because of rounding. ORs are estimated with logistic regression analysis. Mixed AMD= combination of GA + CNV; N.P= not performed. “A” indicates common allele, “a” minor allele. Adjustment for age and gender only in the Rotterdam study.

Chapter 3 20120725.indd 82 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration Appendix 3. Risk of AMD (Subgroup) Cases for SLC2A1 SNP Rs4660687 in Five Replication Populations. No AMD Early AMD GA The Rotterdam study N=2221 N=894 N=72 rs4660687 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 772 (34.8) 279 (31.2) 1 24 (33.3) 1 Heterozygous (Aa) 1091 (49.1) 436 (48.8) 1.14 (0.95-1.36) 34 (47.2) 1.04 (0.61-1.79) Homozygous (aa) 358 (16.1) 179 (20.0) 1.42 (1.13-1.78) 14 (19.4) 1.28 (0.65-2.53) m.a.f(%) 40.7 44.4 43.0 The Franconian AMD study N=608 N=86 N=162 rs4660687 No(%) No(%) OR (95% CI) No(%) OR (95% CI) 3 Genotype Noncarrier (AA) 212 (34.9) 34 (39.5) 1 62 (38.3) 1 Heterozygous (Aa) 296 (48.7) 41 (47.7) 0.86 (0.53-1.41) 79 (48.8) 0.91 (0.63-1.33) Homozygous (aa) 100 (16.4) 11 (12.8) 0.69 (0.33-1.41) 21 (12.9) 0.72 (0.41-1.24) m.a.f(%) 40.8 36.6 37.3 AREDS N=206 N=246 N=167 rs4660687 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 73 (35.4) 91 (37.0) 1 65 (38.9) 1 Heterozygous (Aa) 90 (43.7) 109 (44.3) 0.97 (0.64-1.47) 76 (45.5) 0.95 (0.60-1.49) Homozygous (aa) 43 (20.9) 46 (18.7) 0.86 (0.51-1.44) 26 (15.6) 0.68 (0.38-1.22) m.a.f(%) 42.7 40.8 38.3 Columbia university N=363 N=362 N=208 rs4660687 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 133 (36.6) 119 (32.9) 1 72 (34.6) 1 Heterozygous (Aa) 174 (47.9) 181 (50.0) 1.16 (0.84-1.61) 110 (52.9) 1.17 (0.80-1.70) Homozygous (aa) 56 (15.4) 62 (17.1) 1.24 (0.80-1.92) 26 (12.5) 0.86 (0.50-1.48) m.a.f(%) 39.4 42.1 38.9 The university of Iowa N=379 N=312 N=77 rs4660687 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 127 (33.5) 95 (30.4) 1 27 (35.1) 1 Heterozygous (Aa) 191 (50.4) 158 (50.6) 1.11 (0.79-1.55) 38 (49.4) 0.93 (0.54-1.61) Homozygous (aa) 61 (16.1) 59 (18.9) 1.29 (0.83-2.02) 12 (15.5) 0.92 (0.44-1.95) m.a.f(%) 41.3 44.2 40.3

Chapter 3 20120725.indd 83 23-10-12 19:26 Chapter 3 Appendix 3 (continued). CNV CNV + Mixed cases Mixed AMD The Rotterdam study N=64 N=103 N=39 No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) OR (95% CI)

23 (35.9) 1 37 (35.9) 1 14 (35.9) 1 31 (48.4) 1.01 (0.58-1.75) 47 (45.6) 0.90 (0.58-1.40) 16 (41.0) 0.91 (0.44-1.90) 10 (15.6) 0.93 (0.44-2.00) 19 (18.4) 1.11 (0.63-1.95) 9 (23.1) 1.47 (0.62-3.50) 39.8 41.3 43.6 The Franconian AMD study N=419 N=545 N=126 No(%) OR (95% CI) No(%) OR (95% CI) No(%) OR (95% CI)

152 (36.3) 1 211 (38.7) 1 59 (46.8) 1 205 (48.9) 0.97 (0.73-1.27) 259 (47.5) 0.88 (0.68-1.13) 54 (42.9) 0.66 (0.44-0.99) 62 (14.8) 0.86 (0.59-1.26) 75 (13.8) 0.75 (0.53-1.07) 13 (10.3) 0.47 (0.24-0.89) 39.3 37.5 31.7 AREDS N=312 N=488 N=176 No(%) OR (95% CI) No(%) OR (95% CI) No. (%) OR (95% CI)

113 (36.2) 1 174 (35.7) 1 61 (34.7) 1 132 (42.3) 0.95 (0.64-1.41) 215 (44.1) 1.00 (0.69-1.45) 83 (47.2) 1.10 (0.70-1.73) 67 (21.5) 1.01 (0.62-1.63) 99 (20.3) 0.97 (0.62-1.52) 32 (18.2) 0.89 (0.50-1.57) 42.6 42.3 41.8 Columbia university N=519 No(%) OR (95% CI)

N.P 169 (32.6) 1 N.P 257 (49.5) 1.16 (0.86-1.57) 93 (17.9) 1.31 (0.87-1.95) 42.7 The university of Iowa N=632 N=669 N=37 No(%) OR (95% CI) No(%) OR (95% CI) No. (%) OR (95% CI)

205 (32.4) 1 216 (32.2) 1 11 (29.7) 1 324 (51.2) 1.05 (0.79-1.40) 346 (51.7) 1.07 (0.80 -1.41) 22 (59.4) 1.33 (0.62-2.84) 103 (16.3) 1.05 (0.71-1.54) 107 (16.0) 1.03 (0.70-1.51) 4 (10.8) 0.76 (0.23-2.47) 41.9 41.8 40.5

Chapter 3 20120725.indd 84 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration Appendix 4. Risk of AMD (Subgroup) Cases for SLC2A1 SNP Rs841853 in Five Replication Populations. No AMD Early AMD GA The Rotterdam study N=2217 N=894 N=72 rs841853 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 1027 (46.3) 417 (46.6) 1 35 (48.6) 1 Heterozygous (Aa) 961 (43.3) 377 (42.2) 0.98 (0.83-1.15) 31 (43.1) 0.95 (0.58-1.55) Homozygous (aa) 229 (10.3) 100 (11.2) 1.11 (0.85-1.44) 6 (8.3) 0.77 (0.32-1.85) m.a.f(%) 32 32.3 29.9 The Franconian AMD study N=591 N=83 N=159 rs841853 No(%) No(%) OR (95% CI) No(%) OR (95% CI) 3 Genotype Noncarrier (AA) 265 (44.8) 41 (49.4) 1 75 (47.2) 1 Heterozygous (Aa) 260 (44.0) 36 (43.4) 0.89 (0.55-1.45) 70 (44.0) 0.95 (0.66-1.37) Homozygous (aa) 66 (11.2) 6 (7.2) 0.59 (0.24-1.44) 14 (8.8) 0.75 (0.40-1.41) m.a.f(%) 33.2 28.9 30.8 AREDS N=215 N=253 N=167 rs841853 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 111 (51.6) 117 (46.2) 1 80 (47.9) 1 Heterozygous (Aa) 90 (41.9) 109 (43.1) 1.15 (0.78-1.68) 62 (37.1) 0.96 (0.62-1.47) Homozygous (aa) 14 (6.5) 27 (10.7) 1.83 (0.91-3.67) 25 (15.0) 2.48 (1.21-5.06) m.a.f(%) 27.4 32.2 33.5 Columbia University N=355 N=362 N=206 rs841853 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 158 (44.5) 162 (44.8) 1 107 (51.9) 1 Heterozygous (Aa) 170 (47.9) 159 (43.9) 0.91 (0.67-1.24 76 (36.9) 0.66 (0.46-0.95) Homozygous (aa) 27 (7.6) 41 (11.3) 1.48 (0.87-2.52) 23 (11.2) 1.26 (0.68-2.31) m.a.f(%) 31.5 33.3 29.6 The university of Iowa N=130 N=101 N=28 rs841853 No(%) No(%) OR (95% CI) No(%) OR (95% CI) Genotype Noncarrier (AA) 67 (51.1) 53 (52.5) 1 13 (46.4) 1 Heterozygous (Aa) 49 (37.4) 36 (35.6) 0.93 (0.53-1.63) 14 (50.0) 1.47 (0.64-3.41) Homozygous (aa) 14 (10.7) 12 (11.9) 1.08 (0.46-2.54) 1 (3.6) 0.37 (0.04-3.05) m.a.f(%) 29.6 29.7 28.6

polymorphism; m.a.f = minor allele frequency; GA= geographic athrophy; CNV= choroidal neovascularization; AMD= age- related macular degeneration; CI= confidence interval; OR= odds ratio; SNP= single nucleotide Percentages not always 100% because of rounding. ORs are estimated with logistic regression analysis. Mixed AMD= combination of GA + CNV; N.P= not performed. “A” indicates common allele, “a” minor allele. Adjustment for age and gender only in the Rotterdam study.

Chapter 3 20120725.indd 85 23-10-12 19:26 Chapter 3 Appendix 4 (continued). CNV CNV+ Mixed cases Mixed AMD The Rotterdam study N=64 N=103 N=39 No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) OR (95% CI)

17 (26.6) 1 41 (39.8) 1 24 (61.5) 1 38 (59.4) 2.55 (1.43-4.58) 51 (49.5) 1.33 (0.87-2.02) 13 (33.3) 0.58 (0.29-1.14) 9 (14.1) 2.59 (1.13-5.94) 11 (10.7) 1.20 (0.61-2.38 2 (5.1) 0.37 (0.09-1.59) 43.8 35.4 21.8 The Franconian AMD study N=415 N=538 N=123 No(%) OR (95% CI) No(%) OR (95% CI) No(%) OR (95% CI)

199 (48.0) 1 258 (48.0) 1 59 (48.0) 1 172 (41.4) 0.88 (0.68-1.15) 220 40.9) 0.87 (0.68-1.11) 48 (39.0) 0.83 (0.55-1.26) 44 (10.6) 0.89 (0.58-1.36) 60 (11.1) 0.93 (0.63-1.38) 16 (13.0) 1.09 (0.59-2.01) 31.3 31.6 32.5 AREDS N=325 N=504 N= 179 No(%) OR (95% CI) No(%) OR (95% CI) No(%) OR (95% CI)

139 (42.8) 1 232 (46.0) 1 93 (52.0) 1 144 (44.3) 1.28 (0.89-1.84) 212 (42.1) 1.13 (0.81-1.57) 68 (38.0) 0.90 (0.59-1.37) 42 (12.9) 2.40 (1.25-4.61) 60 (11.9) 2.05 (1.10-3.83) 18 (10.0) 1.53 (0.72-3.25) 35.1 32.9 Columbia University N=515 No(%) OR (95% CI)

N.P. 250 (48.5) 1 N.P. 216 (41.9) 0.80 (0.61-1.07) 49 (9.5) 1.15 (0.69-1.91) 30.5 The university of Iowa N=175 N=187 N=12 No(%) OR (95% CI) No(%) OR (95% CI) No(%) OR (95% CI)

82 (46.9) 1 87 (46.5) 1 5 (41.7) 1 80 (45.7) 1.33 (0.83-2.16) 86 (46.0) 1.35 (0.84-2.17) 6 (50.0) 1.64 (0.47-5.69) 13 (7.4) 0.76 (0.33-1.72) 14 (7.5) 0.77 (0.34-1.72) 1 (8.3) 0.96 (0.10-8.84) 30.2 30.5 33.3

Chapter 3 20120725.indd 86 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration Appendix 5. Comparison of Genotype Frequencies of the Controls and AMD (Subgroup) Cases Between all Study Populations. Controls rs3754219 20.258 df 10 p-value 0.027 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 44 56 121 194 110 711 Heterozygous (Aa) 84 108 187 298 194 1109 Homozygous (aa) 51 48 57 115 79 401 MAF 0.52 0.48 0.41 0.43 0.46 0.43

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.415 0.001 0.014 0.113 0.002 AREDS NA NA 0.060 0.250 0.770 0.124 3 Columbia NA NA NA 0.420 0.151 0.526 Franconian NA NA NA NA 0.534 0.872 IOWA NA NA NA NA NA 0.312 Rotterdam NA NA NA NA NA NA

rs4660687 14.886 df 10 p-value 0.136 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 71 73 133 212 127 772 Heterozygous (Aa) 57 90 174 296 191 1091 Homozygous (aa) 22 43 56 100 61 358 MAF 0.34 0.43 0.39 0.41 0.41 0.41

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.062 0.067 0.017 0.010 0.007 AREDS NA NA 0.246 0.281 0.211 0.154 Columbia NA NA NA 0.829 0.671 0.780 Franconian NA NA NA NA 0.869 0.974 IOWA NA NA NA NA NA 0.881 Rotterdam NA NA NA NA NA NA

rs841853 20.602 df 10 p-value 0.024 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 104 111 158 265 67 1027 Heterozygous (Aa) 62 90 170 260 49 961 Homozygous (aa) 13 14 27 66 14 229 MAF 0.25 0.27 0.32 0.33 0.30 0.32

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.341 0.010 0.007 0.398 0.009 AREDS NA NA 0.256 0.076 0.340 0.126 Columbia NA NA NA 0.165 0.115 0.140 Franconian NA NA NA NA 0.360 0.748 IOWA NA NA NA NA NA 0.438 Rotterdam NA NA NA NA NA NA

Genotype frequencies were compared by using the Pearson χ2 test. Abbreviations: AMD= age-related macular degeneration; GA= geographic athrophy; CNV= choroidal neovascularization; Mixed= combination of GA+CNV; χ2= Chi square; df= degrees of freedom. 87

Chapter 3 20120725.indd 87 23-10-12 19:26 Chapter 3 Appendix 5 (continued). All AMD cases rs3754219 8.493 df 10 p-value 0.581 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 118 289 352 238 338 335 Heterozygous (Aa) 184 437 524 380 532 531 Homozygous (aa) 56 191 215 176 221 203 MAF 0.41 0.45 0.44 0.46 0.45 0.44

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.106 0.218 0.038 0.156 0.361 AREDS NA NA 0.814 0.709 0.882 0.533 Columbia NA NA NA 0.348 0.808 0.745 Franconian NA NA NA NA 0.599 0.242 IOWA NA NA NA NA NA 0.758 Rotterdam NA NA NA NA NA NA

rs4660687 28.303 df 10 p-value 0.002 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 95 330 360 307 338 340 Heterozygous (Aa) 133 400 548 379 542 517 Homozygous (aa) 51 171 181 107 178 212 MAF 0.42 0.41 0.42 0.37 0.42 0.44

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.621 0.691 0.110 0.569 0.726 AREDS NA NA 0.030 0.010 0.010 0.075 Columbia NA NA NA 0.022 0.859 0.155 Franconian NA NA NA NA 0.006 0.000 IOWA NA NA NA NA NA 0.173 Rotterdam NA NA NA NA NA NA

rs841853 8.372 df 10 p-value 0.593 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 150 429 519 374 153 493 Heterozygous (Aa) 168 383 451 326 136 459 Homozygous (aa) 41 112 113 80 27 117 MAF 0.35 0.33 0.31 0.31 0.30 0.32

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.216 0.128 0.152 0.169 0.352 AREDS NA NA 0.471 0.467 0.220 0.648 Columbia NA NA NA 0.992 0.607 0.699 Franconian NA NA NA NA 0.681 0.718 IOWA NA NA NA NA NA 0.443 Rotterdam NA NA NA NA NA NA

Genotype frequencies were compared by using the Pearson χ2 test. Abbreviations: AMD= age-related macular square; df= degrees of freedom. degeneration; GA= geographic athrophy; CNV= choroidal neovascularization; Mixed= combination of GA+CNV; χ2= Chi

Chapter 3 20120725.indd 88 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration Appendix 5 (continued). CNV rs3754219 10.967 df 8 p-value 0.204 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 60 98 NE 135 197 25 Heterozygous (Aa) 91 160 NE 192 318 33 Homozygous (aa) 25 64 NE 93 134 6 MAF 0.40 0.45 NE 0.45 0.45 0.35

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.270 NE 0.081 0.149 0.556 AREDS NA NA NE 0.544 0.959 0.104 Columbia NE NE NE NE NE NE 3 Franconian NA NA NE NA 0.575 0.060 IOWA NA NA NE NA NA 0.072 Rotterdam NA NA NE NA NA NA

rs4660687 12.534 df 8 p-value 0.129 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 52 113 NE 152 205 23 Heterozygous (Aa) 55 132 NE 205 324 31 Homozygous (aa) 26 67 NE 62 103 10 MAF 0.40 0.43 NE 0.39 0.42 0.40

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.821 NE 0.237 0.115 0.614 AREDS NA NA NE 0.045 0.024 0.509 Columbia NE NE NE NE NE NE Franconian NA NA NE NA 0.421 0.985 IOWA NA NA NE NA NA 0.850 Rotterdam NA NA NE NA NA NA

rs841853 17.602 df 8 p-value 0.024 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 65 139 NE 199 82 17 Heterozygous (Aa) 89 144 NE 172 80 38 Homozygous (aa) 22 42 NE 44 13 9 MAF 0.38 0.35 NE 0.31 0.30 0.44

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.380 NE 0.048 0.093 0.324 AREDS NA NA NE 0.322 0.165 0.046 Columbia NE NE NE NE NE NE Franconian NA NA NE NA 0.399 0.006 IOWA NA NA NE NA NA 0.013 Rotterdam NA NA NE NA NA NA

Chapter 3 20120725.indd 89 23-10-12 19:26 Chapter 3 Appendix 5 (continued). CNV + mixed rs3754219 5.630 df 8 p-value 0.689 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 151 167 170 208 38 Heterozygous (Aa) NE 251 248 256 342 51 Homozygous (aa) NE 98 107 120 139 14 MAF NE 0.45 0.44 0.45 0.45 0.38

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA 0.689 0.503 0.967 0.236 Columbia NE NA NA 0.836 0.740 0.244 Franconian NE NA NA NA 0.595 0.137 IOWA NE NA NA NA NA 0.191 Rotterdam NE NA NA NA NA NA

rs4660687 15.592 df 8 p-value 0.049 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 174 169 211 216 37 Heterozygous (Aa) NE 215 257 259 346 47 Homozygous (aa) NE 99 93 75 107 19 MAF NE 0.42 0.43 0.38 0.42 0.41

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA 0.218 0.020 0.026 0.908 Columbia NE NA NA 0.051 0.628 0.751 Franconian NE NA NA NA 0.061 0.460 IOWA NE NA NA NA NA 0.512 Rotterdam NE NA NA NA NA NA

rs841853 6.986 df 8 p-value 0.538 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 232 250 258 87 41 Heterozygous (Aa) NE 212 216 220 86 51 Homozygous (aa) NE 60 49 60 14 11 MAF NE 0.33 0.30 0.32 0.30 0.35

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA 0.427 0.811 0.225 0.379 Columbia NE NA NA 0.680 0.531 0.266 Franconian NE NA NA NA 0.254 0.251 IOWA NE NA NA NA NA 0.441 Rotterdam NE NA NA NA NA NA

Genotype frequencies were compared by using the Pearson χ2 test. Abbreviations: AMD= age-related macular Chi square; df= degrees of freedom. degeneration; GA= geographic athrophy; CNV= choroidal neovascularization; Mixed= combination of GA+CNV; χ2= 90

Chapter 3 20120725.indd 90 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration Appendix 5 (continued). Late AMD rs3754219 8.723 df 10 p-value 0.559 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 95 205 226 212 238 56 Heterozygous (Aa) 132 324 351 338 376 89 Homozygous (aa) 40 138 153 158 154 30 MAF 0.40 0.45 0.45 0.46 0.45 0.43

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.096 0.083 0.027 0.134 0.687 AREDS NA NA 0.982 0.762 0.956 0.579 Columbia NA NA NA 0.804 0.901 0.525 3 Franconian NA NA NA NA 0.566 0.326 IOWA NA NA NA NA NA 0.681 Rotterdam NA NA NA NA NA NA

rs4660687 16.732 df 10 p-value 0.081 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 71 239 241 273 243 61 Heterozygous (Aa) 92 291 367 338 384 81 Homozygous (aa) 34 125 119 96 119 33 MAF 0.41 0.41 0.42 0.37 0.42 0.42

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.797 0.636 0.415 0.489 0.918 AREDS NA NA 0.074 0.022 0.029 0.900 Columbia NA NA NA 0.068 0.929 0.565 Franconian NA NA NA NA 0.048 0.196 IOWA NA NA NA NA NA 0.426 Rotterdam NA NA NA NA NA NA

rs841853 17.742 df 10 p-value 0.059 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 103 312 357 333 100 76 Heterozygous (Aa) 131 274 292 290 100 82 Homozygous (aa) 34 85 72 74 15 17 MAF 0.37 0.33 0.30 0.31 0.30 0.33

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.058 0.008 0.033 0.054 0.459 AREDS NA NA 0.237 0.495 0.052 0.289 Columbia NA NA NA 0.793 0.187 0.293 Franconian NA NA NA NA 0.201 0.455 IOWA NA NA NA NA NA 0.581 Rotterdam NA NA NA NA NA NA

Chapter 3 20120725.indd 91 23-10-12 19:26 Chapter 3 Appendix 5 (continued). Early AMD rs3754219 6.812 df 10 p-value 0.743 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 23 84 126 26 100 279 Heterozygous (Aa) 52 113 173 42 156 442 Homozygous (aa) 16 53 62 18 67 173 MAF 0.46 0.44 0.41 0.45 0.45 0.44

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.144 0.193 0.542 0.328 0.357 AREDS NA NA 0.454 0.816 0.738 0.493 Columbia NA NA NA 0.605 0.376 0.395 Franconian NA NA NA NA 0.992 0.937 IOWA NA NA NA NA NA 0.860 Rotterdam NA NA NA NA NA NA

rs4660687 8.423 df 10 p-value 0.588 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 24 91 119 34 95 279 Heterozygous (Aa) 41 109 181 41 158 436 Homozygous (aa) 17 46 62 11 59 179 MAF 0.46 0.41 0.42 0.37 0.44 0.44

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.446 0.685 0.233 0.929 0.936 AREDS NA NA 0.383 0.457 0.232 0.227 Columbia NA NA NA 0.410 0.733 0.489 Franconian NA NA NA NA 0.195 0.147 IOWA NA NA NA NA NA 0.838 Rotterdam NA NA NA NA NA NA

rs841853 5.132 df 10 p-value 0.882 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) 47 117 162 41 53 417 Heterozygous (Aa) 37 109 159 36 36 377 Homozygous (aa) 7 27 41 6 12 100 MAF 0.28 0.32 0.33 0.29 0.30 0.32

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NA 0.576 0.400 0.936 0.557 0.494 AREDS NA NA 0.925 0.642 0.437 0.955 Columbia NA NA NA 0.497 0.313 0.823 Franconian NA NA NA NA 0.409 0.537 IOWA NA NA NA NA NA 0.444 Rotterdam NA NA NA NA NA NA

Chapter 3 20120725.indd 92 23-10-12 19:26 SLC2A1

and Age-related Macular Degeneration Appendix 5 (continued). GA rs3754219 6.054 df 8 p-value 0.641 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 54 59 42 30 18 Heterozygous (Aa) NE 73 103 82 34 38 Homozygous (aa) NE 40 46 38 15 16 MAF NE 0.46 0.47 0.49 0.41 0.49

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA 0.526 0.368 0.578 0.396 Columbia NE NA NA 0.864 0.291 0.846 3 Franconian NE NA NA NA 0.158 0.954 IOWA NE NA NA NA NA 0.230 Rotterdam NE NA NA NA NA NA

rs4660687 4.207 df 8 p-value 0.838 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 65 72 62 27 24 Heterozygous (Aa) NE 76 110 79 38 34 Homozygous (aa) NE 26 26 21 12 14 MAF NE 0.38 0.39 0.37 0.40 0.43

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA 0.347 0.746 0.829 0.636 Columbia NE NA NA 0.721 0.764 0.339 Franconian NE NA NA NA 0.816 0.416 IOWA NE NA NA NA NA 0.825 Rotterdam NE NA NA NA NA NA

rs841853 7.903 df 8 p-value 0.443 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 80 107 75 13 35 Heterozygous (Aa) NE 62 76 70 14 31 Homozygous (aa) NE 25 23 14 1 6 MAF NE 0.34 0.30 0.31 0.29 0.30

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA 0.512 0.169 0.186 0.338 Columbia NE NA NA 0.360 0.268 0.591 Franconian NE NA NA NA 0.605 0.978 IOWA NE NA NA NA NA 0.641 Rotterdam NE NA NA NA NA NA

Chapter 3 20120725.indd 93 23-10-12 19:26 Chapter 3 Appendix 5 (continued). Mixed rs3754219 2.456 df 6 p-value 0.873 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 53 NE 35 11 13 Heterozygous (Aa) NE 91 NE 64 24 18 Homozygous (aa) NE 34 NE 27 5 8 MAF NE 0.45 NE 0.47 0.43 0.44

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA NE 0.861 0.561 0.867 Columbia NE NE NE NE NE NE Franconian NE NA NE NA 0.438 0.786 IOWA NE NA NE NA NA 0.437 Rotterdam NE NA NE NA NA NA

rs4660687 10.745 df 6 p-value 0.097 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 61 NE 59 11 14 Heterozygous (Aa) NE 83 NE 54 22 16 Homozygous (aa) NE 32 NE 13 4 9 MAF NE 0.42 NE 0.32 0.41 0.44

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA NE 0.046 0.375 0.694 Columbia NE NE NE NE NE NE Franconian NE NA NE NA 0.154 0.120 IOWA NE NA NE NA NA 0.196 Rotterdam NE NA NE NA NA NA

rs841853 3.897 df 6 p-value 0.691 AMRO_NL AREDS Columbia Franconian IOWA Rotterdam χ2 Noncarrier (AA) NE 93 NE 59 5 24 Heterozygous (Aa) NE 68 NE 48 6 13 Homozygous (aa) NE 18 NE 16 1 2 MAF NE 0.29 NE 0.33 0.33 0.22

AMRO_NL AREDS Columbia Franconian IOWA Rotterdam AMRO_NL NE NE NE NE NE NE AREDS NE NA NE 0.663 0.819 0.501 Columbia NE NE NE NA 0.913 0.241 Franconian NE NA NE NE NE NE IOWA NE NA NE NA NA 0.341 Rotterdam NE NA NE NA NA NA

Chapter 3 20120725.indd 94 23-10-12 19:26 Chapter 3 20120725.indd 95 23-10-12 19:26 Chapter 3 20120725.indd 96 23-10-12 19:26 CHAPTER 4 The Complement Component 5 Gene and Age-related Macular Degeneration

Dominique C. Baas, Lintje Ho, Sarah Ennis, Joanna E. Merriam, Michael W.T. Tanck,

Fernando Rivadeneira, Albert Hofman, Cornelia van Duijn, R. Theodore Smith, André G. Uitterlinden, Paulus T.V.M. de Jong, Angela J. Cree, Helen L. Griffiths, Gaetano R. Barile, Theo G.M.F. Gorgels, Johannes R. Vingerling, Caroline C.W. Klaver, Andrew J. Lotery, Rando Allikmets and Arthur A.B. Bergen

Ophthalmology 2010; 117(3):500-511

Chapter 4 20120904.indd 97 23-10-12 22:59 Chapter 4 Abstract

Objective: To investigate the association between variants in the complement component 5 (C5) gene and age-related macular degeneration (AMD). Design: Separate and combined data from three large AMD case-control studies and a prospective population-based study (The Rotterdam Study). Participants: A total of 2599 AMD cases and 3458 ethnically matched controls. Methods: Fifteen single nucleotide polymorphisms (SNPs) spanning the C5 gene were initially genotyped in 375 cases and 199 controls from the Netherlands (The AMRO-NL study population). Replication testing of selected SNPs was performed in the Rotterdam Study (NL) and study populations from Southampton, United Kingdom (UK) and New York, United States (US). Main Outcome Measures: Early and late stages of prevalent and incident AMD,

system of AMD. graded according to (a modification of) the international grading and classification Results C5 SNPs and AMD were found in the AMRO-NL study and this risk appeared independently of CFH : Significant allelic or genotypic associations between eight Y402H, LOC387715 consistently in three replication populations. A69S, age and gender. None of these findings could be confirmed Conclusions: Although the complement pathway, including C5, plays a crucial role in

between C5 SNPs and AMD were found in all studies. The implications for genetic AMD, and the C5 protein is present in drusen, no consistent significant associations screening of AMD are discussed.

Chapter 4 20120904.indd 98 23-10-12 22:59 C5

The gene and Age-related Macular Degeneration Introduction

Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in the elderly in the Western world and is therefore a major public

characterized by the presence of soft drusen and pigmentary changes in the macular health issue [1,2]. Typically, AMD is classified in early and late forms. Early AMD is area. Late AMD is further divided into a “dry” form, geographic atrophy (GA), and a “wet” form, choroidal neovascularization (CNV). The overall prevalence of early and late AMD among Americans over 40 years of age is estimated to be respectively 7.3 million and 1.8 million. The prevalence of late AMD is estimated to increase to 3.0 million in 2020 [1,2]. AMD has a multifactorial etiology. Both environmental and genetic factors contribute to disease susceptibility [3]. Environmental risk factors include cigarette smoking, undue sunlight combined with low antioxidant dietary

studies have been successful in dissecting the genetic susceptibility for AMD in the 4 intake, insufficient physical activity and poor cardiovascular health [4]. Molecular last few years. Genetic association studies for at least four loci (complement factors H (CFH), B (CFB), 2 (C2), 3 (C3)), and the LOC387715 (HTRA1/ARMS2) locus suggested that the complement system and, possibly, oxidative stress pathways play a major role in the molecular pathology of AMD [5-13]. Most importantly, at least 74% of AMD cases can be explained by polymorphisms in CFH, CFB and C2 genes [7-10]. In general, the complement system plays a highly effective role in destructing invading microorganisms and in immune complex elimination. The three activation pathways of complement are the classical, the alternative and the mannose-binding lectin pathway. CFH and CFB are key components of the alternative complement pathway and C2 plays a role in the classical pathway. The three pathways converge where C3- convertase cleaves and activates component C3, creating C3a and C3b. Binding of the latter increases pathogen-immune complex clearance and initiates the formation of the lytic membrane attack complex (MAC), consisting of C5b-C9 [14,15]. Complement component 5 (C5) is cleaved by C5-convertase into the anaphylatoxin C5a and C5b,

mediator [16]. Next to the suggestion that AMD may be a result of local activation with C5a being an effective leukocyte chemoattractant and a powerful inflammatory et al. (2008) showed that genetic variants of CFH could lead to systemic activation of of the alternative pathway provoked by chronic inflammatory processes, Scholl the complement pathway, and thus implying AMD as a systemic disease with a local disease manifestation [17]. Studies on the molecular composition of drusen have also implicated the

drusen, whereas CFH, C3 and MAC (C5b-9) protein complexes were detected in both complement system in AMD. More specifically, CFB and C5 proteins were found in

Chapter 4 20120904.indd 99 23-10-12 22:59 Chapter 4 basal laminar deposits (BLD) and drusen [8, 18-26]. Recently, Nozaki et al. (2006) observed immunolocalization of C3a and C5a in both hard and soft drusen [26]. Preliminary genetic association studies for C3 and C5 have, so far, implicated a role for C3 in the pathogenesis of AMD, but not for C5 [11,13]. In summary, based on the crucial role of C5 in the formation of the MAC complex of

processes, and its localization in drusen, we hypothesized that genetic variants in the complement system, its role as chemoattractant regulating local inflammatory C5 mediate AMD-susceptibility. Therefore, we performed an extensive association analysis to test whether complement C5 gene variants are associated with AMD.

Methods

Cases and controls Altogether, we utilized three case-control studies and one prospective, population- based study, consisting of 2599 AMD cases and 3458 ethnically- and age-matched control subjects. All studies were approved by the Ethics Committees of the Academic Medical Center Amsterdam, the Erasmus Medical Center Rotterdam, Southampton Local Research Ethics Committee (approval no. 347/02/t) and the Institutional Review Board of Columbia University. All studies followed the tenets of the Declaration of Helsinki. All participants provided signed informed consent for participation in the study. The initial population screened for C5 variants, the AMRO-NL study population, consisted of 375 unrelated individuals with AMD and 199 control individuals. All subjects were Caucasian and recruited from the Netherlands Institute of Neuroscience (NIN) Amsterdam and Erasmus University Medical Centre Rotterdam, by newsletters, via patient organizations, and nursing home visits. Controls were at least 65 years old, and were usually unaffected spouses or non- related acquaintances of cases or individuals who attended the ophthalmology department for reasons other than retinal pathology. The second population, the Rotterdam study is a prospective, population-based study of chronic diseases in the elderly [27]. The eligible population comprised all 10 275 inhabitants aged 55 years or older of a middle-class suburb of Rotterdam, the Netherlands, of whom 7983 (78%) participated. Because the ophthalmologic part of the study became operational after the pilot phase of the study had started, 6780 (66%) took part in the ophthalmic examinations. Baseline examinations, including a home interview and physical examinations at the research center, took place from 1990 until 1993 and were followed by three examinations from 1993 through 1994, from 1997 through 1999, and from 2000 through 2004.

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Chapter 4 20120904.indd 100 23-10-12 22:59 C5

The gene and Age-related Macular Degeneration The third population, the United Kingdom (UK) study population, consisted of 564 cases and 640 age matched control subjects from the same clinic population as described elsewhere [28,29]. The fourth population, the Columbia University, United States (US) study population, consisted of 644 unrelated individuals with AMD and 368 unrelated controls of European American descent, recruited at Colombia University as previously described [30].

Diagnosis of AMD Study subjects from the AMRO-NL, Rotterdam and US study population underwent

the macula after pupil dilatation at each visit (Topcon TRV-50VT fundus camera, ophthalmic examination and fundus photography covering a 35° field centered on Topcon Optical Co, Tokyo, Japan). Signs of AMD in those study populations were 4

graded according to (a modification of) the international classification and grading having or not having disease on the basis of the Age-Related Eye Disease Study system for AMD [31]. Cases and controls from the UK study were classified as

(AREDS) classification system [32]. While AREDS uses slightly different criteria for four studies. Controls showed no, classification of early AMD and GA, the grading criteria were very similar for the macular pathology. Early AMD cases had either soft distinct drusen with pigmentary or less than five small hard drusen, and no other irregularities or soft indistinct drusen without pigmentary irregularities and soft indistinct drusen with pigmentary irregularities. Late AMD cases presented with geographic atrophy (GA), choroidal neovascularization (CNV), or a combination of both (mixed AMD).

