|Molecular Vision 2004;
Received 10 August 2003 | Accepted 12 January 2004 | Published 26 January 2004
Linkage analysis for age-related macular degeneration supports a gene on chromosome 10q26
Shannon J. Kenealy,1 Silke Schmidt,2,4 Anita
Agarwal,3 Eric A. Postel,5 Monica A. De La Paz,5
Margaret A. Pericak-Vance,2,4
Jonathan L. Haines1
1Center for Human Genetics Research and 3Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville, TN; 2Center for Human Genetics, 4Department of Medicine, and 5Department of Ophthalmology, Duke University Medical Center, Durham, NC
Correspondence to: Dr. Jonathan L. Haines, Ph.D., Center for Human Genetics Research, Vanderbilt University Medical Center, 519 Light Hall, Nashville, TN, 37232-0700; email: email@example.com
Purpose: Age-related macular degeneration (AMD) is a retinal degenerative disease that is the leading cause of blindness worldwide in individuals over the age of 60. Although the etiology of AMD remains largely unknown, numerous studies have suggested both genetic and environmental influences. A previous study of affected multiplex families identified four chromosomal regions that potentially harbor AMD susceptibility genes. The purpose of our study was to further investigate these regions with additional microsatellite marker coverage in our independent data set.
Methods: We examined regions on chromosomes 1q, 9p, 10q, and 17q for genetic linkage in our 70 multiplex families (consisting of 133 affected sibpairs). Two point heterogeneity LOD score (HLOD) and nonparametric LOD score (MLS) analyses were performed for disease models defined by the most severe status in either eye. Conditional analyses were performed using apolipoprotein E (APOE) alleles as covariates in semiparametric LOD (LOD*) score calculations.
Results: Regions on chromosomes 1q, 9p, and 17q did not provide evidence of linkage in our data set. However, markers D10S1230 and D10S1656 on chromosome 10q26 generated maximum HLOD scores of 1.52 and 1.13, respectively. Marker D10S1230 also generated an MLS score of 1.56 in stage 4 and 5 individuals. Controlling for the potential effect of the APOE-ε4 allele did not substantially alter these scores.
Conclusions: With the inclusion of this study, at least five AMD data sets provide support of genetic linkage to 10q26. Such consistency and confirmation of evidence strongly suggests that this region should be the subject of further detailed genomic efforts for the disease.
Age-related macular degeneration (AMD), or age-related maculopathy (ARM), is a degenerative disease of the retina that causes progressive loss of central vision and is the leading cause of irreversible vision loss in older Americans. The prevalence of the disease increases with age, afflicting 9% of the population over age 65 and 28% over age 75 [1,2]. It is estimated that 25-30 million people in the world are blind as a result of AMD.
Although the etiology of AMD is largely unknown, numerous studies indicate that risk factors include age, gender, ethnicity, smoking, hypertension, and diet. Familial aggregation [3-6], twin [7-9], and segregation analysis [10,11] studies also suggest a significant genetic contribution to the disease. However, subsequent linkage studies attempting to identify these genes have been hindered by the complex and multifactorial nature of AMD (e.g., high prevalence, late onset, and clinical and genetic heterogeneity).
The investigation of genes responsible for macular and retinal dystrophies sharing common features with AMD has primarily used a functional candidate approach that has yielded largely disappointing results. For example, genes ELOVL4 (Stargardt disease) , bestrophin (Best disease) [13,14], TIMP-3 (Sorsby fundus dystrophy) , and peripherin (retinal degeneration) [16-19] have failed to convincingly demonstrate association with AMD. Another candidate that has been extensively studied is the adenosine triphosphate-binding cassette transporter (ABCA4, formerly ABCR) gene involved in the pathogenesis of autosomal recessive Stargardt disease (a common form of inherited juvenile macular degeneration). The original study of ABCA4 suggested that this gene accounts for a significant percentage of AMD cases . However, subsequent studies have failed to identify sequence variants in the ABCA4 gene that confer an increase in AMD risk, suggesting that ABCA4 is not a major susceptibility factor for the disease [21-26].
