testing are mandatory and in consequence reduce the identifiable susceptibility variants to the more common polymorphisms with stronger effects on the phenotype [14].

analyze reasonably large numbers of patients and controls, and should ultimately allow clarification of the role of ABCA4 in AMD predisposition.

2.3Early Findings

Initial candidate gene studies have suggested two possible AMD susceptibility genes. Both genes, the ATPbinding cassette, sub-family A (ABC1), member 4 (ABCA4), and the apolipoprotein E (APOE) were shown to have only marginal impact on the overall disease load; however, they serve as excellent examples for the difficulties arising when analyzing genetic risk factors in complex diseases.

2.3.1The ABCA4 Gene

In 1997, Allikmets and colleagues identified mutations in the ABCA4 gene as a cause for autosomal recessive Stargardt disease, a common form of an early onset hereditary retinal dystrophy [15]. As Stargardt disease shares numerous phenotypic features with AMD, Allikmets et al. subsequently analyzed ABCA4 in AMD patients [5]. They resequenced the 50 exonic regions of the gene in 167 AMD patients to detect probable functional AMD variants, which were then surveyed in a sample of 98 Stargardt patients and 220 healthy controls. Altogether, ten variants were found to be exclusively present in AMD patients [5]. Most of these were rare, and occurred only once or twice in the analyzed patient sample. Only two alterations, namely D2177N and G1961E, were more frequent and were confirmed to be associated with AMD in an extended multicenter study [16]. Subsequent studies reported conflicting results, and demonstrated these variants to be equally frequent in AMD patients and healthy control subjects [17]. Also, segregation of the putative risk alleles with disease was not consistent in several AMD families analyzed [17]. Nevertheless, in these studies the occurrence of non-synonymous coding variants was noticeably enriched in AMD patients in accordance with the concept of the CDRV hypothesis. Unfortunately, most, if not all, studies investigated relatively small sample sizes with insufficient statistical power [18, 19]. Current developments in highthroughput resequencing technology are suited to

2.3.2The APOE Gene

Common variants in the APOE gene are well established to be strongly associated with Alzheimer’s disease (AD), a disorder characterized by age-dependent deposits in the central nervous system [20]. The ageassociation of symptoms as well as the functional role of APOE in lipid transport and metabolism, particularly in the central nervous system, make APOE an excellent candidate for an AMD susceptibility gene.

Three APOE variants (epsilon alleles e2, e3, and e4) are characterized by two non-synonymous coding SNPs (nscSNPs) of the APOE gene. The corresponding protein isoforms (E2, E3, and E4) have been extensively analyzed because of their role in AD and have been shown to reveal explicit functional differences [21]. In 1998, two independent AMD case controlstudies identified the e4 haplotype with significantly lower frequencies in cases than in controls, thus associated the carriers of this haplotype with a reduced risk of developing AMD (Table 2.1) [6, 7]. Several replication studies confirmed the protective nature of the e4 haplotype and further indicated the e2 haplotype to increase AMD risk (e.g., [22–24]). However, these findings were not consistent across published data, again probably reflecting insufficient statistical power. Interestingly, the effects of the e4 haplotype are directly reversed in AMD and AD as e4 carriers are at decreased risk for AMD but at increased risk for AD [20]. The mechanisms underlying these opposing findings are unknown.

2.4CFH: The First Major AMD Susceptibility Locus

In 2005, a GWAS was published concurrent with a functional and two positional candidate gene studies, all of which identified the CFH gene on chromosome 1q31 as the first major AMD susceptibility gene [8–11]. Remarkably, the study by Klein et al. [11] was the first ever GWAS to successfully identify a risk variant for a complex disease by a genome-wide and undirected search, and heralded a new era of genetic analyses in complex diseases. The observed risk effects and

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frequencies of variants within the CFH locus exceeded the expectations of most experts in the field.

