Albert & Jakobiec's Principles & Practice of Ophthalmology, 3rd Edition

CHAPTER 143 - Genetics of Age-related Macular Degeneration

Margaret M. DeAngelis,
Fei Ji

INTRODUCTION

Age-related macular degeneration (AMD) is the most common form of legal blindness in the United States as well as other developed countries. The Eye Disease Prevalence Group estimates that 1.75 million United States citizens have advanced AMD in at least one eye.^[1] Approximately 10% of the population aged 43 and older is affected with some form of the disease and 30% of the population aged 75 and older is affected.^[2] As the population lives longer this number will likely increase. It is estimated that 2.95 million individuals in the US will have advanced AMD by the year 2020.^[1]

Current diagnostic methods focus on the detection of neovascular AMD, since available treatments are directed against this advanced stage of the disease. Although the newest treatments offer some chance of visual improvement, they require invasive delivery methods and have limited ability to prevent or reverse vision loss. Assessments of an individual's risk of developing advanced AMD are based on ocular findings in those who already have the early stages.^[3] Methods are yet to be refined that determine which individuals are at highest risk of vision loss due to AMD prior to the development of any signs of the disease.

Previous studies of genetic and epidemiologic factors have not been in agreement as to predictors of AMD. Cigarette smoking appears to be the only epidemiologic risk factor generally accepted as being associated with an increased risk for AMD,^[4] while an allelic variant in the complement factor H gene (CFH) appears to be the strongest genetic risk factor. Specifically, several independent reports have shown the functional polymorphism Y402H in CFH, where a tyrosine is substituted by a histidine, to be associated with increased risk of both early^[5-9] and late stages of AMD (both neovascular and geographic atrophy).^[6,7,9-12] These findings further suggest that the histidine allele, or disease allele, contributes to almost half of all cases of AMD in the population. Although having these factors may increase one's risk of disease, there are still many individuals who both smoke and have the CFH-associated disease variant but have no signs of AMD. For this reason, the magnitude of risk for AMD probably depends on exposure to other environmental and genetic risk factors in combination with smoking and CFH variation.

Discovering precisely which genes and environmental factors contribute to the pathophysiology of AMD could provide targets that could be modifiable through therapeutic or behavioral intervention, thereby reducing or preventing the incidence of this disease. Knowing which common variants in a handful of genes may predict which individuals in the population are at greatest risk for converting to the more advanced stages of AMD. These genes in turn could serve as biomarkers and hence pharmacological targets in preventing or slowing down progression of disease. This chapter focuses on genetic contributions to the etiology of AMD.

BACKGROUND STUDIES DEMONSTRATING A GENETIC COMPONENT: TWIN AND FAMILIAL AGGREGATION STUDIES

AMD is a challenging disease to study from a genetic perspective because unlike disorders that exhibit Mendelian inheritance patterns (where one gene is responsible for the phenotype observed in a given family), complex diseases like AMD have the following characteristics. Since the disease is common or highly prevalent (with 1.75 million United States citizens having advanced AMD in at least one eye),^[1] it is likely that there is more than one gene or environmental factor in addition to the interactions of these factors that influence an individual's susceptibility to disease. AMD is also a heterogeneous disease and its many subtypes (reviewed in other chapters in this book) present the possibility that there are different disease mechanisms and hence different genes responsible for each subtype.

Although the causes of AMD remain elusive, there is evidence demonstrating that genetics plays an important role. Table 143.1 summarizes many of the studies that have supported a role for genetics in the etiology of AMD. These studies consist of data from both familial aggregation and twin studies. Data from the Rotterdam study showed that first-degree relatives of affected individuals are at 25% greater risk of developing disease than individuals in the general population without any affected family members. It has also been shown that AMD has a tendency to cluster or aggregate within families.^[13] Further, Hammond and colleagues^[14] demonstrated that monozygotic twins with AMD had a higher degree of concordance (both members having AMD) than dizygotic twins, 37% compared to 19%, respectively.

TABLE 143.1 -- Studies Showing Hereditability

Type of Study	Conclusion	Authors
Monozygotic twin case report	Twins shared identical health problems and had similar features of AMD in corresponding eyes	Melrose et al.[89], Meyers et al.[90], Dosso et al.[91]
Twin study	Showed higher concordance in monozygotic twins than in dizygotic, confirmed that stage and type of AMD was similar between twins	Klein et al.[92], Meyers et al.[93] Gottfredsdottir et al.[94] Grizzard et al.[95] Seddon et al.[96] Hammond et al. 2002,[14]
Familial aggregation study	Prevalence of AMD was higher among first-degree relatives of AMD patients	Seddon et al.[13], Klaver et al.[52], Assink et al.[97]

Key Features

A genetic component is supported by the following: . A higher prevalence of AMD is found in identical twins compared to fraternal twins . A higher prevalence of AMD is found in first-degree relatives of AMD patients compared to unrelated individuals from the general population

JUVENILE FORMS (MENDELIAN) OF MACULAR DEGENERATION: CANDIDATE GENES FOR AMD

Because of evidence suggesting a genetic component, some genes known to be associated with juvenile-onset forms of retinal degeneration have been studied for their association with AMD. No studies to date have found an association between mutations in vitelliform macular dystrophy 2 (VMD2) (Best disease),^[15-19] tissue inhibitor of metalloproteinase-3 (TIMP-3) (Sorsby fundus dystrophy),^[20,21] retinal degeneration, slow RDS/peripherin (retinitis pigmentosa),^[22,23] and epidermal growth factor-containing fibulin like extracellular martrix protein 1 (EFEMP1) (Malattia Leventinese/Doyne honeycomb retinal dystrophy)^[24-26] and any type of AMD, either early or late stages. Table 143.2 shows the names of these genes, their genomic locations, the retinal disease associated with the gene and the AMD population studied. Table 143.2 also shows that two genes, the ATP-binding cassette transporter gene, subfamily A, member 4 (ABCA4) (autosomal recessive Stargardt disease) and the elongation of very long chain fatty acids gene (ELOVL4) (Stargardt disease type 3 or autosomal dominant Stargardt disease) are inconsistent with respect to whether or not variations in these genes are associated with AMD.

TABLE 143.2 -- Candidate Genes for AMD Derived from Juvenile Forms of Hereditary Retinal Degenerations

