Ophthalmic Genetics, 2014; 35(3): 151–155 ! Informa Healthcare USA, Inc. ISSN: 1381-6810 print / 1744-5094 online DOI: 10.3109/13816810.2014.921313
RESEARCH REPORT
Genetic Variants in the SKIV2L Gene in Exudative Age-related Macular Degeneration in the Japanese Population
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Seigo Yoneyama1, Yoichi Sakurada1, Fumihiko Mabuchi1, Atsushi Sugiyama1, Takeo Kubota2 and Hiroyuki Iijima1 1
Departments of Ophthalmology and 2Departments of Epigenetics, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan
ABSTRACT Background: To investigate whether genetic variant in superkiller viralicidic activity 2-like (SKIV2L) gene is associated with exudative age-related macular degeneration (AMD) including neovascular AMD, polypoidal choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP). Materials and Methods: A total of 517 patients with exudative AMD comprised of 157patients with neovascular AMD, 333 patients with PCV, and 27patients with RAP, and 205 controls were enrolled in this study. Rs429608 inSKIV2L, rs800292 in complement factor H (CFH), rs10490924 in age-related maculopathy susceptibility2 (ARMS2) gene was genotyped using TaqMan technology. Logistic regression analysis was performed to correlate the risk for exudative AMD with demographic and genetic factors. Results: The A allele frequency of rs429608 in the SKIV2L gene was significantly higher in controls (13.9%) than in those with neovascular AMD (5.7%, p = 0.002), PCV (7.2%, p = 0.003) and RAP (3.7%, p = 0.0345). After adjusting for age, gender, ARMS2 A69S, and CFHI62V, the A allele of rs429608 was significantly protective against neovascular AMD (odds ratio [OR] 0.24, 95% confidence interval [CI] 0.122–0.484, p50.001), PCV (OR 0.43, 95% CI 0.262–0.704, p = 0.001), RAP (OR 0.09, 95% CI 0.014–0.581, p = 0.011). Conclusions: A SKIV2L variant was associated with protection against exudative AMD regardless of subtypes in the Japanese population. Keywords: Age-related macular degeneration; polypoidal choroidal vasculopathy; retinal angiomatous proliferation; superkiller viralicidic activity 2-like gene
INTRODUCTION
neovascularization (CNV). PCV is characterized by a branching vascular network that terminates polypoidal lesions on indocyanine green angiography (ICGA).2 RAP is characterized by proliferation of retinal capillaries and downward progression from subretinal space to CNV.3 In a Japanese clinic-based study, PCV accounts for more than half of presumed exudative AMD.4 Previous reports have described the many similarities and differences between neovascular AMD, PCV and RAP including their manifestations, genetic and clinical background.5–8
Age-related macular degeneration (AMD) is a leading cause of severe vision loss in older people in developed countries.1 The advanced stage of AMD is classified into dry or exudative types. Exudative AMD is subdivided into neovascular AMD, polypoidal choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP). Neovascular AMD is characterized by progressive breakdown of the macula caused by choroidal
Received 12 November 2013; revised 14 March 2014; accepted 11 April 2014; published online 23 May 2014 Correspondence: Yoichi Sakurada, Department of Ophthalmology, Faculty of Medicine, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 409-3898, Japan. Tel: +81 55 273 9657. Fax: +81 55 273 6757. E-mail: [email protected]
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152 S. Yoneyama et al. Two major genetic loci were found which increase the risk of AMD in different races.9–11 These are located in the complement factor H (CFH) gene at 1q32 and age-related maculopathy susceptibility 2(ARMS2) at 10q26. It has been demonstrated that these genetic loci are also associated with PCV and RAP in the Japanese population.5,6 In addition to these loci in genetic susceptibility to exudative AMD, complement component 2 (C2) and factor B (CFB), which encode the regulation of the complement pathway, were found to be associated with protection against AMD in Caucasians although several studies in East Asian showed inconsistent results.12–19 The superkiller viralicidic activity 2-like (SKIV2L) and RD RNA-binding protein (RDBP) genes are located adjacent to the C2/CFB region at 6p21. A recent genome-wide association study revealed that this region is protective against AMD in Caucasians.20 Kondo and co-authors21 reported that rs2075702 in the SKIV2L gene is significantly associated with protection against PCV in the Japanese cohort. Moreover, Liu and colleagues22 investigated the C2-CFB-RDBP-SKIV2L region and reported that rs429608 and rs453821in the SKIV2L gene are associated with neovascular AMD and they also reported that rs429608 is a responsible variant in the C2-CFB-RDBP-SKIV2L region for neovascular AMD in the Chinese cohort though this variant is not associated with PCV. In the present study, we investigated rs429608 in the SKIV2L gene and the association between this variant and neovascular AMD, PCV, and RAP in the Japanese population.