SNP selection Fifteen SNPs were selected to span and tag the entire C5 gene. SNP data were used from the Centre d’Étude du Polymorphisme Humain (CEPH) population (Utah residents with ancestry from northern and western Europe) by use of the International HapMAP Project. Available at: http://www.hapmap.org/, NCBI build 36, dbSNP b126. Accessed January 7, 2009. SNP selection was based on criteria like functional relevance, minor allele frequency (MAF)>10%, coverage of the main linkage disequilibrium (LD) blocks and tagging of the most common haplotypes. Tag SNPs were selected by use of Tagger, an option of Haploview [33] (all SNPs were captured with a LD tagging criteria of r2>0.8).

101

Chapter 4 20120904.indd 101 23-10-12 22:59 Chapter 4 Genotyping Genomic DNA was isolated from peripheral leukocytes after venous puncture according to standard protocols. The AMRO-NL study population was genotyped using an Illumina GoldenGate assay on a BeadStation 500 GX (Illumina Inc., San Diego,CA, US). The Rotterdam Study was genotyped with the Illumina HumanHap 550K array (Illumina Inc., San Diego,CA, US). Quality control was performed using PLINK (version 1.01) [34,35]. The UK study population was genotyped by the KASPar SNP genotyping system. Available at: http://www.kbioscience.co.uk (KBiosciences, Unit 7, Maple Park, Essex Road Hoddesdon, Herts, UK). Accessed December, 2008. The US study population was genotyped with TaqMan SNP Genotyping Assays performed on ABI 7300 Real- Time PCR systems (Applied Biosystems, Foster City, California, US) according to the supplier’s recommendations. Genotyping data from the Rotterdam study, and the UK and US study populations were used as source of replication.

Statistical analysis Baseline characteristics of cases and controls were compared using the independent 2 test statistic for categorical variables. 2 test was also employed to test SNP distributions for conformity with Hardy– samples T- test for continuous variables and χ Weinberg equilibrium (HWE), which was present if the observed homozygote and The χ P<0.05) from the expected frequencies. Allele frequencies between cases and controls were compared using heterozygote frequencies did not differ significantly (

for the risk of early and late AMD were estimated with logistic regression analysis the Fisher’s exact test statistic. Odds ratios (ORs) and 95% confidence intervals (CI) with the major alleles as reference. For the AMRO-NL study population and the Rotterdam study, all ORs were adjusted for age and gender. Interaction with the eight associated C5 SNPs, found in the AMRO-NL study, on early and late AMD was determined for CFH Y402H, LOC387715 A69S, age and gender. All analyses were performed in SPSS for windows (release 16.0; SPSS, Inc). In the Rotterdam study, association testing was performed using logistic regression in the PLINK software package (version 1.01) [34,35].

Results

The AMRO-NL study population: association between C5 SNPs and AMD. Baseline characteristics of the cases and controls of the AMRO-NL study population are given in Table 1. Early AMD was found in 24.8% of all AMD subjects, whereas

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Chapter 4 20120904.indd 102 23-10-12 22:59 C5

The gene and Age-related Macular Degeneration wet AMD, GA and mixed AMD were found in 50.4, 14.7 and 10.1% respectively. The

corrected for in the logistic regression model. The distribution of gender and smoking age distribution was significantly different between cases and controls, which was

OR’s with logistic regression using only the 70+ controls as reference. After all, 65+ were not significantly different. For both early and late AMD, we also calculated controls may be too young and develop late AMD in the future. However, using only 70+ controls did not essentially change the OR’s (data not shown). Initially, 15 SNPs spanning the C5 gene (Figure 1a) were genotyped in 375 unrelated AMD patients and 199 controls of the AMRO-NL study population. The LD plot and the distinct haplotype blocks for the 15 selected SNPs, as generated by Haploview, are presented in Figure 1b [33]. Corresponding LD scores (D’ and R2) for each selected marker of the C5 gene with a MAF>10% are also given (Figure 1c). All genotype frequencies of the controls followed HWE (Table 2), except for one SNP (rs7027797; P= 0.04). However, HWE calculations for this SNP may not be completely 4

accurate since one of the genotype groups contains less than five individuals and [36,37]. We therefore left rs7027797 in the analysis. as multiple HWE tests were conducted this may constitute a false positive finding Allelic P-values and genotype distributions of the association analyses are presented in Table 2 15 C5 SNPs distributed over three LD blocks. The strongest allelic associations were . We found significant allelic associations between AMD and eight out of found for rs17611, rs7026551 and rs7037673 (respectively: P=0.0015, P=0.0065 and P= 0.0009). The corresponding ORs for these eight C5 SNPs in early or late AMD, adjusted for CFH Y402H, age and gender, are given in Table 3. In the early AMD group, we observed association for three SNPs, which are in complete LD (Figure 1c): heterozygous carriers of rs2269066 and rs7026551 had

95%CI 1.01-3.06) compared to the wild type genotype. For rs7037673, we observed a significantly increased risk (respectively OR 2.20; 95%CI 1.12-4.38) and (OR 1.75; a protective effect for the homozygotes (OR 0.34; 95%CI 0.15-0.77). In the late AMD group occur (also) in heterozygous genotypes: heterozygotes for rs17611, rs25681 , we observed protective effects for five SNPs, four of which and rs7040033 had lower risks for late AMD compared to the other genotypes. Rs7037673 also showed a protective effect for late AMD but a lower risk was seen in the homozygotes for the minor allele of this SNP. The ORs for the heterozygotes were respectively 0.53 (95%CI 0.34-0.82), 0.54 (95%CI 0.35-0.84), 0.63 (95%CI 0.41-0.96) and 0.58 (95%CI 0.37-0.90). These 4 SNPs, while tagging 3 different haplotype blocks (Figure 1b and 1c), most likely identify the same protective effect, since they are in almost complete LD (D’>0.92). Homozygotes for the minor alleles of

103

Chapter 4 20120904.indd 103 23-10-12 22:59 Chapter 4 .06† <0.001* 368 (100. 0) (100. 368 NA 396 (61.5) 204 (55.4) 248 (38.5) 164 (44.6) 76.28.7 74.8 7.1 0.540.48 0.34 0.22 UNITED STATES N= 644 N= 368 276 (42.8) 276 (42.8) 92 (14.4) .006† .048† <0.001* 10 640 (100.0) 215 (38.1) 280 (43.7) 349 (61.9) 360 (56.3) 204 (36.2) 275 (43.0) 9 277 (49.1) 291 (45.5) 79 (14.0)0.46 59 (9.2) 0.4 0.38 0.21 78 68 UNITED KINGDOM N= 564 N= 640 243 (43.1) 71 (12.6) 29 (5.1) 221 (39.2) .139† .301† <0.001* 2251(100.0) 424 (41.7) 983 (43.7) 592 (58.3) 1268 (56.3) 351 (35.2) 712 (32.0) 8.9 8.5 423 (42.5) 964 (43.3) 222 (22.3)0.45 549 (24.7) 0.25 0.33 0.18 72 67.8 ROTTERDAM N=1016 N=2251 59 (5.8) 63 (6.2) 36 (3.6) 858 (84.4) .257† .333† <0.001* 6 199 (100. 0) (100. 199 152 (40.5) 89 (44.7) 223 (59.5) 110 (55.3) 87 (23.2) 43 (21.6) 9 0.50.4 0.35 0.18 78 74 AMSTERDAM AMD cases ControlsN= 375 p- value AMD cases N= 199 Controls p- value AMD cases38 (10.1) Controls p- value AMD cases Controls p- value 93 (24.8) A69S, m.a.f. Smoking Table 1. Basic Demographic Characteristics of the Amsterdam, Rotterdam, United Kingdom and United States Study Populations. Study States Kingdom and United United Rotterdam, of the Amsterdam, Characteristics 1. Basic Demographic Table significance represents value p- 100%.* equal not does smokers of % the missings, to Due deviation. standard SD= available; not NA= degeneration; macular related age- AMD= test. significance of Chi square represent test, †p- value of independent samples T- Male Female Never smokersFormer 138 (36.8) 49 (24.6) SD Gender smokersCurrent CFH Y402, m.a.f. LOC387715 44 (11.7) 13 (6.5) Mean No. of subjects (%) No. Diagnosis No AMD AMDNeovascular atrophyGeographic AMD Mixed 189 (50.4) years y, Age. 55 (14.7) Early AMD Early

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Chapter 4 20120904.indd 104 23-10-12 22:59 C5

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C5 SNPs found in the AMRO- rs1468673 showed the lowest statistically significant protective effect for late AMD. NL study with 4 prominent AMD risk factors: CFH Y402H, LOC387715 A69S, age and Interaction studies of the eight significantly associated C5 SNPs with either risk factor. This implies that these risk factors did not modify the relation of one of the gender, did not yield significant interaction for one of the C5 SNPs with AMD and that C5, CFH Y402H, LOC387715 A69S, gender and age are independent risk factors for AMD. Based on the observed allelic and genotypic associations for some of the SNPs in the C5 gene with early and late AMD (Table 2 and 3) replication analyses were performed in one other Dutch population, one UK population and one US population.

Figure 1. Complement Component 5 Single Nucleotide Polymorphisms. (A). Single Nucleotide Polymorphisms (SNPs) screened per study population. (B). Linkage disequilibrium (LD) display in Haploview of SNPs encompassing the complement component 5 (C5) gene with minor allele frequency>10% screened in this study and illustrating the 3 distinct haplotype blocks.

Seven of those SNPs (marked with an asterix) also showed genotypic association with AMD in the Amsterdam All SNPs on the top row showed significant allelic association with age- related macular degeneration (AMD). study population. (C). LD scores (D’ and R2) between markers genotyped. Note D’ above the diagonal and R2 scores below the diagonal.

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Chapter 4 20120904.indd 105 23-10-12 22:59 Chapter 4 Table 2. Allelic Association Between Age-related Macular Degeneration and 15 Selected Complement Component 5 Single Nucleotide Polymorphisms in the AMRO-NL Study Population. No AMD (controls) AMD (cases) Allelic SNP ID AA Aa aa MAF AA Aa aa MAF P- Value HWE number (%) number (%) rs10818495 32 (18.8) 82 (48.2) 56 (32.9) 0.57 79 (22.1) 187 (52.2) 92 (25.7) 0.52 0.1135 yes rs10985126 129 (67.5) 55 (28.8) 7 (3.7) 0.18 228 (62.5) 109 (29.9) 28 (7.7) 0.23 0.0882 yes rs1468673 17 (13.1) 56 (43.1) 57 (43.8) 0.65 66 (21.0) 139 (44.3) 109 (34.7) 0.57 0.0200 yes rs1548782 83 (42.8) 82 (42.3) 29 (14.9) 0.36 142 (38.5) 166 (45.0) 61 (16.5) 0.39 0.3659 yes rs16910280 143 (73.7) 49 (25.3) 2 (1.0) 0.14 267 (72.2) 94 (25.4) 9 (2.4) 0.15 0.5355 yes rs17611 53 (27.7) 99 (51.8) 39 (20.4) 0.46 145 (39.4) 161 (43.8) 62 (16.8) 0.39 0.0015 yes rs1978270 82 (42.5) 82 (42.5) 29 (15.0) 0.36 140 (38.1) 166 (45.2) 61 (16.6) 0.39 0.3651 yes rs2269066 120 (82.8) 23 (15.9) 2 (1.4) 0.09 236 (73.8) 74 (23.1) 10 (3.1) 0.15 0.0268 yes rs25681 54 (28.3) 99 (51.8) 38 (19.9) 0.46 144 (39.0) 164 (44.4) 61 (16.5) 0.39 0.0250 yes rs7026551 132 (69.1) 50 (26.2) 9 (4.7) 0.18 210 (57.5) 127 (34.8) 28 (7.7) 0.25 0.0065 yes rs7027797 160 (83.8) 27 (14.1) 4 (2.1) 0.09 281 (76.2) 77 (20.9) 11 (3.0) 0.13 0.0413 no rs7031128 122 (62.9) 60 (30.9) 12 (6.2) 0.22 218 (59.6) 128 (35.0) 20 (5.5) 0.23 0.6522 yes rs7037673 57 (29.8) 95 (49.7) 39 (20.4) 0.45 156 (42.3) 167 (45.3) 45 (12.5) 0.35 0.0009 yes rs7040033 53 (27.9) 97 (51.1) 40 (21.1) 0.47 140 (38.0) 167 (45.4) 61 (16.6) 0.39 0.0211 yes rs992670 52 (29.9) 79 (45.4) 43 (24.7) 0.47 87 (24.4) 155 (43.5) 114 (32.0) 0.54 0.4988 yes AMD= age- related macular degeneration; SNP= single nucleotide polymorphism; MAF= minor allele frequency; HWE= Hardy-Weinberg Equilibrium. “A” indicates common allele, “a” minor allele. Because of genotyping errors, not all subject data is obtainable. Genotype frequencies are given as a percentage of subjects genotyped. The allelic P 2 test was used to test SNP distributions for conformity with HWE. - value is calculated with Fisher’s exact test. The χ

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The gene and Age-related Macular Degeneration Table 3. Odds Ratios and 95% Confidence Intervals of Early and Late Age-related Macular Degeneration Cases Versus Unrelated Controls of the AMRO-NL Study Population for Single Nucleotide Polymorphisms in the Complement Component 5 Gene. No AMD (controls) Early AMD Late AMD rs1468673 N=130 N=77 N=237 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 17 (13.1) 14 (18.2) 1 52 (21.9) 1 Heterozygous (Aa) 56 (43.1) 31 (40.3) 0.68 (0.29-1.58) 108 (45.6) 0.67 (0.35-1.29) Homozygous (aa) 57 (43.8) 32 (41.6) 0.65 (0.28-1.50) 77 (32.5) 0.46 (0.24-0.90) MAF(%) 0.65 0.62 0.55

rs17611 N=191 N=93 N=275 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 53 (27.7) 33 (35.5) 1 112 (40.7) 1 4 Heterozygous (Aa) 99 (51.8) 46 (49.5) 0.66 (0.37-1.19) 115 (41.8) 0.53 (0.34- 0.82) Homozygous (aa) 39 (20.4) 14 (15.1) 0.50 (0.23-1.10) 48 (17.5) 0.66 (0.38- 1.15) MAF(%) 0.46 0.4 0.38

rs2269066 N=145 N=86 N=234 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 120 (82.8) 58 (67.4) 1 178 (76.1) 1 Heterozygous (Aa) 23 (15.9) 24 (27.9) 2.20 (1.12- 4.38) 50 (21.4) 1.38 (0.78-2.41) Homozygous (aa) 2 (1.4) 4 (4.7) 3.16 (0.54-18.50) 6 (2.6) 1.86 (0.34-10.17) MAF(%) 0.09 0.19 0.13

rs25681 N=191 N=93 N=276 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 54 (28.3) 37 (39.8) 1 112 (40.6) 1 Heterozygous (Aa) 99 (51.8) 42 (45.2) 0.72 (0.40-1.28) 117 (42.4) 0.54 (0.35- 0.84) Homozygous (aa) 38 (19.9) 14 (15.1) 0.54 (0.25-1.17) 47 (17.0) 0.68 (0.39- 1.20) MAF(%) 0.46 0.38 0.38

rs7026551 N=191 N=92 N=273 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 132 (69.1) 49 (53.3) 1 161 (59.0) 1 Heterozygous (Aa) 50 (26.2) 35 (38.0) 1.75 (1.01-3.06) 92 (33.7) 1.32 (0.86-2.03) Homozygous (aa) 9 (4.7) 8 (8.7) 2.22 (0.78-6.31) 20 (7.4) 1.50 (0.63-3.55) MAF(%) 0.18 0.28 0.24

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Chapter 4 20120904.indd 107 23-10-12 22:59 Chapter 4 Table 3 (continued). No AMD (controls Early AMD Late AMD rs7027797 N=191 N=93 N=276 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 160 (83.8) 74 (79.6) 1 207 (75.0) 1 Heterozygous (Aa) 27 (14.1) 17 (18.3) 1.33 (0.67-2.63) 60 (21.7) 1.49 (0.89- 2.49) Homozygous (aa) 4 (2.1) 2 (2.2) 0.98 (0.17-5.60) 9 (3.3) 1.75 (0.49- 6.19) MAF(%) 0.09 0.11 0.14

rs7037673 N=191 N=93 N=276 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 57 (29.8) 38 (40.9) 1 118 (42.8) 1 Heterozygous (Aa) 95 (49.7) 45 (48.4) 0.67 (0.38-1.17) 122 (44.2) 0.63 (0.41- 0.96) Homozygous (aa) 39 (20.4) 10 (10.8) 0.34 (0.15-0.77) 36 (13.0) 0.51 (0.29- 0.92) MAF(%) 0.45 0.35 0.35

rs7040033 N=190 N=93 N=275 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 53 (27.9) 31 (33.3) 1 109 (39.6) 1 Heterozygous (Aa) 97 (51.1) 48 (51.6) 0.77 (0.43-1.38) 119 (43.3) 0.58 (0.37- 0.90) Homozygous (aa) 40 (21.1) 14 (15.1) 0.56 (0.26-1.21) 47 (17.1) 0.65 (0.37- 1.13) MAF(%) 0.47 0.41 0.39

polymorphism; MAF= minor allele frequency. “A” indicates common allele, “a” minor allele. Genotype AMD= age- related macular degeneration; CI= confidence interval; OR= odds ratio; SNP= single nucleotide frequencies are given as a percentage of subjects genotyped. Percentages not always 100% because of rounding. ORs are estimated with logistic regression analysis (with the noncarriers as reference group and respectively early and late AMD as outcome variable). Adjusted for age and gender.

The Rotterdam, UK and US replication populations: replication does not confirm association Baseline characteristics of the cases and controls of the Rotterdam, UK and US replication populations are given in Table 1. In all replication populations, the

AMRO-NL and Rotterdam study we used logistic regression to correct for this. distribution of age was significantly different between cases and controls. In the In the other replication populations (UK and US), this may have reduced power by potentially diluting the control sample with (as yet undeveloped) cases. This

conservative since any observed association tests would be an underestimation. discrepancy would therefore be expected to make the strength of findings more

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The gene and Age-related Macular Degeneration Three C5 SNPs, which were associated with AMD in the AMRO-NL study population, were selected for further screening in the Rotterdam Study: rs17611, rs7026551, and rs7037673. These three gene variants spanned two different haplotype blocks of the C5 gene, and were not in complete LD with each other (Figure 1b and 1c). The results of the replication screening of the three C5 SNPs in early and late AMD cases of the Rotterdam population are presented in Table 4a. Genotypes frequencies for

all SNPs followed HWE (data not shown). None of the significant associations found SNPs. in the AMRO-NL study population could be independently confirmed for these three Two SNPs, rs17611 and rs7037673, which were already screened in both the AMRO- NL and Rotterdam populations, were also screened in the two (case-control) studies from the UK and the US. The results from the genotype analysis in the cases and controls are shown in Table 4b and 4c. Again, all the genotype frequencies followed 4 were found. HWE (data not shown) but no significant associations between the SNPs and AMD

Data pooling of four study populations Pooling of populations with the same genetic background

decreased ORs for carriers of the minor allele of rs17611 and rs7037673 (Table 5). Pooling the data of the two Dutch study populations (still) resulted in significantly We observed a protective effect for the heterozygous genotype for both SNPs with ORs of respectively 0.74 (95%CI 0.59-0.92) and 0.79 (95%CI 0.64-0.99). We also observed a protective effect for homozygotes for the minor alleles of rs7037673 with an OR of 0.67 (95%CI 0.49-0.93).

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Chapter 4 20120904.indd 109 23-10-12 22:59 Chapter 4 110 (30.4) 1 0.46 N=362 105 (28.6) 1 0.48 N=367 Late AMD Late Early Early AMD 0.43 0.45 130 (35.7) 78 (28.5) 1 N=364 N=274 0.46 0.46 120 (32.9) 77 (28.1) 1 N=365 N=274 No AMD c) United States study population study States c) United 0.41 121 (36.3) 1 N=333 0.43 110 (32.8) 1 N=335 Late AMD Late 234 (37.0) 78 (35.8) 1 0.38 0.41 N=633 N=218 213 (33.5) 75 (34.4) 1 0.42 0.42 N=635 N=218 b) United Kingdom study population Kingdom study b) United No AMD AMD Early 100 (62.9) 1 N= 159 0.2 59 (37.6) 1 0.38 N= 157 51 (32.3) 1 0.42 N= 158 Late Late AMD Gene. N= 2247No. (%) N= 858 No. (%) OR (95% CI) No. (%)0.21 OR (95% CI) 0.21 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) 0.41 0.42 N= 2234 N= 849 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) 0.43 0.46 N= 2251 N= 858 a) Rotterdam study a) Rotterdam No AMD AMD Early Table Table 4. Odds Ratios and 95% Confidence Intervals of Age-related Macular Degeneration Early and Late Cases Versus Unrelated in the Controls Nucleotide Polymorphisms for Single Population States of and c) the United Population Kingdom Study b) the United Three Study; a) The Rotterdam Replication Populations: 5 Component Complement rs7026551 Genotype Noncarrier (AA) (Aa)Heterozygous 1384 (61.6) 770 (34.3) 536 (62.5) (aa)Homozygous 1 277 (32.3) 93 (4.1)MAF(%) 0.93 (0.78- 1.11) 54 (34.0) 45 (5.2) 1.00 (0.70-1.43) 1.28 (0.88- 1.86) 5 (3.1) 0.78 (0.30-2.02) Genotype Noncarrier (AA) 781 (35.0) 282 (33.2) 1 Heterozygous (Aa)Heterozygous 1092 (48.9) (aa)Homozygous 415 (48.9) 361 (16.2) 1.06 (0.89- 1.27)MAF(%) 77 (49.0) 152 (17.9) 0.99 (0.69-1.42) 1.15 (0.91- 1.45) 21 (13.4) 311 (49.1) 101 (46.3) 0.76 (0.45-1.28) 0.97 (0.69-1.37) 88 (13.9) 151 (45.3) 39 (17.9) 0.94 (0.70-1.26) 1.33 (0.84-2.10) 156 (42.9) 61 (18.3) 147 (53.6) 1.57 (1.10-2.25) 1.34 (0.90-1.99) 174 (48.1) 78 (21.4) 1.18 (0.79-1.77) 49 (17.9) 1.05 (0.66-1.65) 78 (21.5) 1.32 (0.94-1.84) rs7037673 Genotype Noncarrier (AA) 702 (31.2) 246 (28.7) 1 Heterozygous (Aa)Heterozygous 1141 (50.7) (aa)Homozygous 437 (50.9) 1.10 (0.91- 1.32) 408 (18.1)MAF(%) 82 (51.9) 175 (20.4) 1.04 (0.72- 1.52) 1.21 (0.96- 1.52) 25 (15.8) 316 (49.8) 103 (47.2) 0.82 (0.49-1.36) 0.93 (0.66-1.31) 106 (16.7) 159 (47.5) 40 (18.3) 0.97 (0.72-1.31) 1.07 (0.68-1.68) 157 (43.0) 66 (19.7) 143 (52.2) 1.42 (0.99-2.04) 1.21 (0.82-1.77) 174 (47.4) 88 (24.1) 1.27 (0.90-1.78) 54 (19.7 0.96 (0.61-1.49) 88 (24.0) 1.14 (0.77-1.70) rs17611 AMD= age- related macular degeneration; CI= confidence interval; OR= odds ratio; SNP= single nucleotide polymorphism; MAF= minor allele frequency. “A” indicates common indicates “A” allele minor frequency. MAF= nucleotide polymorphism; single SNP= ratio; OR= odds interval; confidence CI= macular degeneration; age- related AMD= and group as reference (with the noncarriers analysis with logistic regression estimated are ORs because of rounding. 100% not always allele. minor Percentages allele, “a” study. in the Rotterdam age and gender only for Adjustment variable). AMD as outcome and late early respectively

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The gene and Age-related Macular Degeneration Table 5. Odds Ratios and 95% Confidence Intervals of Early and Late Cases Versus Unrelated Controls For Pooled Data of the AMRO-NL Study Population and the Rotterdam Study for Single Nucleotide Polymorphisms in the Complement Component 5 Gene.

No AMD (controls) Early AMD Late AMD rs17611 N=2442 N=951 N=433 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 755 (30.9) 279 (29.3) 1 163 (37.6) 1 Heterozygous (Aa) 1240 (50.8) 483 (50.8) 1.05 (0.89-1.25) 197 (45.5) 0.74 (0.59-0.92) Homozygous (aa) 447 (18.3) 189 (19.9) 1.15 (0.92-1.43) 73 (16.9) 0.76 (0.56-1.02)

rs7026551 N=2438 N=950 N=432 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 1516 (62.2) 585 (61.2) 1 261 (60.4) 1 4 Heterozygous (Aa) 820 (33.6) 312 (32.8) 0.99 (0.84-1.15) 146 (33.8) 1.03 (0.83-1.29) Homozygous (aa) 102 (4.2) 53 (5.6) 1.35 (0.95-1.90) 25 (5.8) 1.42 (0.90-2.25)

rs7037673 N=2425 N=942 N=433 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 838 (34.6) 320 (34.0) 1 177 (40.9) 1 Heterozygous (Aa) 1187 (48.9) 460 (48.8) 1.01 (0.86-1.20) 199 (46.0) 0.79 (0.64-0.99) Homozygous (aa) 400 (16.5) 162 (17.2) 1.06 (0.85-1.33) 57 (13.2) 0.67 (0.49-0.93)

polymorphism; MAF= minor allele frequency. “A” indicates common allele, “a” minor allele. Percentages not AMD= age- related macular degeneration; CI= confidence interval; OR= odds ratio; SNP= single nucleotide always 100% because of rounding. ORs are estimated with logistic regression analysis (with the noncarriers as reference group and respectively early and late AMD as outcome variable). Adjusted for age and gender.

Pooling of populations with the same study design Pooling the data from the Dutch, UK and US case-control studies did not lead to Table 6). Finally, combining all data from the four studies did not result in statistically significant associations for either rs17611 or rs7037673 (

significant association (data not shown).

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Chapter 4 20120904.indd 111 23-10-12 22:59 Chapter 4 Table 6. Odds Ratios and 95% Confidence Intervals of Age-related Macular Degeneration Early and Late Cases Versus Unrelated Controls For Pooled Data of the AMRO-NL, United Kingdom and United States Study Populations for Single Nucleotide Polymorphisms in the Complement Component 5 Gene.

No AMD (controls) Early AMD Late AMD rs17611 N=1191 N=585 N=977 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 386 (32.4) 185 (31.6) 1 327 (33.5) 1 Heterozygous (Aa) 572 (48.0) 292 (49.9) 1.07 (0.85-1.33) 448 (45.9) 0.92 (0.76-1.12) Homozygous (aa) 233 (19.6) 108 (18.5) 0.97 (0.73-1.29) 202 (20.7) 1.02 (0.81-1.30)

rs7037673 N=1188 N=585 N=972 No. (%) No. (%) OR (95% CI) No. (%) OR (95% CI) Genotype Noncarrier (AA) 421 (35.4) 194 (33.2) 1 349 (35.9) 1 Heterozygous (Aa) 562 (47.3) 293 (50.1) 1.13 (0.91-1.41) 448 (46.1) 0.96 (0.80-1.16) Homozygous (aa) 205 (17.3) 98 (16.8) 1.04 (0.77-1.39) 175 (18.0) 1.03 (0.80-1.32)

Discussion

We tested the association between complement C5 gene variants and AMD in four independent studies (three case-control and one prospective, population-based study), consisting of 2599 AMD cases and 3458 ethnically matched controls. Despite the established involvement of the complement system, including C5, in AMD, and C5

association between common SNPs in C5 and AMD in all study populations. protein localization in drusen, [7-11,13,18,21,25] we were unable to find consistent et al. association between C5 SNPs (rs17611, rs7026551 and rs7033790) and AMD [13]. Our data confirms the preliminary data of Yates (2007) who did not find

found in one or more cohort(s), even when it involves very plausible candidate This study also corroborates previous findings that replication of initial associations genes, is essential to determine true genetic disease susceptibility. A limitation of the current study is that we initially selected for relatively common SNPs, with MAFs>10%, in order to haplotype-tag the whole coding region of the C5 gene. Consequently, we could have missed both variants with MAFs<10% and

of the study include the large number of AMD cases and controls screened across outside the coding region which might influence the disease phenotype. Strengths

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The gene and Age-related Macular Degeneration

the cases and controls. populations of European descent, and the relatively uniform clinical classification of

Data pooling of four cohorts

SNPs in C5 and AMD in the AMRO-NL study population, we extended our study After our initial finding of a statistically significant association between several with three other independent cohorts. Although the AMRO-NL study population and the Rotterdam Study have a different design, the populations have the same

genetic background. Therefore, we pooled data from these two populations first. minor allele of rs17611 and rs7037673 (Table 5). The effect seen may be largely Pooling the data resulted in decreased, but still significant ORs for carriers of the

data of the two populations individually did not contradict each other; i.e., the CIs attributable to the findings in the AMRO-NL study population. Nonetheless, the of the associated genotypes overlapped substantially (Table 3, 4a, 5). Even more 4 interestingly, the homozygote minor alleles of rs17611 did not show a protective effect for “late AMD” in both populations if analyzed individually, but almost reached Table 5). The potential AMD risk modifying effect of the rs7026551 allele (AMRO-NL) statistical significance (OR 0.76 with 95% CI 0.56-1.02) in the pooled data set ( disappeared in the pooled data set.

Next, we pooled the AMRO-NL, UK and US data (Table 6), since these studies, despite their different genetic backgrounds, have the same case-control study design. Then, we also added the Rotterdam population data again. Pooling the data in these ways abolished all associations that were initially found in the AMRO-NL study population.

C5 variants and AMD The complement factor 5 is very likely involved in processes leading to AMD, since it is a key component of the (alternative) complement system and is present in

C5 SNPs and AMD among the populations tested. drusen [18,21,25]. Nevertheless, we could not find robust, consistently statistically How can this be explained? significant, association between The simplest explanation chance, and that C5 gene variants do not contribute at all to genetic susceptibility is that the findings in the AMRO-NL population are due to for AMD. In other words, the screened C5 sequence variants may result in a (mildly) altered C5 function but, these possibly functional changes have no role in the etiology of AMD even if they regulate, limit or alter the total activity of the complement cascade.

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Chapter 4 20120904.indd 113 23-10-12 22:59 Chapter 4 But is it that simple? not protective effect for the C5 variant rs17611 in the Dutch population, which cannot If our findings are due to chance (alone), we find a possible be replicated in two UK or USA populations. Interestingly, Hillebrandt et al. (2005) and Gressner et al. (2007) found that this same rs17611 variant is both possibly associated with elevated C5 and Gc globulin serum concentrations in man, and

experimental evidence is lacking, one could speculate that elevated C5 serum levels with enhanced liver fibrosis in mice [38,39]. While, to our knowledge, further increase the risk for AMD through further activation of the complement pathway. In

decrease the risk of advanced AMD, by accelerating scar formation after retinal contrast, increased fibrosis capabilities, if it occurs in the eye also, could perhaps injury. In conclusion, these data suggest that C5 may have multiple roles in AMD pathology, thereby complicating potential correct risk assessment of C5 sequence variants.

Non-replication of C5 variants Whether C5 SNPs are associated with AMD in the Dutch population, or not, our

in one study cannot be replicated in others. So far, only very strong AMD risk factors current study presents another example where initial significant associations found such as CFH [7,9,10] or ARMS2/HTRA1 [6,12] have been consistently replicated. These “strong” risk factors are rather an exception than rule. Initial associations between “weaker” AMD risk factors, such as the TLR4 gene [40], the SERPING1 gene [28], the TLR3 gene1 [41] and even the ApoE2 and 4 alleles [42,43] could not be replicated in several other case-control cohorts [44-47]. Also, in a GWAS on the AREDS study, only 1 (rs2230199; C3) out of 57 initially AMD associated SNPs was

journals are reluctant to publish negative associations, most non-replication studies replicated in a second case-control cohort [45]. Since most investigators or scientific are never published. Non-replications in different populations have been frequently explained by chance, variation in study design, by phenotypic AMD (grading) differences, genotype errors [48,49] or by genetic variability between populations [36]. In our current C5 association study, we used two different study designs which may have affected the outcome in the individual studies. Phenotypic grading differences may not account for a very large variability in our study, since the fundi of all patients and controls

of AMD [31,32] under supervision of the same ophthalmologists (CK, PdJ) in three were graded according to (a modification of) the international classification system out of four populations. Moreover, Colhoun and coworkers (2003) calculated that

5% clinical misclassification has only a small effect, comparable to a reduction of the 114

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The gene and Age-related Macular Degeneration sample size by 10% [50]. Potential genotype errors in our study are also unlikely, since both positive and negative associations between C5 variants and AMD are

Finally, association studies in populations that are genetically heterogeneous can confirmed by at least one second variant in LD with the first one. yield large numbers of spurious associations if population subgroups are unequally represented among cases and controls. Especially case-control designs have

subtypes show variation in their baseline disease risk (the risk not attributable to been described as being susceptible to population stratification when population the gene of interest) and evident heterogeneity in allele frequency for the candidate gene studied. While all participants in our study were of European descent, we did observe marked variation in MAFs of almost all C5 SNPs between the populations. For example, in controls, we found MAFs for rs7037673 varying from 0.38 (UK) to 0.45 (NL). In the AMD cases, we found for the same SNP MAFs of, respectively, 0.35 (NL) to 0.46 (US). Family-based association studies have the advantage that they are less 4 prone to the effect of population structure because of matched genetic background among study participants. Unfortunately, due to the lack of family data from all the case-control populations used here, we cannot correct for this phenomenon. Interestingly, there are at least two (other) marked examples in which geographically determined genetic variation was clearly implicated in differences in disease susceptibility: the CFH Y402H variant confers a very strong risk for AMD in western populations, while it has only a marginal effect in Chinese [51] or Japanese [52,53]. In addition, The Welcome Trust Case Control Consortium [54] suggested, on the basis of a GWA of 14.000 cases and 3000 shared controls, that geographic differences in TLR1 receptor allele frequencies within the UK coincide with natural selection and susceptibility against TLR1 mediated (auto-) immune disease. Apart from differences in C5 more other MAF, population-specific genetic variations in one or also affect AMD disease outcome. These variants may determine the capability of genes of the complement pathway and/or the fibrosis pathways may C5 pathways and thus determine their potential effect on the AMD phenotype. SNPs to influence or regulate the activity of the complement cascade or fibrotic In conclusion, common SNPs in the C5 gene confer either no, or limited risk for AMD, which may be dependent on genetic differences between populations. While, given its crucial role in the complement cascade, and the presence of C5 protein in drusen, C5 is very likely involved in AMD. The tested genetic variation in C5 has a very limited, if any, effect on the AMD phenotype.