Gene Name	Symbol	Location	Function	Disease Association	Study Population	Author
Retinal Degeneration, Slow	RDS	6p21.2- p12.3	Protein binding	Retinitis pigmentosa	56 y.o. index patient w/AMD & his 41 y.o. niece, 42 y.o. index patient w/RP & her 15 y.o. son	Kemp et al.[22]
					3 unrelated families, 12 patients w/AMD or peripheral retinal degeneration, 100 related controls	Gorin et al.[23]
Tissue Inhibitor of metallopeptidase-3	TIMP3	22q12.3	Metalloendopeptidase inhibitor activity	Sorsby's fundus dystrophy	68 sibpairs w/AMD (by AREDs), mean age 71.5 ± 10.2 years, 79 age, gender and ethnically matched controls	De La Paz et al.[20]
					143 German AMD patients (age 48-91), 74 w/other macular dystrophies	Felbor et al.[21]
ATP-Binding Cassette, sub-family A (ABC1), member 4	ABCA4	1p22.1 - p21	ATP binding, ATPase activity, lipid transport & metabolism	Stargardt disease	167 unrelated patients w/drusen, GA, RPE detachment and/or CNV, 220 racially matched controls	Allikmets et al.[27][*]
					182 patients w/AMD (60% w/CNV in at least one eye), 96 unrelated subjects expected to develop AMD	Stone et al.[29]
					212 patients w/intermediate or large soft drusen, RPE detachments, GA and/or CNV (159 familial, 53 sporadic), 56 racially matched controls	De La Paz et al.[30]
					52 unrelated French patients w/CNV, 90 French unrelated self-reported unaffecteds as controls	Souied et al.[31]
					1218 unrelated patients w/dry AMD or CNV from North America and Western Europe, 1258 controls	Allikmets et al.[28][*]
					100 patients w/GA, 100 patients w/CNV, 153 unaffecteds age and geography-matched controls	Rivera et al.[32]
					544 AMD patients (mostly CNV) from US, Australia & Switzerland, 689 controls from the same areas, 62 unaffected Somalians	Guymer et al.[33]
					182 patients w/AMD (Int. ARM Epi. Study Class. System), 96 controls from the same clinic population as AMD patients	Webster et al.[34]
					26 AMD patients ? grade 2 (AREDS scale) from 15 families, 33 unaffected siblings	Bernstein et al.[35]
					165 AMD patients ? grade 3 (Int. ARM Study Class. System) from 70 families, 33 unaffected siblings, 59 unrelated controls	Schmidt et al[36]
Epidemial Growth Factor - containing fibulin-like extracellular matrix protein 1	EFEMP1	2p16	Maintenance of extracellular matrix integrity	Malattia Leventinese (ML)/Doyne Honeycomb Retinal Dystrophy (DHRD)	494 AMD patients, 56 ML/DHRD patients, 477 controls	Stone et al.[24]
					13 index patients w/onset drusen, 15 family members, 54 familial cases of AMD, 150 age and ethnicity-matched controls, all at least 50 y.o.	Guymer et al.[25]
					54 patients w/early onset drusen, 114 unrelated controls ? 60 y.o.	Narendran et al.[26]
Vitelliform Macular Dystrophy 2 (Best disease, bestrophin)	VMD2	11q13	Chloride ion binding & ion channel activity	Best Disease	259 unrelated patients w/drusen, GA, RPE detatchment and/or CNV, 30 patients w/other disorders (1 w/Best)	Allikmets et al.[15]
					41 unrelated patients w/Best's Disease, 200 AMD patients, 140 unaffected controls	Kramer et al.[16]
					321 patients w/drusen and/or atrophy of RPE, 96 unrelated patients w/Best disease, 192 controls at least 40 y.o.	Lotery et al.[17]
					85 Japanese AMD patients, 105 age-matched Japanese controls	Akimoto et al.[18]
					259 AMD patients, 28 patients w/maculopathies other than Best's	Seddon et al.[19]
Elongation of very long chain fatty acids	ELOVL4	6q14	Lipid metabolism & transport	Stargardt Disease, type III	778 AMD patients (Int. Class. System), 551 age and race-matched controls	Ayyagari et al.[39]
					992 Caucasian patients w/extensive drusen, pigmentary changes, GA and/or CNV, 120 unrelated controls	Conley et al.[10][*]
					335 Finnish patients w/more than 5 small drusen (< 63 ?m), at least 10 large drusen (> 125 ?m), GA, or CNV, of which 154 were sporadic cases & 181 familial, 105 controls	Seitsonen et al.[40]

RP - Retinis Pigmentosa; y.o. - years old; AREDS - age-related eye disease study; GA - geographic atrophy; RPE - retinal pigment epithelial; CNV - choroidal neovascularization; Int. ARM Epi. Study Class. System - International Age-Related Maculopathy Epidemiology Study Classification System; Int. Class. System - International Classification System; ML - Malattia Leventinese; DHRD - Doyne Honeycomb Retinal Dystrophy.

*	Denotes studies finding positive associations.

ABCA4

ABCA4 is located on the short arm of chromosome 1 (1p22.1-p21) and functions in lipid metabolism and transport. Mutations in ABCA4, also known as ABCR, cause Stargardt disease, a juvenile form of macular degeneration characterized by deposits of lipofuscin in and beneath the retinal pigment epithelium. In 1997, Allikmets et al found an association of heterozygous ABCA4 alleles with AMD.^[27] In particular, G1961E (glycine is substituted by glutamic acid) and D2177N (aspartic acid is substituted by asparagine), the two most common variants, along with rare variants in ABCA4 were reported to be associated with ?4% of AMD cases (mostly the dry form). Further, in a larger screen of more than 1200 patients with both dry and neovascular AMD, Allikmets et al in 2000 reported that ABCA4 variants could probably explain up to 8% of AMD.^[28] However, several groups have tried to replicate these findings and were unable to conclude that variants in ABCA4 were associated with the dry or more advanced stages of AMD.^[29-36] It could be that if variation in ABCA4 increases susceptibility to AMD, the risk of disease would only be small or modest, or there exists multiple susceptibility genes for AMD, not necessarily expressed in every patient with AMD.

ELOVL4

Like ABCA4, the protein product of ELOVL4 is involved in lipid transport and metabolism. Unlike ABCA4, ELOVL4 is located in a region (6q24-q14) previously identified from genome-wide scans to harbor AMD susceptibility genes.^[37,38] A five base pair deletion in this gene has been found to be associated with Stargardt disease 3, which has phenotypic similarity to the dry or atrophic form of AMD. A recent report has suggested that the M299V variant, where a methionine is substituted by a valine in the protein sequence of ELOVL4, could increase risk of neovascular AMD,^[10] although an earlier study did not find this association for either the early or more advanced stages of AMD.^[39] Similarly, a recently published study of a Finnish population also did not find an association between the M299V variant in ELOVL4 and patients with large drusen, neovascular, or atrophic AMD.^[40] It appears that more studies may need to be conducted before the contribution of the ELOVL4 gene to the etiology of AMD can be confirmed.

Key Features

Of the six genes responsible for juvenile forms of retinal degeneration (VMD2, TIMP-3, RDS/peripherin, EFEMP1, ABCA4, ELOVL4), only ABCA4 and ELOVL4 possibly influence susceptibility to AMD. Both ABCA4 and ELOV4L4 function in lipid metabolism and transport

GENOME-WIDE SCANS

A genetic component is further supported by the multiple genome-wide scans which have identified similar chromosomal locations harboring AMD susceptibility genes. The purpose of a genome-wide scan is to search the 22 pairs of autosomes (no sex chromosomes) with the goal of identifying regions significantly linked to the disease under investigation. Although linkage studies are powerful and can uncover novel disease genes, the significant or linked region could harbor hundreds of these disease loci or genes making the identification of one locus a challenge. Another disadvantage of a linkage study is that regions that contain genes contributing modest or subtle effects to a given phenotype may be missed.

Linkage refers to the physical proximity of the disease locus with the marker locus. The degree of linkage is determined by the logarithm of the odds ratio (lod score). A lod score of 3 translates to 1000 to 1 odds in favor of linkage (P = 0.0001) and a lod score of -3 is 1000 to 1 against the genotype being linked to the disease.

For complex diseases such as AMD generally no assumption regarding the mode of inheritance is made a priority - this is called model-free linkage analysis. Model-free linkage analysis employs statistical tests that calculate the number of alleles shared between related individuals with and without disease. Markers called microsatellites or single nucleotide polymorphisms (SNPs) can be used for this purpose. A microsatellite is a tandemly repetitive unit of base pairs in DNA. The repetitive units usually consist of 2, 3, or 4 base pairs and it is the number of these repetitive units that varies between individuals. Because of the high variation or heterozygosity in the number of these repetitive units for a given microsatellite between individuals in a population, they are very informative for linkage studies as they are highly polymorphic. The disadvantage is that microsatellites are not evenly distributed throughout the genome. An SNP is a single base change, located every 1000-3000 base pairs in DNA sequence and therefore they are abundant in the genome. Over the last few years the discovery rate of SNPs throughout the genome has accelerated, thus making them useful for genome-wide scans; however, they are not as polymorphic and many more are needed for linkage analysis. Almost all of the genome-wide scans conducted to date are linkage studies using microsatellite markers, except Klein's scan which is an association study employing SNPs in the identification of CFH (Fig. 143.1 and Table 143.3).