The apolipoprotein E gene (APOE) is another functional candidate due to its role in lipid transport and distribution, high expression levels in the retina, and involvement in processes that may lead to drusen formation. Our growing understanding of APOE's critical role in neurodegeneration in multiple diseases suggests it has a pervasive effect [27-29]. Case-control studies for AMD have consistently demonstrated a protective effect for the APOE-ε4 allele on disease risk in Caucasian populations [30-35]. A few studies have also suggested a modest increase in disease risk with the ε2 allele [31,34,35], with one of these studies reporting a sex-specific effect in males .
The locational candidate approach is an alternative to the functional candidate approach that is currently being used in an attempt to identify susceptibility genes for AMD. This method requires studying multiplex families and performing a genomic screen to identify regions of interest for disease loci. The largest genomic screen conducted to date for AMD utilized 391 families and identified four regions yielding multipoint heterogeneity LOD scores or Sall scores >2.0: 1q31, 9p13, 10q26, and 17q25 . The aim of the present study is to further investigate these four regions with additional microsatellite marker coverage in our data set of 70 multiplex families.
The multiplex families (more than one affected family member) used for this study were ascertained in the southeastern United States by Duke University Medical Center (DUMC) and Vanderbilt University Medical Center (VUMC). Stereoscopic fundus photographs were available for all cases. All protocols were approved by the appropriate Institutional Review Boards and all individuals provided informed consent before participating in the study.
Grading of disease severity in patients was determined using a slightly modified version of established classification systems . Severity was assessed on a scale of 1-5, with grade 1 individuals lacking any AMD features; grade 2 individuals having only small or non-extensive intermediate drusen; grade 3 ("early" AMD) individuals having extensive intermediate drusen (deposits between 63 and 125 μm totaling or exceeding the area of a circle with a 350 μm diameter), large drusen, and/or drusenoid retinal pigment epithelium (RPE) detachments; grade 4 ("atrophic" AMD) individuals exhibiting geographic atrophy; and grade 5 individuals with neovascular/exudative disease. The entire data set consisted of 70 multiplex families with 133 affected sibpairs (Table 1).
Microsatellite markers were selected to create denser maps surrounding the four peak regions . Fifteen markers (seven from the Weeks et al. screens [36,37] and eight new) were selected and genotyped in all 70 families (Table 2).
Genomic DNA was extracted from blood using standard protocols and the Puregene system (Gentra Systems, Minneapolis, MN). Marker primer sequences were obtained from the Genome Database or designed with Primer3 software  and synthesized by Invitrogen Life Technologies (Carlsbad, CA). Amplification was performed in a PCR Express machine (ThermoHybaid, Needham Heights, MA) with the following conditions: 94 °C for 4 min; 94 °C for 15 s, AT for 30 s, 72 °C for 45 s (35 cycles); 72 °C for 4 min. PCR products were denatured for 3 min at 95 °C and run on a 6% polyacrylamide gel (Sequagel-6® from National Diagnostics, Atlanta, GA) for about an hour at 75 W. Gels were stained with a SybrGold® rinse (Molecular Probes, Eugene, OR) and scanned with the Hitachi Biosystems FMBIOII laser scanner (Brisbane, CA). APOE genotypes were determined using one of two standard methods (; Roche APOE Lightcycler kit, Indianapolis, IN).
Laboratory personnel were blinded to pedigree structure, affection status, and location of quality control samples. Duplicate quality controls samples were placed both within and across 96-well plates and equivalent genotypes were required for all quality control samples to ensure accurate genotyping. Hardy-Weinberg calculations were performed for each marker and Mendelian inconsistencies were identified using PedCheck . Suspect genotypes were re-read or re-run. All microsatellites were required to have >90% of possible genotypes.
Genotyping data were analyzed for two different disease models defined by the most severe status in either eye: stages 3, 4, and 5; and stages 4 and 5. Too few families existed with stage 5 alone to warrant analysis. The primary hypothesis of the study was linkage to AMD stages 3, 4, and 5. The subset of stages 4 and 5 was examined solely to determine if results were restricted to the most severe stages of the disease.
Two point heterogeneity LOD score (HLOD) analyses were computed using FASTLINK and HOMOG [41-43]. Because the mode of inheritance for AMD is unknown, parametric analyses were performed using both autosomal dominant and autosomal recessive models with disease allele frequencies of 0.01 and 0.14 to model a common susceptibility allele . Nonparametric affected sibpair LOD score (MLS) analyses were computed using ASPEX (version 1.81). Microsatellite marker allele frequencies were obtained from the data set by counting all independent chromosomes. Power calculations were done using SIMLINK .