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Chapter 4 20120904.indd 115 23-10-12 22:59 Chapter 4 Acknowledgements

and Dohme and the Nederlandse Vereniging ter Voorkoming van Blindheid (both to This study was in part financed by an unrestricted research grant from Merck Sharpe A.A.B.) The generation and management of GWAS genotype data for the Rotterdam Study is

(nr. 175.010.2005.011, 911-03-012). This study is funded by the Research Institute supported by the Netherlands Organisation of Scientific Research NWO Investments for Diseases in the Elderly (014-93-015; RIDE2), the Netherlands Genomics Initiative

060-810, and funding from the Erasmus Medical Center and Erasmus University, (NGI)/Netherlands Organisation for Scientific Research (NWO) project nr. 050- Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. The US study is supported in part by the grants from the National Eye Institute EY13435 and EY017404; the Macula Vision Research Foundation; Kaplen Foundation; Wigdeon Point Charitable Foundation and an unrestricted grant to the Department of Ophthalmology, Columbia University, from Research to Prevent Blindness, Inc.

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The gene and Age-related Macular Degeneration References

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the formation of drusen in the aging eye. Am J Ophthalmol 134: 411-431. 18. Anderson DH, Mullins RF, Hageman GS, Johnson LV (2002) A role for local inflammation in 19. Crabb JW, Miyagi M, Gu X, Shadrach K, West KA, et al. (2002) Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci U S A 99: 14682-14687.

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Chapter 4 20120904.indd 117 23-10-12 22:59 Chapter 4 20. Hageman GS, Luthert PJ, Victor Chong NH, Johnson LV, Anderson DH, et al. (2001) An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res 20: 705-732. 21. Johnson LV, Ozaki S, Staples MK, Erickson PA, Anderson DH (2000) A potential role for immune complex pathogenesis in drusen formation. Exp Eye Res 70: 441-449. 22. Johnson LV, Leitner WP, Staples MK, Anderson DH (2001) Complement activation and

Eye Res 73: 887-896. inflammatory processes in Drusen formation and age related macular degeneration. Exp 23. Lommatzsch A, Hermans P, Weber B, Pauleikhoff D (2007) Complement factor H variant Y402H and basal laminar deposits in exudative age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol 245: 1713-1716.

reactions involved in exudative age-related macular degeneration? Morphological and 24. Lommatzsch A, Hermans P, Muller KD, Bornfeld N, Bird AC, et al. (2008) Are low inflammatory immunhistochemical analysis of AMD associated with basal deposits. Graefes Arch Clin Exp Ophthalmol 246: 803-810. 25. Mullins RF, Russell SR, Anderson DH, Hageman GS (2000) Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. FASEB J 14: 835-846. 26. Nozaki M, Raisler BJ, Sakurai E, Sarma JV, Barnum SR, et al. (2006) Drusen complement components C3a and C5a promote choroidal neovascularization. Proc Natl Acad Sci U S A 103: 2328-2333. 27. Hofman A, Breteler MM, van Duijn CM, Krestin GP, Pols HA, et al. (2007) The Rotterdam Study: objectives and design update. Eur J Epidemiol 22: 819-829. 28. Ennis S, Jomary C, Mullins R, Cree A, Chen X, et al. (2008) Association between the SERPING1 gene and age-related macular degeneration: a two-stage case-control study. Lancet 372: 1828-1834.

in age-related macular degeneration and cutis laxa. Hum Mutat 27: 568-574. 29. Lotery AJ, Baas D, Ridley C, Jones RP, Klaver CC, et al. (2006) Reduced secretion of fibulin 5 30. Hageman GS, Anderson DH, Johnson LV, Hancox LS, Taiber AJ, et al. (2005) A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci U S A 102: 7227-7232. 31. Bird AC, Bressler NM, Bressler SB, Chisholm IH, Coscas G, et al. (1995) An international

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The gene and Age-related Macular Degeneration 34. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, et al. (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81: 559-575. 35. Richards JB, Rivadeneira F, Inouye M, Pastinen TM, Soranzo N, et al. (2008) Bone mineral density, osteoporosis, and osteoporotic fractures: a genome-wide association study. Lancet 371: 1505-1512. 36. Attia J, Ioannidis JP, Thakkinstian A, McEvoy M, Scott RJ, et al. (2009) How to use an article about genetic association: B: Are the results of the study valid? JAMA 301: 191-197. 37. Trikalinos TA, Salanti G, Khoury MJ, Ioannidis JP (2006) Impact of violations and deviations in Hardy-Weinberg equilibrium on postulated gene-disease associations. Am J Epidemiol 163: 300-309. 38. Gressner O, Meier U, Hillebrandt S, Wasmuth HE, Kohl J, et al. (2007) Gc-globulin concentrations and C5 haplotype-tagging polymorphisms contribute to variations in serum activity of complement factor C5. Clin Biochem 40: 771-775. 39. Hillebrandt S, Wasmuth HE, Weiskirchen R, Hellerbrand C, Keppeler H, et al. (2005) 4 humans. Nat Genet 37: 835-843. Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and 40. Zareparsi S, Buraczynska M, Branham KE, Shah S, Eng D, et al. (2005) Toll-like receptor 4 variant D299G is associated with susceptibility to age-related macular degeneration. Hum Mol Genet 14: 1449-1455. 41. Yang Z, Stratton C, Francis PJ, Kleinman ME, Tan PL, et al. (2008) Toll-like receptor 3 and geographic atrophy in age-related macular degeneration. N Engl J Med 359: 1456-1463. 42. Baird PN, Guida E, Chu DT, Vu HT, Guymer RH (2004) The epsilon2 and epsilon4 alleles of the apolipoprotein gene are associated with age-related macular degeneration. Invest Ophthalmol Vis Sci 45: 1311-1315. 43. Baird PN, Richardson AJ, Robman LD, Dimitrov PN, Tikellis G, et al. (2006) Apolipoprotein (APOE) gene is associated with progression of age-related macular degeneration (AMD). Hum Mutat 27: 337-342. 44. Despriet DD, Bergen AA, Merriam JE, Zernant J, Barile GR, et al. (2008) Comprehensive analysis of the candidate genes CCL2, CCR2, and TLR4 in age-related macular degeneration. Invest Ophthalmol Vis Sci 49: 364-371. 45. Edwards AO, Fridley BL, James KM, Sharma AK, Cunningham JM, et al. (2008) Evaluation of clustering and genotype distribution for replication in genome wide association studies: the age-related eye disease study. PLoS One 3: e3813. 46. Park KH, Ryu E, Tosakulwong N, Wu Y, Edwards AO (2009) Common variation in the SERPING1 gene is not associated with age-related macular degeneration in two independent groups of subjects. Mol Vis 15: 200-207. 47. Allikmets R, Bergen AA, Dean M, Guymer RH, Hageman GS, et al. (2009) Geographic atrophy in age-related macular degeneration and TLR3. N Engl J Med 360: 2252-2254. 48. Kang SJ, Gordon D, Finch SJ (2004) What SNP genotyping errors are most costly for genetic association studies? Genet Epidemiol 26: 132-141.

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Chapter 4 20120904.indd 119 23-10-12 22:59 Chapter 4 49. Miller MB, Schwander K, Rao DC (2008) Genotyping errors and their impact on genetic analysis. Adv Genet 60: 141-152. 50. Colhoun HM, McKeigue PM, Davey SG (2003)Problems of reporting genetic associations with complex outcomes. Lancet 361: 865-872. 51. Lau LI, Chen SJ, Cheng CY, Yen MY, Lee FL, et al. (2006) Association of the Y402H polymorphism in complement factor H gene and neovascular age-related macular degeneration in Chinese patients. Invest Ophthalmol Vis Sci 47: 3242-3246. 52. Gotoh N, Yamada R, Hiratani H, Renault V, Kuroiwa S, et al. (2006) No association between complement factor H gene polymorphism and exudative age-related macular degeneration in Japanese. Hum Genet 120: 139-143. 53. Okamoto H, Umeda S, Obazawa M, Minami M, Noda T, et al. (2006) Complement factor H polymorphisms in Japanese population with age-related macular degeneration. Mol Vis 12: 156-158. 54. Wellcome Trust Case Control Consortium (2007) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447: 661-678.

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Rando Allikmets, Michael Dean, Gregory S. Hageman, Paul N. Baird, Caroline C. Klaver, Arthur A.B. Bergen, Bernhard H. Weber and the International AMD Genetics Consortium

Members of the International AMD Genetics Consortium: Joanna E. Merriam, R. Theodore Smith, Gaetano R. Barile, Julie Sawitzke, Karen M. Gehrs, Jill L. Hageman, Norma J. Miller, Mary L. Howard, Robyn H. Guymer, Andrea Richardson, Lintje Ho, Johannes R Vingerling, Andre G. Uitterlinden, Paulus T. de Jong, Dominique C. Baas, Lars G. Fritsche, Claudia N. Keilhauer

The Lancet 2009; 374(9693):875-876

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SERPING1) and Two recent studies have provided conflicting evidence about the association between the gene encoding serpin peptidase inhibitor, clade G, member 1 ( SERPING1 age-related macular degeneration (AMD). An article in The Lancet [1] suggested that a variant (rs2511989) in intron 6 of conferred protection against the development of AMD in case-control studies from the UK and USA. A follow-up study SERPING1 [2] showed the absence of any association in two US case-control studies, including the AREDS cohort [3]. As a member of the classical complement pathway, CFH C2/CFB C3 is a plausible candidate gene for AMD since several genes encoding proteins involved in the innate immune response ( [4-7], [8], [9,10]), are robustly associated with the development of this disorder. To test this hypothesis further, we genotyped rs2511989 in seven large case-control studies involving individuals of European descent (4881 patients with AMD and 2842 matched controls). Samples from all cohorts in the seven centers were collected and genotyped Table 1 and Figure 1 independently to minimize data selection, analysis, or interpretation bias. Data from the seven studies are summarized in . They show, both SERPING1 independently and collectively, that there is no significant association between the rs2511989 variant in the gene and AMD. In fact, data from no single Table 1 study suggested even a trend towards protective association (p>0·3 in all cohorts; ); one cohort (AREDS) suggested an opposite, marginally significant (p=0·03), trend towards increased susceptibility. However, the study by Park and colleagues [2] found no association after genotyping more AREDS samples, thereby suggesting that the marginal association detected in our study is due to a limited sample size. The minor allele frequency (MAF) for the rs2511989 single nucleotide polymorphism (SNP) varied substantially between studies, from 0·35 and 0·45 both in AMD patients (average 0·40) and in controls matched for age and ethnic group (average 0·40). However, there was almost no difference in the MAF between patients and controls within all, except for the AREDS, studies. When summarizing the data across cohorts, the MAF of the rs2511989 SNP was practically identical in Table 1 2842 controls and in 4881 patients with AMD (0·40), clearly showing no association (p=0·99; odds ratio 1, ). The same lack of significance was seen for all AMD subphenotypes, early AMD, geographic atrophy, and choroidal neovascularization, when analyzed separately. Since the other SNPs (eg, rs2509879, rs2511988, etc) reported in the study by Ennis and colleagues are in high linkage disequilibrium with the rs2511989 variant [1], the MAFs for those confirmed the rs2511989 data (not Figure 1 shown) [2], as did a combined analysis of all three studies by the Mantel-Haenszel fixed effects model ( ).

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The Gene and Age-related Macular Degeneration Table 1. Genotype Counts and Association Analysis of the SERPING1 Rs2511989 Variant in Seven Studies. GG GA AA MAF HWE p (allele) p (genotype) Columbia

0·62 0·39 AMD (n=1004) 449 (44·7%) 431 (42·9%) 124 (12·4%) 0·34 0·45 Iowa Control (n=363) 151 (41·6%) 171 (47·1%) 41 (12·3%) 0·35 0·79 0·57 0·68 AMD (n=368) 116 (31·5%) 178 (48·4%) 74 (20·1%) 0·44 0·91 Amsterdam Control (n=115) 37 (32·2%) 59 (51·3%) 19 (16·5%) 0·42 0·89 0·81 0·62 AMD (n=338) 107 (31·7%) 184 (54·4%) 47 (13·9%) 0·41 0·08 Rotterdam Control (n=257) 84 (32·7%) 131 (51·0%) 42 (16·3%) 0·42 0·74 0·85 0·53 AMD (n=1017) 328 (32·3%) 518 (50·9%) 171 (16·8%) 0·42 0·37 AREDS Control (n=842) 285 (33·8%) 407 (48·3%) 150 (17·9%) 0·42 0·97 0·03 0·04 AMD (n=415) 133 (32·0%) 188 (45·3%) 94 (22·7%) 0·45 0·20 Australia Control (n=213) 90 (42·3%) 81 (38·0%) 42 (19·7%) 0·39 0·02 0·28 0·43 AMD (n=741) 251 (33·9%) 367 (49·5%) 123 (16·6%) 0·41 0·83 Germany Control (n=327) 105 (32·1%) 157 (48·0%) 65 (19·9%) 0·44 0·90 0·71 0·80 5 AMD (n=998) 377 (37·8%) 485 (48·6%) 136 (13·6%) 0·38 0·58 Total Control (n=725) 284 (39·2%) 341 (47·0%) 100 (13·8%) 0·37 0·99 0·99 0·79 AMD (n=4881) 1761 (36·1%) 2351 (48·2%) 769 (15·7%) 0·40 0·94 Control (n=2842) 1036 (36·5%) 1347 (47·4%) 459 (16·1%) 0·40 0·83 AMD= age-related macular degeneration; MAF= minor allele (A allele) frequency; HWE= Hardy-Weinberg equilibrium.

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Figure 1. Forest Plot of the Combined Analysis of Ten Case–control Cohorts from the Three Studies.

Horizontal lines represent 95% CIs, and various-sized squares−15 correspond to significance levels of p values. Test for heterogeneity was highly significant (p=3·1×10 ), suggesting that calculation of a combined odds ratio is not valid.

Excluding experimental errors discussed by Park and colleagues [2], the difference between our collective findings and those published by Ennis and colleagues could be explained by the two following scenarios: (1) Whereas most methods used in our study closely match those used by Ennis and colleagues (genotyping methods, selection criteria for AMD patients, etc), the selection and matching of controls in the seven centers in this study were more rigorous by age and, in some centers (Germany, the Netherlands), also by ethnicity. The significantly higher MAFs in controls in both cohorts used in Ennis

and colleagues’ study (average−6 0·46 vs. 0·40 in this study and the one by Park and colleagues [2], p=8×10 ) could be explained by the fact that controls were younger than AMD patients [1]. (2) Other possible sources for the difference in results could be non-random sampling of study populations or population substructure (stratification). For within example, although there was no significant difference in MAFs between any of the between AMD and control cohorts any one study, the same numbers varied significantly some studies (MAF 0·35 in AMD cases from New York vs. >0·41 in all other cohorts), probably originating from varied ethnic composition within populations of European descent. The New York (Columbia University) study population originates mainly from eastern Europe, whereas that from Iowa from western Europe and

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Scandinavia. At the same time it is of interest that one of the two cohorts in Ennis and colleagues’ study (248 AMD cases) was collected at exactly the same location, the University of Iowa, as was one of the seven cohorts in our study (368 AMD cases). However, whereas the frequencies of the minor allele in Iowa controls were very similar in the two studies (0·44 vs. 0·42), they differed significantly in patients SERPING1 with AMD (0·35 vs. 0·44; p=0·001). In summary, analysis of the rs2511989 variant in in seven large, well characterised case-control studies did not confirm an association between this SERPING1 variant and AMD. Since there is also no evidence of a functional role for this variant, it is unlikely that the gene has a significant, if any, role in the etiology of AMD.

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Chapter 5.indd 127 23-10-12 19:41 Chapter 5 C Germany 725 132 (13.2) 145 (14.5) Australia Australia Germany 58 (7.8) 103 (13.9)580 (78.3)0 183 (18.3) 538 (53.9) AREDS 213 741 327 998 80 (19.3) 146 (35.2) 65 (15.7) Rotterdam 859 (84.5) 124 (29.9) 64 (6.3) 36 (3.5) Amsterdam Amsterdam Rotterdam AREDS 49 (14.5) 34 (10.1) Iowa Iowa 0 76.6 (8.6) 74.8 (7.1) 79.2 (7.8) 78.3 (8.1) 77.9 (7.7) 74.9 (6.7) 72.8 (8.7) 77.0 (5.5) 79.5 (5.3) 76.9 (4.6) 78.1 (7.7) 78.6 (7.7) 76.3 (6.8) 76.4 (5.2) 346 (34.5) 162 (44.6) 121 (32.9) 40 (34.8) 132 (39.1) 112 (43.6) 424 (41.7) 314 (37.3) 187 (45.1) 100 (46.9) 261 (35.2) 132 (40.4) 353 (34.8) 288 (38.5) 658 (65.5) 201 (55.4) 247 (67.1) 75 (65.2) 206 (60.9) 145 (56.4) 688 (58.3) 528 (62.7) 228 (54.9) 113 (53.1) 480 (64.8) 195 (59.6) 660 (65.2) 460 (61.5) 1004 363 368 115 338 257 1017 842 415 213 741 327 1013 748 Columbia AMD C AMD C AMD C AMD C AMD C AMD C AMD Columbia AMD C AMD C AMD C AMD C AMD C AMD C AMD C 1004 363 368 115 338 257 1017 842 415 269 (26.8) 166 (45.1) 91 (26.9) 170 (16.9) 44 (11.9) 524 (52.2)39 (3.9) 158 (42.9) 164 (48.5) 58 (5.7) Diagnosis N eAMD GA CNV Mixed Average Average age (s.d.) Male Gender (%) Female N Case-control series Table 2. Disease Status (Percentile) of Subjects in the Seven Studies. of Subjects in the Seven 2. Disease Status (Percentile) Table one Mixed= neovascularization; CNV= choroidal atrophy; geographic stage AMD; GA= eAMD= early C= controls; macular degeneration; AMD= age-related the other CNV. GA, eye Studies. 3. Disease Status, Gender and Age of Subjects in the Seven Table macular degeneration. N= number of subjects; AMD= age-related C= controls;

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The Gene and Age-related Macular Degeneration Materials and Methods

Study Cohorts

The consortium includes seven international cohorts of patients diagnosed with AMD and controls matched by age and ethnicity, all of whom have been extensively characterized and described in previous genetic and epidemiological studies of AMD as follows: from Columbia University, New York, USA [5,8,11]; University of Iowa, Iowa City, USA [5,8]; AREDS cohort, NEI/NIH, USA [12]; Rotterdam Study, Erasmus University, Rotterdam, The Netherlands [13,14]; the Netherlands Institute of Neuroscience (NIN), Amsterdam, The Netherlands [11,13]; University of Würzburg, Würzburg, Germany [15,16]; and the Centre for Eye Research Australia, University Tables 2 and 3 of Melbourne, Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Australia [17,18]. Details of the case-control series are provided in . All study subjects provided written informed consent before participating. The study was conducted at all sites in strict adherence to the tenets of the Declaration of Helsinki and was approved by the respective Institutional Review Boards at Columbia University and University of Iowa, by the Age-Related Eye Disease Study (AREDS) 5 Access Committee, Data Protection Authorities and National and Institutional Ethics Committees (at Amsterdam, Rotterdam and Würzburg sites) and according to the National Health and Medical Research Council of Australia’s statement on ethical conduct in research involving humans (revised in 1999) at the Melbourne site. Grading

All study subjects underwent clinical examination and stereoscopic color fundus photography after pharmacologic mydriasis, and were graded according to the International Classification and Grading System for AMD [19] (Melbourne and Würzburg sites), the Rotterdam modification of the International classification [14,20] (Columbia, Iowa, Amsterdam and Rotterdam sites), or the classification established by the AREDS study [3] (for the AREDS study). The detailed description of each cohort has been provided in earlier publications, as described in the previous section. In short, AMD patients were subdivided into phenotypic categories: early AMD (eAMD), geographic atrophy (GA) and choroidal neovascularization (CNV) AMD Tables 2 and 3 based on the classification of their most severe eye at the time of their entry into the study. The average age of subjects was ~75 years in all cohorts ( ). i.e. Subjects were graded as ‘unaffected controls’ only when fundus photographs showed no signs of AMD; , they had clear fundi at >60 years of age or <5 small

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hard drusen (grade 1 by AREDS classification or grade 0 by International/Rotterdam classification). In the Rotterdam Study the selection criteria for controls were made even more stringent by raising the age threshold to >70 and by extending the follow- up period to several years (Rotterdam Study). Genotyping

The rs2511989 SNP was genotyped by the Applied Biosystems (Foster City, CA) Taqman® 5’ nucleotidase assay (Columbia, AREDS, Würzburg and Amsterdam studies), by the Illumina HumanHap arrays (Rotterdam Study) or, in Iowa and Melbourne studies, by a combination of SSCP, direct sequencing and Molecular Inversion Probe Genotyping (ParAllele Biosciences/Affymetrix). Statistical analysis

Genotypes were tabulated in Microsoft Excel and presented to SPSS for contingency table analysis as described previously [5,8]. Allele tests were performed with Chi- square analysis and the G-test was used to assess genotype distributions. Compliance to Hardy-Weinberg equilibrium was checked using SAS/Genetics (SAS Institute), et al et al and all cohorts in both cases and controls survived a cutoff of p>0.05. The combined analysis of all three studies (Ennis ., Park ., and ours) was performed using R statistical package. The Mantel-Haenszel fixed effects model was used to estimate individual odds ratios, summary of pooled effect, and test for heterogeneity. Acknowledgements

The authors thank Sarah Schlotterbeck, Kendal Lemon, Melinda Cain, Andrea Richardson, Pascal Arp, Mila Jhamai, Dr Fernando Rivadeneira, Dr Michael Moorhouse, Marijn Verkerk, Nancy Parmalee and Sander Bervoets for technical assistance. Supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research and SAIC-Frederick under contract #NO1-CO-12400 and with grants from the National Eye Institute EY13435 (RA) and EY017404 (RA, GSH); the Macula Vision Research Foundation (RA); Kaplen Foundation (RA); Widgeon Point Charitable Foundation (RA); unrestricted grants to the Department of Ophthalmology, Columbia University, and the Department of Ophthalmology and Visual Sciences, University of Iowa from Research to Prevent Blindness; German Research Foundation (DFG) WE1259/18- 1 and WE1259/19-1, the Ruth and Milton Steinbach Foundation (BHW, RA), the Alcon Research Institute (BHW, RA), the Netherlands Genomics Initiative (NGI)/

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Netherlands Organization for Scientific Research (NWO; nr. 050-060-810); NWO Investments (nr. 175.010.2005.011, 911-03-012); Research Institute for Diseases in the Elderly (RIDE2; 014-93-015); Netherlands Organization for the Health Research and Development (ZonMw); Ministry of Education, Culture and Science, Ministry for Health, Welfare and Sports, European Commision (DG XII), Municipality of Rotterdam; the Netherlands Macula Society, Netherlands National Foundation for the Blind (LSBS), the Netherlands National Society for Prevention of Blindness (ANVVB), National Health and Medical Research Council, Australia, Career development fellowship (RHG), J A COM Foundation (PNB).

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Chapter 5.indd 131 23-10-12 19:41 Chapter 5 References

1. Ennis S, Jomary C, Mullins R, Cree A, Chen X, et al. (2008) Association between the SERPING1 gene and age-related macular degeneration: a two-stage case-control study. Lancet 372: 1828-1834. 2. Park KH, Ryu E, Tosakulwong N, Wu Y, Edwards AO (2009) Common variation in the SERPING1 gene is not associated with age-related macular degeneration in two independent groups of subjects. Mol Vis 15: 200-207. 3. (1999) The Age-Related Eye Disease Study (AREDS): design implications. AREDS report no. 1. Control Clin Trials 20: 573-600. 4. Edwards AO, Ritter R, III, Abel KJ, Manning A, Panhuysen C, et al. (2005) Complement factor H polymorphism and age-related macular degeneration. Science 308: 421-424. 5. Hageman GS, Anderson DH, Johnson LV, Hancox LS, Taiber AJ, et al. (2005) A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci U S A 102: 7227-7232. 6. Haines JL, Hauser MA, Schmidt S, Scott WK, Olson LM, et al. (2005) Complement factor H variant increases the risk of age-related macular degeneration. Science 308: 419-421. 7. Klein RJ, Zeiss C, Chew EY, Tsai JY, Sackler RS, et al. (2005) Complement factor H polymorphism in age-related macular degeneration. Science 308: 385-389. 8. Gold B, Merriam JE, Zernant J, Hancox LS, Taiber AJ, et al. (2006) Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet 38: 458-462. 9. Maller JB, Fagerness JA, Reynolds RC, Neale BM, Daly MJ, et al. (2007) Variation in complement factor 3 is associated with risk of age-related macular degeneration. Nat Genet 39: 1200-1201. 10. Yates JR, Sepp T, Matharu BK, Khan JC, Thurlby DA, et al. (2007) Complement C3 variant and the risk of age-related macular degeneration. N Engl J Med 357: 553-561. 11. Despriet DD, Bergen AA, Merriam JE, Zernant J, Barile GR, et al. (2008) Comprehensive analysis of the candidate genes CCL2, CCR2, and TLR4 in age-related macular degeneration. Invest Ophthalmol Vis Sci 49: 364-371. 12. (2001) A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E and beta carotene for age-related cataract and vision loss: AREDS report no. 9. Arch Ophthalmol 119: 1439-1452. 13. Despriet DD, Klaver CC, Witteman JC, Bergen AA, Kardys I, et al. (2006) Complement factor H polymorphism, complement activators, and risk of age-related macular degeneration. JAMA 296: 301-309. 14. van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT (2003) The risk and natural course of age-related maculopathy: follow-up at 6 1/2 years in the Rotterdam study. Arch Ophthalmol 121: 519-526. 15. Fritsche LG, Loenhardt T, Janssen A, Fisher SA, Rivera A, et al. (2008) Age-related macular degeneration is associated with an unstable ARMS2 (LOC387715) mRNA. Nat Genet 40: 892-896.

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The Gene and Age-related Macular Degeneration

16. Rivera A, Fisher SA, Fritsche LG, Keilhauer CN, Lichtner P, et al. (2005) Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum Mol Genet 14: 3227-3236. 17. Baird PN, Islam FM, Richardson AJ, Cain M, Hunt N, et al. (2006) Analysis of the Y402H variant of the complement factor H gene in age-related macular degeneration. Invest Ophthalmol Vis Sci 47: 4194-4198. 18. Richardson AJ, Islam FM, Guymer RH, Baird PN (2009) Analysis of rare variants in the complement component 2 (C2) and factor B (BF) genes refine association for age-related macular degeneration (AMD). Invest Ophthalmol Vis Sci 50: 540-543. 19. Bird AC, Bressler NM, Bressler SB, Chisholm IH, Coscas G, et al. (1995) An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group. Surv Ophthalmol 39: 367-374. 20. Klaver CC, Assink JJ, van LR, Wolfs RC, Vingerling JR, et al. (2001) Incidence and progression rates of age-related maculopathy: the Rotterdam Study. Invest Ophthalmol Vis Sci 42: 2237- 2241. 5

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Chapter 5.indd 133 23-10-12 19:41 Chapter 5.indd 134 23-10-12 19:41 CHAPTER 6 Geographic Atrophy in Age-related Macular Degeneration and TLR3 (Letter)

Rando Allikmets, Arthur A. Bergen, Michael Dean, Robyn H. Guymer, Gregory S. Hageman, Caroline C. Klaver, Kari Stefansson, Bernhard H. Weber and the International AMD Genetics Consortium

Members of the International AMD Genetics Consortium: Joanna E. Merriam, R. Theodore Smith, Gaetano R. Barile, Karen M. Gehrs, Lisa S. Hancox, Norma J. Miller, Mary L. Howard, Paulus T. de Jong, Dominique Baas, Lintje Ho, Johannes R. Vingerling, Andre G. Uitterlinden, Lars G. Fritsche, Claudia N. Keilhauer, Paul N. Baird, Julie Sawitzke, Kristinn P. Magnusson, Gudmar Thorleifsson, Unnur Thorsteinsdottir

The New England Journal of Medicine 2009; 360(21):2252-2254

Chapter 6.indd 135 23-10-12 19:42 Chapter 6 Yang et al. report an association between the F412L (rs3775291) variant of TLR3 and protection against the development of geographic atrophy (GA), a phenotype of late-stage age-related macular degeneration (AMD). The International Age-related Macular Degeneration Genetics Consortium genotyped rs3775291 in eight well- known case–control studies involving data from a total of 1080 patients of European descent with GA and 2669 matched controls. Data from the eight studies are summarized in Table 1. The studies show, both

an association between the TLR3 rs3775291 single nucleotide polymorphism individually and collectively, neither a significant association nor a trend toward Table 1). The difference in the minor allele frequency (MAF) between patients with GA and controls did not (SNP) and protection against GA (P≥0.29 in all cohorts) ( exceed 4% in any study and, summarizing the data across studies, the MAF of the rs3775291 SNP was identical in the 2669 controls than in the 1080 patients with

GA. Moreover, the MAF of rs3775291 did not differ significantly between any of the et al. GA groups or between any of the control groups, making population stratification an Two other explanations remain. Random variability of this SNP in the general unlikely explanation for the difference between our findings and those of Yang

the difference can be explained by experimental error. We and Yang et al. screened population can result in a chance finding in relatively small cohorts. Alternatively,

samples from the AREDS cohort [1] and obtained significantly different results. Since Repositories), there should be substantial overlap and concordance between data there were only 237 subjects with verified GA in the AREDS cohort (in the Coriell Cell generated in the two studies. Although the MAF in AREDS controls was very similar in our study and in the study by Yang et al. (0.30 and 0.31, respectively), in AREDS

and 0.21, respectively; P=0.02). The reasons underlying these differences could be patients with GA, the MAF differed significantly between the two studies (0.28 resolved by a direct comparison of the genotypes obtained in the AREDS subjects in the two studies. We conclude that it is incorrect to describe TLR3 as being associated with dry AMD and therefore inappropriate to suggest revising therapeutic strategies on the basis of the available data.

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Geographic Atrophy in Age-related Macular Degeneration and C C GA 0.59 1.03 (0.92-1.15) Australia GA C C Australia Australia Total GA 0.35 0.8 (0.5-1.27) AREDS GA C C AREDS GA 0.46 0.89 (0.64-1.22) C Iceland GA GA 0.75 1.05 (0.77-1.45) C C Germany GA GA 0.29 0.86 (0.65-1.14) C C Rotterdam GA GA 0.36 1.20 (0.821.75) 6 C C Amsterdam Rotterdam Germany Iceland GA 0.53 1.12 (0.78-1.63) Amsterdam GA C 152 41 136 28 422 105 191 102 90 82 101 30 70 546 1366 108 40 103 29 341 63 139 96 65 71 82 21 76 450 1047 35 8 25 7 80 16 36 12 14 10 21 6 17 84 256 C Iowa Iowa GA 0.94 0.99 (0.70-1.40) Iowa GA C 365 102 295 89 264 64 843 184 366 210 169 163 204 57 163 1080 2669 204 53 133 37 28 12 C Columbia GA 211 105 93 13 0.28 0.26 0.3 0.3 0.31 0.29 0.33 0.3 0.26 0.29 0.29 0.28 0.28 0.3 0.29 0.33 0.29 0.29 0.44 1.12 (0.86-1.47) GA 209139 (66.5) 365 201 (55.2)70 (33.5) 64 (62.7)79.6 (8.6) 164 (44.8) 187 (63.4) 102 38 (37.3) 74.6 (7.1) 54 (61.0) 108 (36.6) 144 (54.5) 76.8 (7.7) 35 (39.0) 35 (54.7) 80.9 (8.6) 295 120 (45.5) 314 (37.2) 78.7 (7.6) 127 (66.1) 29 (45.3) 74.8 (6.7) 240 (62.5) 529 (62.8) 69 (32.9) 65 (33.9) 76.4 (8.3) 89 96 (56.8) 77.0 (5.5) 144 (37.5) 93 (57.1) 141 (67.1) 78.5 (6.2) 108 (52.9) 73 (43.2) 78.8 (5.4) 35 (64.8) 70 (42.9) 84.4 (7.4) 95 (52.3) 264 96 (47.1) 92.6 (2.0) 19 (35.2) 79.5 (5.3) 68 (41.7) 76.9 (4.6) 70.5 (9.8) 64 70.9 (9.8) 843 192 384 210 169 163 204 54 163 *AREDS= Age-related Eye Disease Study; GA= geographic atrophy; CI= confidence interval; C= controls. C= controls. CI= confidence interval; atrophy; geographic GA= Disease Study; Eye *AREDS= Age-related group. with the control as compared group atrophy in the geographic of the TLR3 variant the frequency for are ** Odds ratios N= number of study subjects; GA= geographic atrophy; C= controls. C= controls. atrophy; geographic subjects; GA= N= number of study N Gender (%) Female Male age (s.d.) Average Table 1. Genotyping and Association Analysis of the TLR3 Rs3775291 Variant in Eight Cohorts* in Eight Variant of the TLR3 Rs3775291 1. Genotyping and Association Analysis Table Value Table 2. Disease Status, Gender and Age of Subjects in Eight Cohorts. 2. Disease Status, Gender and Age of Subjects in Eight Table seriesCase-control Columbia Total no. of subjects no. Total Genotype (no. of subjects) Genotype (no. CC CT TT Minor allele (T allele) frequency P value equilibriumHardy-Weinberg 0.54 0.58 0.36 0.09 0.89 0.67 1 0.63 0.33 0.33 0.24 0.93 0.56 0,8 0.82 0.82 For difference in allele difference For frequency Odds ratio (95% CI)** Odds ratio 137

Chapter 6.indd 137 23-10-12 19:42 Chapter 6 Materials and Methods

Patients Study cohorts The consortium included eight international cohorts of patients with GA and matched by age and ethnicity controls, all of which have been extensively characterized and described in previous genetic and epidemiological studies of AMD as follows: from Columbia University, New York, USA [2-4]; University of Iowa, Iowa City, USA [3,4]; AREDS cohort, NEI/NIH, USA [5]; Rotterdam Study, Erasmus University, Rotterdam, The Netherlands [6,7]; The Netherlands Institute for Neurosciences (NIN), Amsterdam, The Netherlands [2,6]; University Eye Clinic, Reykjavik, Iceland [8]; University of Würzburg, Würzburg, Germany [9,10]; and the Center for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Australia [11,12]. The details of case-control series are provided in Table 2. All study subjects provided written informed consent before participating. The study was conducted at all sites in strict adherence to the tenets of the Declaration of Helsinki and was approved by the respective Institutional Review Boards at Columbia University and University of Iowa, by the Age-Related Eye Disease Study (AREDS) Access Committee, Data Protection Authorities and National and Institutional Ethics Committees (at Amsterdam, Rotterdam, Reykjavik and Würzburg sites) and according to the National Health and Medical Research Council of Australia’s statement on ethical conduct in research involving humans (revised in 1999) at the Melbourne site.