FIGURE 143.1 The number of genome wide scans for AMD performed each year since the first, Klein et al, in 1998. Note that none were completed in 1999 nor have there been any thus far in 2006.

TABLE 143.3 -- Summary of genes uncoved by genome wide scans and candidate gene studies for AMD

Author	Region	Band	LOD	P value	AMD population	Grading scale
Klein et al.[41]	D1S466 - D1S413	1q31	3.00[?]	0.0001	21 individuals from a family W/ARMD	Wis
Weeks et al.[42]	D1S1660	1q31	2.46	0.0004	630 individuals w/either GA and/or CNV from 364 families	modified
Majewski et al.[44]	D1S518	1q31	2.07	0.001[§]	70 families w/258 individuals w/advanced AMD, stratified according to age and dry vs. wet	None
Seddon et al.[45]	D1S1589	1q24	1.33	0.0070	490 affected individuals ? grade 3, 608 relative pairs	AREDS
Abecasis et al.[47]	D1S549	1q41	1.55	0.0040	113 families, 331 individuals w/any type of AMD	Intnl ARM Epid
Iyengar et al.	D1S202	1q31	1.02[?]	0.0149	297 individuals w/CNV or GA from 34 families, 349 sib pairs, corrected for age	Wis
Weeks et al.[43]	D1S1660	1q31	2.72	0.0610	1274 individuals w/large drusen, either GA and/or CNV from 530 families	modified
Klein et al.[6]	rs380390	1q31	>5.5[?]	0.00000004	96 individuals w/GA and/or CNV, 50 controls, all from AREDS	AREDS
	rs1329428		4.77[?]	0.00000140
Fisher 2005	D1S202 - D1S425	1q31.1 - q32	0.90[?]	0.0209	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Fisher 2005	D1S2705 - D1S202	1q23.3 - q31.1	1.16[?]	0.0104	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Summary region		1q23.3 - q31.1
Seddon et al.[45]	D2S1391	2q33	1.81	0.0020	490 affected individuals ? grade 3, 608 relative pairs	AREDS
Abecasis et al.[47]	D2S1780	2p25.3	1.84	0.0020	113 families, 331 individuals w/any type of AMD (69% w/GA and/or CNV)-only CNV patients	Intnl ARM Epid
Iyengar et al. 2004	D2S1356	2p21	1.52[?]	0.0041	297 individuals w/CNV or GA from 34 families, 349 sib pairs, corrected for age	Wis
Fisher 2005	D2S297 - D2S2312	2p25.1 - p23.2	0.76[?]	0.0308	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Fisher 2005	D2S2312 - D2S2251	2p23.2 - p16.2	0.84[?]	0.0244	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Fisher 2005	D2S2251 - D2S139	2p16.2 - p12	0.59[?]	0.0495	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Summary region		2p25.3 - p12
Majewski et al.[44]	D3S1304	3p13	2.19	0.0007	70 families w/258 individuals w/advanced AMD, samples limited to individuals w/predominantly dry AMD	None
Schick et al.[37] ^	D3S1763	3q26.1	1.25[?]	0.0081	Beaver Dam Eye Study (105 families w/258 individuals having scores ? 4.5 ? No late AMD), adjusted for age	Wis
Summary region		6q14
Weeks et al.[48]	D9S925 - D9S301[*] - D9S1120	9q21-q22	1.87	0.0017	212 individuals w/either GA and/or CNV from 225 families	modified
Weeks et al.[42]	D9S1118	9p13	1.79	0.0020	630 individuals w/either GA and/or CNV from 364 families	modified
Majewski et al.[44]	D9S934	9q33	2.07	0.0010	70 families w/258 individuals w/advanced AMD, stratified according to age and dry vs. wet	None
Abecasis et al.[47]	D9S938	9q31.1	1.78	0.0020	113 families, 331 individuals w/any type of AMD	Intnl ARM Epid
Iyengar et al. 2004	D9S1871	9p24	1.46[?]	0.0048	297 individuals w/advanced AMD from 34 families, 349 sib pairs, corrected for age	Wis
Iyengar et al. 2004	D9S938	9q31	1.26[?]	0.0079	297 individuals w/advanced AMD from 34 families, 349 sib pairs, corrected for age	Wis
Summary Region		9q31
Weeks et al.[48]	D10S1230	10q26	1.42	0.0053	212 individuals w/either GA and/or CNV from 225 families	modified
Weeks et al.[42]	D10S1230	10q26	2.00	0.0400	630 individuals w/either GA and/or CNV from 364 families	modified
Majewski et al.[44]	D10S1230	10q26	3.06	0.00009	70 families w/258 individuals w/advanced AMD, stratified according to age and dry vs. wet	None
Seddon et al.[45]	D10S1222	10q26	1.61	0.0030	490 affected individuals ? grade 3, 608 relative pairs	AREDS
Iyengar et al. 2004	D10S1248	10q26	1.55[?]	0.0037	297 individuals w/advanced AMD from 34 families, 349 sib pairs, corrected for age	Wis
Kenealy et al.[49]	D10S1230	10q26	1.52		133 affected sib pairs (graded 3, 4, or 5)	modified
Weeks et al.[43]	D10S1230	10q26	1.91	0.0600	1274 individuals w/either GA and/or CNV from 530 families	modified
Fisher 2005	D10S1690 - D10S1483	10q23.33 - q26.13	1.39[?]	0.0057	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Fisher 2005	D10S1483 - qter	10q24.31 - q26.13	2.63[?]	0.0003	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Summary region		10q26
Fisher 2005	D12S318 - D12S1349	12q23.2 - q24.31	0.76[?]	0.0305	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Schick et al.[37]	D12S1300	12q23- 12q24	1.35[?]	0.0063	Beaver Dam Eye Study (105 families w/258 individuals having scores ? 4.5 ? No late AMD)	Wis
Schick et al.[37]	D12S346	12q22- 12q23	1.53[?]	0.0040	Beaver Dam Eye Study (105 families w/258 individuals having scores ? 4.5 ? No late AMD)	Wis
Iyengar et al. 2004	D12S2078	12q23	1.88[?]	0.0016	297 individuals w/CNV or GA from 34 families, 349 sib pairs, corrected for age	Wis
Iyengar et al. 2004	D12S297	12q13	1.33[?]	0.0066	297 individuals w/CNV or GA from 34 families, 349 sib pairs, corrected for age	Wis
Summary region		12q23 - q24.31
Schmidt et al.[38]	D14S608, D14S599	14q13	3.20	0.0020	62 families: 2+ sampled relatives w/early or late AMD in 28 families w/lower-than-average IOP values	Wis
Jun et al.[51]	D14S1007	14q32.33	1.97[?]	0.0013	346 sib pairs from FARMS w/advanced AMD	Wis
Summary: No overlapping regions found
Schick et al.[37] ^	D15S659	15q11.1- 15q14	0.60[?]	0.0479	Beaver Dam Eye Study (105 families w/258 individuals having scores ? 4.5 ? No late AMD)	Wis
Schick et al.[37] ^	D15S816	15q25- 15q26	0.63[?]	0.0447	Beaver Dam Eye Study (105 families w/258 individuals having scores ? 4.5 ? No late AMD)	Wis
Iyengar et al. 2004	GATA50C03	15q14	3.94[?]	0.00001	297 individuals w/advanced AMD from 34 families, 349 sib pairs, corrected for age	Wis
Iyengar et al. 2004	D15S1012	15q13-15q15	5.34[?]	0.00000035	297 individuals w/advanced AMD from 34 families, 349 sib pairs, corrected for age	Wis
Summary region		15q13 - q15
Schick et al.[37] ^	D16S769	16p12.1	1.23[?]	0.0086	Beaver Dam Eye Study (105 families w/258 individuals having scores = 4.5 ? No late AMD)	Wis
Iyengar et al. 2004	D16S769	16p12.1	1.10[?]	0.0120	297 individuals w/advanced AMD from 34 families, 349 sib pairs, corrected for age	Wis
Schmidt et al.[38]	D16S403	16p12	2.90	0.0400	32 families (late AMD) w/higher than average BMI, SBP & IOP values	Wis
Fisher 2005	D16S3103 -? D16S415	16p13 - q12.2	0.92[?]	0.0195	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Fisher 2005	D16S415 - D16S516	16q12.2 - q23.1	0.94[?]	0.0187	Meta-analysis of Abecasis, Iyengar, Majewski, Schick, Seddon and Weeks	n/a
Jun et al.[51] [*]	D16S2616	16p13.13	1.91[?]	0.0015	346 sib pairs from FARMS w/advanced AMD	Wis
Summary region		16p12 - p12.1
Weeks et al.[42]	D17S928	17q25	3.53	0.0070	630 individuals w/either GA and/or CNV from 364 families 1274 individuals w/either GA and/or CNV from 530 families	modified
	D17S928	17q25	1.56	0.0037		modified
Summary region		17q25
Iyengar et al. 2004	GATA178F11	18p11	1.60[?]	0.0033	297 individuals w/advanced AMD from 34 families, 349 sib pairs, corrected for age	Wis
Jun et al.[*][51]	D19S245	19q13.31	2.35[?]	0.0005	225 sib pairs from BDES & 346 from FARMS w/advanced AMD	Wis
Iyengar et al. 2004	D20S451	20q13	1.42[?]	0.0052	297 individuals w/advanced AMD from 34 families, 349 sib pairs, corrected for age	Wis
Jun et al.[51] [*]	D21S2052	21q21.2	1.31[?]	0.007	346 sib pairs from FARMS w/advanced AMD	Wis
Seddon et al.[45]	D22S1045	22q12 - q13	2.00	0.0010	490 affected individuals ? grade 3, 608 relative pairs	AREDS
Abecasis et al.[47]	AGAT055Z	22q12.1	2.55	0.0003	113 families, 331 individuals w/any type of AMD (69% w/GA and/or CNV) - only CNV patients	Intnl ARM Epdiem
Summary region		22q12 - q13