The most prominent causative effect of the CFH locus is attributed to a single haplotype, which next to other variants is tagged (i.e., uniquely identifiable) by the nscSNP rs1061170 (CFH:Y402H). This SNP has a minor allele frequency (MAF) of 31–40% in Caucasian populations and is markedly enriched in AMD patients with a frequency of 54–63% (Table 2.1). Heterozygous individuals of the risk variant reveal a range of odds ratios between 2.0 and 3.0, homozygous individuals odds ratios between 3.3 and 11.6. Besides this risk haplotype, two common protective haplotypes were also observed and most likely represent independent risk modifiers [25]. One of these is tagged by the nscSNP rs800292 (CFH:I62V). This SNP has a lower MAF in the Caucasian population than rs1061170 and reveals a comparable but reversed risk effect (Table 2.1). The second protective haplotype was found to be perfectly correlated with a large copy number polymorphism (CNP) telomeric to CFH [25]. This CNP was observed with a frequency of 14–24% in controls and 8–11% in AMD patients. Its minor allele represents a common 85-kb deletion that encompasses two CFH- related genes, CFHR3 and CFHR1 [25, 26]. Another interesting variant at this locus is the synonymous coding SNP rs2274700 (CFH:A473A) whose minor allele almost exclusively occurs on the two common protective haplotypes consequently conferring a protective effect. The observed MAF of rs2274700 in the general population is 40–45%, which is higher than that of rs1061170, rendering rs2274700 one of the most important risk determinants of the CFH gene locus. Noteworthily, all CFH risk variants account for an altered risk of both forms of late-stage AMD, geographic atrophy (GA) and choroidal neovascularization (CNV). A comparison of Caucasian and Asian populations revealed distinct differences in the MAFs of rs1061170, rs800292, and rs2274700. Especially rs1061170 was observed with markedly lower frequencies in Asians and hence should only play a minor role in this ethnic group (Table 2.1).

2.4.1Functional Implications

The discovery of AMD-associated CFH variants was a major breakthrough in AMD genetics. Subsequent studies of the functional consequences of the CFH risk

variants provided the first direct insight into the molecular mechanisms leading to AMD.

The CFH gene encodes two functional proteins, namely CFH and the isoform FHL-1, both of which feature the risk variant CFH:Y402H at codon 402 in their amino acid sequence, and both of which have been shown to act as negative regulators of complement activation [27, 28]. In individuals homozygous for the CFH risk variant His402, CFH serum levels were found to be unaltered when compared to controls; however, several complement activators were elevated in the choroid and in blood plasma [29, 30]. In further studies, the CFH and FHL-1 risk variant exhibited a decreased binding affinity to C-reactive protein (CRP), heparin, as well as bacterial and RPE cell surfaces [31–34]. As a consequence, the risk variant at codon 402 obviously alters the capacity of CFH and FHL-1 to regulate the complement system.

The impact of the two protective CFH haplotypes on the complement system is still unclear although the CFH:I62V exchange is likely to have functional consequences as it occurs within a binding site for C3b [9], an activator of the complement system. Similarly, the precise mechanism of protection in the case of a homozygous deletion of CFHR1 and CFHR3 is not understood. Both encoded proteins, CFHR1 and CFHR3, are likely involved in complement regulation similar to CFH, based on their ability to bind specific complement components and bacterial surfaces [35]. While the biological function of CFHR-3 still needs to be resolved, CFHR-1 was recently shown to inhibit the late steps of the complement cascade [36]. At first sight, it appears counterintuitive that a loss of CFHR1 and CFHR3 should be beneficial with an associated decreased AMD risk. Possibly, CFHR1 which is known to compete with the complement inhibitor CFH for binding sites at C3b, heparin, and cell surfaces, may simply reduce CFH activity [36], which in turn might explain the protective nature of CFHR1 and CFHR3 loss.

