To determine whether polymorphisms in the ARMS2 ( LOC387715 ) gene and the lysyl oxidase–like 1 ( LOXL1 ) gene are associated with age-related macular degeneration (AMD) in Japanese patients.
Clinically relevant laboratory investigation.
Forty-one unrelated Japanese subjects with dry AMD, 50 subjects with exudative (wet) AMD, and 60 subjects with polypoidal choroidal vasculopathy (PCV) were studied. The single nucleotide polymorphisms (SNPs), p.Ala69Ser of the ARMS2 gene and p.Arg141Leu of the LOXL1 gene, were amplified by polymerase chain reaction, directly sequenced, and genotyped.
For the ARMS2 gene, the genotype frequency of the p.Ala69Ser single nucleotide polymorphism in eyes with dry AMD was not significantly different from that in the controls ( P = .04), but the frequency was significantly higher in the exudative AMD group ( P = 3.1 × 10 −8 ) and PCV group ( P = 6.9 × 10 −3 ). For the LOXL1 gene, the genotype frequency of the p.Arg141Leu single nucleotide polymorphism was not statistically higher in the dry AMD and PCV groups than in the control group (dry AMD, P = .05; PCV, P = .16), but was statistically higher in the exudative AMD group ( P = 6.8 × 10 −3 ). Regression analyses showed significant associations between the ARMS2 gene and LOXL1 gene in patients with exudative AMD.
The p.Ala69Ser polymorphism of the ARMS2 gene is strongly associated with exudative AMD and PCV and is associated marginally with dry AMD. The polymorphisms in the LOXL1 gene did not predispose the individual to dry AMD and PCV. These findings suggest that there is a significant association between the ARMS2 gene and LOXL1 gene in exudative AMD.
Age-related macular degeneration (AMD) is the most frequent cause of irreversible blindness in the elderly in developed countries. AMD is a complex disorder that is genetically associated with multiple susceptibility loci. Both genetic predispositions and environmental factors, such as smoking, play important roles in the pathogenesis of AMD. AMD is broadly classified as either dry, nonneovascular or wet, or exudative neovascular AMD. The primary clinical sign of dry AMD is the presence of drusen with indistinct margins that are located between the retinal pigment epithelium and Bruch membrane. Drusen are small yellow or white accumulations of extracellular material. The proteins in drusen include apolipoproteins and members of the complement system. The primary clinical sign of exudative AMD is choroidal neovascularizations (CNVs) that develop from new blood vessels beneath the retina in the subretinal space.
Phenotypic and genetic heterogeneity makes the determination of the cause of AMD difficult. In the United States and Europe, approximately 85% to 90% of the patients diagnosed with AMD have dry AMD, with a high prevalence of eyes with drusen. However, the exudative, or wet type, of AMD is more common in Japanese persons. Earlier studies have shown that there are substantial differences in the phenotype and frequencies of single-nucleotide polymorphisms (SNPs) between the 2 ethnic groups.
Recently, 2 major genes, the complement factor H ( CFH ) gene on chromosome 1q31 and ARMS2/HTRA1 gene on chromosome 10q26, were shown to be significantly associated with a distinct component of the AMD phenotype in 2 different biological pathways. ARMS2 is located in the ellipsoid, a mitochondria-concentrated part of human photoreceptor cells, and HTRA1 is a serine protease gene. Polymorphisms in ARMS2 were associated with a decrease in the stability of the mRNA of the ARMS2 gene. HTRA1 seems to regulate the degradation of extracellular matrix proteoglycans. CFH is a component of an innate system that modulates inflammation through the C3 component, and it influences the formation of drusen that characterize dry AMD. However, ARMS2/HTRA1 influences the formation of CNVs, the hallmark of exudative AMD.
Thorleifsson and associates used a genome-wide scan to show a strong association between SNPs in the lysyl oxidase–like 1 ( LOXL1 ) gene and pseudoexfoliation syndrome (XFS; OMIM:177650). XFS is a generalized disorder of the extracellular matrix characterized by the pathologic accumulation of abnormal fibrillar material in the anterior segment of the eye. The LOXL1 gene is a member of the lysyl oxidase family of proteins that catalyzes the oxidative deamination of the lysine residues of tropoelastin, and the homeostasis of elastic fibers requires the lysyl oxidase-like 1 protein. Thus, the lysyl oxidase family of proteins plays important roles in elastogenesis.