Note: All maps Marshfield unless ARMD-Age Related Macular Degeneration, Wis-Wisconsin, GA-Geographic Atrophy, CNV-Choroidal Neovascularization, AMD-Age-related Macular Degeneration, AREDS-Age Related Eye Disease Study, Intnl ARM Epid-International Age Related Maculopathy Epidemiology, BDES-Beaver Dam Eye Study, FARMS-Family Age Related Maculopathy Study, IOP-Intaocular Pressure, BMI-Body Mass Index, SBP-Systolic Blood Pressure, n/a-non applicable.

*	= not indicated and ^ = Harvard Partners, Marshfield and Weizmann.

§	indicates P value estimated by LOD score reported

?	indicates LOD score estimated by P value reported.

Table 143.3 illustrates results from the AMD genome-wide scans to date including regions found to be significant, microsatellite markers used to identify the region, microsatellite markers/SNPs, chromosomal band, LOD score, P value, as well as the AMD population studied. A summary of chromosomal regions found by more than one genome-wide scan to be significantly linked to AMD is shown in Table 143.3 and includes 1q23.3-q31.1,^[6,41-46] 2p25.3-p12,^[46,47] 6q14,^[37,38] 9q31,^[46,47] 10q26,^{[42-46,48,49]} 12q23-q24.31,^[37,46] 15q13-q15,^[37,46] 16p12-p12.1,^[37,38,46] 17q25,^[42,43] and 22q12.1.^[45,47] A recent pooled analysis of several of these genome-wide scans that used linkage analysis and examined all types of AMD confirmed two of the most consistently reported chromosomal regions (1q23.3-q31.1 and 10q26) while identifying others that were not initially significant in the separate studies alone.^[50] This pooled analysis or meta-analysis also confirmed two regions that had been reported to be significant by only individual studies: 3p14.1-p25.3^[50,51] and 4q32.^[44,50] Many of the AMD genome-wide scan studies illustrated in Table 143.3 have resulted in the identification of various chromosomal regions which have not been replicated by other studies. Differences across studies in definition and measurement of AMD type and grade coupled with simultaneous analysis of different subtypes within study populations may make it difficult to compare and interpret findings. For example, sibling pairs with advanced AMD examined from the family age-related maculopathy study (FARMS) (Wisconsin grading scale) showed significance to 5q33.3, 14q32.33, 16p13.13, 19q13.31, 21q21.2,^[51] while other investigators who also studied populations characterized by advanced AMD did not report significant linkage to these same regions.^[42-44,46]Moreover, another group's finding of a significant association between neovascular AMD and 16p12.1 was dependent on factors such as body mass index (BMI) and hypertension,^[38] although the 16p12 region has been shown by other studies to be significantly associated with advanced AMD independent of these factors.^[46] In summary, genome-wide scans have helped to identify candidate genes for AMD as well as to highlight the need for further evaluation of candidate regions.

Key Features

Genome-wide scans can be an important tool in finding genes associated with disease. Genome-wide scans applying either linkage or association methods are an important approach in finding genes associated with disease. Because AMD is a multifactorial disease with both genetic and environmental factors influencing susceptibility, genes with modest or subtle effects may go undetected by linkage studies, yet may be uncovered by an association study. For this reason, it is important to have multiple approaches in elucidating the genetic etiology of a complex or multifactorial phenotype such as AMD. In genome-wide scans, there is the problem of multiple testing issue, which can lead to false positive results. The point-wise significance level for linkage analysis (lod = 3, P = 0.0001) and association analysis (P = 0.05) need to be set to more stringent values to obtain a reasonable family-wise significance.

CANDIDATE GENES

Based on the evidence for a genetic component, a variety of candidate genes have been evaluated either for their roles in diseases that share phenotypic similarity to AMD and/or because they were identified in genome-wide scans that searched for areas of the genome harboring AMD susceptibility genes as described earlier in this chapter. The comprehensive discussion that follows includes the genes that have been found by at least one study to be statistically associated with either increased or decreased risk of AMD. Table 143.4 summarizes these findings.