Grading All study subjects underwent clinical examination and stereoscopic color fundus photography after pharmacologic mydriasis, and were graded according to the

International Classification and Grading System for AMD [13] (Reykjavik, Melbourne [7,14] (Columbia, Iowa, Amsterdam and Rotterdam sites), or according to the and Würzburg sites), Rotterdam modification of the International classification

detailed description of each cohort has been provided in earlier publications, classification established by the AREDS study [1] (for the AREDS cohort). The as described in the previous section. In summary, cases with GA (disease stage 4

atrophy of the retinal pigment epithelium (RPE) and choriocapillaris with diameter according to all classifications) were defined as presenting a well-defined area of

or pigmentary abnormalities) AMD in the other eye, or with distinct bilateral GA. >= 175 μm in one (more severely affected) eye and early stage (soft drusen and/ Patients with atrophy due to other causes, such as RPE scarring and post choroidal

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Geographic Atrophy in Age-related Macular Degeneration and neovascularization (CNV)- associated degeneration were excluded from the GA category, as were patients with mixed (GA and CNV) phenotype in the same eye or in contralateral eyes. The average age of subjects with GA was ~75 years in all cohorts. Subjects were graded as ‘unaffected controls’ only when fundus photographs showed absolutely no signs of AMD; i.e., they had clear fundi at >60 years of age or

<5 small hard drusen (grade 1 by AREDS classification or grade 0 by International/ made even more stringent by either raising the age threshold (>70 in the Rotterdam Rotterdam classification). In some cohorts the selection criteria for controls were Study and >90 in the Iceland cohort), or by extending the follow-up period to several years (Rotterdam Study).

Genotyping The rs3775291 SNP was genotyped by the Applied Biosystems (Foster City, CA) Taqman® 5’ nucleotidase assay (Columbia, AREDS, Würzburg and Amsterdam cohorts), by the Illumina HumanHap arrays (Iceland and Rotterdam Study cohorts) or, in Iowa and Melbourne cohorts, by a combination of SSCP, direct sequencing and Molecular Inversion Probe Genotyping (ParAllele Biosciences/Affymetrix).

Statistical analysis Genotypes were tabulated in Microsoft Excel and presented to SPSS for contingency table analysis as described previously [3,4]. Compliance to Hardy-Weinberg 6 equilibrium was checked using SAS/Genetics (SAS Institute), and all cohorts in both cases and controls survived a cutoff of P>0.05. For the combined analysis of all studies we calculated the weighted Mantel-Haenszel estimate for the odds ratio, and

obtained similarly non-significant values. Acknowledgements

The authors thank Sarah Schlotterbeck, Kendal Lemon, Melinda Cain, Andrea Richardson, Pascal Arp, Mila Jhamai, Dr Fernando Rivadeneira, Dr Michael Moorhouse, Marijn Verkerk, and Sander Bervoets for technical assistance and Haraldur Sigurdsson and Fridbert Jonasson for phenotyping. Supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research and SAIC-Frederick under contract #NO1-CO-12400 and with grants from the National Eye Institute EY13435 (RA) and EY017404 (RA, GSH); the Macula Vision Research Foundation (RA); Wallach Foundation (RA); Elyachar Foundation (RA); Kaplen Foundation (RA); Wigdeon

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Chapter 6.indd 139 23-10-12 19:42 Chapter 6 Point Charitable Foundation (RA); an unrestricted grant to the Department of Ophthalmology, Columbia University, from Research to Prevent Blindness; German Research Foundation (DFG) WE1259/18-1 and WE1259/19-1, the Ruth and Milton Steinbach Foundation (BHW, RA), the Alcon Research Institute (BHW, RA), the

Research (NWO; nr. 050-060-810); NWO Investments (nr. 175.010.2005.011, Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific 911-03 012); Research Institute for Diseases in the Elderly (RIDE2; 014-93-015); Netherlands Organization for the Health Research and Development (ZonMw); Ministry of Education, Culture and Science, Ministry for Health, Welfare and Sports, European Commision (DG XII), Municipality of Rotterdam; the Netherlands Macula Society, Netherlands National Foundation for the Blind (LSBS), the Netherlands National Society for Prevention of Blindness (ANVVB), National Health and Medical Research Council, Australia, Career development fellowship (RHG), J A COM Foundation (PNB).

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Geographic Atrophy in Age-related Macular Degeneration and References

1. (1999) The Age-Related Eye Disease Study (AREDS): design implications. AREDS report no. 1. Control Clin Trials 20: 573-600. 2. Despriet DD, Bergen AA, Merriam JE, Zernant J, Barile GR, et al. (2008) Comprehensive analysis of the candidate genes CCL2, CCR2, and TLR4 in age-related macular degeneration. Invest Ophthalmol Vis Sci 49: 364-371. 3. Gold B, Merriam JE, Zernant J, Hancox LS, Taiber AJ, et al. (2006) Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet 38: 458-462. 4. Hageman GS, Anderson DH, Johnson LV, Hancox LS, Taiber AJ, et al. (2005) A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci U S A 102: 7227-7232. 5. (2001) A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E and beta carotene for age-related cataract and vision loss: AREDS report no. 9. Arch Ophthalmol 119: 1439-1452. 6. Despriet DD, Klaver CC, Witteman JC, Bergen AA, Kardys I, et al. (2006) Complement factor H polymorphism, complement activators, and risk of age-related macular degeneration. JAMA 296: 301-309. 7. van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT (2003) The risk and natural course of age-related maculopathy: follow-up at 6 1/2 years in the Rotterdam study. Arch Ophthalmol 121: 519-526. 8. Magnusson KP, Duan S, Sigurdsson H, Petursson H, Yang Z, et al. (2006) CFH Y402H confers similar risk of soft drusen and both forms of advanced AMD. PLoS Med 3: e5. 6 9. Fritsche LG, Loenhardt T, Janssen A, Fisher SA, Rivera A, et al. (2008) Age-related macular degeneration is associated with an unstable ARMS2 (LOC387715) mRNA. Nat Genet 40: 892-896. 10. Rivera A, Fisher SA, Fritsche LG, Keilhauer CN, Lichtner P, et al. (2005) Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum Mol Genet 14: 3227-3236. 11. Baird PN, Islam FM, Richardson AJ, Cain M, Hunt N, et al. (2006) Analysis of the Y402H variant of the complement factor H gene in age-related macular degeneration. Invest Ophthalmol Vis Sci 47: 4194-4198. 12. Richardson AJ, Islam FM, Guymer RH, Baird PN (2009) Analysis of rare variants in the

macular degeneration (AMD). Invest Ophthalmol Vis Sci 50: 540-543. complement component 2 (C2) and factor B (BF) genes refine association for age-related 13. Bird AC, Bressler NM, Bressler SB, Chisholm IH, Coscas G, et al. (1995) An international

degeneration. The International ARM Epidemiological Study Group. Surv Ophthalmol 39: classification and grading system for age-related maculopathy and age-related macular 367-374. 14. Klaver CC, Assink JJ, van LR, Wolfs RC, Vingerling JR, et al. (2001) Incidence and progression rates of age-related maculopathy: the Rotterdam Study. Invest Ophthalmol Vis Sci 42: 2237- 2241. 141

Chapter 6.indd 141 23-10-12 19:42 Chapter 6.indd 142 23-10-12 19:42 CHAPTER 7 The Dynamic Nature of Bruch’s Membrane

Judith C. Booij, Dominique C. Baas, Juldyz Beisekeeva, Theo G.M.F. Gorgels and Arthur A.B. Bergen

Progress in Retinal and Eye Research 2010; 29:1-18

Chapter 7.indd 143 23-10-12 19:44 Chapter 7 Abstract

Bruch’s membrane (BM) is a unique pentalaminar structure, which is strategically located between the retinal pigment epithelium (RPE) and the fenestrated choroidal capillaries of the eye. BM is an elastin- and collagen-rich extracellular matrix that acts as a molecular sieve. BM partly regulates the reciprocal exchange of biomolecules,

general circulation. Accumulating evidence suggests that the molecular, structural nutrients, oxygen, fluids and metabolic waste products between the retina and the and functional properties of BM are dependent on age, genetic constitution, environmental factors, retinal location and disease state. As a result, part of the properties of BM are unique to each human individual at a given age, and therefore uniquely affect the development of normal vision and ocular disease.

The changes occurring in BM with age include increased calcification of elastic glycosaminoglycans. In addition, advanced glycation end products (AGEs) and fat fibres, increased cross-linkage of collagen fibres and increased turnover of accumulate in BM.

These age-related changes may not only influence the normal age-related health pigmentosa (RP) and age-related macular degeneration (AMD). Undoubtedly, BM is of photoreceptor cells, but also the onset and progression of diseases like retinitis

the site of drusen development. Confluent drusen and uncontrolled activation of the adhesive interactions between the RPE and BM are instrumental in the development complement cascade are most likely the first signs of AMD. Furthermore, the nature of

actively involved in a range of other retinal disorders such as Pseudoxanthoma of retinal detachments and proliferative retinal disease. Finally, BM is passively or

Here, we review the dynamic nature of Bruch’s membrane, from molecule to man, elasticum (PXE), Sorsby’s Fundus Dystrophy and Malattia Leventinese. during development, aging and disease. We propose a simple and straightforward

nomenclature for BM deposits. Finally, we attempt to correlate recently published functional properties of BM. Our review may shed light on the complex involvement mRNA expression profiles of the RPE and choroid with molecular, structural and of BM in retinal pathology, notably age-related macular degeneration.

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Chapter 7.indd 144 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane Introduction

In the past, many investigators considered Bruch’s membrane (BM) a relatively boring and simple sheet of extracellular matrix, merely occupying space between the retinal pigment epithelium (RPE) and the choroid. Recently, however, interest in BM has increased exponentially; and understandably so, given its strategic location between the retina and the general circulation, and its crucial role in retinal function, aging and ocular disease. The pentalaminar BM structure forms a single functional unit with the RPE and choriocapillaris. It is involved in the essential exchange of numerous biomolecules, oxygen, nutrients and waste products in between these tissues. Given its unique location and structure, BM plays a crucial role in cell- cell communication, cellular differentiation, proliferation or migration, tissue remodelling and in shaping pathologic processes [1-4]. The nature of BM is highly dynamic, and depends on genetic factors, environmental burden, the topographic position in the retina, age and disease [5-11]. It has also become clear that BM is the

focal point of local and systemic risk factors for initial stages of the most frequent (AMD). In addition, BM is primarily or secondarily involved in a number of additional untreatable blinding disorder known to man: age-related macular degeneration genetically determined ophthalmic disorders such as proliferative vitreoretinopathy (PVR)[12,13], pseudoxanthoma elasticum (PXE) [14,15], Marfan syndrome [16-18], Sorsby’s fundus dystrophy [19-21] and others.

Here, we review the existing knowledge on BM, with a focus on its normal structure and function and the role of BM in normal aging and early AMD pathology. Finally, we provide an outlook on possible AMD disease prevention or treatment. Bruch’s Membrane: molecular composition, structure and 7 normal physiology

Development of BM The retina is a derivative of the neuroectoderm of the diencephalon. Around the 4th

develops, which forms the future optic cup. Upon invagination of the optic cup in the week of gestation, a secondary eye bubble, surrounded by ectoderm and mesenchyme,

can be distinguished. In the next stage, neural crest-derived mesenchyme, which will sixth week of development, the future RPE and the undifferentiated neural retina form the future choroid, starts to condensate around the optic cup. At the same time, the primitive retina, by now consisting of an RPE cell layer, an outer nuclear zone and

an acellular marginal zone flanked by basal membranes, continues to differentiate. 145

Chapter 7.indd 145 23-10-12 19:44 Chapter 7 The outer RPE basal membrane becomes incorporated in the future BM. Analysis

of the developing chick retina showed that, at the tenth week of gestation, collagen fibrils are deposited beneath the basal RPE lamina. The elastic fibre layer can be detected 3-4 weeks later. Full differentiation of the elastic fibre layer to a perforated sheet is achieved by mid-term. In chicken, BM most likely continues to mature over membranes of the RPE and choroid develop, followed by the development of the the next weeks, months or even years [2,22]. In normal mice, initially the basal

about the molecular and cellular events that regulate the early developmental collagen layers, and, finally the central elastin layer [23]. Relatively little is known phases of BM in man. However, it is not hard to imagine that deposition of collagen and elastin proteins is preceded by upregulation of the expression of corresponding genes in the adjacent tissues. Once BM formation has started, the ECM layers may affect cell-cell communication directly, thereby possibly creating the opportunity to further direct their own formation and differentiation. Although it is not exactly clear how BM is formed, gene expression data on adult RPE and choroid from our own lab (Figure 1) [11, 24], indicate that both the choroid and RPE cells are, in principle, capable of synthesizing the major components of BM. In conclusion, available evidence suggests that BM is (ultimately) formed or maintained from both the RPE and choroidal side, perhaps in a coordinated or stochastic fashion.

Ultrastructure and protein content of BM layers

Figure 1 According to the classification of Hogan in the early 1960’s [25], BM consists of five layers ( ). From the RPE toward the choroid, the following layers can be distinguished histologically: the basement membrane of the RPE, the inner Figure 1). collagenous layer (ICL), the elastin layer (EL), the outer collagenous layer (OCL) and, finally, the basement membrane of the choriocapillaris (

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Figure 1. Schematic Drawing of Proteins Present in Bruch’s Membrane and the Corresponding Genes Expressed in Adjacent Cells. COL4A3, FNDC5, TIMP3) with higher expression levels in the RPE than in the choroid, and 17 genes (remainder) with higher expression levels in the choroid than in the RPE. A qualitative impression We identified three genes ( of the gene expression is given. Gene expression levels were determined by RNA microarray study comparing gene expression levels from two adjacent tissue types from the same donor. Experiments were performed in

triplicate (on three different healthy older human donor eyes)[24]. bm RPE: basement membrane of the RPE, bm choroid: basement membrane of the choroid. The abbreviation for basement membrane is not used in the 7 accompanying text. A color version of this figure can be found in the color figures section. The basement membrane of the RPE

The basement membrane of the RPE is a continuous BM layer approximately 0.14- membranes in the body [27,28]. The RPE basement membrane contains many 0.15 µm in thickness in the young [26]. It resembles in many aspects other basement

components similar to the basement membrane of the choriocapillaris: collagens dermatan sulphate [32] (Figure 1). In contrast, collagen type VI is not present in the type IV [29], laminin [30], fibronectin [31], heparan sulphate and chondroitin/ basement membrane of the RPE.

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Chapter 7.indd 147 23-10-12 19:44 Chapter 7 The inner collagenous layer (ICL)

The inner collagenous layer is approximately 1.4 µm in diameter. The ICL consists of 60 nm thick striated fibres of collagen type I, III, and V, organized in a multilayered biomolecules, such as glycosaminoglycans (chondroitin sulphate, dermatan sulphate grid-like structure [33]. The collagen grid is embedded in a mass of interacting and hyaluronic acid) [32] and components of the coagulation and complement system.

The elastin layer (EL)

The elastin layer (EL) consists of several stacked layers of linear elastin fibres of varying shapes and sizes. The fibres form a perforated sheet with interfibrillary from the edge of the optic nerve to the pars plana of the ciliary body [33]. In addition spaces of about 1 µm. The sheet is about 0.8 mm thick in the young and extends

to elastin fibres, the EL contains collagen type VI, fibronectin and other protein- associated substances. Recently, Chong and co-workers [34] found, by examining 121 human donor eyes, that the EL is three to six times thinner in the macula than in the periphery in all studied age groups. Collagen fibres from the ICL and OCL frequently cross the EL [33]. The outer collagenous layer (OCL)

The OCL is less thick than the ICL (0.7 µm in the young) but the structure and components are largely identical to those of the ICL (see above) [33]. The basement membrane of the choriocapillaris The basement membrane of the choriocapillaris is an non-continuous, interrupted BM layer due to the so-called intercapillary columns of the choroid. The basement

composed of laminin, heparan sulphate and collagens type IV, V and VI. Aisenbrey et membrane has an average thickness of 0.14 mm in the young eye and is predominantly al

. (2006) found that the RPE synthesizes laminins that preferentially adhere BM to glycosaminoglycan in the BM. The (two) HS polysaccharide side chains bind to a the RPE through interaction with integrins [30]. Heparan sulphate (HS) is a common variety of protein ligands in BM and regulate a range of biological activities. Roberts

choriocapillaris may inhibit endothelial cell migration into the BM. Collagen type and Forrester [22] proposed that collagen type IV in the basement membrane of the V is present in most types of connective tissue, particularly in pericellular spaces and near basement membranes, and plays a role in platelet aggregation, epithelial

cell migration and binding of interstitial collagen fibrils [35]. Type VI collagen is the 148

Chapter 7.indd 148 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane

membrane. It may be involved in anchoring BM to the capillary endothelial cells major structural component of microfibrils, and specific for the choroidal basement of the choroid [36]. Collagen VI possibly interacts with collagen I [22] which is

feature of the choriocapillaries adjacent to the choroidal basement membrane is the abundant in both the BM-OCL and the choroidal matrix. A remarkable structural endothelial fenestrations, or pores, that are permeable to macromolecules [37].

Molecular composition of BM considering gene expression of RPE and choroid

choroidal cells for the production of most of its extracellular matrix constituents Since BM is an acellular layer, it most likely depends on the adjacent RPE and

[9]. Furthermore, a large number of biomolecules and waste products from the RPE and choroid pass through BM and can get trapped there influencing both the age and there is an extensive turnover of ECM molecules, driven by matrix metallo structure and function of BM. Finally, the molecular composition of BM changes with proteinases (MMPs), during life.

relevant to the molecular composition of BM [9] (Figure 1; [24]). In this regard, we We hypothesized that the RPE and choroidal gene expression profiles are potentially observe that 1) both the RPE and choroid are capable of producing BM proteins,

such as collagens, fibronectin and heparan sulphate containing proteoglycans; and 2) for those proteins known to be present in BM, the choroidal cells (endothelial RPE does. In summary, approximately sixteen different proteins (subunits) were cells, fibroblasts, smooth muscle cells) appear to contribute more to BM than the

previously assigned to BM by immunohistochemistry. Two (COL4A3, TIMP3; are synthesized both by the RPE and choroidal cells, and the majority (i.e. numerous 12.5%) of these are predominantly synthesized by the RPE, two (FN, HSP; 12.5%) 7

cells (Figure 1 collagens, elastin; 75%) of the known BM proteins is mainly synthesized by choroidal ). These data support the hypothesis of Sivaprasad and co-workers interest to note that both the RPE and choroidal cells produce mRNA of many more who suggested a common origin of BM and the vascular intima [38]. Finally, it is of collagen (subunits) and other ECM molecules, that have not (yet) been assigned to BM [9, 24].

Structure and molecular composition of BM in the macula and retinal periphery Evidence exists that BM is structurally different in the macular area compared

since investigators use eyes from both human donors and animal models. Also the to the retinal periphery. Interpretation of the literature in this respect is difficult ages of the studied eyes vary considerably and frequently retinal punches are used

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Chapter 7.indd 149 23-10-12 19:44 Chapter 7

from undefined or various retinal locations. Nevertheless, in sheep, topographical differences in BM thickness were observed as early as 1983 [39]. In human donor eyes of all ages, Chong and colleagues [34] found that the EL of porous than in the retinal periphery. RPE microarray studies in human donor eyes of BM in the macular area was three to six times thinner, and two to five times more age 17-36 years, performed by van Soest et al. [9] provided evidence that RPE gene

and a number of proteoglycans) was lower in the macular area than in the retinal expression of at least 33 structural BM proteins (collagens, laminins, fibronectins periphery, while two genes showed an inversed expression pattern (COMP and

no regional differences in RPE gene expression was observed [9, Bergen and van THBS4). For collagen type IV chains, most fibronectin types, as well as elastin, Soest, unpublished results]. These data suggest that the topographical differences et al. [34] may not be due to the higher or lower transcriptional activity of the elastin gene. in EL thickness observed by Chong

Taken together, these data may suggest that region-specific structural and functional of the genes ALDH1A3, cKIT, FLJ36353, NADH dehydrogenase, RTBND2, TIMM17B, properties and/or turnover rates of components of BM exist. With the exception and WFDC1

[9,40,41], these gene expression data await further verification from other topographical studies on mRNA and protein level. Confirmation of additional modelling of BM, and correlation of observed molecular, structural and functional differentially expressed genes will enable definite molecular and bioinformatic differences.

BM functions The three primary functions of BM include 1) regulating the diffusion of (bio-) molecules between choroid and RPE, 2) providing physical support for RPE cell adhesion, migration and perhaps differentiation, and 3) acting as a division barrier, restricting choroidal and retinal cellular migration (Figure 2). Obviously, the functional aspects are closely related to the local structure and molecular composition of BM (in the macula or in the periphery).

Diffusion properties As BM is located between the RPE and choroid, its passive transport function

biomolecules between the retina and the choroid. Given the acellular nature of BM, is obvious. BM acts as a semi-permeable filter for the reciprocal exchange of diffusion is primarily regulated by passive processes. Diffusion across BM depends

on its molecular composition, which, in turn, is influenced by several factors like

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Figure 2. Schematic Drawing Showing the Normal Structure and Functions of Bruch’s Membrane. Transmission Electron Microscopic image of BM courtesy of Prof. J. Marshall. BM allows for the transport of biomolecules across the membrane, it attaches RPE cells to the membrane and it acts as a physical barrier to 7 prevent the migration of RPE cells and choroidal cells across the membrane. A color version of this figure can be found in the color figures section.

age and location in the retina. Indeed, Marshall and co-workers found a relationship between BM porosity and water flow: The EL showed the greatest pore size between the more or less randomly organized fibres and the largest water conductivity. The Diffusion through the BM also depends on hydrostatic pressure on both sides of ICL had the smallest pores and the lowest conductivity [33].

Biomolecules trying to pass through the BM from the choroid to the RPE include BM and on rescue and concentration of specific biomolecules and anorganic ions. nutrients, lipids, pigment precursors, vitamins (vitamin A), oxygen, minerals, antioxidant components, trace elements and (other) serum constituents [33,42,43]

BM, since they are needed for optimal function of the photoreceptor RPE complex All these molecules bind to BM, or are taken up by the RPE from the bloodstream via

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Chapter 7.indd 151 23-10-12 19:44 Chapter 7 and also the neural retina.

(Bio-)molecules trying to pass BM from RPE to choroid, include CO2, water, ions, oxidized lipids, oxidized cholesterol, and other waste products cleared by the RPE. The waste products consist of metabolic, visual cycle or electrophysiological waste from the photoreceptors or the RPE, as well as waste products from partly digested, partly oxidized, membranous fragments of shed photoreceptor outer segments

its structure and molecular composition and vary from the macula to the periphery. (POS) [33,42-44]. Obviously, the filtration properties of BM are closely related to

the build up of hydrophobic lipids (lipid wall) and membranous debris in the aging BM’s permeability to water is influenced by age-related collagen cross-linking, and BM [45]. Using Ussing chambers, Moore et al. [46], and Statira et al. [47,48] measured the movement of water across BM-choroid samples experimentally. They found an exponential decrease in permeability to water with age, measured over time- et al. intervals of 9.5 to 15 years; these findings were later confirmed by Hillenkamp the macular area than in the retinal periphery [48]. Interestingly, most of the loss [49]. The rate of loss of water permeability was largest in the ICL and larger in of hydraulic permeability appears to occur in early life, long before BM debris can

pathogenic processes, the molecular changes in BM precede the changes that can be be visualized [50]. The latter finding suggests that, like many other biological and visualized histologically [46,51].

RPE cell adhesion and differentiation

attachment site for the RPE [52,53]. BM may also act in RPE differentiation [54] In addition to having filtration properties, BM also provides support and acts as an

attachment of RPE cells than BM from old donors [57]. Not all layers of BM show and wound healing [55,56]. BM from young donors is much more efficient in the

layer that RPE cells normally adhere to [52]. equally strong adhesion properties. For example, the RPE basal lamina of BM is the RPE-BM adhesion is mediated by integrin cell surface receptors. Integrins are a group of (RPE basal) membrane proteins that is capable of binding to a number of extracellular and BM matrix components such as laminin isoforms and type IV collagen in so-called anchoring plaques [58]. Indeed, cultured RPE cells overexpressing integrins show more adhesion to BM than normal RPE cells [59]. The RPE continues to develop until approximately 6 months after birth. After that, the RPE is generally considered to be post-mitotic. However, in the event of mechanical or light-induced damage to the RPE cell layer, the RPE cells can

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Chapter 7.indd 152 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane proliferate in a manner similar to that of other epithelial tissues. Small defects are corrected by migration of cells from the edges of the wound, in larger defects, there

In is also proliferation of cells. Wound healing is more efficient in the presence of the vitro basal lamina of the RPE than when this layer of BM is absent or damaged [60]. wound healing is impaired upon inhibition of integrins. It is known that BM thickens and calcifies with age, which may also impede the attachment of the RPE to BM. Finally, the process of wound healing appears to be disturbed in AMD patients, that viable RPE cells from AMD patients did not grow well in culture. Moreover, possibly due to alterations in the RPE and/or BM. This is exemplified by the fact dissection of a choroidal neovascularization (CNV) membrane in AMD patients was not followed by complete restoration of the RPE cell layer [61].

Division barrier for cell migration The outer blood-retina barrier (oBRB) is formed by RPE cells that are connected to each other by tight junctions. The oBRB prevents transport of molecules larger than

supported by BM, that acts as a semi-permeable molecular sieve. The inner blood- 300 kDa into and out of the retina [62]. The barrier function of the RPE is physically retina barrier (iBRB) is comprised of a single layer of non-fenestrated retinal

vascular endothelial cells connected by tight junctions [62]. If both leukocytes and in mice and rats showed that bone marrow-derived cells cannot easily pass the iBRB endothelial cells are normal, leukocytes do not cross the iBRB [62-64]. Experiments as these cells could not be detected in the retina one year after injection into the

normal retina despite an apparently intact iBRB [62] and may pass BM and the oBRB. circulation [65,66]. Nonetheless, lymphocytes have been shown to infiltrate the 7 BRB, enabling sampling of the retinal environment and possibly further recruitment If lymphocytes are activated, they are able to initiate a transient breakdown in the

of inflammatory cells [62]. The aging Bruch’s membrane and AMD pathology

Normal aging of BM and AMD pathology The distinction between normal aging and pathology in, for example, AMD, is not

However, features of aging and disease may overlap, may be different for different clear cut. Interestingly, aging itself is the strongest risk factor for developing AMD.

example, some investigators view aging itself as a pathology, that can ultimately be cell types involved, and may even raise an almost philosophical discussion. For cured, while others do not.

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Chapter 7.indd 153 23-10-12 19:44 Chapter 7 Here, we consider “normal aging” those changes that occur in the majority of individuals continuously from adulthood to old age, without direct clinical consequences. It is clear, however, that these age-related, non-pathogenic changes

state. We regard “pathogenic changes” as those changes that cause local loss of may affect the overall fitness of cells and tissues, predisposing them to a pathogenic function, leading to clinical consequences. Since these categories certainly overlap, it may be useful to illustrate our line of thought with a number of examples. We consider the continuously decreasing vitality of RPE, photoreceptors and

choroidal cells part of normal aging. This decrease in cellular fitness continues observations of Curcio et al. in man [67,68]. Interestingly, the rate of spontaneous or is accelerated when specific pathological processes set in. This is in line with photoreceptor cell loss with age may be genetically determined, since different wild type mouse strains have different rates of spontaneous photoreceptor loss [69]. We also consider the formation of lipofuscin in RPE as part of normal aging. However, we classify excessive lipofuscin accumulation, leading to local loss of function, cell

Also, the deposition of lipids in BM can be seen as a normal aging phenomenon, until damage and cell death, as (AMD) pathology [70]. the point where the build up of the lipid wall starts to affect local RPE function.

drusen development, and the controlled involvement of the complement system We also think that the formation of basal laminar deposits as well as (subclinical)

to clear BM debris is a normal aging phenomenon. In 90 out of 100 apparently healthy Dutch donor eyes, age between 70 and 80, we observed subclinical macular drusen after histology and PAS staining (Bergen and co-workers, unpublished (pathogenicity). However, in our view, pathology only sets in when the involvement results). Many investigators consider the appearance of drusen a hallmark of AMD of the complement system becomes uncontrolled, and abnormal loss of local cellular function, cell damage and cell death occur.

age, RPE and BM change continuously. Normal BM aging can insidiously change In summary, we support the early views of Marshall and co-workers that, with into (AMD) pathology [33]. Consequently, the processes underlying normal aging

fashion below. Our views are illustrated in Figure 3. and AMD pathology are difficult to separate, and are discussed in a comprehensive

Structural and molecular changes of the aging BM

throughout the larger part of life. The molecular composition and physiological The pentalaminar structure of BM, identified at birth, undergoes age-related changes

aspects of BM change dramatically. In general, there is an overall increase in thickness 154

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Figure 3. Changes in Bruch’s Membrane with Age and its Relation to AMD Pathology. The upper half of the picture shows the changes that occur in BM with age. The lower half of the picture show how the age related changes can progress into AMD pathology, either through uncontrolled activation of the complement system, through the occurence of neovascularisation or as an indirect result of the aging of BM.

and a reduced filtration capacity due to molecular modification and reconfiguration level, each causing functional changes (see Figure 4). [50,71,72]. Remodelling of BM with age occurs at the biochemical and histological

Increased cross-linkage of collagen

on the permeability of BM and changes the nature of the extracellular matrix. Cross- With age, increased collagen cross-linking occurs in BM. This has a negative effect 7

linkage increases the strength and density of the collagen network but it decreases its gradually becomes (more) inaccessible for the RPE collagenases which results in a elasticity, flexibility and perhaps filtration capabilities. The dense collagen network less effective turnover of BM components (see Figure 4) [72].

Turnover of BM proteoglycans Proteoglycans (PG) are heavily glycosylated glycoproteins that are “the glue”

to glycosaminoglycan (GAG) chains. Individual functions of proteoglycans are of extracellular matrices such as BM. PG contain a core protein covalently linked determined by both the type of core protein and the type of GAG chains. Evidence from RPE cell culture experiments [32] and immuno-electron microscopy stainings [73] suggest that the RPE predominantly synthesizes heparan sulphate, which

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Figure 4. Schematic Overview of the Changes Occuring in the Aging BM, the Functional Consequences and the Subsequent Pathology.

is incorporated into BM. Indeed, 58% of the PGs in BM are of the heparan sulfate

two percent of BM PGs are chondroitin sulphate or dermatan sulphate, which are type, which is primary located at the basal lamina of the RPE and choroid. Forty-

Interestingly, newly synthesized PG’s consist of 25% heparan sulphate and for 75% uniquely associated with collagen fibrils [32,74].

chondroitin/dermatan sulphate [32]. Since the ratio between heparan sulphate and suggested that the turnover of heparan sulphate is slower than that of chondroitin chondroitin/dermatan sulphate in BM remains unchanged during life, these data

suggested that proteoglycan turnover rate is tightly controlled [11]. Nonetheless, sulphate/dermatan sulphate [32,74]. RPE gene expression data of several individuals

inability of cells to normally digest the PG core protein [32]. Eyes from a limited after the age of 70 years, there is a slight shift toward larger PG’s, indicating the number of donors with retinitis pigmentosa and diabetic retinopathy revealed relatively high heparan sulphate levels (55% - 64%) in BM compared with healthy controls (23%) [32,75].