In the pathogenesis of AMD, the age-related degradation of the elastic lamina of the Bruch membrane may permit the growth of CNVs. It recently was reported that the elastic lamina of the Bruch membrane in LOXL1 -deficient mice was fragmented and less continuously than in controls, and these alterations led to more aggressive CNV growth after laser photocoagulation. It was also suggested that the elastin gene ( ELN ) was a susceptibility gene for polypoidal choroidal vasculopathy (PCV) and that a different pathogenic process may be involved in the phenotypic expression of neovascular AMD and PCV. However, we are not aware of any study that has examined the relationship between the common polymorphisms in the LOXL1 gene and the presence of AMD.
The relative homogeneous and well-characterized Japanese population provides a unique opportunity to evaluate the possible association between AMD and the mRNA stability-related ARMS2 gene and the elastin-related LOXL1 gene. In addition, the factors that explain why some individuals develop the more aggressive exudative AMD, whereas others have the slowly degenerating dry AMD, have not been determined. Thus, the purpose of this study was to investigate the association between the SNPs of the ARMS2 and LOXL1 genes and the phenotypes of Japanese patients with dry AMD, exudative AMD, and PCV.
We studied 41 unrelated Japanese patients with dry or geographic atrophic AMD (grade 4 24 ; 34 men and 7 women; mean age, 73.3 ± 9.2 years), 50 unrelated Japanese patients with exudative AMD (grade 5 24 ; 40 men and 10 women; mean age, 71.3 ± 8.2 years), 60 unrelated Japanese patients with PCV (48 men and 12 women; mean age, 70.3 ± 9.2 years), and 138 Japanese controls who represent normal individuals of older age (101 men and 37 women; mean age, 68.6 ± 7.4 years) with no signs of macular disease (stage 0). All of the subjects were from the Ophthalmology Clinic of the Tohoku University Hospital, Miyagi, Japan, and standard ophthalmic examinations were performed on all subjects.
Genomic DNA was extracted from the leukocytes of peripheral blood and was purified with the Qiagen QIAamp Blood Kit (Qiagen, Valencia, California, USA), and the SNPs rs10490924 or p.Ala69Ser of the ARMS2 gene and rs1048661 or p.Arg141Leu of the LOXL1 gene were amplified by polymerase chain reaction, directly sequenced, and genotyped. The 2 primer sets for rs10490924 of ARMS2 were 5′-TTC AAA TCC CTG GGT CTC TG-3′ and 5′-CTG CTG CTG CTC AGT TTC CT-3′, and the sequencing primer was 5′-GAC CTC TGT TGC CTC CTC TG-3′. For rs1048661 of exon 10f LOXL1 , the 2 primer sets were 5′-CTC AGC GCT CCG AGA GTA G-3′ and 5′-ACA CGA AAC CCT GGT CGT AG-3′.
These primers were used under standard polymerase chain reaction conditions. The amplifications of the 2 SNPs were performed at 60 C annealing temperature. The polymerase chain reaction fragments were purified by ExoSAP-IT (USB, Cleveland, Ohio, USA) and were sequenced with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, California, USA) on an automated DNA sequencer (ABI PRISM 3100 Genetic Analyzer; Perkin-Elmer).
Ophthalmic Examination, Definitions, and Subtype Classification of Age-Related Macular Degeneration
All of the subjects underwent a complete ophthalmic examination, including visual acuity measurement, slit-lamp biomicroscopy of the fundi, color fundus photography, optical coherence tomography, fluorescein angiography, and indocyanine green angiography. The type and status of the AMD was made by 2 retina specialists (R.W. and T.A.) before the genetic analyses.
The dry AMD subjects had many large, soft drusen, geographic atrophy with adjacent soft drusen without neovascularization, or both. The exudate AMD subjects had clear vascular CNV networks or diffuse staining of CNV membranes in the fluorescein angiography or indocyanine green angiography images, or both. The PCV patients had characteristic abnormal vascular network of choroidal vessels with polyp-like dilations at the terminals of the branches in the fluorescein angiography or indocyanine green angiography images, or both.
The AMD subtypes were diagnosed and classified using the criteria of the Age-Related Eye Disease Study Research Group. The inclusion criteria were: age 50 years or older, diagnosis of AMD in one or both eyes, and no other retinochoroidal diseases, for example, high myopia (> −6 diopters, spherical equivalent), angioid streaks, central serous chorioretinopathy, and possible ocular histoplasmosis. The control subjects were confirmed not to have clinical evidence of AMD by the findings in the same complete ophthalmologic examination that was used to identify the AMD patients.