TABLE 143.4 -- Candidate Genes for AMD

Gene name	Symbol	Location	Function	Previously Identified Region?	Study Population	Study
APOlipoprotein E	APOE	19q13.2	Lipid metabolism and transport		88 AMD patients (Int. Class. System), 901 controls from the Netherlands ? 55 y.o.	Klaver et al.[52]
					116 unrelated French patients w/CNV, 168 age and sex-matched controls ? 60 y.o.	Souied et al.[53]
					39 Chinese patients w/disciform scarring or CNV, 133 normal controls	Pang et al.[59][*]
					230 patients ? grade 3 by Int. Class. System-61 independent familial cases, 101 sporadic cases & 68 familial cases used as sporadic, 333 spouses used as controls	Schmidt et al.[55][*]
					87 Italian patients w/AMD (Int. ARM Epi. Study), 47 age-matched controls, 1287 individuals from the general population	Simonelli et al.[54]
					Pooled analysis of 4 independent case-control data sets - 377 patients w/any extensive, intermediate or large soft drusen, GA or CNV, 198 ethnically-matched controls of similar age	Schmidt et al.[56]
					259 affected in 56 families w/AMD, 207 unaffected from these families, 104 unrelated affected, 88 unrelated controls (AMD = GA, CNV and/or extensive drusen)	Schultz et al.[61][*]
					322 Anglo-Celtic patients w/drusen > 63 ?m, CNV or dry AMD, 123 unrelated controls matched for ethnicity	Baird et al.[64]
					85 unrelated Japanese patients w/CNV, 82 unrelated Japanese controls ? 50 y.o.	Gotoh et al.[83][*]
					632 white unrelated patients w/GA, CNV, coarse RPE changes or large drusen, 206 controls	Zareparsi et al.[57]
					992 Caucasian patients w/extensive drusen, pigmentary changes, GA and/or CNV, 120 unrelated controls	Conley et al.[10][*]
					377 unrelated patients w/extensive intermediate or soft drusen, GA and/or CNV (221 ever smokers), 198 unrelated controls (90 ever smokers)	Schmidt et al.[60][*]
					95 patients w/white drusen with or without pigmentation, CNV or geographic AMD, 65 spouses as controls	Asensio-Sanchez et al. 2006
					160 patients w/GA and/or CNV, 227 controls - 133 screened unaffecteds, 94 volunteers with a younger mean age	Bojanowski et al. 2006
SuperOxide Dismutase 2, mitochondrial	SOD2	6q25.3	Regulator of oxidative stress		102 Japanese patients w/CNV, 200 controls	Kimura et al.[67]
					94 patients from Northern Ireland w/CNV, 95 controls	Esfandiary et al.[65][*]
ParaoxONase 1	PON1	7q21.3	Lipid metabolism and transport		72 unrelated Japanese patients w/CNV, 140 age and sex-matched controls	Ikeda et al.[63]
					62 Anglo-Celtic patients w/late AMD (Int. ARM Epi. Study), 115 controls matched for age and ethnicity	Baird et al.[64][*]
					94 patients from Northern Ireland w/CNV, 95 controls	Esfandiary et al.[65][*]
CySTatin C	CST3	20p11.21	Protease inhibitor		167 German patients w/CNV (Int. ARM Epi. Study), 517 unrelated controls from Germany, Switzerland, Italy & USA	Zurdel et al.[68]
Angiotensin I Converting Enzyme 1	ACE	17q23.3	Regulator of systemic blood pressure regulation		173 patients w/CNV or extensive small (< 63 ?m) or intermediate (>125 ?m) drusen, 189 age-matched controls	Hamdi et al.[69]
					992 Caucasian patients w/extensive drusen, pigmentary changes, GA and/or CNV, 120 unrelated controls	Conley et al.[10][*]
Hemicentin-1	HMCN1	1q25.3 - q31.1	Maintenance of extracellular matrix integrity	Klein 1998, Weeks 2001, Majewski 2003, Seddon 2003 & Iyengar 2004	100 families w/3 or more living AMD patients, 188 sporadic cases of AMD (Wisconsin Grading Scale), 174 controls matched for age, sex and ethnicity	Schultz et al.[61]
					368 patients ? grade 2 (Int. Class. System), equal matched unaffected controls	Hayashi et al.[77][*]
					508 patients ? Grade 3 (Rotterdam Study class), 25 possibly affected and 163 controls	McKay et al.[78][*]
					992 Caucasian patients w/extensive drusen, pigmentary changes, GA and/or CNV, 120 unrelated controls	Conley et al.[10][*]
					335 Finnish patients w/large drusen, GA, or CNV, of which 154 were sporadic cases & 181 familial, 105 controls	Seitsonen et al.[40][*]
Chemokine (C-X3-C motif) Receptor 1	CX3CR1	3p21.3	Regulator of immune acute phase response	Majewski 2003 & Jun 2005	85 patients w/GA or CNV in at least one eye, 105 controls (Clinically diagnosed group), 21 patients w/CNV, 19 w/areolar AMD & 171 controls (pathologically diagnosed group)	Tuo et al.[70]
Fibulin-5	FBLN5	14q32.1	Maintenance of extracellular matrix integrity	Jun 2005 & Schmidt 2004	402 patients w/drusen, disruption or atrophy of RPE or CNV, 429 controls (80% of both groups Caucasian)	Stone et al.[79]
					805 European patients w/AMD (Int. Class. System), 279 unrelated controls ? 65 y.o.	Lotery et al.[80]
Toll-Like Receptor 4	TLR4	9q32 - q33	Regulator of innate immunity	Majewski 2003	667 unrelated AMD patients (Int. ARM Epi. Study), 439 unrelated controls, all Caucasian	Zareparsi et al.[81]
Complement component 2	C2	6p21.3	Regulator of alternative-complement-pathway		898 unrelated patients ? 60 y.o. w/AMD (Int. ARM Epi. Study), 389 unrelated age and ethnicity matched controls, all of European-American descent	Gold et al. 2005
Complement Factor B	CFB	6p21.3	Regulator of alternative-complement-pathway		898 unrelated patients ? 60 y.o. w/AMD (Int. ARM Epi. Study), 389 unrelated age and ethnicity matched controls, all of European-American descent	Gold et al. 2005
Complement Factor H	CFH	1q32	Regulator of alternative-complement-pathway and innate immunity	Klein 1998, Weeks 2001, Majewski 2003, Seddon 2003 & Iyengar 2004	992 Caucasian patients w/extensive drusen, pigmentary changes, GA and/or CNV, 120 unrelated controls	Conley et al.[10]
					400 patients w/extensive drusen or macular pigmentary abnormalities, 202 controls w/no more than 4 small, hard drusen	Edwards et al.[5]
					954 unrelated AMD patients 60 or older, 406 age- and ethnicity-matched controls	Hageman et al.[9]
					Patients w/CNV and/or GA - 1443 affected from 594 families, 196 sporadic cases, 296 unrelated controls	Jakobsdottir et al.[71]
					96 white patients with large drusen, GA and/or CNV, 50 white controls	Klein et al.[6]
					1166 German AMD patients (Int. Class. System), 945 unrelated controls	Rivera et al.[72]
					2 unrelated CNV groups from France, 60 sporadic and 81 familial cases compared w/91 controls	Souied et al.[11]
					616 white patients w/CNV, GA and/or large drusen, 275 white controls at least 68 y.o.	Zareparsi et al.[81]
					The Rotterdam Study - 2387 patients, of whom 49 had soft drusen, GA or CNV, remainder controls	Despriet et al. 2006
					146 unrelated Japanese patients w/CNV, 105 controls	Gotoh et al.[62][*]
					163 Chinese patients w/CNV, 232 age-matched controls	Lau et al. 2006
					Patients w/soft drusen, GA and/or CNV, 1016 from Iceland and 431 from the US, 1108 unaffected Icelandic relatives, 431 controls	Magnusson et al. 2006
					96 Japanese patients w/CNV, 89 matched controls between 50-85 y.o.	Okamoto et al. 2006
					647 patients ? grade 3 (AREDS scale) and at least 55 y.o., 163 controls grade 1 or 2	Postel et al. 2006
					111 white males w/drusen, RPE changes, atrophy, hypertrophy, RPE detachment, GA, subretinal CNV membrane and/or disciform scars, 401 male controls	Schaumberg et al. 2006
					335 Finnish patients w/large drusen, GA, or CNV, of which 154 were sporadic cases & 181 familial, 105 controls	Seitsonen et al.[40]
					443 unrelated English patients w/GA or CNV (mean number of pack years of cigarette smoking = 15.4±18.8), 262 spouses as controls (pack years =10.7±14.5)	Sepp et al.[12]
					104 unrelated Italian AMD patients (Int. Class. System) and 131 unrelated controls	Simonelli et al. 2006
Very Low Density Lipoprotein Receptor	VLDLR	9p24	Lipid metabolism and transport	Iyengar 2004	992 Caucasian patients w/extensive drusen, pigmentary changes, GA and/or CNV, 120 unrelated controls	Conley et al.[10][*]
					Two independent data sets - 314 affected and 97 unaffected from the family dataset, 399 affected and 159 unaffected from the case-control dataset. Affecteds had extensive drusen (? 63-125 ?m), GA or CNV	Haines et al.[66]
PLEcKstrin Homology domain containing, family A (phosphoinositide binding specific) member 1	PLEKHA1	10q26.13	Phospholipid binding indirectly immune response	Weeks 2001, Majewski 2003, Seddon 2003, Iyengar 2004 & Kenealy 2004	Patients w/CNV and/or GA - 1443 affected from 594 families, 196 sporadic cases, 296 unrelated controls	Jakobsdottir et al.[71]
					1166 German AMD patients (Int. Class. System), 945 unrelated controls	Rivera et al.[72][*]
					810 white unrelated patients w/extensive intermediate or large (? 125 ?m) drusen, GA or CNV (39% reported smokers), 259 unrelated controls	Schmidt et al.[73][*]
LOC387715	LOC 387715	10q26.13	Unknown	Weeks 2001, Majewski 2003, Seddon 2003, Iyengar 2004 & Kenealy 2004	Patients w/CNV and/or GA - 1443 affected from 594 families, 196 sporadic cases, 296 unrelated controls	Jakobsdottir et al.[71]
					1166 German AMD patients (Int. Class. System), 945 unrelated controls	Rivera et al.[72]
					810 caucasion unrelated patients w/extensive intermediate or large (? 125 ?m) drusen, GA or CNV (39% reported smokers), 259 unrelated controls	Schmidt et al.[73]
HtrA serine peptidase 1	HTRA1	10q26	Unknown	Weeks 2001, Majewski 2003, Seddon 2003, Iyengar 2004 & Kenealy 2004	96 patients with neovascular AMD and 130 age-matched controls of Southeast Asian descent	Dewan et al., 2006
					442 AMD cases (265 CNV and 177 with soft confluent drusen) and 309 normal controls from a Caucasian population in Utah	Yang et al., 2006
Vascular Endothelial Growth Factor	VEG-F	6p12	Regulator of angiogenesis		Two independent data sets - 314 affected and 97 unaffected from the family dataset, 399 affected and 159 unaffected from the case-control dataset. Affecteds had extensive drusen (? 63-? 125 ?m), GA or CNV	Haines et al.[66]
Low density lipoprotein Receptor-related Protein 6	LRP6	12p13 - p11	Lipid metabolism and transport		Two independent data sets - 314 affected and 97 unaffected fromthe family dataset, 399 affected and 159 unaffected from the case-control dataset. Affecteds had extensive drusen (? 63-? 125 ?m), GA or CNV	Haines et al.[66]
Excision Repair Cross-complementing rodent repair deficiency, Complementation group 6	ERCC6	10q11.23	DNA repair		460 advanced AMD cases and 269 age-matched controls	Tuo et al.[76]