PGs have important structural and filtration properties in BM, and may play a role in the anti-inflammatory response. PG molecules form structural networks through 156

Chapter 7.indd 156 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane interaction with their side chains. These interactions occur not only among the

different types of PGs, but also with collagen fibres in the OCL and ICL, and, most likely, with hyaluronic acid in the interphotoreceptor space. bind water and positively charged cations such as sodium, potassium and calcium. In terms of filtration properties, the negatively charged PG side chains cause PGs to In addition, they cause PGs to form a barrier for the passage of negatively charged macromolecules [32,76]. Thus, by physical, electrostatic and biochemical means, PGs regulate the movement of molecules through the BM matrix. Evidence also reveals that PGs can affect the activity and stability of proteins and signalling molecules

et al. [77] showed that heparan sulphate interacts within the matrix [76]. Finally, PGs, and especially heparan sulphate, may have anti- with complement factor H, an important regulator of the complement cascade, and inflammatory properties. Meri regulator of the local immune response.

can modulate complement activity, by inhibition of the cleavage of complement Conversely, Kelly and co-workers [78] showed that BM-associated heparan sulphate factors B and C3 to, respectively Bb and C3b. Given the major involvement of the complement system and the innate immune system in the development of AMD

[79], the interaction between heparan sulphate PG(s) and CFH may be one of the key molecular switches that turn normal RPE aging into AMD pathology. Mineralization of BM Calcification of BM As in other soft tissues in the body, calcium can be deposited in the connective tissue of BM. In post mortem eyes, the deposits can readily be demonstrated, for example with the von Kossa staining method. Van der Schaft et al. (1992) studied 182 human 7 maculae older than 33 years of age, and found calcium deposition in BM in 59%

correlated with age, but not with AMD. However, a later study by Spraul et al. (1999) of the samples [71]. The presence and extent of the calcification was positively

The potential correlation with neovascular AMD agrees well with the notion that did demonstrate a significant correlation between BM calcification and AMD [80].

calcification of BM renders the membrane more brittle and more susceptible to breaks, allowing faster neovascularization. This finding was corroborated by the pathology observed in PXE, an autosomal recessive disease characterized by soft connective tissue calcification [81,82]. For reasons yet unknown, BM is a preferred site for these ectopic calcifications and PXE patients often also develop eye pathology. In these patients, extensive calcification of the elastic fibres of BM makes the membrane brittle, and prone to breaks. The 157

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breaks are visible upon funduscopy as angioid streaks that radiate from the macula in these patients resulting in loss of visual acuity [83]. toward the periphery [33]. Ultimately, the breaks in BM lead to neovascularisation

interest, especially in the cardiovascular system. These studies reveal a multitude of The mechanism of soft tissue calcification is currently the focus of intense scientific

molecules and processes that can influence this process. It has become increasingly clear that control of calcification involves a delicate balance between pro-calcific pyrophosphate, and several proteins such as fetuin A, Matrix Gla Protein (MGP) and and anti-calcific mediators [84]. Anti-calcific factors include molecules such as

matrix and cell death. Many different environmental and genetic factors may be vitamin K. Pro-calcific factors include high phosphate levels, damaged extracellular

causative Abcc6 involved in BM calcification. For PXE, a mouse model was made by disrupting the gene. Among other things, the PXE mice develop ectopic calcification in BM [85]. Further elucidation of the calcification process holds the promise that soft tissue and BM calcification can perhaps be influenced by drugs or by dietary mice [87]). means [86] (own unpublished observations in PXE mice, dietary influences in DCC

Iron depositions

It is well known that (ab-)normal levels of iron ions contribute to various disease retinal degeneration [88]. The subject of iron homeostasis and toxicity in retinal of aging, including atherosclerosis, Alzheimer’s disease, Parkinson’s disease and degeneration was recently and excellently reviewed elsewhere [89]. In summary, iron is essential for many metabolic processes, but can also cause damage through

inducing (local) oxidative stress. An entire network of molecules, including metal receptors/transporters and ceruloplasmin, try to maintain (local) iron homeostasis: in the body. The resulting iron overload in the retina and RPE can cause retinal the balance between benefit and damage. However, with age, iron accumulates degeneration. Within the RPE cell, iron and related ferruginous compounds play an important role in the lysosome mediated build up of lipofuscin, and in general

cellular oxidative stress, aging and apoptosis [90,91]. At the surface of BM, iron and indirectly affecting the inhibition the complement cascade [92]. other metal ions may play a role in the oligomerization of CFH molecules, thereby

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Chapter 7.indd 158 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane Zinc depositions

In 1988, Newsome and co-workers [93] found a beneficial effect for oral zinc year follow-up study of the AREDS population [94]. In parallel, millimolar amounts supplementation in AMD. These findings were corroborated by a subsequent 6.5 of zinc were found in sub-RPE deposits and BM [95,96]. While the true relationship between the presence of zinc and AMD is undoubtedly very complex, Nan and

co-workers [92] suggested, on the basis of a series of elegant experiments, that complement regulation. Zn is involved in the oligomerization of CFH, and consequently in AMD related

Advanced glycation end products (AGEs) in BM

Advanced glycation end products (AGEs) are chemically modified glycosylated or oxidized fats and proteins. Outside the body, they are produced by smoking or levels, AGE accumulation in tissues, and ultimately total body damage by (per-) cooking. The dietary intake of exogenous AGEs may be related to the AGE serum oxidative stress. Inside the body, AGEs can be produced by the combined metabolism of fat, proteins and sugar [97,98]. In general, cellular proteolysis of AGE releases AGE by-products into the serum which can be excreted in the urine. However, extracellular matrix proteins in the body are resistant to proteolysis. Consequently, AGEs accumulate preferentially

on structural proteins, like collagens in BM, where they inhibit protein function tissues activate the AGE receptor (RAGE), which is present on multiple cells in the and cause age related damage [99,100]. High concentrations of AGEs in serum or

body. Local activation of RAGEs frequently aggravates diseases like atherosclerosis, 7 diabetic nephropathy, and neurodegeneration through inflammatory and other mechanisms [101,102]. AGE accumulations containing pentosidine and carboxymethyllysine (CML) form Yamada et al age-promoting structures in BM, basal deposits, and choroid [103-106]. Indeed, for the AGE receptors RAGE and AGER1 in areas containing basal deposits than in . (2006) found that the RPE showed more intense immuno-staining

and AGE receptors are locally present in BM, basal deposits or drusen, and promote areas of normal BM [105]. In summary, these data suggests that both specific AGEs

aging and/or the development of AMD [105]. Accumulation of lipids in BM As age increases, there is a progressive accumulation of lipids (phospholipids, triglycerides, fatty acids and free cholesterol) in BM, especially in the macular area

[107]. 159

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Lipoprotein-like particle (LLPs) composition in BM resembles plasma LDL more mainly derived from the RPE. Only a small proportion is of extracellular choroidal than it does photoreceptor membrane composition [108]; moreover, these lipids are

origin [45,109-111]. In young eyes, lipid inclusions, such as LLPs, small granules and membrane-like structures, are associated with fine elastin and collagen filaments in the ICL, EL and OCL. Huang and co-workers (2008) found that, once the EL and ICL were filled with particles, LLP continued to accumulate near the RPE, but did not increase in the OCL anymore [111]. Thus, with age, these lipid inclusions filled the interfibrillary spaces Holz et al. (1994) observed that the macula of the elderly contained seven times of the EL and accumulated in the ICL, forming the so-called lipid wall [26,45]. higher concentrations of cholesterol esters than the retinal periphery. However,

these findings are not undisputed and perhaps the lipid wall thickness and content observed that the ratio of phospholipids to neutral fats varies per individual, perhaps may even differ per individual [109]. Interestingly, Holz and others [107,109] also in part depending on diet. The accumulation of lipids with increasing age, impairs

the capacity of Bruch’s membrane to facilitate fluid and macromolecular exchange retinal function. between the choroid and the RPE or vice versa [50], which is essential for normal With age, small and large extracellular deposits, such as basal deposits and drusen (discussed below) slowly but surely appear in BM. These deposits contain large

based biomolecules. amounts of (un-) esterified cholesterol, oxy-cholesterols, and many other lipid-

BM thickening Throughout life, the ‘normal’ BM almost doubles in size [71]. In a comparative study et al

of 120 human donor eyes, Ramrattan . (1994) found that the overall thickness of BM shows a positive linear relationship with age. BM thickness increased from 2 µm in the first decade to 4.7 µm 80 years later [72]. The largest part of BM thickening where RPE gene expression of most structural components of BM appears to be occurs in the ICL, followed by the OCL. This process starts in the retinal periphery, higher than in the macular area [9,112].

In general, BM thickening is caused by increased deposition and cross-linking of (less (oxidized) waste products of RPE metabolism. There is an age-related accumulation soluble) collagen fibres and increased deposition of biomolecules, the majority being

of granular, membranous, filamentous and vesicular material eventually resulting several functional changes, such as changes in elasticity and hydraulic permeability. in focal deposits and drusen. Obviously, the thickening of BM eventually leads to

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Chapter 7.indd 160 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane Functional changes of the aging BM Decreased elasticity and recoil The elasticity of the BM-choroid complex decreases with age while recoil capacity does not [33]. As discussed above, BM may loose much of its elasticity during life

AGE mediated oxidative stress damage. Overall, the decrease in elasticity and recoil because of increased collagen cross-linking, calcification of the elastic fibres and is not exacerbated in AMD [33].

Decreased hydraulic permeability The normal diffusion properties of BM are discussed above. Age-related changes in BM, such as accumulation of (neutral) lipids, turnover of

BM. Indeed, the overall water permeability of BM decreases with age primarily due proteoglycans, as well as calcification most likely alter the biophysical properties of to the changing properties of the inner half of BM [33].

Basal deposits and drusen Nomenclature and classification Basal deposits Basal deposits are accumulations of waste material between the RPE and BM [113]. Two types of basal deposits exist, basal laminar deposits and basal linear deposits. Basal laminar deposits, located internal to the basement membrane of the RPE cells, contain granular material with collagen type IV, laminin, glycoproteins, glycosaminoglycans (chondroitin- and heparan sulphate), carbohydrates (N-acetyl- galactosamine), cholesterol, and apolipoproteins B and E [33,113-115]. Basal linear 7

deposits are located in the ICL and are electron-dense, containing phospholipid drusen and AMD than basal laminar deposits [113]. granules [115,116]. Basal linear deposits are stronger markers for progression to The nomenclature of basal deposits is confusing and authors have used several different terms for numerous deposits in different layers of BM in the past (reviewed by Marshall in 1998) [33]. The frequently used term “sub-RPE deposit” [117] is also unclear, since it does not clarify the exact location of the deposit below the RPE.

In our view, the most straightforward and simple classification is to use the term based on the layer(s) in which, or in between which, the deposits are detected: OCL deposits, ICL deposits, ICL-RPE-basement membrane deposits, etc. If the layer BM deposit or basal deposit. containing a deposit cannot be defined, it seems appropriate to simply use the term

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Chapter 7.indd 161 23-10-12 19:44 Chapter 7 The presence of basal deposits and subclinical drusen have been reported as early as in the third decade [118].

Drusen Drusen are extracellular deposits that form below the RPE in BM. Drusen initially appear in the macular area, but certainly also occur in the retinal periphery. Several

point of view. types of drusen exist, they can be defined from a clinical, histological and molecular

Clinically, drusen are defined according to their location, size and shape: ophthalmologists usually make a distinction between macular and peripheral drusen, small and large drusen, or drusen with defined (hard) and less well defined borders (soft and confluent drusen) [118,119]. The presence of soft, confluent visible by ophthalmoscopy, normal aging of the RPE and BM insidiously progresses drusen is a major risk factor for AMD [118]. When (confluent) drusen become to AMD pathology. Histologically, drusen can be described by their size, shape and PAS staining [113,114]. They can be seen as small yellow patches, initially in the macular area

under the RPE. In the case of well-defined hard drusen, histological staining usually reveals local atrophy of the photoreceptors over clearly defined mounds beneath the RPE, solidly stained by PAS. In the case of less well defined soft drusen, which may become coalescent (confluent drusen), linear granular bands can be observed locally, with a light PAS staining [113]. Lengyel and co-workers suggested that so called auto-fluorescent drusen are strongly associated with the lateral walls of the intercapillary pillars of the choriocapillaris [120]. The molecular classification of drusen is discussed below. Molecular composition of drusen Drusen contain acute phase proteins, C-reactive protein, complement components, complement inhibitors, apolipoproteins, lipids and many more proteins [113]. They

vary in fat and cholesterol content, with a stable ratio between esterified cholesterol (EC) and unesterified cholesterol (UC). Frequently, drusen proteins are post- Initially, a number of complement proteins were immunolocalized to drusen by translationally modified [119]. et al Table 1) [6]. Sixty- Hageman and co-workers [121]. Using a proteomics approach, Crabb . (2002) subsequently identified 129 different proteins in drusen ( AMD donor eyes. The most common proteins in drusen of non-affected eyes were five percent of these proteins were present in drusen from both non-affected and

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Chapter 7.indd 162 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin. The

or lipids (docosahexaenoate-containing) in drusen suggested that oxidative stress presence of crystallin, and oxidatively modified proteins (TIMP3 and vitronectin) is critical for drusen formation [6]. Next, a number of additional proteins were assigned to drusen, including the amyloid beta protein [122]. Given their origin, location, and pathobiological involvement, one could possibly consider drusen a mixture between atherosclerotic plaques [123] and Alzheimer

of these three extracellular deposits (see Figure 5) showed that they only share (AD) plaques [124,125]. However, comparison of the known molecular constituents

seven proteins (amyloid (beta, P), APOE, C3, CLU, FGG and VTN). Drusen and AD plaques have 24 known molecular constituents in common. In contrast, drusen share hypothesize that drusen resemble AD plaques more than atherosclerotic plaques. only 10 known molecular components with atherosclerotic plaques. Therefore, we

Table 1. Origin of Drusen Proteins: Gene Expression in the Choroid or the RPE and the Presence of Proteins in Serum. Primary Sequence Name Sequence Code Protein Gene expression Protein in drusen chor>RPE RPE>chor in serum ACTB + ACTG1 + NM_001101 ACTN1 + NM_001614 ALB + + NM_001102 ALDH1A1 + + + NM_000477 AMBP + NM_000689 ANXA1 + + + NM_001633 ANXA2 + + + NM_000700 ANXA5 + + + NM_004039 ANXA6 + NM_001154 APCS + 7 NM_001155 APOA1 + NM_001639 APOA4 + NM_000039 APOE + NM_000482 APP + + NM_000041 ATP5A1 + + NM_000484 BFSP1 + NM_004046 BFSP2 + NM_001195 BGN + NM_003571 C3 + + + NM_001711 C5 + + NM_000064 C6 + NM_001735 C7 + NM_000065 C8B + NM_000587 C9 + NM_000066 NM_001737

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Chapter 7.indd 163 23-10-12 19:44 Chapter 7 Table 1 (continued). Primary Sequence Name Sequence Code Protein Gene expression Protein in drusen chor>RPE RPE>chor in serum CKB + + CLU + + NM_001823 COL1A2 + + + NM_001831 COL6A1 + + + NM_000089 COL6A2 + + + NM_001848 COL8A1 + + + NM_001849 CRYAA + NM_001850 CRYAB + + + NM_000394 CRYBA1 + NM_001885 CRYBA4 + NM_005208 CRYBB1 + NM_001886 CRYBB2 + NM_001887 CRYGB + NM_000496 CRYGC + NM_005210 CRYGD + NM_020989 CRYGS + NM_006891 CTSD + + + NM_017541 DIP2C + NM_001909 EFEMP1 + + + NM_014974 ELN + NM_004105 EPHX2 + NM_000501 FBLN5 + + + NM_001979 FGG + + NM_006329 FN1 + + + NM_021870 FRZB + + + NM_054034 GAPDH + + NM_001463 GPNMB + + NM_002046 H3F3A + NM_002510 HBA1 + + + NM_002107 HBA2 + + + NM_000558 HIST1H1E + NM_000517 HIST1H2AE + + + NM_005321 HIST1H2BJ + + NM_021052 HIST1H2BL + + NM_021058 HIST1H4H + + NM_003519 HIST2H2AA3 + + + NM_003543 HIST2H2BE + NM_003516 HIST4H4 + NM_003528 HP + BC111093.1 IGHA1 + + NM_005143 IGHG1 + + + AF067420 IGHG2 AAH62335 + BC037361 IGHG3 AAH33178 + + IGHG3 + IGKC + + ENST00000319391 LAMB2 + + BC073779.1 LMNA + + + NM_002292 NM_005572 164

Chapter 7.indd 164 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane Table 1 (continued). Primary Sequence Name Sequence code Protein Gene expression Protein in drusen chor>RPE RPE>chor in serum LTF + LUM + + NM_002343 LYZ + NM_002345 MFAP4 + + + NM_000239 MYH9 + + + NM_002404 MYL6 + NM_002473 OGN + + + NM_079425 ORM1 + NM_033014 PLA2G2A + + NM_000607 PLG + NM_000300 PRDX1 + NM_000301 PRELP + + NM_002574 PSMB5 + NM_002725 RBP3 + + NM_002797 RGR + + + NM_002900 RNASE4 + + + NM_002921 RPS14 + NM_002937 S100A7 + NM_005617 S100A8 + + NM_002963 S100A9 + + + NM_002964 SAA1 + + + NM_002965 SEMA3B + + NM_000331 SERPINA1 + NM_004636 SERPINA3 + + + NM_000295 SERPINF1 + + + NM_001085 SMC6 + + NM_002615 SPP2 + NM_024624 SPTAN1 + NM_006944 THBS4 + + NM_003127 TIMP3 + + + NM_003248 TNC + NM_000362 7 TUBA1A + NM_002160 TUBA1B + NM_006009 TUBA1C + NM_006082 TUBA3C + NM_032704 TUBB + NM_006001 TUBB2C + + NM_178014 TUBB3 + + NM_006088 TYRP1 + + NM_006086 UBB + NM_000550 VIM + + + NM_018955 VTN + NM_003380 NM_000638 et al Genes corresponding to proteins identified by Crabb .[6] and confirmed by Ingenuity analysis [126] to have choroid compared to the RPE (chor>RPE) of the same donor eye, in triplicate microarray measurements from a sequence code (see also Figure 6), ‘+’ = Gene expression levels were found to be at least 2.5-fold higher in the three older healthy humans [24]. Gene expression levels at least 2.5-fold higher in the RPE than the choroid are found in the column RPE>chor indicated by a ‘+’ [24]. ‘+’ in the serum indicates genes with expression in serum

identified by Ingenuity analysis. 165

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Figure 5. Overlap in Protein Content of Alzheimer Plaques, AMD Drusen, and Atherosclerotic Plaques.

Alzheimer plaque picture courtesy of Dr. I. Huitinga, Netherlands Brain Bank, donor number nhb:2006- We analyzed 121 proteins based on the article by Crabb et al. [6] and additional literature searches. We obtained 060,VU:S06/189. Atheroslcerotic plaque picture courtesy of Dr. P Sampaio Gutierrez. NM numbers for 85 of the proteins characterized by Crabb, and an additional 36 proteins were added based on

the recent literature [167]. Note that drusen share 20% of their protein content with AD plaques and less than (a). Amyloid deposits, (b). Cell nucleus, (c). Enthorinal cortex, (d). Retinal Pigment Epithelium, (e). Bruch’s 10% with atherosclerotic plaques. Membrane, (f). Druse, (g). Choroid, (h). Choroidal bloodvessel, (i). Intima with atheroslerotic plaque, (j). Tunica media, (k l section. ). Fibrous cap, ( ). Lipid + necrotic core. A color version of this figure can be found in the color figures

Drusen: where do they come from?

A priori, drusen constituents are most likely derived from (modified) fats and proteins produced by the 1) photoreceptor cells, and/or 2) RPE cells, and/or 3) from serum constituents. As discussed above, the majority of lipids in drusen, choroidal cells (endothelial cells, fibroblasts, smooth muscle cells), or 4) derived including (oxidized) cholesterol are derived from the RPE and photoreceptors cells, and only a small part from the serum.

et al. To gain insight into the question: “where do drusen proteins come from?”, we compared drusen protein content (modified from Crab 2002) [6] with triplicate mRNA expression profiles from human photoreceptor cells, RPE cells and choroidal cells166 [24] and a proteomics profile from human serum generated by the Ingenuity

Chapter 7.indd 166 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane Figure 6 and Table 1).

knowledge database [126] (Bergen unpublished results) ( By doing so, we can track, within obvious limitations, the main potential origin of the in serum [6]. Thirty-six (32%) drusen proteins are potentially (also) synthesized by drusen proteins. At least 23 (20%) of 113 proteins identified in drusen are present local choroidal cells. Thirteen (12%) drusen proteins are potentially (also) derived

from RPE cells and three (3%) (also) from the photoreceptor cells. For the sake of of mRNA expression in cells adjacent to BM is, in general, linear with the amount argument, we assumed - and of course this is an oversimplification- that the amount of protein produced and subsequently transported to drusen. In that case, it is

choroidal cells and or serum, and not from the photoreceptors (Figure 6). remarkable that the larger part of drusen proteins appear to be derived from the In summary, and perhaps surprisingly, human drusen consist of 1) lipids primarily derived from the photoreceptor cells and serum, and 2) proteins apparently

that the type and relative amount of molecular constituents of drusen, and patho- primarily derived from choroidal cells and serum. Finally, it must be pointed out

fraction of serum-derived molecules from the complement cascade can have large biological effects frequently do not have a linear relationship. For example, a minor functional or pathological consequences.

Why do drusen develop preferentially in the macular area?

It is currently not known why drusen develop mainly in the macular area. However, a combination of specific structural, molecular and functional properties may predispose the macula to develop drusen. First, the extremely high density of phagocytosis of photoreceptor outer segments (POS) by the RPE causes a highly photoreceptors, particularly in the perifoveal ring, may play a role [127]. Local 7

waste products. focussed and localized peak of oxidative stress, and focal build-up of membraneous

play a role. As discussed above, BM in the macula has a thinner elastic layer and In addition, the specific structural properties of BM in the macular area may also a more open maze compared to the periphery. Initially, in the still healthy eye, the macular RPE and BM may get rid of an excess of oxidized molecules and neutral

fats (by transporting them toward the bloodstream rapidly. Most likely, however, a subset of these proteins may get physically or chemically trapped in BM, thereby additional proteins reach BM from the choroidal side. After oxidative modification,

initiating the first events of drusen formation in the macular area. Finally, local functional macular RPE properties, as annotated by gene expression profiles, according to van Soest (2007) and Booij (2009) may also play a role [9,11]. 167

Chapter 7.indd 167 23-10-12 19:44 Chapter 7

Figure 6. Schematic Drawing of Proteins in Drusen and the Corresponding Genes Expressed in Adjacent Cell Layers.

et al.[6]. Thirty six of these genes had higher expression levels in choroid compared to RPE We could annotate 113 genes with genbank codes (using Ingenuity) which correspond to drusen proteins (chor>RPE), thirteen genes had higher expression levels in RPE than in choroid (RPE>chor) and only three genes identified by Crabb had higher expression levels in photoreceptors than RPE (phot>RPE). Gene expression levels were determined by RNA microarray study comparing gene expression levels from two adjacent tissue types from the same donor. Experiments were performed in triplicate (on three different healthy older human donor eyes) [24]. Details of

the 113 genes can be found in Table 1. A color version of this figure can be found in the color figures section. Most importantly, we compared the data of van Soest [9] and Crabb [6]. Van Soest and

expressed in macular RPE compared to RPE in the retinal periphery. Crabb et al. coworkers identified 438 genes (out of 22.000), that were significantly differentially

overlap between these two datasets consists of 16 genes, while by chance alone this identified 129 proteins in drusen using a proteomics approach [6]. Interestingly, the overlap would be no greater than 2.5 genes (Bergen et al. manuscript in preparation).

168

Chapter 7.indd 168 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane Bruch’s membrane and AMD pathology Oxidative stress in the RPE and its effects on BM Multiple studies in man, AMD animal models and (RPE) cell lines point toward an important role of oxidative stress in the development of AMD [119].

Epidemiological studies in man demonstrated that smoking is associated with increased risk of AMD. Smoking is a well known source of oxidative stress [118]. as lutein and zeaxanthin or vitamin C and beta-carotene [128,129]. Animal models A decreased risk of AMD was found with the use of high dietary antioxidants, such SOD1 ERCC6

susceptible to oxidative stress, like -/- [130] and -/- knock-out mice cell lines, several investigators showed that oxidative stress is implicated in retinal [131] show remarkable signs of retinal degeneration, if not AMD. Finally, using RPE degeneration [132,133]. In the macula, where photoreceptor density is high and incoming light is focussed, the RPE and BM are highly susceptible to high levels of oxidative stress. Sources of local oxidative stress include the high metabolic rate of photoreceptors required to sustain their normal function and structural renewal, the exposure to light, the high local oxygen pressure and the high metabolic rate of the RPE due to processing of photoreceptor outer segments (POS) [79]. The combination of high levels of oxidative stress and segmental POS digestion in the

as cholesterol [134] and docosahexanoic fatty acid that accumulate in drusen or are RPE most likely results in the oxidative modification of lipid-related molecules, such exported to the bloodstream through BM. In addition, a vast number of molecular constituents that can neither be digested, nor exported across the plasma membrane,

bisretinoid A2E, an indigestible remnant of POS, and an important constituent of accumulate inside the RPE cell. A well known example is the accumulation of the 7 or indigestible residual molecules basolaterally, where they accumulate in BM, BM intracellular lipofuscin [135,136]. Finally, the RPE cells attempt to export unneeded

BLDs, or drusen, or diffuse to the bloodstream [33]. Complement activation, inflammation and the immune response

complement system (CFH C2/FB and C3 [79,141,142]) established the The recent finding of genetic associations between AMD and genes from the long suspected role of the innate immune system in AMD [121,143]. The detailed role [137-140], of the regulation of the complement system and all complement factors individually has recently been reviewed elsewhere [119,144].

Multiple immune-related cells, including macrophages, fibroblasts, and lymphocytes have been implicated in RPE atrophy, the breakdown of BM, and neovascularization 169

Chapter 7.indd 169 23-10-12 19:44 Chapter 7 in AMD [143]. In the healthy and balanced situation (i.e. in the absence of AMD), the

(alternative) complement system is activated just sufficiently by foreign antigens to clear up debris in BM, while at the same time invoking relatively little RPE cell et al. found that intermediates cellular damage through the membrane attack complex. But what is the trigger that activates this pathway? Zhou antigens, and activated the complement cascade [145]. Alternatively, Johnson et al. of lipofuscin in the RPE, like the bisretinoid pigment A2E, were recognized as non-self co-localized “the Alzheimer protein” amyloid beta which activated complement

(Aβ) [122]. Recently, the latter data were substantiated by functional studies by Wang components in drusen, and suggested that Aβ invokes a local inflammatory response

and colleagues who showed that Aβ interaction with complement factor I activated the complement cascade [146]. Subsequently, Hollyfield and co-workers showed abundantly present in the retina, resulted in a unique fragment, carboxyethylpyrrole, that oxidative modification of docosahexanoic acid, a polyunsaturated fatty acid

Scholl et al. reported that not only local factors, but also systemic complement that can also invoke a local immune response [147]. To further complicate the issue,

serum factors can activate the alternative complement pathway in the RPE/BM in serum, while others only occur in serum [148]. [148]. Complement factors, such as CFH, and C3 are expressed in the RPE and occur

In summary, it is likely that multiple non-self antigen triggers can invoke a local may therefore initially be determined by both the type and the amount of non-self- complement/immune response leading to AMD. The local complement response antigen present. In addition to the type and amount of trigger, the actual activity of the complement cascade is regulated by both genetic variation in, and biochemical

interaction between a number of regulatory proteins, such as CFH, MCP (CD46), C3 and Factor I. These proteins contain complement control repeats (CCPs) that can sulphate present at the surface of BM. Through more or less effective binding of bind to other complement factors and/or substances like CRP, heparin and heparan these regulatory protein domains, due to DNA sequence variation, metal ion traces,

proteins, the actual activity of the complement system is controlled [144]. post translational protein modifications or simply by bio-availability of regulatory It is of interest to note here that BM’s proteoglycan turnover and content appears to et al. found an increased heparan sulphate content in retinas of animal models affected by retinal change with age (discussed above) and disease. For example, Landers degeneration [149]. These changes may modify the natural binding characteristics

surface of BM with age. This may be one of the factors that determine the rate of and inhibitory complement capacity of CFH or other complement molecules at the

170

Chapter 7.indd 170 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane photoreceptor cell loss during aging and neural degeneration. The interaction of local and systemic complement factors, their regulatory binding to extracellular matrix components, growth factors, and other molecules at the BM interface is, so

far, poorly understood, and currently subject to thorough investigation [147,150]. Breaking the barrier: choroidal neovascularization (CNV) The whole process of choroidal neovascularization (CNV) has recently been reviewed elsewhere [151]. In summary, CNV is a process whereby new vessels sprout from the choroid and penetrate BM. In many aspects, CNV resembles normal wound healing

CNV is controlled by local pro- an anti-angiogenic factors. Among these factors is a in the skin [151].

combination of proteins secreted by the RPE and/or choroidal cells, including well studied growth factors like VEGF-A and PEDF. Another factor is the physical barrier embodied by the RPE/BM complex. Leukocytes, lymphocytes, macrophages and collagen, thereby facilitating neovascularization [152]. Nevertheless, new vessels can endothelial cells may directly or indirectly degrade BM through the breakdown of

phagocytic series (MPS) cells, have been implicated in the development of new also penetrate the intact BM [152]. Specific subtypes of macrophages, mononuclear

vessels in healthy and AMD eyes [153]. Furthermore, multiple neovascularization studies in normal and genetically modified mouse models support the notion that induction of CNV. To illustrate the complexity of this issue, we present in Table 2 additional factors, such as the breakdown of BM integrity, are essential for the the genes that are expressed at higher levels in the RPE than in the choroid with the functional annotation ‘angiogenesis’ [126]. (Bergen and Booij; unpublished results). Analysis of our data showed that at least 23 genes may be involved in this process. If the local balance between pro- an anti-angiogenic factors is disturbed substantially 7

purposes, Table 3 shows the genes that are expressed at higher levels in the choroid in favour of VEGF, CNV or enhanced fibrosis may occur [154]. For further illustration than in the RPE with the functional annotation ‘angiogenesis’ [24].

171

Chapter 7.indd 171 23-10-12 19:44 Chapter 7 Table 2. The Overlap between Genes Associated with the Term Angiogenesis (Ingenuity) and Genes Identified in Triplicate RNA Microarray Measurements from Older Healthy Human Donor Eyes [24] with Expression Levels Higher in the RPE than the Choroid. Gene name Sequence code BAI1 EGF NM_001702 EPAS1 NM_001963 EPHA2 NM_001430 FLT1 NM_004431 HGF NM_002019 HMMR BC022308 IGF1R NM_012484 IGHG1 NM_000875 IL2 AF035027 IL18 NM_000586 INSR NM_001562 MAPK8 NM_000208 MFGE8 AK125150 MMP9 NM_005928 NOS1 NM_004994 NOS3 NM_000620 PLG NM_000603 PTHLH M31157 NM_000301 SERPINF1 SOD1 NM_002615 TP73 NM_000454 VEGFA NM_005427 NM_003376

172

Chapter 7.indd 172 23-10-12 19:44 The Dynamic Nature of Buch’s Membrane Table 3. The Overlap between Genes Associated with the Term Angiogenesis (Ingenuity) and Genes Identified in Triplicate RNA Microarray Measurements from Older Healthy Human Donor Eyes [24] with Expression Levels Higher in the Choroid than the RPE. Gene name Sequence code ALOX5 ANPEP NM_000698 APOE NM_001150 C3 NM_000041 C5 NM_000064 C3AR1 NM_001735 CCL13 NM_004054 CFB NM_005408 COL18A1 NM_001710 CSF2 NM_030582 CXCL12 NM_000758 CYR61 NM_000609 EFNA1 NM_001554 HGF NM_004428 HMMR NM_000601 ICAM1 BC035392 IGF1 NM_000201 IGF2 NM_000618 IGFBP3 NM_000612 IGHG1 NM_000598 IL13 BC037361 INHBA NM_002188 ITGB2 NM_002192 LEP NM_000211 MAPK8 NM_000230 MYC AL137667 NR3C1 NM_002467 NRP1 NM_000176 PLAU NM_003873 7 PTGS2 NM_002658 S100A4 NM_000963 SCYE1 NM_002961 THBS2 NM_004757 TIMP2 NM_003247 TNF NM_003255 TP53 NM_000594 VCAM1 NM_000546 VEGFC NM_001078 NM_005429

173

Chapter 7.indd 173 23-10-12 19:45 Chapter 7 Outlook and perspectives: Toward rational, genomics-driven, molecular therapies for AMD

Summarizing the events leading up to early AMD The molecular pathology of AMD has recently been reviewed extensively elsewhere [119]. In summary, Ding et al aspects of AMD, as well as the use of mouse models for potential AMD therapy. In . (2009) reviewed clinical, epidemiological, and genetic contrast, we here reviewed the central role of BM in normal retinal aging, in drusen formation and in the early stages of AMD. Obviously, the normal function and pathology of BM can only be understood in the context of the molecular and cellular events involving the adjacent cell layers (photoreceptor, RPE and choroid) as well as systemic factors (from serum). As

discussed, many normal aging processes affect BM, such as thickening of its layers Clearly, these normal (subclinical) aging events may predispose BM and the RPE to due to fat deposition, calcification of the EL, oxidative stress and drusen formation.

development of age-related macular degeneration. Its extracellular matrix, heavily disease, especially in the macular area. BM is the key acellular tissue involved in the dominated by heparan sulphate, appears to be the regulatory playground of both local and systemic interactions involving complement activators, proteoglycans,

chemokines, cytokines, growth factors and, above all, toxic waste products. pattern, which maintains the local homeostasis. This local homeostasis in each In the healthy, non-AMD, situation these molecular interactions may follow a fixed individual probably depends on, and is limited by environmental factors, genetic constitution and local anatomy of the neural retina, RPE and BM. However, fuelled by changes due to normal aging, such as prolonged oxidative stress and immune activation, the molecular interactions at the surface of BM change. These changes may be accommodated until local homeostasis cannot be maintained anymore, which ultimately leads to the devastating clinical manifestations of AMD.

Prevention and therapy of AMD; is there a role for BM biology?

So, with the current state-of-the-art knowledge, can we “prevent” or “cure” AMD? as molecular, cellular, and even systemic events underlying AMD has grown Over the last decades our understanding of environmental risk factors as well

tremendously. In summary, environmental risk factors now include smoking, diet and perhaps light exposure [118]. Dietary intake of saturated fats increases the risk of AMD [155]. Intake of anti-oxidants (lutein, zeaxanthin, beta carotene, vitamin C), omega-3 polyunsaturated fatty acids (nuts, fish) and zinc supplements may 174

Chapter 7.indd 174 23-10-12 19:45 The Dynamic Nature of Buch’s Membrane

several AMD disease genes (APOE, CFH, C3, C2/BF, HTRA1/ARMS2) [148], or to be beneficial [94]. Genetic association studies were successfully used to implicate identify potential candidate disease genes (CXCR3, Il-8, ERCC6) [119]. Genetic and functional studies were instrumental to the discovery of functional pathways in AMD. The most important are, as discussed above, fat metabolism, oxidative stress, complement activation, and STAT3/VEGF induced neovascularization. So far, prevention and therapeutic efforts have largely focussed on these four pathways. By far the best option is, of course, to prevent AMD; that is to aim to postpone its

onset or to slow its progression. Most likely, for a large majority of individuals, this can be done by avoiding risk factors for AMD. Although not every individual would benefit equally due to differences in their genetic constitution [156], one should quit oxidants and unsaturated fatty acids. smoking, wear sunglasses, and change to a diet that contains sufficient zinc, anti-

negative and positive developments can be noted. Previously, serum, lipid lowering Alternatively, is it possible to influence the progression of AMD by drugs? Several

variable [157]. In addition, the obvious idea of local manipulation of the complement drugs, like statins, were used to treat AMD, but the outcome of clinical trials were system in AMD by complement inhibitors may reduce neovascularization [158], but

is not without risk: It may turn an essentially useful chronic inflammation at the RPE-BM interface into a harmful acute inflammation. On the positive side, a CNTF trial is ongoing that aims to supplement the In this case, the photoreceptors remain more viable, which may delay disease onset. photoreceptors with small quantities of CNTF over a prolonged period of time [159]. In addition, for dry AMD, a drug called fenretinide, which halts the accumulation of retinol-related toxins, thought to be involved in vision loss, shows promise 7

in treating the wet form of AMD [162]. [160,161]. Finally, VEGF based treatments have, of course, been relatively successful These new (experimental) therapies focus on cells (photoreceptors, RPE) or on systemic factors (e.g. statins). However, aside from studies by Del Priore et al. [163] and Marshall (unpublished), curiously enough, so far, little attention has been paid to

normal aging of the photoreceptor-RPE-BM-choroid complex, but is also essentially BM, the prime site of AMD development. As discussed, BM not only plays a key role in involved in pathogenic effects of fat metabolism, oxidative stress, complement activation, and neovascularization. Even a number of structural BM genes were Fibl-3 (EFEMP-1), Fibl-5, Fibl-6, CTRP5 two of these genes, a corresponding animal model (ctpr5 Fibl-3 [165] identified as AMD genes ( ) [79]. For at least -/-) [164], showing AMD like features, is available. At least, in terms of local BM therapy, there 175

Chapter 7.indd 175 23-10-12 19:45 Chapter 7

are two options: 1) removal of pathogenic or non-self compounds from BM which the use of microbial enzymes to augment or restore missing, or failing, metabolic slowly accumulate during aging and disease; or 2) “medical bioremediation” [166]: functions. However, both approaches have been largely unsuccessful up till now.