The genotypes were in Hardy–Weinberg equilibrium. The significance of differences in the genotype frequencies among the cases and controls was tested by the Fisher exact test, depending on the cell counts. P values and odds ratios (approximated to relative risk) were calculated as a measure of the association between the ARMS2 and LOXL1 genotype and the phenotype of AMD, with the effects of the mutant allele assumed to be dominant (wild/wild vs wild/mutant and mutant/mutant combined). For each odds ratio, the P values and 95% confidence intervals were calculated using the SNPAlyze program version 4.0 (Dynacom, Yokohama, Japan). The significance of associations was determined by contingency table analysis using the chi-square or Fisher exact test. The Hardy–Weinberg equilibrium was determined by using gene frequencies obtained by simple gene counting and the chi-square test with Yates’ correction for comparing observed and expected values. Two-locus analyses were performed for the rs10490924 SNP of ARMS2 and the rs1 048661S NP of LOXL1 by comparing each genotypic combination with the baseline of homozygosity for the common allele at both loci using logistical regression (JMP software version 7.0.2; SAS Institute Inc., Cary, North Carolina, USA).
Distribution of p.Ala69Ser Single-Nucleotide Polymorphism of ARMS2 in Dry Age-Related Macular Degeneration, Exudative Age-Related Macular Degeneration, Polypoidal Choroidal Vasculopathy, and Control Subjects
The allelic frequencies for the p.Ala69Ser SNP of the ARMS2 gene for dry AMD, exudative AMD, PCV, and control subjects are presented in Table 1 . For the ARMS2 gene, the genotype frequency of the rs10490924 or p.Ala69Ser SNPs was significantly higher in the dry AMD and PCV groups (minor allele frequency, T = 0.476 in dry AMD and T = 0.467 in PCV) than in the controls (T = 0.326). However, the degree of significance was less in the dry AMD versus the control group ( P = .01) and higher in the PCV versus control group ( P = 7.7 × 10 −3 ). The T allele of the rs10490924 SNP was detected at a significantly higher frequency in patients with exudative AMD than in control subjects ( P = 8.1 × 10 −10 ).
|SNP||p.A69S (rs10490942 G/T)||P Value a||Odds Ratio (95% CI)|
|Dry AMD (n = 41)||0.476||0.524||.01||0.53 (0.32 to 0.88)|
|Exudative AMD (n = 50)||0.680||0.320||8.1 × 10 –10||4.39 (2.69 to 7.17)|
|PCV (n = 60)||0.467||0.533||7.7 × 10 –3||1.80 (1.17 to 2.80)|
|Control (n = 138)||0.326||0.674|
The genotype frequencies for the 2 ARMS2 SNPs were compared among dry AMD, exudative AMD, PCV, and control groups ( Table 2 ). For the ARMS2 gene, the genotype frequency of the p.Ala69Ser SNP was significantly higher in the dry AMD ( P = .04), the exudative AMD ( P = 3.1 × 10 −8 ), and PCV ( P = 6.9 × 10 −3 ) groups than in the control group ( Table 2 ). The SNP adhered to the Hardy–Weinberg expectations ( P > .05).
|ARMS2 p.A69S Variant||Dry AMD (n = 41)||Exudative AMD (n = 50)||PCV (n = 60)||Control (n = 138)|
|T/T||8 (19.5%)||24 (48.0%)||18 (30.0%)||16 (11.6%)|
|T/G||23 (56.1%)||20 (40.0%)||20 (33.3%)||58 (42.0%)|
|G/G||10 (24.4%)||6 (12.0%)||22 (36.7%)||64 (46.4%)|
|P value a||.04||3.1 × 10 –8||6.9 × 10 –3|
Distribution of p.Arg141Leu Single-Nucleotide Polymorphism of LOXL1 in Dry Age-Related Macular Degeneration, Exudative Age-Related Macular Degeneration, Polypoidal Choroidal Vasculopathy, and Control Subjects
The allelic frequencies of the LOXL1 SNP were compared among the dry AMD, exudative AMD, PCV, and control subjects ( Table 3 ). The T allele of rs1048661 and Arg141Leu was not detected at a statistically higher frequency in patients with dry AMD ( P = .21) and PCV ( P = .86) than in control subjects. In exudative AMD, the frequency of the T variant was higher than that of controls, but the frequency was statistically marginal (major allele frequency T = 0.620 in exudative AMD and T = 0.507 in controls).
|SNP||p.R141L (rs1048661 G/T)||P Value a|
|Dry AMD (n = 41)||0.585||0.415||.21|
|Exudative AMD (n = 50)||0.620||0.380||.05|
|PCV (n = 60)||0.517||0.483||.86|
|XFS b (n = 54)||0.947||0.053||1.5 × 10 –12|
|Control (n 26 138)||0.507||0.493|