Int. Class System-International Classification System, CNV-Choroidal Neovascularization, GA-Geographic Atrophy, Int. ARM Epi. Study-International Age Related Maculopathy Epidemiology Study, RPE-Retinal Pigment Epithelial, Int. Class. System-International Classification System, y.o.-years old, AREDS-Age Related Eye Disease Study.

*	Indicates no association found.

APOLIPOPROTEIN GENE (APOE)

Like ELOVL4, APOE functions in lipid transport and metabolism. APOE was the first candidate gene investigated for a role in AMD outside of the genes responsible for the juvenile forms of macular degenerations. The role of apolipoprotein gene (APOE) in susceptibility to Alzheimer's disease is well established, i.e., APOE is a component of the amyloid plaques that are a hallmark of this aging degenerative disease of the central nervous system. Three alleles E2, E3, E4 (E3 is the most frequent allele in the general population) in the last exon, exon 4, of APOE influence disease risk for Alzheimer's. The combination of these three alleles makes up six genotypes: E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, and E4/E4. The E4 allele, and in particular the E4/E4 genotype, is thought to be associated with increased risk of an earlier onset of Alzheimer's disease while the E2 allele is thought to be protective for development of Alzheimer's disease. The E4 allele of APOE was first reported by the Rotterdam group from the Netherlands in 1998 to be associated with a 'decreased' risk of AMD.^[52] A trend toward increased risk of development of AMD was observed with the E2 allele but this finding was not statistically significant. This study further demonstrated histologically the presence of APOE in large soft drusen from eyes of patients with AMD as well as the absence of this protein from small hard drusen. Many other studies have supported the finding that the E4 allele is associated with a decreased risk of either the early form or more advanced stages of AMD.^[53-58] Although many of these studies have shown a trend toward an association of the E2 allele with increased risk of AMD,^[52,54,56] this finding did not reach statistical significance with the exception of Baird et al who in 2004 showed that the presence of the E2 allele was statistically significantly associated with an earlier onset of AMD in women with the neovascular form of AMD.^[58] Conversely, Pang et al,^[59] Schmidt et al,^[60] Schultz et al,^[61] and Gotoh et al^[62] found no evidence for either a protective effect of APOE E4 or a causal effect of APOE E2 on AMD risk. Further supporting the findings of a lack of association of APOE and AMD is that of the many genome-wide scans conducted, none has shown the 19q13.2 region to be linked to AMD. Moreover, a study that performed a genome-wide scan on 34 families with advanced stages of AMD comprising 346 sibling pairs showed linkage to 19q13.31,^[51] a region in close proximity to APOE, and concluded that the signal was not due to the effect of the APOE gene. Specifically, this group investigated all six APOE genotypes in conjunction with the linkage findings from the FARMS cohort. This resulted in diminished significance of the linkage peak on 19.31, suggesting that a gene in this region, other than APOE, may be responsible for risk of AMD. Furthermore, in a study that examined the effects of APOE genotypes along with smoking, APOE genotype alone was not found to influence risk of AMD;^[60] however, any association observed between APOE and AMD was dependent on smoking history. For example, risk of neovascular AMD increased if an individual was a carrier of the E2 allele and had a history of smoking compared to individuals who had the E3/E3 genotype and never smoked.^[60] This finding may suggest that if APOE is involved in AMD pathogenesis, it may function as a weak modifier on risk for either early or more advanced stages of AMD.