NPL scores were used as weights in calculations for conditional analyses. Two different analyses were performed using the APOE alleles as covariates. Semiparametric LOD (LOD*) scores  were generated conditioning on the proportion of the APOE-ε4 allele in affected family members. Analysis 1 conditioned on the proportion of APOE-ε4 alleles in the affected individuals in each family, while Analysis 2 conditioned on the proportion of APOE non-ε4 alleles in the affected individuals in each family.
Across all analyses, only two markers yielded LOD scores >1.0 (Table 2). D10S1230 generated an HLOD score of 1.52 and an MLS of 1.56 in stage 4 and 5 individuals. D10S1656 generated an HLOD score of 1.13 in stage 3, 4, and 5 individuals (Table 2). Our study failed to detect even suggestive evidence of linkage in the remaining regions, including those for which the Weeks et al.  study generated its highest multipoint HLOD scores of 2.46 (between D1S1660 and D1S1647) and 3.16 (at D17S928). Incorporating the APOE alleles as covariates for analysis of each region did not substantially change the results, with the highest LOD* score being 1.04 for D10S1656 (data not shown).
In their initial genomic screen, Weeks et al.  found evidence of linkage to chromosomes 5, 9, 10, and 12. However, after follow-up of these regions, only chromosome 10 near D10S1230 continued to provide evidence of linkage across all diagnostic models in their data set (LOD scores >1.30). An expanded genomic screen conducted by this group also continued to provide evidence of linkage in this region, generating a maximum multipoint Sall LOD score of 2.10 between D10S1237 and D10S1230 under their strictest diagnostic model .
The positive result we obtained for markers on chromosome 10q26 provides further support for an AMD gene locus in this region. Weeks et al.  genotyped 225 families in their initial screen, providing the first evidence of linkage to this region. An additional 190 families in their expanded genomic screen replicated this result . Recently published screens by Majewski et al. (70 families) and Seddon et al. (158 families) also identify 10q26 as a region of interest [47,48]. With the inclusion of our data set, at least five data sets to date provide positive results for chromosome 10q26. This level of consistency and confirmation is unusual in complex diseases and strongly suggests that this region should be the subject of detailed genomic efforts.
While the result for 10q26 is encouraging, caution should be used in interpreting the lack of positive results on chromosomes 1q, 9p, and 17q in our data set. Weeks et al.  genotyped 452 sibpairs in their expanded genomic screen, generating a maximum LOD score for any marker under any model of 3.16. With less than one-third as many sibpairs, our data set has substantially less power and would not be expected to demonstrate scores of the same magnitude. In fact, assuming a dominant or recessive model with 50% heterogeneity, our data set has 41% and 74% power, respectively, to obtain a LOD score >1.00 at 5% recombination. In addition, replication of linkage results in any complex disease may be difficult due to underlying locus heterogeneity. Even if linkage to a true locus is observed, replication of the result may require data sets many times larger than the original data set .
Conditional linkage analysis using APOE as a covariate did not substantially change the results seen in the unconditional analyses, suggesting that APOE is not substantially influencing these linkage results. However, given the potential role of the APOE-ε4 allele in AMD, conditioning on APOE should still be considered for other linkage data sets and candidate gene studies to determine if moderate effects may be present.
We would like to express our appreciation to all of the families who generously participated in this study. We thank Ann Saunders for providing APOE genotyping on some samples; Lan Jiang for performing many of the analyses; Krista Stanton and Erin Hennessey for diligently genotyping many of the markers; and Ruth Domurath, Molly Klein, Jennifer Caldwell, and Katie Haynes for their tireless work in ascertaining many of the families used in this study. We would also like to thank the following clinics and clinicians for referring individuals to the study: Southern Retina, LLC (Charles Harris, MD); Vitreo-Retinal Surgeons (Michael E. Duan, MD and Christopher J. Devine, MD); Georgia Retina, PC; and The Retina Group of Washington. This study was supported by NIH/NEI grant EY12118.
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