Further dissection and definition of the molecular events involved in age-related BM changes is therefore warranted to successfully develop drugs: To end with a considered BM to be a relatively boring and simple sheet of extracellular matrix, sentence from the beginning of this article: For too long many investigators have merely occupying space between the retinal pigment epithelium (RPE) and the choroid. This is about to change.

Acknowledgements

The authors thank the ‘Algemene Nederlandse Vereniging ter Voorkoming van Stichting voor Blinden en Slechtzienden’, the ‘Rotterdamse Blinden Belangen’, the Blindheid’ (ANVVB), the Macula Foundation of the Netherlands, the ‘Landelijke ‘Blindenpenning’, the ‘Gelderse Blinden Vereniging’, and ‘Stichting Uitzicht’ for their

financial contributions.

176

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Chapter 7.indd 187 23-10-12 19:45 Chapter 7.indd 188 23-10-12 19:45 CHAPTER 8 Reduced Secretion of Fibulin 5 in Age- related Macular Degeneration and Cutis Laxa

Andrew J. Lotery, Dominique C. Baas, Caroline Ridley, Richard P.O. Jones, Caroline

Wang, Arthur A.B. Bergen and Dorothy Trump C.W. Klaver, Edwin Stone, Tomoyuki Nakamura, Andrew Luff, Helen Griffiths, Tao

Human Mutation 2006; 27(6):568-574

Chapter 8.indd 189 23-10-12 19:45 Chapter 8 Abstract

Background: Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the Western world, affecting approximately 25 million

(FBLN5) have been reported in association with AMD. people worldwide. The pathogenesis is complex and missense mutations in fibulin 5 Methods European study of the gene FBLN5 in AMD (using 2 European cohorts of 805 AMD : We have investigated the role of fibulin 5 in AMD by completing the first patients and 279 controls) and by determining the functional effects of the missense FBLN5 genotype with the AMD phenotype. mutations on fibulin 5 expression. We also correlated the Results: We found two novel sequence changes in AMD patients that were absent in controls and expressed these and the other nine reported FBLN5 mutations associated with AMD and two associated with the autosomal recessive disease cutis

laxa. Fibulin 5 secretion was significantly reduced (P<0.001) for four AMD (p.G412E, Conclusions: These results suggest that some missense mutations associated with p.G267S, p.I169T, and p.Q124P) and two cutis laxa (p.S227P, p.C217R) mutations.

AMD lead to decreased fibulin 5 secretion with a possible corresponding reduction between FBLN5 in elastinogenesis. This study confirms the previous work identifying an association mutations and AMD and for the first time suggests a functional FBLN5 mutations can be associated with different phenotypes of AMD (not limited mechanism by which these mutations can lead to AMD. It further demonstrates that

lead to the development of novel therapies for this common disease. to the previously described cuticular drusen type). Such knowledge may ultimately

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the commonest cause of irreversible visual loss in the Western world [1,2] affecting Age-related macular degeneration (AMD; MIM#s 603075, 153800, and 608895) is approximately 25 million people worldwide (http://AMDalliance.org). The progressive loss of central vision in AMD occurs due to degenerative and

neovascular changes within the retina at the macula. Specifically, extracellular deposits of protein and lipid develop beneath the retinal pigment epithelium (RPE) and within an elastin-containing structure known as Bruch’s membrane and can be seen clinically as yellowish white spots in the retina [3]. The RPE degenerates and choroidal neovascularization can spread through the RPE into the retina with disastrous consequences for central vision [3]. FBLN5 Both environmental and genetic risk factors are known to be associated with AMD [4,5]. Sequence changes in the , complement factor H, human leukocyte antigen (HLA), and perhaps the toll-like receptor 4 genes have all been associated FBLN5 with AMD [6-12]. Previously a comprehensive analysis of fibulin 5 ( ; MIM#604580) in a cohort in FBLN5 of 402 patients with AMD and age-matched controls identified missense mutations the high prevalence of AMD this equates to many hundreds of thousands of AMD in 1.7% of AMD patients recruited from the United States [11]. Due to patients worldwide with FBLN5 mutations.

The fibulins are a family of extracellular matrix (ECM) proteins [13] and are characterized by tandem arrays of epidermal growth factor–like (EGF-like) supramolecular structures with binding sites for several proteins including domains. Fibulins are widespread components of ECM and participate in diverse

tropoelastin, fibrillin and proteoglycans. Phenotypes previously associated with mutations in fibulin genes include Malattia Leventinese and Doyne honeycomb retinal dystrophy (fibulin 3) [14], cutis laxa (fibulin 5) [15,16], and AMD in a single 8 family (fibulin 6) [17]. Fibulin 5 is a 66-kDa secreted protein of 448 amino acids [18]. It shares around 90% amino acid identity with other fibulin species. It is one of the shorter fibulins and has a central segment of five calcium binding EGF-like modules and a carboxy- terminal domain III of sequence identity with the other fibulins [13]. It is widely FBLN5 expressed throughout the body including the RPE [11] and choroid (www.ncbi.nlm. nih.gov/UniGene). The missense variants identified in the AMD patients [11] are spread along the protein with one lying very close to the arg-gly-asp (RGD) integrin binding domain (p.V60L), one affecting a calcium-binding EGF-like domain (p.I169T), and three affecting the region thought to bind to LOXL1 (p.R351W,191 p.A363T, and p.G412E) [19] .

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Two groups have published a description of a fibulin 5 −/− mouse [20,21]. In both abnormalities, and emphysematous lungs, demonstrating that FBLN5 is essential for cases the mice exhibited a severe form of elastinopathy with loose skin, vascular

multilayered structure that contains a central layer of elastin, and abnormalities in elastinogenesis. Of note, Bruch’s membrane, lying between the RPE and choroid, is a

We chose to investigate FBLN5 this membrane play a key role in the pathogenesis of AMD. variants in the European population, identified novel patients (United Kingdom) and 288 patients (Netherlands). Fibulin 5 is a secreted sequence variants, and determined their prevalence in two European cohorts of 514

protein which functions in the extracellular matrix [13]. We thus evaluated whether these novel sequence variants and previously identified variants impaired secretion genotype correlations. of mutant fibulin 5 in cell culture experiments. We also determined phenotype–

Materials and methods

patients from the same clinic population were screened. All patients and control At the University of Southampton a cohort of 514 AMD patients and 188 control patients were over the age of 50 years and had undergone a dilated retinal examination. Mutation analysis of FBLN5 was performed using a combination of single stranded conformation polymorphism and direct sequencing as reported

previously [22-24]. Ethical permission for this study was obtained from the Southampton and Southwest Hants Local Research Ethics Committee (approval no. 347/02/t). The Dutch cohort consisted of 288 unrelated Caucasian patients and 149 ethnically- and age-matched controls. Subjects were primarily recruited from the ophthalmic departments in Amsterdam and Rotterdam. All subjects underwent fundus photography. Transparencies were graded according to a modified version of the drusen and pigmentary irregularities, soft indistinct or reticular drusen, geographic International Classification System [25]. Cases were subjects with soft distinct atrophy, or neovascular macular degeneration (i.e., age-related maculopathy stages

2–4). Controls were subjects aged 65 years and older with no or only a few small hard drusen and no other macular pathology (age-related maculopathy stage 0). Control ophthalmology department for reasons other than retinal pathology. A combination subjects were either unaffected spouses of cases, or subjects who attended the

sequencing was used for mutation analysis. of DHPLC analysis (Transgenomic, Omaha, NE; www.transgenomic.com) and direct

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Chapter 8.indd 192 23-10-12 19:45 Reduced Secretion of Fibulin 5 in AMD and Cutis Laxa Computer-based predictions of structure

effects of missense mutations on protein folding. Threader assesses whether a test The protein fold recognition program “Threader 3” [26] was used to predict the

primary amino acid sequence (in this case a fibulin 5 sequence) can align with and divided dynamically into small segments. Threading of the test sequence upon each fold like known protein structures from a library. For threading, the test sequence is

also the solvation energy sums. Threader generates Z-scores, which are quantitative known structure is based on optimization of the pairwise (folding) energy sums and measures of the quality of matches: Large, positive Z-scores point toward a successful prediction. Threading was not constrained by providing any predicted secondary

structure for fibulin 5. Expression of fibulin 5 in COS7 cells To investigate the expression of wild-type (WT) and mutant FBLN5 we generated FBLN5 FBLN5 mutations. These included those mutations previously described as being associated expression constructs in pEF6-ssFLAG vector for WT and 13 different

with AMD (p.V60L, p.R71Q, p.P87S, p.I169T, p.R351W, p.A363T, and p.G412E) [11], the mutations we have identified as being associated with AMD (p.G267S, p.Q124P), one missense change we identified in a control patient without AMD as a control (p.G202R), a mutation identified in both AMD and control patients (p.V126M), and two mutations previously described as causing cutis laxa (p.S227P) [15] and (p.C217R) [27]. Mutations were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA; www. stratagene.com) as described [28] and the presence of each mutation was confirmed by DNA sequencing. The pEF6-ssFLAG constructs were used to transfect COS-7 cells and protein expression was induced. Nontransfected COS-7 cells do not express fibulin 5 (data not shown). 8 Cell culture and transfection

All experiments were carried out in triplicate. COS-7 cells were grown and transfected for Western blot analysis whole cell lysates were prepared as previously described as described previously using 0.5 μg of DNA [28]. After 72 hr cells were harvested, and

and blotted [28]. Samples of the medium (nonconcentrated) in which the cells had grown were also blotted to detect secreted fibulin 5. The expressed tagged fibulin 5 was detected by Anti-FLAG antibody (Sigma, Gillingham, Dorset, England; www. sigmaaldrich.com) and the bands were quantified using Fluor-S Max MultiImager (Biorad, Hercules, CA; www.bio-rad.com). 193

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5 in the cell lysate, thus allowing us to determine a ratio for each sample. We did For each sample in all experiments we compared the secreted fibulin 5 to fibulin not compare absolute measurements between samples as we were using transient

transfections but, instead, we compared the ratios of cell lysate and medium fibulin 5 for each mutant fibulin 5 construct with the ratio for the WT fibulin 5 constructs. Results

Two novel heterozygous missense mutations were identified solely in three United patients). No sequence variations were found in patients alone in the Netherlands Kingdom patients (p.Q124P in one patient and p.G267S in two apparently unrelated cohort (Table 1

). Two missense changes were identified in controls (p.V126M suggesting these changes are polymorphic sequence variants. found in 5 controls and 1 AMD patient and p.G202R found in a control individual),

protein folding of these missense sequence changes together with those previously We used the protein fold recognition program Threader 3 to predict the effects on

published in AMD patients [11], the missense changes identified in control individuals (p.V126M, p.G202R) and two mutations previously described in cutis laxa (p.S227P, p.C217R) [15,27]. The structure 1l0qa0 (391 residues) was the best match both 1) when the WT fibulin 5 sequence (448 residues) was threaded using default parameter values against each of the 6251 structures in the reference library (Z = 6.8, statistically very significant); and 2) when the top 15 matches from (1) positive matches and eliminate false-positives. A prediction of the effects on folding were subjected to randomization tests (using the option r50), which identify true- of each mutation was obtained indirectly by comparing the results from threading the mutant against 1l0qa0 with those from threading the WT against the same structure. Table 2 presents the threading energies, the Z-scores (which indicate the

quality of matches), and the alignments for WT and each mutation. Of note, both the magnitude of the pairwise (folding) energy sum by >2.5%, and reduce the the cutis laxa mutation p.C217R and the AMD-associated change p.Q124P reduce

(Table 2 corresponding Z-score. For p.Q124P, compensation for the change in folding energy ; column 2) by an opposing change in solvation energy (column 4) leads to virtually no change in the total energy (column 6) and its Z-score (column 7). For p.C217R, the changes in pairwise and solvation energies are synergistic, leading to a reduction of the magnitude of total energy by >2.5%. The two mutations p.G267S and p.R351W improve the stability on folding by >2.5%, and with reduced percentage alignments. Changes in protein folding might interfere with secretion of the protein and to test this we expressed each of the sequence variants and WT fibulin 5 in COS7

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Chapter 8.indd 194 23-10-12 19:45 Reduced Secretion of Fibulin 5 in AMD and Cutis Laxa cells together with those previously published in AMD patients [11], the missense

changes identified in control individuals (p.V126M, p.G202R) and two mutations previously described in cutis laxa (p.S227P, p.C217R) [15,27]. Western blot analysis in whole cell lysates and in the medium from the cell culture (Figure 1A following harvesting of the transfected cells indicated WT fibulin 5 was present both ), confirming that fibulin 5 is secreted by these cells. The ratio of fibulin 5 detected in the medium Figure 1B) indicating a reduction in secretion compared to that in the whole cell lysate for 6 of the 11 disease-associated mutations was significantly reduced (P<0.001) ( for these mutant forms of fibulin 5 but no change in secretion was noted for the two sequence variants identified in controls. Four of these mutations are associated with Figure 1B). AMD (p.G412E, p.G267S, p.I169 T, and p.Q124P) and two have been found to cause

cutisTable 1.laxa Sequence (p.S227P, Variations p.C217R) in the FBLN5 ( Gene* Nucleotide Effect or Degree of change sequence conservation** change Amino acid changing variants United Kingdom data AMD (n = 188) 1 0Controls 0(n = 514) 1 Exon 84 c.371A>C p.Gln124Pro 2 0 8/8, chicken NetherlandsExon 6 data c.604G>A p.Gly202Arg AMD 11/11, chicken c.799G>A p.Gly267Ser (n = 291) (n = 91) 10/10, chicken 1 5Controls 7/8, bovine Synonymous coding variants UnitedExon 4 Kingdom data c.376G>A p.Val126Met AMD affected (n = 188) Exon 5 1 0Controls Exon 9 (n = 514) Exon 10 c.484C>T p.Leu162Leu 19 10 Exon 10 c.945T>C p.Ile315Ile2 206 076 Netherlands data c.1122C>T p.Tyr374Tyr AMD c.1134T>C p.Tyr378Tyr (n = 291) (n = 91) Exon 7 2 0Controls Exon 9 99 Exon 10 c.735C>T p.Cys245Cys 18 7 Intronic variants c.945T>C p.Ile315lle 37 c.1122C>T p.Tyr374Tyr United Kingdom data AMD (n = 188) 1 0Controls 5(n = 514) 1 8 NetherlandsIntron 3 data c.125-53G>A AMD Intron 3 c.125-45G>C (n = 291) (n = 91) 1 0Controls 21 8 5’UTR c.1-33C>A 0 1 Intron 1 c.18-28A>G 1 0 3’UTR c.1347+97G>A 15 28 3’UTR c.1347+121C>T 7 3’UTR c.1347+352G>C 18 3’UTR*Degree of conservation values c.1347+423T>Care the number of nonhuman species6 that share this residue with humans. 3’UTR c.1347+514A>G 38 The species with the greatest phylogenic distance from humans with a sequence that is homologous to that of

humans is also given. cDNA numbering is taken from Ref Seq # NM_006329.2 where +1 corresponds to the A of the ATG initiation codon. ** Number of conserved variations/total number of variations

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Chapter 8.indd 195 23-10-12 19:45 Chapter 8 Table 2. “Threading” of the Fibulin 5 Mutants Against the Structure 1I0qa0* 1 2 3 4 5 6 7 8 9 10 11 12 13 WT →

−1026 6.8 −7.81 2.41 −1214 6.14155.9 157.3 359 92.3 80.4 → V60L** −1021 6.77 −7.82 2.42 −1210 6.14 359 92.3 80.4 IE → R71Q** −1026 6.8 −7.77 2.41 −1213 6.13158.5 156.9 359 92.3 80.4 IC → P87S** −1031 6.84 −7.812.78 2.44 −1222 6.21 359 92.3 80.4 IE ↓↓ Q124P** −970 6.39 −9.75 −1207 6.1 174.8 359 92.3 80.4 IE → V126M** −1010 6.74 −7.77 2.41 −1197 6.12157.2 160 359 92.3 80.4 IE ↓ I169T** −1018 6.77 −8.462.51 2.54 −1224 6.16 359 92.3 80.4 IE → G202R*** −1026 6.84 −8.20 −12255.88 6.27 155.2158.6 359 92.3 80.4 IC ↓↓ C217R**** −986 6.62 −6.76 2.23 −1148 159 359 92.3 80.4 IE ↓↓ S227P**** −1026 6.87 −7.772.85 2.4 −1211 6.13 35990.8 92.3 7980.4 IC ↓ G267S** −1036 6.93 −10.03 2.75 −1279 6.56175.8 190.4 354 91.8 79.9 IC → R351W** −1026 6.76 −9.55 −1258 6.34 357 IC → A363T** −1026 6.79 −8.06 2.44 −1221 6.15 158.4 359 92.3 80.4 IC ↓ G412E** −1026 6.83 −7.81 2.42 −1215 6.18 157.3 359 92.3 80.4 O

*Threading of the test fibulin 5 sequences (column 1) upon 1l0qa 0 is based on optimization−1 of the pairwise

energy sums and the solvation energy sums. The raw pairwise energy sum (kcal mol−1 , column 2) is adjacent to its corresponding Z-score (column 3). The raw solvation energy sum (kcal mol , column 4) is adjacent to Threader documentation, is given with its Z-score (column 7). The high sequence-similarity scores (column its Z-score (column 5). The sum of pairwise and solvation energies (column 6), weighted as described in the

8) might indicate common ancestry between 1l0qa0 and fibulin 5. In addition, the number of residues (column 9) and the exceptionally high percentages of 1l0qa 0 (column 10) and fibulin 5 (column 11) that were aligned are listed. Column 12 lists whether a mutation is inside (I) or outside (O) the alignment, and the 1l0qa0 secondary structure (E, extended; C, coil) at that point. Column 13 summarizes the secretion the alignments are shown in italics. Z-scores are quantitative measures of the quality of matches. Large and data (Fig. 1). Deviations >2.5% from figures obtained for wild-type fibulin 5 are shown in bold. Changes in

positive Z-scores point toward a successful prediction: Z>4.0, very significant, indicating probably a correct prediction; 4.0>Z>3.5, significant with a good chance of being correct; 3.5>Z>2.7, borderline; 2.7>Z>2.0, without AMD. ****Mutations associated with cutis laxa. poor; 2.0>Z, very poor. **Mutations associated with AMD. ***Missense change identified in a control patient

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Chapter 8.indd 196 23-10-12 19:45 Reduced Secretion of Fibulin 5 in AMD and Cutis Laxa

Figure 1. Secretion of Fibulin 5. (A

). Secretion of WT and mutant forms of fibulin 5. WT and mutant fibulin 5 were expressed in transfected COS-7 cells. At 72 hr after transfection, cell lysate and medium samples were prepared and separated by10% SDS-polyacrylamide gels followed by immunoblotting. These included WT, two mutations previously associated with cutis laxa (p.C217R and p.S227P), mutations previously associated with AMD (p.R71Q, p.P87S, p.I169 T, B). . For WT and p.V60L, p.A363 T, p.R351W, and p.G412E), novel mutations we identified in AMD patients (p.Q124P, p.G267S), and two control missense mutations (p.V126M, p.G202R). ( Quantification of secreted fibulin 5 each mutation the ratio of secreted fibulin 5 to fibulin 5 in the cell lysate was calculated. These were compared between WT and each mutations and results analyzed using Student’s t-test. *P<0.001. 8 Genotype–phenotype correlation

Two of the three Southampton patients had a clinical phenotype of cuticular drusen. Patient 1 (p.Q124P) had small round uniform drusen but no choroidal neovascularization and no evidence of pigment epithelial detachments. Patient 2 (p.G267S) had pure choroidal neovascularization in one eye but no drusen. Patient 3 (Figure 2). (p.G267S) had cuticular drusen and central localized pigment epithelial detachments

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Figure 2. Retinal Photographs of Three Patients. (A). and (B). present. (C Retinal Photographs of Patient 1 (p.Q124P). Small round (cuticular) drusen are symmetrically pigment epithelium (*). (D). and (E). ). Optical coherence tomogram of patient 1 demonstrates nodular thickening of the retinal neovascularization in one eye only. (F). and (G Fluorescein angiograms of patient 2 (p.G267S) demonstrate choroidal demonstrate cuticular drusen with central areas of focal retinal pigment epithelial detachment (*). A color ). Fluorescein angiogram photographs of patient 3 (p.G267S)

version of this figure can be found in the color figures section.

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Chapter 8.indd 198 23-10-12 19:45 Reduced Secretion of Fibulin 5 in AMD and Cutis Laxa Discussion

FBLN5

This study identified novel heterozygous mutations in 3 out of 514 UK found no FBLN5 FBLN5 patients with AMD, a prevalence of 0.6%. In the Netherlands cohort, however, we sequence variants specific to patients. Previously, missense these mutations were proposed to result in an increased susceptibility to AMD. The mutations were identified in 7 out of 402 U.S. patients with AMD (1.7%) [11] and

patients. The results from the UK cohort support the hypothesis that a number of work we describe here is the first similar study in a different population of AMD rare FBLN5 variants are unique for AMD patients, which suggests these mutations

may affect fibulin 5 function. The results from the Netherlands cohort might reflect the lower number of patients studied or genetic founder effects specific for the Dutch between a gene and a disease often overestimates the fraction of disease caused by population. It is well recognized that the first report [11] describing an association mutation in the gene in question [29] and these results would suggest this may be a factor here. Again this emphasizes the need for subsequent studies such as ours, both to assess the effect in further populations and also to further investigate the biological consequences of the genetic mutations. The statistical association of sequence variants with a multifactorial condition such

a secreted extracellular matrix protein that has a role in elastinogenesis through as AMD is only the first step in determining any causative relationship. Fibulin 5 is

interactions with integrins [21], tropoelastin [30], and LOXL1 [19]. Missense mutations affecting protein folding might interfere with fibulin 5 moving through the secretory pathway or with subsequent protein interactions. Interference with protein trafficking and secretion is an increasingly recognized mechanism by which missense mutation leads to abnormal protein folding, thus inducing the “unfolded missense mutations cause genetic disease [31]. It is thought to occur when the

of the mutant protein; and it can occur with missense changes at different points in protein response” in the endoplasmic reticulum, leading to intracellular degradation 8 We hypothesized that the missense mutations in FBLN5 may lead to susceptibility a protein [31].

to AMD due to reduced fibulin 5 secretion; as an initial approach to investigating results suggested some sequence variants might affect folding and thus potentially this we modeled the effects of mutations on protein folding using Threader 3. These

secretion of mutant protein. We therefore expressed WT and mutant forms of fibulin 5 and compared secretion. Six sequence changes did indeed interfere with secretion changes were also predicted to change protein folding but did not change secretion. and four of these were predicted to interfere with folding. In contrast, other sequence 199

Chapter 8.indd 199 23-10-12 19:45 Chapter 8 This may indicate protein folding changes that did not activate the unfolded protein

Mutations leading to autosomal recessive cutis laxa caused a dramatic reduction in response or, alternatively, reflect the limitations of computer modeling.

in secretion was observed for four of the nine mutations associated with AMD secretion of fibulin 5 when compared with WT (P<0.001) and a similar reduction In vitro expression studies such as these

(p.G412E, p.G267S,p.I169T, and p.Q124P). are often the first indication of missense mutations reducing protein secretion and et al., personal communication] thus causing a phenotype. Fibulin 5 is moderately expressed by the human RPE and not by the photoreceptors [Simone van Soest and therefore fibulin 5 through its role in elastinogenesis may contribute to the reduction in secretion might interfere with this. maintenance of the integrity of Bruch’s membrane in the eye. Correspondingly, a

et al Several members of the fibulin gene family have been associated with macular degeneration. In previous studies by Stone . [2004] and other groups [17], variations in other fibulins (fibulin-1, 2, 4, and 6) were also reported specifically severe form of macular degeneration (earlier onset), Malattia Leventinese or associated with AMD patients. In addition, a mutation in fibulin-3 causes a more

Doyne honeycomb retinal dystrophy with high similarities to AMD [14]. Although no fibulin-3 mutation has been found in AMD, the fibulin 3 protein accumulates in FBLN5 Bruch’s membrane in AMD [32]. Secretion of mutant fibulin 3 is also significantly new mutations in different AMD cohorts but not in control populations (this study reduced [32]. The gene is not highly polymorphic and the identification of et al.

and the previous one by Stone , 2004) suggests that the missense sequence relationship between FBLN5 and the pathogenesis of AMD rather then this being a changes have a functional effect on fibulin 5 function and that there is a causal chance association. This study further demonstrates that FBLN5 mutations can be associated with different phenotypes of AMD (not limited to cuticular druse type).

Our results suggest that some missense mutations associated with AMD lead to hypothesis is that this reduced secretion in turn results in a corresponding reduction decreased fibulin 5 secretion. Since fibulin 5 is essential for elastinogenesis, our

and requires further investigation. in elastinogenesis. However, this is as yet unconfirmed for the AMD related mutations

It is particularly noteworthy that one of the AMD associated mutations (p.Q124P) laxa in which elastinogenesis is disrupted [15, 27]. This raises the possibility that leads to a similar reduction in secretion to the two mutations known to cause cutis patients with this form of cutis laxa may have early onset AMD, and their parents

(heterozygous for these mutations) may themselves be at a higher risk of AMD than 200

Chapter 8.indd 200 23-10-12 19:45 Reduced Secretion of Fibulin 5 in AMD and Cutis Laxa the general population. Furthermore, these AMD FBLN5 sequence variants may lead to cutis laxa if inherited as homozygous mutations and the AMD patients in whom FBLN5

we have identified a sequence variant in may be “carriers” for cutis laxa. We have found reduced secretion of fibulin 5 for both cutis laxa mutations and for some and disease severity. Homozygous mutations may interfere with elastinogenesis and AMD mutations. This suggests there may be a relationship between dose of fibulin 5 result in the early onset cutis laxa phenotype, whereas heterozygous mutations may

result in a later onset phenotype of AMD. Environmental stresses such as smoking investigating the effects of these variants and gene–environment interactions will may also influence the development of the AMD late onset phenotype. Further studies

determine whether we should give lifestyle advice for the parents of patients with be important. Not only to clarify how they may affect fibulin 5 function, but also to cutis laxa and consider additional ophthalmologic supervision and possible early

FBLN5 treatment for these individuals with vitamin supplements [33]. phenotype of cuticular drusen [11]. We have observed this same phenotype in Previously, associated AMD has been associated with a specific retinal two of our three patients with novel FBLN5 mutations. However, interestingly, two

patients with the same p.G267S sequence change demonstrated markedly different FBLN5 AMD can therefore also be a cause of phenotypes. Such variation in retinal phenotype has been well described before choroidal neovascularization in the absence of drusen. in monogenetic retinal diseases [34]. FBLN5

This study identifies a mechanism by which mutations can lead to AMD. Such FBLN5 knowledge should ultimately lead to the development of novel therapies for this devastating disease. It also expands the retinal phenotypes associated with information will be valuable for future gene directed therapies. mutations and identifies the prevalence of AMD in two European cohorts. Such Acknowledgements 8

Research nurse support was provided by the Southampton Wellcome Trust Clinical Research Facility. Kay Kielty and Adam Huffman are acknowledged for providing access to, and assistance with, computing facilities in the Wellcome Trust Centre for Cell-Matrix Research, The University of Manchester. Angela Cree, University of Southampton, is acknowledged for technical support.

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Chapter 8.indd 201 23-10-12 19:45 Chapter 8 References

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16. Markova D, Zou Y, Ringpfeil F, Sasaki T, Kostka G, et al. (2003) Genetic heterogeneity of cutis laxa: a heterozygous tandem duplication within the fibulin-5 (FBLN5) gene. Am J Hum Genet 72: 998-1004. 17. Schultz DW, Klein ML, Humpert AJ, Luzier CW, Persun V, et al. (2003) Analysis of the AMD1 locus: evidence that a mutation in HEMICENTIN-1 is associated with age-related macular degeneration in a large family. Hum Mol Genet 12: 3315-3323. 18. Nakamura T, Ruiz-Lozano P, Lindner V, Yabe D, Taniwaki M, et al. (1999) DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries. J Biol Chem 274: 22476-22483. 19. Liu X, Zhao Y, Gao J, Pawlyk B, Starcher B, et al. (2004) Elastic fiber homeostasis requires lysyl oxidase-like 1 protein. Nat Genet 36: 178-182. 20. Yanagisawa H, Davis EC, Starcher BC, Ouchi T, Yanagisawa M, et al. (2002) Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo. Nature 415: 168-171. 21. Nakamura T, Lozano PR, Ikeda Y, Iwanaga Y, Hinek A, et al. (2002) Fibulin-5/DANCE is essential for elastogenesis in vivo. Nature 415: 171-175. 22. Lotery AJ, Munier FL, Fishman GA, Weleber RG, Jacobson SG, et al. (2000) Allelic variation in the VMD2 gene in best disease and age-related macular degeneration. Invest Ophthalmol Vis Sci 41: 1291-1296. 23. Lotery AJ, Namperumalsamy P, Jacobson SG, Weleber RG, Fishman GA, et al. (2000) Mutation analysis of 3 genes in patients with Leber congenital amaurosis. Arch Ophthalmol 118: 538- 543. 24. Lotery AJ, Malik A, Shami SA, Sindhi M, Chohan B, et al. (2001) CRB1 mutations may result in retinitis pigmentosa without para-arteriolar RPE preservation. Ophthalmic Genet 22: 163- 169. 25. Bird AC, Bressler NM, Bressler SB, Chisholm IH, Coscas G, et al. (1995) An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group. Surv Ophthalmol 39: 367-374. using threading methods biased by sequence similarity and predicted secondary structure. 26. Jones DT, Tress M, Bryson K, and Hadley C (1999) Successful recognition of protein folds

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27. Fischer J, Jobard F, Oudotth T, Genevièverd D, Mégarbané A, et al. (2004) Identification of novel mutations in elastin and fibulin 5 in three patients affected with cutis laxa. Third European Symposium Elastin. 30 June-3 July 2004, Manchester, UK. 28. Wang T, Waters CT, Rothman AM, Jakins TJ, Romisch K, et al. (2002) Intracellular retention of mutant retinoschisin is the pathological mechanism underlying X-linked retinoschisis. Hum Mol Genet 11: 3097-3105. 29. Ioannidis JP, Ntzani EE, Trikalinos TA, and Contopoulos-Ioannidis DG (2001) Replication validity of genetic association studies. Nat Genet 29: 306-309. 30. Tsuruga E, Yajima T, and Irie K (2004) Induction of fibulin-5 gene is regulated by tropoelastin gene, and correlated with tropoelastin accumulation in vitro. Int J Biochem Cell Biol 36: 395-400.

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31. Aridor M and Hannan LA (2000) Traffic jam: a compendium of human diseases that affect intracellular transport processes. Traffic 1: 836-851. 32. Marmorstein LY, Munier FL, Arsenijevic Y, Schorderet DF, McLaughlin PJ, et al. (2002) Aberrant accumulation of EFEMP1 underlies drusen formation in Malattia Leventinese and age-related macular degeneration. Proc Natl Acad Sci U S A 99: 13067-13072. 33. (2001) A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch Ophthalmol 119: 1417-1436. 34. Weleber RG, Carr RE, Murphey WH, Sheffield VC, and Stone EM (1993) Phenotypic variation including retinitis pigmentosa, pattern dystrophy, and fundus flavimaculatus in a single family with a deletion of codon 153 or 154 of the peripherin/RDS gene. Arch Ophthalmol 111: 1531-1542.

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Chapter 9.indd 207 23-10-12 19:47 General Discussion In the developed world the average life expectancy is constantly increasing. It is one of modern medicine’s aims, that a high quality of life is preserved during these additional days of old age and that these will not be crippled by disease. Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the industrialized countries and one of the major threats for independence and activity in old age. Many factors, including environmental and genetic factors, are associated with the disease. Previous studies have generated strong hypotheses on genes and pathways potentially involved in the etiology of AMD. These include oxidative stress

and inflammatory pathways. In addition, a role for the extracellular matrix (ECM) this thesis was to further enlighten the genetic background of AMD, based on these as a barrier for choroidal neovascularization (CNV) has been proposed. The aim of

this thesis in the light of the current literature, discusses strengths and weaknesses formerly implicated molecular pathways. This chapter discusses the main findings of of the studies performed, and suggests future directions.