2.4.2Further AMD-Associated Genes of the Complement Cascade

Focusing on other members of the complement cascade, nscSNPs within the complement factor B gene (CFB) were found to also influence AMD risk. Similarly, associated variants were identified in the complement component 2 gene (C2), which is located

in direct chromosomal proximity to CFB (Table 2.1) [37–41]. CFB and C2 are activators of the complement system by regulating C3 production, and both are expressed in the retina, the RPE, and the choroid. Two nscSNPs in CFB, rs4151667 (L9H) and rs641153 (R32Q), appear to have functional relevance [37]. On the one hand, the Gln32 protein isoform was shown to have reduced hemolytic activity compared to the more frequent Arg32 protein isoform. This was ascribed to a lower binding affinity of Gln32 to C3b [42]. On the other hand, the L9H variant affects the signal peptide of the protein, and although functional studies are not yet available, this likely affects CFB secretion. At the C2 locus, AMD-associated SNPs rs9332739 (E318D) and rs547154 (intronic) are highly correlated with the SNPs rs4151667 and rs641153 at the CFB locus and consequently have been associated with protective effects similar to CFB (Table 2.1). An independent role of the C2 SNPs in AMD risk alteration has not been shown so far. In fact, such a role may not exist, in particular since the present functional data indicate that an abnormal CFB variant alone may significantly influence the activity of the alternative complement pathway.

The various pathways of the complement system all converge in complement component 3 (C3), which regulates the formation of the membrane attack complex and ultimately cell lysis. A candidate study focusing on the C3 gene identified two highly correlated nscSNPs, rs2230199 (R102G) and rs1047286 (P314L), to be associated with an increased risk for AMD [41, 43, 44] (Table 2.1). Already over 30 years ago, a functional consequence of these variants was established by demonstrating different binding capacities of the C3 isoforms to complement receptors [45]. Functionally, it was suggested that the C3 isoforms differ in their tertiary structure likely affecting the accessory binding sites [46].

Based on the findings in CFH, CFB and C3, a new and most promising focus in AMD research has emerged addressing the pathological pathway of the alternative complement cascade in AMD etiology. Therefore, a major theme of current research follows the candidate gene approach by testing the individual players of the complement cascade for an association with AMD. The success of this approach was shown in the recent work by Fagerness et al. [47] who identified AMD-associated SNPs near the complement factor I (CFI) gene. It will further help to elucidate the key

mechanisms and effects of complement activation in AMD pathogenesis.

2.5ARMS2/HTRA1: The Second Major AMD Susceptibility Locus

Refinement of a prominent linkage signal on chromosome 10q26 initially identified a region spanning two genes, namely PLEKHA1 (pleckstrin homology domain containing family A member 1) and ARMS2 (age-related maculopathy susceptibility 2, alias hypothetical LOC387715) [48, 49]. In particular, a nonsynonymous coding variant rs10490924 (A69S) in the ARMS2 gene sufficiently explained the association signal at 10q26 [49]. The MAF was 19–24% in controls and about 36–52% in AMD patients, resulting in a range of odds ratios between 2.1 and 3.2 for heterozygous and between 5.7 and 10.3 for homozygous individuals. Compared to the CFH risk variant rs1061170, rs10490924 revealed a slightly higher effect size with a slightly lower frequency, leading to a similar contribution for disease load. Consequently, those findings identified the 10q26 region as a second major AMD susceptibility locus [49] (Table 2.1).

The two major risk variants, rs1061170 at 1q31 and rs10490924 at 10q26, contribute independently and additively to AMD risk. Similar to CFH risk variant rs1061170, association of rs10490924 with AMD was observed in GA as well as CNV. Further association studies revealed additional correlated and strongly associated variants that were also found in the first exon of the downstream HTRA1 (HtrA serine peptidase 1) gene [39, 50, 51]. A comprehensive fine-mapping association study finally defined an approximately 23-kb candidate region encompassing the complete gene locus of ARMS2 and the 5’ region of HTRA1 including its regulatory sequences, the first exon of the gene, and part of intron 1. Resequencing of the risk haplotype defined a total of 15 highly correlated variants, each being similarly associated with AMD [52]. It should be noted, however, that a delineation of the 15 variants by statistical means, i.e., the definition of the most likely causal variant, would likely require the analysis of thousands or even ten thousands of AMD patients and controls, and thus would be highly impracticable. This leaves the option of functional analyses of