PAROXONASE 1 GENE (PON-1)

Paroxonase 1 gene (PON-1) like APOE encodes for a protein that regulates both lipid metabolism and transport. The three studies to date that have been published are not in agreement as to the contribution of PON1 to the etiology of AMD. Specifically the M55L (a methionine is substituted by leucine) and Q192R (a glutamine is substituted by arginine) variants were reported to be associated with neovascular AMD in 72 individuals from Japan.^[63] Further the affected individuals also had significantly higher levels of low-density lipoprotein in the plasma.^[63] Two other studies, however, found no association between these variants in patients diagnosed with late AMD of Anglo-Celtic origin^[64] or patients with neovascular AMD from Northern Ireland.^[65] Again, it is possible that there exists multiple susceptibility genes for AMD, that are not necessarily expressed in every patient with AMD, possibly due to ethnic heterogeneity.

VERY-LOW-DENSITY LIPOPROTEIN RECEPTOR GENE (VLDLR)

Like APOE and PON-1, VLDLR is also involved in lipid metabolism and transport. There is a lack of agreement between the two studies conducted to date as to the association of VLDR and AMD. VLDR is located on the short arm of chromosome 9 (9p24), a region found to be significantly associated with late AMD in one genome-wide scan (Tables 143.3 and 143.4).^[46] While one study found a significant association of this gene with all types of AMD in Caucasian patients including those with large, extensive drusen, neovascularization, and geographic atrophy using SNPs,^[66] another large study on Caucasian patients with similar AMD diagnosis did not find a significant association with VLDR.^[10] More studies may need to be done to validate the role of VLDR in the pathophysiology of AMD.

LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 6 GENE (LRP6)

Another gene involved in lipid me tabolism and transport is LRP6. Using a combination of linkage and association analysis on family-based and case-control populations, respectively, Haines et al demonstrated that LRP6 was associated with advanced AMD in Caucasian patients.^[66] Although LRP6 is located in a region, 12p11-p13, yet to be identified by genome-wide scans as significant, its recent discovery as a candidate gene in the etiology of AMD may warrant further study.

MANGANESE SUPEROXIDE DISMUTASE 2 GENE (SOD-2)

SOD2, localized to 6q25.3, encodes an enzyme with antioxidant properties and is also expressed in the retina. The substitution of an alanine for a valine homozygously in exon 2 of this gene was found to be associated with a 10-fold increased risk of neovascular AMD in 102 Japanese patients when compared to 200 ethnically matched controls.^[67] Like in the case of PON1, the variant in SOD2 could not be found in patients with neovascular AMD from Northern Ireland at a statistically significant level when compared to controls.^[65] Again, it is possible that there exist multiple susceptibility genes for AMD, not necessarily expressed in every patient with AMD, possibly due to ethnic heterogeneity.

CYSTATIN C GENE (CST3)

CST3, similar to TIMP3 (Sorsby's fundus dystrophy), is a protease inhibitor. CST3 is localized to 20p11.21, a region as yet to be reported as being linked to any type of AMD. The homozygous variant in the 5' untranslated region of CST3 state was demonstrated to be associated with almost 7% of German patients with neovascular AMD.^[68] Further study of this gene in the etiology of AMD is warranted as this is the only investigation to date.

ANGIOTENSIN I CONVERTING ENZYME GENE (ACE)

The role of ACE as a regulator of blood pressure has been established. ACE is localized to the long arm of chromosome 17 (17q23.3). The polymorphism in this gene that was found to be associated with 'decreased' susceptibility to AMD is a repetitive element called an Alu.^[69] This polymorphism was found to be significantly associated homozygously with a fivefold decreased risk from the early dry or atrophic forms of AMD.^[69] This association was found after studying patients with extensive small (>63 ?m) drusen, intermediate (<125 ?m) drusen, geographic atrophy as well as neovascular AMD. However, another study that examined a similar spectrum of AMD phenotypes concluded that there was no association of ACE with either a decreased or increased risk of AMD.^[10]

CHEMOKINE (C-X3-C MOTIF) RECEPTOR 1 GENE (CX3CR1)

CX3CR1 functions as a regulator of the immune response in the acute phase.

CXCR1 is localized to the short arm of chromosome 3 (3p21.3), a region found to be significantly linked to the advanced stages of AMD by two independent genome-wide scans (Tables 143.3 and 143.4).^[44,51] Two polymorphisms CX3CR1, V249I (a valine is substituted by isoleucine) and T280 M (a threonine is substituted by methionine), were found both homozygously and heterozygously to be associated with cases of advanced AMD.^[70] Further, this study also demonstrated decreased level of expression of this gene in the maculae of a patient with the T280 M variation in the homozygous state, when compared to the maculae from a control individual. Given that CX3CR1 is in a previously linked region to AMD, the findings of a positive association with risk of AMD and decreased level of expression of this gene in an AMD retina should warrant further study.

PLECKSTRIN HOMOLOGY DOMAIN CONTAINING FAMILY A (PHOSPHOINOSITIDE BINDING SPECIFIC) MEMBER 1 GENE (PLEKHA 1), HYPOTHETICAL LOC387715 GENE (LOC387715), AND HTRAA SERINE PEPTODASE 1 (HTRA1)

PLEKHA1, LOC387715, and HRTA1 are localized to the long arm of chromosome 10 (10q26.13), a region consistently found by many genome-wide scans to be linked to both early and the more advanced stages of AMD (Tables 143.3 and 143.4).^{[42-46,48,49]} Meta-analysis or pooled findings from six genome-wide scans demonstrated that the 10q26 demonstrated the greatest significance for linkage compared to other significant regions, such as the 1q25-q32 region.^[50] PLEKHA1 is believed to participate in phospholipid binding and more indirectly in the immune response, whereas LOC387715, a gene that is not well conserved across species, has no attributable function. Although the function of HTRA1 is unknown, it ahs been reported that the encoded protein is upregulated in aging and downregulated in certain tumors. Using a combination of linkage and association employing SNPs specific to the 10q26 region, Jakobsdottir et al demonstrated that while both PLEKHA1 and LOC387715 were significantly associated with both neovascular and atrophic AMD, the association was stronger for PLEKHA1. Over 1400 affected individuals comprised of both familial and sporadic cases of AMD were examined.^[71] Rivera et al^[72]extended these findings by demonstrating that a specific variant within the LOC387715 locus was associated with increased AMD risk after examining a cohort that consisted of both early and advanced stages. Similar findings were replicated in a Caucasian population that consisted of advanced cases, including large drusen (=125 ?m), geographic atrophy and neovascularization.^[73] These two studies concluded that LOC387715, and not PLEKHA1, was associated with AMD. More recently, it was demonstrated that another variant or SNP in the HTRA1 promotor, in linkage disequilibrium (LD) with the LOC 387715 variant, is likely the functional polymorphism responsible for increased AMD risk.^[74,75] Given that 10q26 is a region that has been consistently linked with AMD by many genome-wide scans and that PLEKHA1, LOC387715, and TRA1 are located adjacent to one another, teasing out precisely which gene in this region is associated with AMD may require further investigation.

VASCULAR ENDOTHELIAL GROWTH FACTOR GENE (VEGF)

VEGF is localized to the short arm of chromosome 6 (6p12), a region as yet to be identified in any genome-wide scan (Table 143.2). The role of VEGF as a regulator of angiogenesis is well established. Further, VEGF is a target for therapeutic interventions, such as Macugen and Lucentis. Only one study to date has examined the genetic contribution of VEGF to AMD.^[66]

Employing a combination of linkage (family-based) and association (sporadic) analysis using SNPs, common variation in this gene was significantly found to be associated with AMD.^[66] The affected individuals consisted of those with drusen only (63-125 ?m) geographic atrophy or neovascularization.