The role of oxidative stress

Oxidative stress refers to cellular or molecular damage caused by reactive oxygen species (ROS) and has been implicated in aging and many age-related diseases

to high levels of oxidative stress [3]. Sources of local oxidative stress include the [1,2]. The retina, and especially the retinal pigment epithelium (RPE), is exposed high metabolic rate of photoreceptors required to sustain their normal function and structural renewal, the exposure to light, the high local oxygen pressure and the high

metabolic rate of the RPE due to the processing of photoreceptor outer segments damage, which may be susceptibility factors for the development of AMD. Indeed, (POS) [4]. All these processes may lead to molecular (DNA, RNA, protein) and cellular a number of studies in the literature showed that human donor eyes affected by

compared with non-AMD eyes, which implicates that oxidative stress is involved in AMD contain higher levels of oxidatively modified proteins, carbohydrates and lipids AMD [5,6]. Oxidative stress may be caused by both environmental and genetic factors. Inside the cell, mitochondria produce the majority of ROS. Genetic variation in genes

AMD pathology. In this context, it is of interest that a major susceptibility locus for related to mitochondrial function may thus influence oxidative damage mediated AMD has been mapped to the chromosome 10q26 region by several genome-wide linkage studies [7-9]. This locus contains two genes. Since the physical distance between these genes is only about 6 kb, they are in strong linkage disequilibrium

208(LD). Consequently, it is not yet clear which of these (or both) genes is involved in

Chapter 9.indd 208 23-10-12 19:47 General Discussion AMD. The two genes are age-related maculopathy susceptibility 2 (ARMS2), also known as LOC387715 [10,11] and high temperature requirement factor A1 (HTRA1) [12-14]. One of these genes, the ARMS2 gene, may play a role in mitochondrial function [13]. The ARMS2 protein was initially localized at the mitochondrial outer membrane [15].

to the cytosol [16] and the group of Kortvely described ARMS2 as a constituent of However, this finding is not undisputed: Wang and coworkers localized the protein

the ECM [17]. Genetically, association odds ratios up to 8.21 for homozygous cases, of the ARMS2 gene and AMD [11]. Several subsequent studies suggested that also were observed between the A69S single nucleotide polymorphism (SNP) in exon 1 ARMS2 gene increase susceptibility to oxidative damage, possibly in association with smoking [10,18]. other SNPs in the Besides autosomal ARMS2

SNPs, combinations of neutral polymorphisms in the to AMD onset or progression [19]. Mitochondrial haplogroups (H, J, U and T) were mitochondrial DNA genome, defined as mtDNA haplogroups, may also contribute previously associated with AMD in case-control studies from the United States and Australia [19-23]. Haplogroup H appears to be protective. It was associated with a reduced (early and late) AMD prevalence and large distinct and indistinct soft drusen. In contrast, haplogroup J was associated with increased risk of large soft distinct drusen. Haplogroup U was associated with higher prevalence of retinal

haplogroup, was reported as an independent predictor of AMD [23]. pigment abnormalities [20-22]. Finally, A4917G, a defining polymorphism for the T Antioxidants are bio-molecules that protect the cell against oxidative damage. So far, studies, which investigated the potential human genetic association between variants in antioxidant genes (CAT, CYP, GPX1, PON1, SOD2 and SOD1) and AMD,

CAT), several cytochrome P450 gene were inconclusive. Esfandiary and coworkers investigated the potential role of superfamily members (CYPs), glutathione peroxidase 1 (GPX1) and paraoxonase SNPs in antioxidant genes such as catalase ( 1 (PON1) but reported no association with AMD [24]. In contrast, Ikeda and PON1 Kimura et al SOD2 coworkers did find an association between SNPs in the gene and CNV [25]. in a Japanese population. However, two additional studies, one also in a Japanese . [26] reported a significant association between SNPs in and CNV 9 evidence. Interestingly, Sod1 (-/-) knock-out mice [28] showed pathological features population [27] and one in a Northern Irish cohort [24], failed to corroborate this resembling AMD. These features included drusen, thickened Bruch’s membrane

oxidative stress does play a role in AMD. (BM) and CNV [29]. Obviously, these observations in mice strongly suggest that

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Environmental factors, such as smoking and diet, also point at the role of oxidative stress in AMD. Numerous studies robustly confirm tobacco smoking as one of the fold in ever smokers and from 1.9 to 4.5-fold in current smokers [30-32]. Indeed, major risk factors for AMD: by itself, it increases the risk for AMD from 1.7 to 3.2-

oxidants present in cigarette smoke have numerous relevant effects: they reduce the plasma concentration of antioxidants and the choroidal blood flow, and increase platelet adhesiveness, hypoxia, and the release of free radicals [33]. Epidemiological evidence further suggested that dietary antioxidant supplementation (vitamins C et al. [35] described that the risk of developing advanced AMD is greatly reduced and E, beta-carotene and zinc) decreases the incidence of AMD [34]. Van Leeuwen by high dietary intake of nutrients with antioxidant properties. In 2011, the same group reported that antioxidants reduce the risk of early AMD in those with a high genetic risk [36]. Yet other evidence for an important role of oxidative stress in the pathogenesis of AMD comes from in vitro studies. Lipofuscin is a photoinducible free radical generator and plays an important role in the oxidative stress theory. Lipofuscin accumulates in many metabolically active post-mitotic cells during aging. The

oxidized proteins and lipids and probably derives, at least in part, from oxidatively lipofuscin present in the RPE is composed of a complex heterogeneous mixture of

retinoid N-retinylidene-N-retinylethanolamine, which arises as toxic byproduct of damaged photoreceptor outer segments [3]. RPE lipofuscin contains A2E, the bis-

and confers susceptibility to blue light induced generation of ROS [37-40]. Indeed, phototransduction. A2E impairs RPE phagocytosis, induces complement activation blue light mediated accumulation of lipofuscin has been implicated in oxidative

To further investigate the causative role of oxidative stress in the pathogenesis of stress related RPE cytotoxicity [41] and the development of AMD [42].

cross-complementation group 6 (ERCC6) (chapter 2) and the solute carrier family 2 AMD we focused in this thesis on two potentially relevant genes: the excision repair (facilitated glucose transporter) (SLC2A1) (chapter 3). Both are potentially involved in oxidative stress and oxidative damage related pathways. In chapter 2 gene ERCC6 , we studied the potential association between SNPs in the DNA repair ERCC6 expression in and AMD. Previously, Tuo and colleagues had reported that the C6530G lymphocytes from healthy donors bearing the ERCC6 SNP was associated with AMD [43]. They also found enhanced ERCC6 C6530G alleles [43]. We did not find an association between variants and AMD. Using GWAS, Chen and ERCC6 coworkers recently confirmed our results: they did not find an association between the C6530G, or other SNPs and AMD [44]. In addition, no associations were 210

Chapter 9.indd 210 23-10-12 19:47 General Discussion ERCC6 gene and (neovascular) AMD [45]. found between copy number variations (CNVs) in the Although a genetic association between ERCC6 and AMD is unlikely, our study did suggest a functional role for ERCC6 of ERCC6 in AMD: We found that RPE expression levels genotype. In addition, Ercc6 knock-out mice are very sensitive to oxidative stress were significantly reduced in early AMD affected RPE, independent of and develop retinal degeneration [46]. Further functional studies are needed to determine the exact role of ERCC6 in the etiology of AMD. SLC2A1 gene, the main glucose transporter in the retina. SLC2A1 could potentially be involved in oxidative stress As a second target, we tested the role of SNPs in the

SLC2A1 mediated AMD pathology by changing the bioavailability of glucose in the RPE and in retina and brain is altered in several pathophysiological conditions related to neural retina and thereby altering the local oxidative burden. Expression of oxidative stress like hypoxia [47,48], Alzheimer’s disease [49] and epilepsy [50]. In a

between SLC2A1 multi-cohort association analysis, we could not find a consistent genetic association of effect across the study populations. Interestingly, Kortvely and coworkers (2012) SNPs and AMD. This was possibly due to significant heterogeneity very recently genotyped nine SLC2A1 SLC2A1 SNPs in three independent cohorts and studies regarding the role of SLC2A1 detected a significant correlation of SNP rs3768029 and AMD [51]. Further In summary, we investigated two oxidative stress genes (ERCC6 and SLC2A1 SNPS and AMD are therefore needed. ). No ERCC6 consistent genetic association between SNPs in these genes and AMD was found. might be functionally involved in AMD. More studies are required to further assess Nevertheless, our findings on expression in the RPE did suggest that this gene the genetic contribution to the oxidative stress component of AMD.

The role of the immune response

of AMD came initially from immunocytochemical studies of the composition of Strong indications that inflammation plays an important role in the pathogenesis drusen. Several studies illustrated the presence of complement components and

complement regulatory proteins in drusen and nearby RPE of AMD patients [5,52- 9 57]. Final proof for the inflammatory pathogenesis of AMD came from recent genetic (CFH), B (CFB), I (CFI), 2 (C2), 3 (C3)) consistently suggested that the complement studies. Genetic association studies for at least five genes (complement factors H system plays a major role in the molecular pathology of AMD. Genetic variation in the complement regulator CFH accounts for the strongest association [58-64] (Figure 1).

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Chapter 9.indd 211 23-10-12 19:47 G�� D��

Figure 1. The complement pathway. The components of the complement system that have been genetically associated with increased or decreased

permission from Donoso et al. risk of drusen, geographic atrophy (GA), and choroidal neovascularization (CNV), are circled. Reprinted with [65]. A color version of this figure can be found in the color figures section. The complement system is part of the host innate immune system and enables a number of essential functions, including (1) opsonization and lysis of

(4) regulation of antibody production and (5) removal of immune complexes [66]. microorganisms, (2) recruitment of inflammatory cells, (3) removal of dead cells, The complement system can be activated by three pathways, i.e., the classical, the mannose-binding lectin, and the alternative pathway. In short, the classical pathway

is triggered by antigen-antibody complexes and surfaced-bound C-reactive protein interact with surface sugar molecules on microorganisms. The alternative pathway (CRP). The lectin pathway is triggered when mannose-binding lectins or ficolin is triggered by non-self antigens, such as intermediates of the lipofuscin biosynthesis

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Chapter 9.indd 212 23-10-12 19:47 General Discussion or oxidized lipids or proteins. The pathway subsequently sustains a continuous low-

level state of activation through the spontaneous hydrolysis of C3 into C3a and C3b.

C3 is abundantly present in the blood plasma [66,67]. All complement pathways Figure 1). come together at component C3, and lead eventually to the formation of the lytic As mentioned above, genetic association studies revealed that the Y402H membrane attack complex (MAC), consisting of C5b-C9 [68,69] ( polymorphism of the CFH gene was associated with greatly altered susceptibility

to all forms of AMD. This SNP results in the substitution of histidine for tyrosine streptococcal M6 protein [70]. Since CFH is a negative regulator of the alternative at codon 402 in a domain of CFH that contains binding sites for CRP, heparin, and complement pathway [71], the risk allele leads to inadequate inhibition of the

complement cascade and eventually induces chronic damage to the RPE, BM and/or choroid via the MAC complex. This is consistent with the observation that the CFH protein is present in drusen [72]. Very recently, a potentially important functional et al. present evidence that malondialdehyde (MDA), a naturally clarification for the strong association of the Y402H polymorphism with AMD was provided: Weisman occurring product of lipid peroxidation, is an important ligand for CFH present on apoptotic cells. MDA epitopes allow CFH to bind to the surface of the apoptotic cells and neutralize the pro-inflammatory properties of the MDA epitopes. This occurs through iC3b generation and closing down complement activation. The Y402H polymorphism reduces the capacity of CFH to bind MDA and thereby decreasing the present on the surface of in vitro generation of anti-inflammatory iC3b fragments. MDA epitopes were shown to be choroid and BM including drusen of AMD lesions [73]. - generated necrotic RPE cells and throughout the Another complement component genetically linked to AMD is C3 plasma protein, a common component of all three complement pathways and critical . C3 is an important

C3 gene and AMD was found in two independent for the downstream formation of the terminal MAC that leads to cell lysis. Initially, association between SNPs in the The associated C3 AMD case-control cohorts with English (discovery) and Scottish (replication) origin. SNP rs2230199 resulted in an amino acid change with at least some functional consequences: The change influenced binding to pathogenic cell 9 surfaces and enhanced complement activation, which, in the end, leads to RPE cell [59,74]. A Dutch study by Despriet et al. (2009) showed that not only rs2230199, lysis and AMD [58]. Multiple subsequent studies replicated these initial findings

of AMD [75]. but also another variant, rs1047286, significantly increased the risk of all subtypes

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Chapter 9.indd 213 23-10-12 19:47 General Discussion In 2006, association was found between AMD and genetic variants in yet two C2 and CFB, respectively activators of the classic and alternative complement pathways, which exhibited a protective instead of risk other complement components:

largely due to the CFB variants, which are in strong LD with C2. Structural variants increasing effect [62]. Gold and his colleagues hypothesized that the significance is in CFB may lead to impairment in the complement activating function of CFB and

of the C2/CFB haplotypes and CFH variants showed that variation in the two loci may thus provide a lower risk of chronic complement activation. Combined analysis

American populations [62,67]. can predict the clinical outcome in 74% of all AMD cases in European and North Subsequently, Fagerness et al. (2009) provided yet additional evidence for the involvement of the complement system in AMD. They found association between common variants near the CFI gene on chromosome 4q25 and advanced AMD [63]. CFI gene, rs10033900, yielded

A SNP located 2781 bp upstream of the 3’ UTR of the the statistically most significant evidence. CFI is a serine protease that inhibits all complement pathways by degrading activated complement components C3b and C4b. To complicate matters, C3b inactivation by CFI is regulated by CFH. CFH inhibits C3 activation by binding to C3b and acting as a co-factor for CFI-mediated cleavage of C3b and has decay-accelerating activity for the alternative pathway C3 convertase, C3bBb [63]. Multiple complement genes are locally expressed by the RPE and choroid, which, However, Scholl et al. (2008) showed that markers of chronic complement activation, in theory, would be sufficient to invoke and sustain local chronic inflammation.

like Ba and C3d, and regulator of the alternative pathway of complement, factor D, AMD is, at least in part, a systemic disease with a local disease manifestation [76]. are abundantly present in the plasma of AMD patients. This finding implied that CX3CR1, may

Next to complement genes, yet another immune system-related gene, is macrophage and microglia recruitment. Three independent studies showed that be associated with AMD. The function of the chemokine receptor protein CX3CR1 CX3CR1 variants were moderately associated with AMD [77-79]. However, a recent

GWAS study and a recent study in a large population in France could not corroborate Based on the crucial role of the immune system in AMD (Figure 1, Donoso et al.), this finding [80-81]. we further tested the contribution of other complement system players to AMD by extensively genotyping the complement component 5 (C5) gene (chapter 4) and a member of the classical complement pathway, serpin peptidase inhibitor, clade G, member 1 (SERPING1) (chapter 5). Our C5 study showed that, in spite of the

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Chapter 9.indd 214 23-10-12 19:47 General Discussion

C5 crucial role of C5 in the complement cascade and the presence of C5 protein in The negative outcome of our association analysis may depend on genetic or clinical drusen, no consistent significant association between SNPs and AMD was found.

complement activation molecules, elevated in peripheral blood of AMD patients differences between our study populations. C5a is, among many other alternative

[76]. In 2006, Nozaki provided evidence that bioactive fragments of complement components C3 and C5 (C3a and C5a) accumulate in drusen of patients with AMD et al. (2008) found an association between AMD and genetic variants and promote CNV [57]. in the SERPING1 Initially, Ennis SERPING 1 gene in seven large gene, encoding the complement component 1 inhibitor (C1inh) protein [82]. We tested the rs2511989 SNP in the Park et al case-control studies. We found no evidence that this variant plays a role in AMD. between SERPING1 et al. shows . in 2009 and Carter and Churchill in 2011 also failed to find an association SERPING1 SNPs and AMD [83,84]. Yet, a study in 2012 by Gibson significantly higher plasma levels of C1inh, the protein product of the (rs2649663), located 7.7 kb of SERPING1 gene, in AMD cases versus controls. This group also shows evidence that one SNP by differential expression of SERPING1 or its proxies may influence C1inh levels et al. [82]. [85]. This SNP was not associated with AMD In this thesis (chapter 6) we furthermore tested the role of toll-like receptor 3 susceptibility by Ennis (TLR3), known to be involved in the innate immune system, in geographic atrophy (GA) cases and controls in eight well-known cohorts. Yang and coworkers (2008) found that variation in TLR3 (rs3775291) was associated with protection against

we illustrate that it is unlikely that TLR3 variant rs3775291 has a major effect in GA, possibly through suppression of RPE apoptosis [86]. However, in this thesis,

89]. Interestingly, for another member of the TLR family, TLR4 the etiology of AMD. Our findings were confirmed by several other studies [87- results exist. A study by Zareparsi et al. reported that the D299G polymorphism , also conflicting leads to increased AMD risk [90] whereas Despriet et al TLR3 and TLR4 interact, and need to be tested . could not find a significant together in future studies. association [91]. Perhaps SNPs in In conclusion, we tested genetic variation in C5, SERPING1 and TLR3 for association with AMD. The relationship between these genes and AMD is controversial to date 9 and requires additional studies in diverse and larger populations.

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Chapter 9.indd 215 23-10-12 19:47 General Discussion The role of the extracellular matrix

BM is a multi-layered ECM complex that functions as a molecular sieve. BM partly

metabolic waste products between the retina and the general circulation. In chapter regulates the reciprocal exchange of biomolecules, nutrients, oxygen, fluids and 7 of this thesis we provided a review of BM. With age, many functional properties of BM change, leading not only to normal age-related changes in the RPE and photoreceptor cells, but also to the onset and/or progression of diseases like AMD.

We showed that the changes occurring in BM with age include increased calcification turnover of glycosaminoglycans. In addition, lipids and advanced glycation end of elastic fibres, increased cross-linkage of collagen fibres and an alteration in the products (AGEs) accumulate in BM. In particular, diffuse thickening and progressive deposition of material in BM may result in a barrier to normal metabolic exchange between the choroid and the RPE or vice versa [92]. Extracellular deposits slowly

deposits beneath the RPE that include basal laminar deposits and basal linear but surely appear in BM. They include drusen and basal deposits (flat and diffuse deposits) [93]. Undoubtedly, BM is the primary site of drusen development in the early stage of AMD [93,94]. Biochemically, drusen contain (oxidation products of) proteins, lipids and carbohydrates [5,94-96]. The oxidized components in drusen

and immune responses [97]. Besides the complement system, also multiple may be recognized as non-self epitopes by the human body and trigger inflammatory immune-related cells, including dendritic cells, macrophages and lymphocytes,

and, ultimately, neovascularization in AMD [98]. In chapter 7, we provided a list as well as fibroblasts have been implicated in RPE atrophy, the breakdown of BM, of genes that are expressed at higher levels in the RPE than in the choroid with the functional annotation “angiogenesis”. These data indicate that at least 23 genes may be involved in this process, which clearly illustrates the complexity of this matter. Next to the aging changes and its relation to AMD pathology, one could hypothesize that mutations in genes encoding ECM molecules result in disturbance of the transport or the barrier function of BM (and the RPE), which may also contribute to AMD. Genetic variants in ECM genes such as TIMP3, COL8A1, COL10A1, FBLN5, VEGFA and possibly HTRA1 support involvement of the ECM in the pathogenesis of AMD. As illustrated below, variation in these genes was linked to the pathology of AMD. The tissue inhibitor of metalloproteinases-3 (TIMP3) is an inhibitor of MMP activity, i.e. an inhibitor of enzymatic degradation of the ECM. Mutations in this gene cause Sorsby fundus dystrophy (SFD) [99]. The clinical picture of SFD strikingly resembles

between SNPs in TIMP3 and AMD, recently a SNP near this gene was associated with early onset AMD. Although initial investigations [99,100] did not find an association 216

Chapter 9.indd 216 23-10-12 23:06 General Discussion AMD [44]. Two other ECM genes, the collagen matrix pathway genes COL8A1 and COL10A1, were recently implicated in advanced AMD using GWAS studies [101,102]. As early as 2004, Stone et al. FBLN5) in patients with AMD [103]. Besides mutations, several other lines of evidence described rare missense mutations in fibulin 5 ( support a role of FBLN5 in AMD: FBLN5 is located in an AMD associated GWAS locus on chromosome14q32.1 [104]. FBLN5 encodes an ECM protein that plays a role in maintaining the integrity of elastic lamina, such as that found in BM. Mullins et al.

(deposits) and choriocapillaris in normal eyes and to pathologic basal deposits as [105] examined fibulin 5 expression in human donor eyes and localized it to BM well as some small drusen in AMD eyes. These data suggested a role for FBLN5 in extracellular deposit formation in AMD [105]. FBLN5 has also similarities with FBLN3. Mutations in FBLN3 cause a rare, early-onset form of macular degeneration known as malattia leventinese (Doyne’s honeycomb dystrophy), a disease characterized by the presence of BM deposits with similarities to drusen in AMD [106]. Additionally, the Fbln5 (-/-) knock-out mice showed abnormalities in normal elastinogenesis [107], which may increase susceptibility to accumulate abnormal extracellular deposits and/or CNV later in life. Finally, Albig and Schiemann [108], showed interaction between vascular endothelial growth factor (VEGF) and FBLN5. VEGFA is a member of the VEGF family and functions to increase vascular permeability, angiogenesis, cell growth and migration of endothelial cells. Activation of VEGFA mediates CNV [109,110]. While association between VEGFA and AMD remains controversial [111,112], two SNPs, one in the VEGFA promoter region (rs2010963) and one (rs4711751) near VEGFA, may be associated with AMD [113,102]. In chapter 8 we replicated the study of Stone et al. (2004) and revealed the FBLN5 missense mutations possibly implicated in AMD. We further demonstrated in vitro that these genetic variants identification of two novel heterozygous

corresponding reduction in elastinogenesis. Mullins et al. (2007) hypothesized that may lead to AMD susceptibility due to reduced fibulin 5 secretion resulting in a the FBLN5 missense mutations described in our study [114] could indeed potentially FBLN5 and VEGF. Both mechanisms could possibly modify the regulation of VEGF [105]. Bhutto lead to reduced fibulin 5 secretion or to decreased affinity between et al. suggested that if the local balance between pro- and anti-angiogenic factors is disturbed substantially in favor of VEGF, BM may become more vulnerable, thereby 9 creating an environment permissive for CNV [115,116]. Additionally, a very recent paper describes an important functional role for HTRA1 in altered elastogenesis in HTRA1 is a key modulator of proteoglycans degradation in the ECM and one of the two genes located on the major AMD BM through fibulin 5 cleavage [117].

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Chapter 9.indd 217 23-10-12 23:06 General Discussion susceptibility locus on 10q26. The HTRA1 protein was localized in drusen of AMD patients [118]. Accumulation of the protein in drusen could possibly compromise the integrity of the BM and so allow growth of choroidal capillaries and result in CNV. Vierkotten et al. (2011) showed that overexpression of HTRA1 in the mouse leads to AMD like features by fragmentation of the elastic layer in BM due to decreased [117]. In conclusion, we conducted an extended review of BM, which showed that many fibulin 5 aging processes affect BM, such as thickening of its layers due to lipid deposition,

these normal (subclinical) aging events may predispose BM and the RPE to calcification of the elastin layer, oxidative stress and drusen formation. Clearly, disease. Furthermore, mutations in genes encoding ECM molecules might result in disturbance of the structural and/or functional properties of the BM, which may ultimately lead to the devastating clinical manifestations of AMD.

Strenghts and weaknesses of our studies; non-replication

Both genome-wide linkage analysis and genome-wide association studies (GWAS) have been proven useful in detecting susceptibility genes of AMD. Numerous genome- wide scans repeatedly showed linkage between AMD and markers on the long arms of chromosomes 1 and 10 [9,119-124]. Later, association studies discovered two major risk variants, the Y402H polymorphism of the CFH gene on 1q32, and, subsequently, the ARMS2/HTRA1 locus on 10q26. Beside genome wide linkage and

screen SNPs in functional candidate genes for a disease. For the studies performed GWAS, an additional approach to find disease genes for complex disorders is to in this thesis, candidate disease genes were selected in two ways: First, based on their previously reported association with AMD in the literature or second, based on the outcome of an Illumina SNP genotyping assay containing 1,536 SNPs. Potential AMD disease genes for this genotyping assay were selected on the basis of AMD associated gene expression studies of the RPE carried out by our research group. Subsequently, the results of the genetic association studies found in the initial discovery cohorts were replicated in a study population from the University of Southampton, Southampton, United Kingdom and/or (a subset of) populations of the International Age-related Macular Degeneration Genetics Consortium which includes eight international cohorts (see introduction section of this thesis) of patients and controls matched by age and ethnicity (all of European descent). One of the greatest challenges in interpreting genetic association studies (not only in this thesis), is the lack of consistent reproducibility. The association between AMD and CFH Y402H is an example of the common disease-common variant

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Chapter 9.indd 218 23-10-12 23:06 General Discussion hypothesis, which means that a relatively common allele consistently predisposes to a common disease [125,126]. This can be understood because of the vital role of the complement system in innate immunity and the critical role of CFH in protecting host cells from the excessive attack of complement. The role of CFH and the complement system is fundamental and wide-spread (common) in many pathological processes, including AMD. Yet, these “strong” common risk factors are rather an exception than rule. Initial associations between putative “weaker” AMD risk factors, such as genetic variants in ERCC6, TLR3 and SERPING1 could not be consistently replicated in several other case-control cohorts [127-129]. Replication has become the gold standard for evaluating statistical results from genetic association studies. In general, the results of many reported studies can neither be replicated nor corroborated. Lohmueller et al. [130] and Hirschhorn et al. [131] reviewed large numbers of reported associations between common gene variants and disease and found that the majority is not robust. Inconsistent replication in genetic association studies can be caused by numerous factors including chance, variation in study design, phenotypic AMD (grading) differences, genotype errors, population substructure

Many of these factors were already discussed in previous chapters. The effect of (stratification), environmental interactions and inadequate sample size [132-134].

AMD. A limitation of our studies is that we selected for relatively common SNPs, with these factors is stronger in small cohorts, such as defined by clinical subtypes of MAFs >10%. Consequently, we could have missed variants with MAFs <10% which

(prospective and case-control) were used, which may have affected the outcomes. might influence the disease phenotype. Also, for this thesis, 2 different study designs Conversely, phenotypic grading differences may not have accounted for a large variability in our studies since all study subjects underwent clinical examination and stereoscopic color fundus photography after pharmacologic mydriasis, and the grading criteria were very similar for the studies. Study subjects were either graded

according to the International Classification and Grading System for AMD (Reykjavik, Melbourne and Würzburg sites), Rotterdam modification of the International classification (Columbia, Iowa, Amsterdam and Rotterdam sites), or according to issue that possibly may have played a crucial role in the outcomes of our studies the classification established by the AREDS study (AREDS and Southampton). An is that in genetic association studies the allele frequencies of SNPs may differ due 9 to sampling error or population differences. Several studies showed that extensive differences in genetic variation across different ethnic populations exist in terms of allele frequency, LD and haplotype structure [135-137]. Even across Europe, subtle geographic variation in allele frequencies exist [138-140]. Indeed, although all our

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Chapter 9.indd 219 23-10-12 23:06 General Discussion cohorts were from European descent (which can be considered a strength), in almost all of our studies we observed extensive differences in MAFs of SNPs from the tested genes between the studied populations. In our studies, this probably originated from varied ethnic composition within the populations of European descent. The Columbia study population originates mainly from Eastern Europe, whereas that from Iowa from Western Europe and Scandinavia. Replication failure of statistically

populations differ and when the polymorphism under study interacts with one or significant independent genetic effects might occur when allele frequencies among more other polymorphisms at another locus [141,142]. For example, Greene et al. (2009) illustrated that under an epistatic model (gene-gene interaction), successful

a MAF change of less than 0.1 of an interacting SNP (with heritability’s ranging replication of a significantly associated SNP can decrease from 80% to 20% with from 0.025 to 0.4)[141]. They also showed that allele frequency differences under an epistatic model could reverse the allelic outcomes (i.e., a risk increasing effect becomes a protective effect after replication). In chapters 3 and 5 of this thesis, we

in highly comparable populations. This pattern of allelic reversal has been noted in were not able to replicate initial findings and we found such opposite associations replication studies of other candidate SNPs [130]. In a recent study investigating et al. [142] suggested that

the potential causes of this “flip-flop” phenomenon, Lin effects and variation in inter-locus correlations. flip-flop associations can occur due to variation in LD, or to unaccounted multi-locus

Future directions

We expect even more advances within the next couple of years. GWAS and whole Our knowledge of the genetics of AMD has increased significantly in the past years.

susceptibility and offer us a better understanding of the molecular basis of AMD. genome sequencing will allow us to find other loci and genes associated with disease As mentioned above, one of the pitfalls in genetic association studies is lack of consistent reproducibility. We need to interpret associations in the perspective of differences in allele frequencies, LD and haplotype structure that occur in different populations or as a result of sample heterogeneity. The effect of one locus on disease risk may be inconsistent or missed entirely if we fail to examine it together with other known disease variants. Hence, we propose that SNPs that fail to replicate should be tested for interactions with other polymorphisms, predominantly when samples are collected from groups with distinct ethnic backgrounds or different geographic regions. Yet, the power to identify the interactions is limited particularly in the case of GWAS where the number of potential comparisons is enormous. Epistasis analysis

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Chapter 9.indd 220 23-10-12 23:06 General Discussion would also require adjustment for multiple comparisons to avoid the introduction of false-positives. Recently, Marchini et al et al. (2006) [144], demonstrated that, even considering a conservative multiple testing burden, it was . (2005) [143] and Evans possible to identify interacting loci that increased odds of disease using realistic sample sizes (approximately 1500 individuals).

of gene-environment interactions. To achieve this objective, large cohorts would Other strategies to elucidate the genetic influence on AMD may include the study be required, with the emphasis on harmonization of descriptions (e.g., primary features, age of onset, environmental factors) of AMD cases. This would allow for

stratification into well-characterized phenotypes, which need to be standardized across multiple populations, such as GA and CNV or primary features, such as soft body mass index. Since AMD is such a complex disease, individual gene effects may drusen. Environmental factors for stratification might include cigarette smoking and

only be exposed within patient subgroups with specific environmental exposures. MAF>5%). Recent studies have revealed that common variants explain only a small GWAS was designed to detect associations between disease and common SNPs (e.g., fraction of the heritability [145]. Some studies suggest that not common, but rare variants are more likely to be pathological variants [146,147]. In order to capture rare variants, it will be necessary to sequence whole genome sections of cases. An effective approach seems to be next-generation sequencing, which holds great promise in cost-effectively detecting rare variants. Genetic epidemiology provided us with the names of major molecular players in AMD and will reveal more in the next couple of years. Although this methodology paves the way to population and individual (genetic) risk assessments, it does not provide detailed mechanistic insight. Further functional studies are needed to

itself. Hopefully this will lead to intervention in the course of this devastating disease specify the role of the identified players and reveal the pathophysiological process and improve the quality of life for AMD patients.

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144. Evans DM, Marchini J, Morris AP, Cardon LR (2006) Two-stage two-locus models in genome- wide association. PLoS Genet 2: e157. 145. Schork NJ, Murray SS, Frazer KA, Topol EJ (2009) Common vs. rare allele hypotheses for complex diseases. Curr Opin Genet Dev 19: 212-219. 146. Pritchard JK (2001) Are rare variants responsible for susceptibility to complex diseases? Am J Hum Genet 69: 124-137. 147. Pritchard JK, Cox NJ (2002) The allelic architecture of human disease genes: common disease-common variant...or not? Hum Mol Genet 11: 2417-2423.

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Ch 10 diversen.indd 235 23-10-12 19:48 Summary Age-related macular degeneration (AMD) is a devastating disease of the retina and the most common cause of irreversible visual impairment in the developed world. This degenerative disease has a large impact on the quality of life of affected individuals. AMD has a multifactorial etiology and results from the interplay between numerous genetic susceptibility factors and environmental components. The studies conducted in this thesis explored (new) genes and molecular pathways of retinal aging and disease, based on previously implicated pathobiological processes in order to further unravel the genetic background of AMD.

Part 1. Aspects of oxidative stress

In chapter 2 we investigated the role of oxidative stress in AMD. The retina, and especially the retinal pigment epithelium (RPE) are exposed to high levels of oxidative stress: The combination of high oxygen consumption, intense light exposure and the presence of photosensitizers such as lipofuscin, may locally lead to excessive oxidative damage, for example of the DNA. Therefore, the ERCC6/AMD manuscript of Tuo and coworkers (2006) caught our attention. This group reported that a single nucleotide polymorphism (SNP; rs3793784) in the promoter region of the ERCC6 DNA repair gene was associated with AMD. ERCC6 causes Cockayne syndrome, in which patients age rapidly and, among others, develop retinal degeneration. We assessed the potential genetic association of this gene with AMD. Additionally, we examined a possible functional relationship by determining both rs3793784 dependent as well as normal ERCC6 mRNA expression levels in healthy and early AMD affected RPE isolated from human donor eyes. Our results showed no positive replication of the reported association between SNPs in ERCC6 and ERCC6 expression in the RPE did suggest that ERCC6 may be functionally involved in AMD. ERCC6 expression in the human RPE AMD. Nevertheless, our findings on did not depend on rs3793784 genotype, but, interestingly, on AMD status: Early AMD-affected donor eyes had a 50% lower ERCC6 expression than healthy donor eyes (p=0.018). We hypothesized that the AMD-related reduced ERCC6 expression may be caused by diverse, small and heterogeneous genetic and/or environmental determinants. In chapter 3 we tested the role of another gene potentially involved in oxidative stress mediated AMD pathology. The sodium independent glucose transporter SLC2A1 is the main glucose transporter in the retina. DNA sequence variations or altered expression levels in SLC2A1 profoundly affect local oxidative stress. Based on this hypothesis, we carried out a may influence glucose delivery to the retina and thereby multicenter cohort association study. Three (rs3754219, rs4660687 and rs841853)

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Ch 10 diversen.indd 236 23-10-12 19:48 Summary out of 22 SLC2A1 with AMD in the AMRO-NL discovery cohort. However, replication of these three SNPs tested showed a significant allelic and genotypic association

analysis revealed no overall association between SLC2A1 and AMD. For all three SNPs in five independent cohorts yielded inconsistent association results. Meta-

SCL2A1 SNPs SNPs, we observed significant heterogeneity of effect and high inconsistency across provide further evidence for population-dependent genetic risk heterogeneity in the study populations. The heterogeneous findings on these three AMD.

Part 2. Aspects of the immune system

In chapter 4 we provide an extensive association analysis to discuss the role of complement component 5 (C5) gene variants in AMD susceptibility. The complement system is part of the innate immune system and plays an important role in the defense against microorganisms. Based on the crucial role of C5 in the complement pathway and the formation of the membrane attack complex, its role

in drusen, we hypothesized that genetic variants in C5 would mediate AMD as chemoattractant regulating local inflammatory processes, and its localization susceptibility. We genotyped 15 C5 SNPs in our AMD discovery cohort (AMRO-NL). C5 SNPs and AMD were found in the AMRO-NL cohort. The strongest allelic associations were found for SNPs Significant allelic or genotypic associations between eight rs17611, rs7026551 and rs7037673. Although the complement pathway, including C5, plays a crucial role in AMD, replication testing of these three SNPs could not corroborate an association between these C5 SNPs and AMD. The negative outcome of our replication study may depend on genetic or clinical differences between the study populations. In chapter 5 we discussed the association of a classical complement pathway member, SERPING1, with AMD. Previously, an association between SERPING1 SNPs, and especially, rs2511989, and AMD was found in 2 UK and USA populations. In view of possible population-based genetic risk heterogeneity in AMD, we retested this potential association, in seven additional large case-control studies involving individuals of European descent (4881 patients with AMD and 2842 matched controls). We found no evidence that this SERPING1 variant plays a role in AMD. Since there is no indication of a functional role for this SNP, it is unlikely that SERPING1

In chapter 6 we genotyped toll-like receptor 3 (TLR3) variant, rs3775291, in eight plays a significant role in the etiology of AMD across populations. well-known case-control studies involving patients from European descent to investigate the alleged protective association of this gene with geographic atrophy,

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Ch 10 diversen.indd 237 23-10-12 19:48 Summary a phenotype of late-stage AMD. TLR3 is known to be involved in the innate immune

that this TLR3 variant, rs3775291, has a major effect on the risk of AMD. system. Our studies did not find a significant association and it is therefore unlikely

Part 3. Aspects of the extracellular matrix

In chapter 7 we provided an extended review of the Bruch’s membrane (BM). The BM is strategically located between the RPE and the choroidal blood vessels. The BM plays an essential role in maintaining the normal function of the outer retina and is implicated in many retinal disorders such as AMD. Accordingly, we reviewed the molecular, structural and functional properties of BM and attempted to correlate

Moreover, we described the formation and composition of drusen and their relation these to recently published mRNA expression profiles of the RPE and choroid.

to these gene expression profiles. We showed that during aging, many functional photoreceptor cells, but also the onset and/or progression of retinal disorders like properties of BM change. This may influence not only the normal aging of RPE and retinitis pigmentosa (RP) and AMD. In chapter 8 (FBLN5) gene in AMD. Fibulin 5 is a secreted extracellular matrix protein we conducted the first European study describing the role of the with a role in elastinogenesis through interactions with integrin. We determined fibulin 5

FBLN5 genotype with the AMD phenotype and expanded the retinal phenotypes the functional effects of missense mutations on fibulin 5 expression, correlated the associated with FBLN5 FBLN5 mutations. Our study identified two novel heterozygous secretion with a possible corresponding reduction in elastinogenesis. Our study missense mutations and suggested that they lead to decreased fibulin 5 extends previous work identifying an association between rare FBLN5 variants and AMD.

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Ch 10 diversen.indd 241 23-10-12 19:48 Nederlandse Samenvatting Leeftijdsgebonden macula degeneratie (LMD) is een ernstige aandoening van het netvlies en de belangrijkste oorzaak van blindheid in de Westerse wereld. Bij LMD is het centrale deel van het netvlies, genaamd macula of gele vlek, aangetast. LMD heeft een multifactoriële oorzaak en is het resultaat van samenspel tussen meerdere genetische- en omgevingsrisicofactoren. Het doel van dit proefschrift was om de kennis omtrent de genetische oorzaak van LMD te vergroten. Op basis van eerder onderzoek wisten we dat 3 mechanismen een belangrijke rol spelen bij het ontstaan

extracellulaire matrix (Bruch’s membraan, BM). In dit proefschrift hebben we van 6 van LMD: Oxidatieve stress, het immuunsysteem en de afbraak of opbouw van de genen, die bij deze mechanismen een rol spelen, bepaald of ze (genetisch) betrokken zijn bij LMD. Bovendien beschrijven we een literatuur review over de rol van het BM bij normale veroudering en het ontstaan van LMD.

Deel 1. Aspecten van oxidatieve stress

We hebben twee genen onderzocht die een rol spelen bij oxidatieve stress. In hoofdstuk 2 beschrijven we het onderzoek naar de relatie tussen het ERCC6 gen en AMD. In het netvlies worden veel vrije radicalen geproduceerd, omdat dit weefsel blootgesteld wordt aan intensieve belichting, veel lipofuscine bevat, en zich bevindt in een zuurstofrijk milieu. Vrije radicalen zijn de belangrijkste veroorzakers van schade aan cellen en biomoleculen, zoals bijvoorbeeld het DNA in de celkern. Het werk van Tuo en anderen trok daarom onze aandacht. In het artikel van Tuo werd beschreven dat een variant (rs3793784) in de promotor regio van het ERCC6 gen geassocieerd zou zijn met LMD. Dit gen is belangrijk voor het herstel van oxidatieve DNA schade. Mutaties in ERCC6 leiden tot het Cockayne syndroom. Dit is een syndroom waarbij patiënten snel verouderen en waarbij ook afwijkingen ontstaan in het netvlies. Wij hebben de mogelijke relatie tussen dit gen en LMD onderzocht in een uitgebreide associatie studie. Onze resultaten konden de eerder beschreven associatie tussen genetische varianten in het ERCC6 gen en LMD niet bevestigen. Vervolgens hebben we de ERCC6 mRNA expressie niveaus gemeten in het RPE van gezonde en met beginstadium LMD aangedane humane donor ogen. Onze metingen suggereren dat ERCC6 misschien functioneel wél betrokken is bij LMD. ERCC6

expressie bleek namelijk niet afhankelijk te zijn van het rs3793784 genotype, maar 50% lagere RPE expressie niveaus van ERCC6 dan gezonde donor ogen (p=0.018). was wél afhankelijk van de LMD status. Donor ogen met beginstadium LMD hadden Mogelijk wordt dit veroorzaakt door een groot aantal heterogene en verschillende genetische of omgevingsfactoren.

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Ch 10 diversen.indd 242 23-10-12 19:48 Nederlandse Samenvatting In hoofdstuk 3 bestudeerden wij de rol van het SLC2A1 gen bij het ontstaan van LMD. SLC2A1 is de belangrijkste glucose transporter in de retina. Verandering in de expressie van SLC2A1 of genetische variatie in dit gen zouden kunnen leiden tot verschillen in de beschikbaarheid van glucose voor het retinaal pigment epitheel (RPE) en de retina. Dit kan resulteren in lokale veranderingen in de mate van oxidatieve stress. Wij hebben een uitgebreide genetische associatie analyse uitgevoerd in verschillende cohorten. Voor drie varianten (rs3754219, rs4660687

en rs841853) werd er in de AMRO-NL studie significante associatie aangetoond met geen consistente relatie aan met LMD. Een meta-analyse van de cohort onderzoeken LMD. Replicatie van deze drie SNPs in vijf onafhankelijke cohorten toonde echter liet geen associatie zien tussen SLC2A1 varianten en LMD. Er bleek wel grote heterogeniteit te zijn tussen de verschillende studies. Deze bevindingen bevestigen

het ontstaan van LMD. het bestaan van populatieafhankelijke heterogeniteit voor het genetische risico op

Deel 2. Aspecten van het immuunsysteem

We hebben drie genen onderzocht die betrokken zijn bij het immuunsysteem. In hoofdstuk 4 beschrijven we een uitgebreide associatie studie naar de rol van genetische varianten in het complement component 5 (C5) gen bij het ontstaan van LMD. Het complement systeem speelt een belangrijke rol bij de afweer tegen micro- organismen en is onderdeel van het aangeboren immuunsysteem. C5 is cruciaal voor de vorming van het zgn. ‘membrane attack complex’ (MAC) van het complement systeem en het komt voor in ogen die met LMD zijn aangedaan. Vijftien genetische varianten in het C5 tussen acht C5 varianten en LMD in onze AMRO-NL studiepopulatie. De drie meest gen werden getest. Er werd een significante associatie gevonden

significant geassocieerde varianten werden geselecteerd voor replicatie analyse in C5 gen en LMD. Dit is waarschijnlijk drie grote onafhankelijke cohorten. Deze replicatie testen konden geen consistente te wijten aan genetische of klinische verschillen tussen de verschillende populaties. significante relatie aantonen tussen variatie in het In hoofdstuk 5 rapporteren wij het onderzoek naar de rol van het SERPING1 gen bij het ontstaan van LMD. Recente studies, die het genetisch risico beschrijven van het SERPING1 gen op LMD, spreken elkaar tegen. Om de werkelijke bijdrage van dit gen aan het ontstaan van deze oogziekte te onderzoeken, hebben wij de eerder beschreven associatie tussen SERPING1 variant rs2511989 en LMD getest in zeven grote case-control studies (4881 patiënten met LMD en 2842 controles). Wij konden geen relatie aantonen tussen deze variant en LMD. Aangezien er geen aanwijzingen

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Ch 10 diversen.indd 243 23-10-12 19:48 Nederlandse Samenvatting zijn voor een functionele rol van deze variant, is het onwaarschijnlijk dat SERPING1

Hoofdstuk 6 bevat de beschrijving van de genetische associatie analyse tussen de een significante rol speelt in het ontstaan van LMD. toll-like receptor (TLR3) variant rs3775291 en LMD in acht bekende internationale case-control studies. TLR3 is een signaal molecuul dat onderdeel is van het aangeboren immuunsysteem. In een eerdere publicatie werd beschreven dat de rs3775291 variant in TLR3 laat stadium fenotype van LMD, zou verlagen. Onze omvangrijke studies konden het risico op het ontstaan van geografische atrofie, een TLR3 variant waarschijnlijk geen groot effect op het risico op LMD. echter geen significante associatie aantonen en daarom heeft de

Deel 3. Aspecten van de extracellulaire matrix

In hoofdstuk 7 geven we een uitgebreide beschrijving van de normale structuur en functie van het BM. Het BM is strategisch gelokaliseerd tussen het RPE en het choroid (vaatvlies). Het BM speelt een essentiële rol in het behouden van de functie van het RPE en de neurale retina en is betrokken bij de allereerste verschijnselen van LMD. We hebben de moleculaire, structurele en functionele eigenschappen van het BM beschreven op basis van de huidige literatuur en we hebben geprobeerd deze eigenschappen te correleren aan onze recent gepubliceerde mRNA expressie

profielen van het RPE en choroid. Daarnaast beschrijven we het ontstaan en de zien dat tijdens veroudering veel functionele eigenschappen van het BM veranderen samenstelling van drusen en hun relatie tot deze genexpressie profielen. We laten en dat dit niet alleen leidt tot normale leeftijdsgerelateerde veranderingen in het RPE en de fotoreceptor cellen, maar ook tot het ontstaan en/of progressie van aandoeningen zoals LMD. In hoofdstuk 8 5 (FBLN5) gen bij het ontstaan van LMD. Fibuline 5 is een eiwit dat noodzakelijk is beschrijven we de eerste Europese studie naar de rol van het fibuline voor de vorming van elastine vezels. We hebben twee nieuwe, zeldzame heterozygote missense mutaties in FBLN5 gevonden. We hebben tevens vastgesteld wat het effect

is van missense mutaties op de expressie van fibuline 5. We suggereren dat de resulteren in een afname in de vorming van elastine vezels. door ons gevonden mutaties leiden tot verminderde secretie van fibuline 5. Dit kan Tenslotte hebben we ook het FBLN5 genotype gecorreleerd aan het LMD fenotype. Onze studie borduurt voort op resultaten van eerder onderzoek waarin een eerste potentiële associatie werd gevonden tussen FBLN5 mutaties en LMD.

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Ch 10 diversen.indd 247 23-10-12 19:48 Ch 10 diversen.indd 248 23-10-12 19:48 List of Publications List of Publications

1. Sofat R, Casas JP, Webster AR, Bird AC, Mann SS, Yates JR, Moore AT, Sepp T, Cipriani V, Bunce C, Khan JC, Shahid H, Swaroop A, Abecasis G, Branham KE, Zareparsi S, Bergen AA, Klaver CC, Baas DC, Zhang K, Chen Y, Gibbs D, Weber BH, Keilhauer CN, Fritsche LG, Lotery A, Cree AJ,

Weeks DE, Ortube MC, Ferrell RE, Jakobsdottir J, Conley YP, Rahu M, Seland JH, Soubrane G, Griffiths HL, Bhattacharya SS, Chen LL, Jenkins SA, Peto T, Lathrop M, Leveillard T, Gorin MB, Topouzis F, Vioque J, Tomazzoli L, Young I, Whittaker J, Chakravarthy U, de Jong PT, Smeeth L, Fletcher A, Hingorani AD. Complement factor H genetic variant and age-related macular

2012 Feb;41(1):250-62. degeneration: effect size, modifiers and relationship to disease subtype. Int J Epidemiol. 2. Baas DC, Ho L, Tanck MW, Fritsche LG, Merriam JE, van Het Slot R, Koeleman BP, Gorgels TG, van Duijn CM, Uitterlinden AG, de Jong PT, Hofman A, Ten Brink JB, Vingerling JR, Klaver CC, Dean M, Weber BH, Allikmets R, Hageman GS, Bergen AA. Multicenter cohort association study of SLC2A1 single nucleotide polymorphisms and age-related macular degeneration. Mol Vis. 2012;18:657-74. 3. Baas DC, Despriet DD, Gorgels TG, Bergeron-Sawitzke J, Uitterlinden AG, Hofman A, van Duijn CM, Merriam JE, Smith RT, Barile GR, ten Brink JB, Vingerling JR, Klaver CC, Allikmets R, Dean M, Bergen AA. The ERCC6 gene and age-related macular degeneration. PLoS One. 2010 Nov 1;5(11):e13786. 4. Baas DC HL, Rivadeneira F, Hofman A, van Duijn C, Smith RT, Barile GR, Gorgels TG, Vingerling JR, , Ho L, Ennis S, Merriam JE, Tanck MW, Uitterlinden AG, de Jong PT, Cree AJ, Griffiths Klaver CC, Lotery AJ, Allikmets R, Bergen AA. The complement component 5 gene and age- related macular degeneration. Ophthalmology. 2010 Mar;117(3):500-11. 5. Booij JC, Baas DC, Beisekeeva J, Gorgels TG, Bergen AA. The dynamic nature of Bruch’s membrane. Prog Retin Eye Res. 2010 Jan;29(1):1-18. 6. Allikmets R, Dean M, Hageman GS, Baird PN, Klaver CC, Bergen AA, Weber BH, Merriam JE, Smith RT, Barile GR, Sawitzke J, Gehrs K, Hageman JL, Miller NJ, Howard ML, Guymer RH, Richardson A, Ho L, Vingerling JR, Uitterlinden AG, de Jong PT, Baas DC, Fritsche LG, Keilhauer CN. The SERPING1 gene and age-related macular degeneration. Lancet. 2009 Sep 12;374(9693):875-6; author reply 876-7. 7. Allikmets R, Bergen AA, Dean M, Guymer RH, Hageman GS, Klaver CC, Stefansson K, Weber BH, Merriam JE, R. Smith RT, Barile GR, Gehrs KM, Hancox LS, Miller NJ, Howard ML, de Jong PT, Baas DC, Ho L, Vingerling JR, Uitterlinden AG, Fritsche LG, Keilhauer CN, Baird PN, Sawitzke J, Magnusson KP, Thorleifsson G, Thorsteinsdottir U. Geographic atrophy in age-related macular degeneration and TLR3. N Engl J Med. 2009 May 21;360(21):2252-4; author reply 2255-6. 8. Lotery AJ, Baas DC H, Wang T, Bergen AA, Trump D. , Ridley C, Jones RP, Klaver CC, Stone E, Nakamura T, Luff A, Griffiths degeneration and cutis laxa. Hum Mutat. 2006 Jun;27(6):568-74. Reduced secretion of fibulin 5 in age-related macular

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Ch 10 diversen.indd 251 23-10-12 19:48 Dankwoord Dankwoord

Het werk is af!! Wat heb ik uitgekeken naar dit moment. De afgelopen 8 jaren heeft er altijd wel een stemmetje in mijn achterhoofd gezeten dat me zelfs op de meest ontspannende momenten herinnerde aan mijn proefschrift. Dit proefschrift was niet tot stand gekomen zonder de bijdrage en inzet van velen. Ik wil iedereen dan ook heel erg bedanken voor alle hulp en adviezen, de kritische commentaren en voor de getoonde interesse in mijn proefschrift.

Op de eerste plaats wil ik de AMD patiënten en controles bedanken voor hun deelname aan de studies. Zonder hun bijdrage was dit proefschrift er nooit gekomen.

Inmiddels heb ik de oogheelkunde alweer drie jaar achter me gelaten en ben ik met heel veel enthousiasme en plezier werkzaam in de virologie bij het RIVM onder Prof. dr. M. Koopmans. Dit had ik echter nooit kunnen bereiken zonder de ervaringen, contacten en leerpunten die ik tijdens mijn promotietraject heb opgedaan. Ik ben hiervoor zeer dankbaar en ik wil dan ook graag mijn promotor Prof. dr. Bergen en mijn co-promotor Dr. Theo Gorgels hiervoor zeer hartelijk bedanken. Arthur, bedankt voor je begeleiding tijdens de lange weg door de wetenschap. Jouw visie op AMD was verhelderend en leerzaam. De laagdrempeligheid waarmee altijd overleg

naambordje naast je kamerdeur als ik weer eens naar binnen liep voor vragen. plaats kon vinden vond ik erg fijn. Hoe vaak heb ik je naam wel niet gelezen op het Dank je wel ook voor je kritische opmerkingen (die de vaak pijnlijke waarheid in een dataset of experiment onthulden). Beste Theo, bedankt voor je scherpzinnige blik, maar ook voor je leerzame en eerlijke commentaar op mijn stukken. Ik heb er veel van opgestoken voor de rest van mijn wetenschappelijke carrière. Je kon mijn vele (soms ook onbenullige) vragen vaak helder beantwoorden en moeilijke materie duidelijk uitleggen. Bedankt ook voor je enthousiasme en het bespreken van onze ideeën over mijn onderzoek.

Prof. dr. Baas, Prof. dr. Hoyng, Prof. dr. Kamermans, Prof. dr. Klaver, Prof. dr. Schlingemann en Prof. dr. Zwinderman wil ik bedanken voor de bereidheid plaats te nemen in mijn promotiecommissie en voor de tijd en moeite die jullie hebben genomen voor het deskundig beoordelen van mijn proefschrift. Ook wil ik alle co-auteurs bedanken voor hun hulp, inzet en waardevolle commentaar. Thanks to all co-authors of the papers included in this thesis for your expertise,

critical comments and contribution to the final contents of this thesis.

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Ch 10 diversen.indd 252 23-10-12 19:48 Dankwoord Michael Tanck wil ik in het bijzonder bedanken voor zijn statistische input en zijn eindeloze geduld tijdens het schrijfproces. Michael bedankt dat je altijd bereid was me te helpen met al mijn “reviewer” vragen. Je kwam dan met een gedegen uitleg waar ik echt iets mee kon.

Al mijn oud-collega’s van het NIN (Wendy Aartsen, Arne Bakker, Helma de Bruin, Martijn Dahlhaus, Frederike Dijk, Ralph Florijn, Ria Grimbergen, Joop van Heerikhuize, Paulus de Jong, Wendy Hettema, Maarten Kamermans, Willem Kamphuis, Jan Klooster, Willem Loves, Youssef Moutaouakil, Cecile Ottenheim, Astrid Plomp, Mary van Schooneveld, Simone van Soest, Danielle Versteeg, Inge Versteeg, Gerard de Wit en Nataliya Yeremenko) wil ik bedanken voor de inhoudelijke bijdrage en/of hun collegialiteit en gezelligheid. In het bijzonder wil ik Jacoline ten Brink en Anke Essing noemen. Bedankt voor jullie hulp bij het tot stand komen van mijn proefschrift. Dank voor jullie bijdrage aan de experimenten en voor het wegwijs maken in de verschillende labtechnieken. Dank jullie wel ook voor de gezellige persoonlijke gesprekken en het feit dat ik tegen jullie aan mocht klagen.

Het secretariaat van het NIN, altijd stonden jullie voor me klaar en jullie wisten steeds weer een beetje orde te scheppen in de soms dreigende chaos. Jullie zijn onmisbaar.

Judith, een betere paranimf had ik me niet kunnen wensen. Wat hebben wij steun aan elkaar gehad als we het even echt niet meer zagen zitten of gewoon even onze frustraties kwijt wilden. Door jouw positieve blik kwam er bij tegenslag altijd weer een lichtpuntje in zicht. Bedankt voor aaaaaaaallllllllll je adviezen en tips. Ik heb heeeel wat centimetertjes van het tapijt op jouw kamer versleten met ijsberen en vragen om hulp. Jij was er altijd om mijn “problemen” te relativeren. Ook naast het

werk hebben we een fijne band op weten te bouwen en ik ben blij dat die er nog ik hoop dat we nog veel contact zullen blijven houden en onze lange mailtjes zullen steeds is. Ik vind het ontzettend fijn je zo gelukkig te zien met Martijn en Casper en blijven voortzetten.

Mijn lieve naaste collega’s van het RIVM: ik wil jullie allen bedanken voor de

RIVM heb mogen werken. Ik heb met heel veel enthousiasme en plezier gewerkt persoonlijke betrokkenheid en de 3 fijne jaren die ik samen met jullie voor het aan o.a. B-cel immuniteit (Rob/Hinke, bedankt voor de leuke gesprekken en alles wat jullie me in die korte tijd hebben geleerd) en voedselgerelateerde infecties (ENT groep en Mariska bedankt!!). In het bijzonder wil ik mijn collegaatjes van de

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Ch 10 diversen.indd 253 23-10-12 19:48 Dankwoord AIO kamer noemen (Era, Faizel, Giovana, Gudi, Janko, Marcel, Sabine (en hier hoort

en steunen tijdens alle AIO perikelen en andere vraagstukken. Ook al vonden mijn Linda stiekem ook een beetje bij). Het was fijn dat we elkaar konden stimuleren AIO werkzaamheden zich buiten RIVM werktijden plaats, ik kon er altijd met jullie over praten en het was goed om het ook eens vanuit een ander perspectief dan de oogheelkunde te benaderen. Vragen over het een of ander werden altijd wel door iemand op de kamer beantwoord. Gelukkig was er zeker niet alleen maar ellende en hebben we ook een hele hoop lol gemaakt op onze drukke, gezellige, “soms bijna onmogelijk om te werken”, Black Eyed Peas AIO kamer.

In het bijzonder wil ik mijn RIVM collega Wilma Witkamp bedanken voor de geweldige hulp en creativiteit bij de design en lay-out van dit proefschrift. We leerden elkaar kennen voor een heel ander doel, maar jij zei dat je het heel leuk zou vinden mij te helpen met mijn proefschrift en daar ben ik ontzettend blij mee! Het was een lange tocht en ik ben heel erg dankbaar dat je ondanks dat het soms niet meeviel met alle tabellen, je toch altijd met enthousiasme en goede tips voor me klaarstond. Wat is het mooi geworden hè?

Een proefschrift schrijf je niet alleen met collega’s maar ook zeker met vrienden en familie. Lieve allemaal, het lijkt me voor een buitenstaander soms heel lastig te begrijpen waar het nou eigenlijk allemaal om gaat. Ik kan me zo goed voorstellen dat jullie vaak hebben gedacht: “zo’n artikel schrijven hoeft toch niet zooooo lang te duren”?

radiostilte van de afgelopen maanden hoop ik vanaf 2013 weer overal bij te zijn!! Zie Jawel dus ( - ; Veel dank voor de benodigde afleiding en gezelligheid. Ondanks de hier het resultaat voor jullie liggen waar het al die jaren over is gegaan.

In het bijzonder wil ik mijn dierbaarste vriendinnetjes Elke, Grace, Roos Boer en Sacha bedanken. Binnenkort eindelijk weer meer tijd om leuke dingetjes te gaan doen. Mooie dineetjes, leuke uitjes en lekker chillen. Met jullie kan ik dat als geen ander! Bedankt ook altijd voor de interesse in de voortgang van mijn proefschrift en andere perikelen. El, kunnen we eindelijk op het nieuwe jaar (in jullie nieuwe huisje natuurlijk) proosten zonder het goede voornemen om de promotie af te ronden ( - ; EINDELIJK!!

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Ch 10 diversen.indd 254 23-10-12 19:48 Dankwoord Lieve Heleentje, jou wil ik eventjes apart bedanken. Er was naast Kimmie niemand die het zoooooo goed begreep als jij. Jij stond bijna alweer 2 jaar geleden waar ik nu sta en ik was zoooo supertrots op je. Als ik het zo kan afronden als jij, dan mag ik wel onwijs trots zijn. Je was er altijd om mijn gezeur aan te horen, maar we hebben ook ontzettend veel gelachen. Ik kan met jou echt over alles praten, maar dan ook ECHT alles…. Zullen we nog heel veel jaartjes zulke goede vriendinnetjes blijven? Nu eerst maar eens op naar Jamaica! Zum wohl!

Lieve pipje en mama, aan jullie misschien wel mijn allergrootste woord van dank. Bedankt voor jullie geweldige motivatie in alle stappen die ik heb gemaakt en jullie geloof in mijn kunnen. Dankzij jullie onvoorwaardelijke liefde en vertrouwen ben ik in staat geweest om te komen waar in nu ben. Jullie hebben het mogelijk gemaakt dit proefschrift af te ronden. Dank voor alle super mooie, lieve en gezellige dingen die we hebben gedaan wanneer jullie me weer eens meenamen om te relaxen, jullie relativerende woorden, humor, geduld, wijze raad en bovenal jullie gezelligheid. Het is altijd heerlijk om bij jullie beiden in de tuin te zitten en even weer helemaal bij te komen.

Lieve Kimmie, jij dan als allerlaatste…. Ik weet zeker dat jij de allerbelangrijkste persoon voor me bent geweest tijdens mijn promotie en dat je dat verder ook altijd zult zijn. Bedankt lief zusje dat je er altijd voor me was, bent en zult zijn. Ik kan altijd bij je terecht voor een eerlijk advies en wijze raad, maar ook voor een knuffel en het echte ontspannen. Wij hebben de gehele promotieperiode samen doorgemaakt en ik kan niet wachten tot ook jij je promotie hebt afgerond en dat we ein-de-lijk lekker kunnen gaan reizen, wandelen, feesten en genieten. Je bent de beste tweelingzus ever!! Lapland, here we come!!!!!!!!!!!

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Ch 10 diversen.indd 257 23-10-12 19:48 Ch 10 diversen.indd 258 23-10-12 19:48 Curriculum Vitae Curriculum Vitae

Dominique Baas werd geboren op 23 september 1975 te Naarden. Na het behalen van haar VWO- diploma aan het Gooisch Dag en Avond College te Hilversum, begon ze in 1996 aan de studie Biologische Gezondheidswetenschappen aan de Universiteit van Maastricht. Haar afstudeerstage heeft ze verricht in het Academisch Ziekenhuis Maastricht bij de afdeling Medische Microbiologie. Onder begeleiding van Prof. dr. C.A. Bruggeman onderzocht zij de rol van het C-reactief eiwit in combinatie met Chlamydia pneumoniae infectie in atherosclerose. Ze behaalde haar doctoraal in 2002 en begon met werken bij het Academisch Medisch Centrum in Amsterdam bij de afdeling Inwendige Geneeskunde. Bij de onderafdeling Infectieziekten, Tropische geneeskunde en AIDS werkte zij onder begeleiding van Prof. dr. J.T. van der Meer aan het opzetten van een richtlijn voor de behandeling van een Staphylococcus aureus bacteriëmie. Zij vervolgde haar werkzaamheden bij de Inwendige Geneeskunde bij de onderafdeling Vasculaire Geneeskunde onder begeleiding van Prof. dr. J.P. Kastelein, waar ze gedurende 1 jaar drieledig onderzoek deed naar pseudoxanthoma elasticum (PXE). Zij werkte hiervoor samen met het Nederlands Instituut voor Neurowetenschappen (NIN) in Amsterdam. Van december 2004 tot oktober 2009 heeft zij hier als promovenda gewerkt bij de afdeling Klinische en Moleculaire Ophthalmogenetica onder begeleiding van Prof. dr. A.A.B. Bergen en Dr. T.G.M.F. Gorgels. Haar promotieonderzoek richtte zich op genetische studies voor leeftijdsgebonden macula degeneratie. De resultaten hiervan zijn beschreven in dit proefschrift. Sinds oktober 2009 is ze werkzaam als wetenschappelijk onderzoeker bij het Rijksinstituut voor Volksgezondheid en Milieu (RIVM) in Bilthoven bij de afdeling Laboratorium voor Infectieziekten Screening (LIS) onder begeleiding van Prof. dr. M. Koopmans.

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Ch 10 diversen.indd 261 23-10-12 19:48 Color Figures Chapter 1: General Introduction

Figure 1. Normal Macular Anatomy (left) and Normal Macular Fundus Appearance (right). Reprinted with permission from The Macular Degeneration Research, a program of the American Health Assistance Foundation © 2012. http://www.ahaf.org/macular.

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Stage De�inition 0 - No signs of AMD - Hard drusen (<63 µm)only 1 - - Soft distinct drusen (≤63 µm) only 2 Pigmentary abnormalities only, no soft drusen (≤63 µm) - - Soft indistinct drusen (≤125 µm) or reticular drusen only 3 Soft distinct drusen (≤63 µm) with pigmentary abnormalities with pigmentary abnormalities Soft indistinct drusen (≤125 µm) or reticular drusen 4 Geographic atrophy or choroidal neovacularization

Figure 2. Fundus Photographs Illustrating The Signs of AMD Graded According To The International Classi�ication and Grading System for AMD. (A). Stage 0; (B). Stage 1; (C). Stage 2; (D). Stage 3; (E). Stage 4 (geographic atrophy); (F). Stage 4 (choroidal

permission from Redmer van Leeuwen et al. [17]. neovascularization). Definitions of the stages are given in the text. Fundus photographs reprinted with

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Phot

COL4A3 FNDC5 TIMP3 RPE [COL 4A1, 4A2, 4A3, 4A4, 4A5, 5] LAMHSPG bm RPE [COL1, 3] FN inner collagen elastin layer BM [COL 4, 6, 18] ELNTIMP3* [COL1, 3] FN outer collagen [COL 4A1, 4A2, 5] LAM HSPG bm choroid COL 1A1, 1A2, 3A1, 4A1, LAM A2, FN1, 4A2, 5A1, 5A2, 6A1, 6A2, A4, B1, FNDC3A 18A1 C1, C3 Chor

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biomolecules RPE RPE

BMP ICL xx

OCL

BMC

biomolecules Choroid

Figure 2. Schematic Drawing Showing the Normal Structure and Functions of Bruch’s Membrane. Transmission Electron Microscopic image of BM courtesy of Prof. J. Marshall. BM allows for the transport of biomolecules across the membrane, it attaches RPE cells to the membrane and it acts as a physical barrier to prevent the migration of RPE cells and choroidal cells across the membrane.

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7 (6%) shared proteins: Amyloid (beta, P), APOE, C3, CLU, FGG, VTN

Alzheimer plaque AMD drusen Atherosclerotic plaque

a j

d f k e i b l c g h

24 (20%) shared proteins: 10 (8%) shared proteins: Alpha B-Crystallin, Amyloid (beta, Amyloid (beta, P), APCS, P), APOE, ATP5A1, complement Apolipoprotein (A1, E), comple- (C3, C7), CLU, FGG, HP, IGHG1, ment C3, CLU, FGG, PLG, VTN IGKC, OGN, PRDX1, Serpin (A1, A3) TIMP3, Tubulin (A1A, A1C, A3C, B2C, B3), UBB, VTN

Figure 5. Overlap in Protein Content of Alzheimer Plaques, AMD Drusen, and Atherosclerotic Plaques. Alzheimer plaque picture courtesy of Dr. I. Huitinga, Netherlands Brain Bank, donor number nhb:2006- 060,VU:S06/189. Atheroslcerotic plaque picture courtesy of Dr. P Sampaio Gutierrez. We analyzed 121 proteins based on the article by Crabb et al. [6] and additional literature searches. We obtained NM numbers for 85 of the proteins characterized by Crabb, and an additional 36 proteins were added based on the recent literature [167]. Note that drusen share 20% of their protein content with AD plaques and less than 10% with atherosclerotic plaques. (a). Amyloid deposits, (b). Cell nucleus, (c). Enthorinal cortex, (d). Retinal Pigment Epithelium, (e). Bruch’s Membrane, (f). Druse, (g). Choroid, (h). Choroidal bloodvessel, (i). Intima with atheroslerotic plaque, (j). Tunica media, (k). Fibrous cap, (l). Lipid + necrotic core.

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3 genes

Phot

13 genes RPE

113 proteins

41 genes

Chor

36 proteins

Figure 6. Schematic Drawing of Proteins in Drusen and the Corresponding Genes Expressed in Adjacent Cell Layers. We could annotate 113 genes with genbank codes (using Ingenuity) which correspond to drusen proteins et al.[6]. Thirty six of these genes had higher expression levels in choroid compared to RPE (chor>RPE), thirteen genes had higher expression levels in RPE than in choroid (RPE>chor) and only three genes identified by Crabb had higher expression levels in photoreceptors than RPE (phot>RPE). Gene expression levels were determined by RNA microarray study comparing gene expression levels from two adjacent tissue types from the same donor. Experiments were performed in triplicate (on three different healthy older human donor eyes) [24]. Details of the 113 genes can be found in Table 1.

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Figure 2. Retinal Photographs of Three Patients. (A). and (B). Retinal Photographs of Patient 1 (p.Q124P). Small round (cuticular) drusen are symmetrically present. (C). Optical coherence tomogram of patient 1 demonstrates nodular thickening of the retinal pigment epithelium (*). (D). and (E). Fluorescein angiograms of patient 2 (p.G267S) demonstrate choroidal neovascularization in one eye only. (F). and (G). Fluorescein angiogram photographs of patient 3 (p.G267S) demonstrate cuticular drusen with central areas of focal retinal pigment epithelial detachment (*).

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Figure 1. The complement pathway. The components of the complement system that have been genetically associated with increased or decreased risk of drusen, geographic atrophy (GA), and choroidal neovascularization (CNV), are circled. Reprinted with permission from Donoso et al. [65].

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