EXCISION REPAIR CROSS-COMPLEMENTATION GROUP 6 GENE (ERCC6)

ERCC6 is localized to 10q11.23 and functions in the repair of DNA as well as aging. A SNP in the 5'-untranslated region of this gene was reported to be significantly associated in the homozygous state with advanced AMD.^[76] Further, increased expression of this gene was observed in lymphocytes from individuals that were homozygous for his variation. To date this is the only study showing an association of this gene with AMD.

HEMICENTIN-1 GENE (FIBULIN 6, FIBL-6)

In 1998 Klein (Oregon) described a family linked to the long arm of chromosome 1 (1q25-1q32).^[41] Subsequently, this same region has been found by many studies to be linked or associated with both early and advanced stages of AMD (Table 143.3).^[6,42-46,50] In 2003, Schultz et al analyzed this same multigenerational family and found that a mutation in hemicentin-1 (Fibl-6) was associated with the early stage of AMD.^[77] Specifically, this group found that a Gln5345Arg change (a glutamine is replaced by arginine) in exon 104 segregated with the disease. Based on these results, it was suggested that hemicentin-1 was the gene responsible for the phenotype observed in this family.^[76] Since this time, there have been no other confirmatory reports that the Gln5345Arg variant in hemicentin-1 is associated with any type of AMD (Table 143.3).^{[10,40,78,79]} However, Iyengar et al^[46] reported that this gene could be involved in the etiology of AMD because linkage was observed for four SNPs in this gene. Iyengar then further concluded that although they did not find the specific Gln5345Arg that Schultz did, that maybe other variants in FIBL-6, as yet to be discovered, could be associated with AMD.^[46]

FIBULIN 5 (FBLN5)

FBLN5, located on the long arm of chromosome 14 (14q32.1), was chosen as a candidate gene because of its similarity to Fibulin 3, for which a mutation in this gene is associated with Doyne's honeycomb dystrophy, a disease with some overlapping phenotype to AMD (Table 143.2). More recently, the 14q32 region has been reported by one genome-wide scan to be linked to advanced AMD.^[51] Like hemicentin-1, FIBLN5 is an extracellular matrix protein that functions in maintaining the integrity of elastic lamina, similar to what is found in Bruch's membrane. Various mutations in FIBLN5 were reported to be associated with AMD in 7 out of 40 AMD patients in a case-control study.^[80] Phenotypically, the seven patients all had a distinct and unusual pattern of drusen in common. Three out of the seven patients also had neovascularization. None of these mutations were found in unaffected individuals. Stone et al concluded that fibulin 5 accounts for 1.7% of all cases of AMD.^[80] A recent study has replicated these as well as showed that two additional novel variants in FIBLN5 are associated with other subtypes of AMD.^[80] This study further demonstrated in vitro that there is a reduction in secretion of FIBLN5 in COS7 cells from patients expressing these variants.^[81]

TOLL-LIKE RECEPTOR 4 GENE (TLR4)

TLR4 is localized to the long arm of chromosome 9 (9q32-q33) a region previously linked to patients with the advanced forms of AMD.^[44] A variant in this gene which functions in innate immunity and lipid transport and metabolism efflux has been previously shown to be associated with a 'decreased' risk of arteriosclerosis. This same variant, D299G (asparagine is substituted by glycine), was found to be associated with a 2.6-fold 'increase' in risk of AMD.^[82] Further, this study concluded that an individual's risk is increased even more when this variant is present in the same individual along with variants in APOE and/or ATP-binding cassette transporter-1 gene (ABCA1) gene. Given the suggestive findings of linkage and association between TLR4 and AMD further studies are warranted.

COMPLEMENT FACTOR H GENE (CFH)

The last year in the field of AMD genetics has focused on the exciting discovery of the association of CFH with AMD (Fig. 143.2 and Table 143.4). As stated in the beginning of this chapter, numerous independent reports have shown that the functional polymorphism Y402H in CFH, where a tyrosine is substituted by a histidine, to be associated with a two-to sevenfold increased risk of both early and/or late stages of AMD.^[5-12,83] Only one study has found no association of the CFH gene to AMD.^[83] This study examined the Y402 variant in a Japanese cohort with neovascular AMD.^[84] This negative finding may be possibly due to the fact that other AMD susceptibility genes may explain the phenotype in this population. CFH is localized to 1q32, a region found by both linkage^[6,42-46,50] and by a study of association^[6] to be associated with any subtype of AMD. CFH functions in regulation of the alternative complement pathway as well as innate immunity. Some groups have reported that common variation in CFH, other than Y402H, is associated with both early and advanced forms of AMD.^[9,74,83] Interestingly, some of these same variants (or a combination of them), including Y402H, are associated with increased risk for other diseases such as myocardial infarction,^[85] hemolytic uremic syndrome (HUS),^[86] and membranoproliferative glomerulonephritis (MPGN).^[87] This by no means indicates that CFH does not play an important role in the pathophysiology of AMD but underscores the importance of examining common variation in multiple susceptibility genes as well as contributing environmental factors simultaneously to get a better estimate of an individual's risk of AMD. Several groups have begun to do this with respect to the contribution of CFH along with other reported genetic and epidemiological risk factors for AMD. Some of these studies include, CFH, LOC387715 and smoking,^[73] CFH and smoking,^[12] CFH, Complement Component 2 Gene (C2), Complement Factor B Gene (CFB),^[87] and CFH, C2, CFB, and LOC387715.

FIGURE 143.2 The number of studies to determine the association between the candidate genes and AMD. With the number of candidate genes increasing, the number of studies performed has also increased each year. Because of recent findings of association of AMD with the CFH gene, a large number of studies were performed in 2005 and 2006.

COMPLEMENT COMPONENT 2 GENE (C2) AND COMPLEMENT FACTOR B GENE (CFB)

C2 and CFB are both part of a major histocompatibility complex that, like CFH, regulates the alternative complement pathway. Recently, Gold et al postulated that because variation in CFH had been significantly associated with AMD, genes in the alternative complement pathway may also play a role in the pathophysiology of AMD.^[88] This study demonstrated that combinations of SNPs in these two genes decreased an individual's risk of AMD. Specifically, the protective effect on an individual in having variation in these genes was most significant in those who had two histidine alleles in the CFH gene. In other words individuals who were homozygous for the Y402H variation in exon 9 of CFH were afforded the most protection from risk of AMD.^[87] These findings were recently confirmed in a published report that examined simultaneously CFH, C2, CFB, and LOC387715 on risk of advanced AMD.

Key Features

. A role for Inflammation, lipid transport and metabolism as well as oxidative stress in the pathophysiology of AMD may be supported by findings that show that candidate genes associated with these pathways may be involved in the early or late stages of AMD . The CFH Y402H variant is the most consistently associated genetic risk factor for AMD . Genome-wide scans and candidate gene association studies are complementary methods

In summary, genes could potentially be used as biological markers in the early or presymptomatic diagnosis of the neovascular form of AMD. Genes or biological markers that are identified can then be used as possible disease targets for the development of preventative therapies for AMD. Further the findings of candidate gene associations with risk of AMD point to possible mechanisms or pathways involved in the etiology of AMD. Studying the hypothesized mechanisms involving oxidative stress, inflammation, and lipid metabolism from both an epidemiologic (reviewed in detail elsewhere in this book) and genetic perspective is necessary to further elucidate the role of these pathways in AMD. Given the multifactorial and heterogeneous nature of AMD, complementary methods are necessary to detect weak to moderate associations to identify the contribution epidemiologic and genetic risk factors make independently and in combination in order to more accurately determine the overall risk of AMD. Both population stratification between cases and controls as well as heterogeneity in phenotype among cases, can confound findings in analysis of data. Inconsistency in replication of findings among studies could possibly be due to these factors.

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