MATERIALS AND METHODS Consecutive patients with exudative AMD including 333 patients with PCV, 157 patients with neovascular AMD and 27 patients with RAP were recruited from the Macular Clinic, Department of Ophthalmology, University of Yamanashi Hospital. A total of 205 patients who had no retinal or choroidal vascular pathology were recruited from the Department of Ophthalmology, University of Yamanashi Hospital and served as controls. This study was reviewed and approved by the Ethics and Gene Analysis Committee of the Faculty of Medicine, University of Yamanashi and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from each subject. All patients with exudative AMD including neovascular AMD, PCV, and RAP underwent comprehensive ophthalmic examination including best-corrected visual acuity, slit-lamp biomicroscopy with a +78D lens, color fundus photography, fluorescein angiography (FA), indocyanine angiography (ICGA) using a fundus camera
(TRC-50LX/IMAGENET2000, Topcon, Tokyo, Japan) or a confocal laser scanning ophthalmoscope HRA2 (Heidelberg Engineering, Dossenheim, Germany) and optical coherence tomography (OCT) using SD-OCT Spectralis (Heidelberg Engineering) or Cirrus HD-OCT system (Carl Zeiss Meditec, Dublin, California, USA). The diagnosis of PCV was mainly based on the ICGA findings, which indicated clusters of polypoidal dilation of the vessels with or without abnormal vascular networks in the superficial choroid in eyes with PCV. Eyes with neovascular AMD exhibited classic CNV or occult CNV in FA without polypoidal lesions in ICGA. The diagnosis of RAP is based on OCT and angiography including FA and ICGA, which showed retinal–retinal anastomosis and retinal–choroidal anastomosis. Peripheral blood was collected from the study participants. Genomic DNA was purified using a PUREGENE DNA Isolation Kit (Gentra Systems, Minneapolis, MN). We genotyped three single nucleotide polymorphisms (SNPs) including rs429608 in the SKIV2L gene, rs800292 in the CFH gene, and rs10490924 in the ARMS2 gene. Genotyping was performed using TaqMan genotyping assays with a 7300/7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) in accordance with the manufacturer’s recommendations. Statistical analyses were performed using DR SPSS for Windows (SPSS Inc., Tokyo, Japan). Differences of categorical variables including gender distribution, genotypic and allelic frequencies between cases and control subjects were estimated by chi-square test. Differences of continuous variables including age between cases and controls were compared using the Mann-Whitney U test. A chi-square test of the Hardy-Weinberg equilibrium for each variant was performed for cases and controls. A p value less than 0.05 was statistically significant. Logistic regression analysis was also performed to reveal the demographic and genetic risk factors for each subtype of exudative AMD.
RESULTS Table 1 showed the age and gender distribution in the study participants. Mean age was significantly younger in the control subjects than in neovascular AMD and RAP patients (p50.001) though the mean age of the PCV patients was not significantly different from that of PCV (p = 0.285). The proportion of females is significantly higher in control subjects than in PCV patients and neovascular AMD patients (both p50.001) though there was not a significant difference in gender distribution between controls and patients with RAP (p = 0.18). Ophthalmic Genetics
SKIV2L Gene in Exudative AMD 153 TABLE 1. Demographic characteristics of controls and patients with exudative age-related macular degeneration.
Age Gender male
Controls (n = 205)
nAMD (n = 157)
p Value (versus control)
PCV (n = 333)
p Value (versus control)
RAP (n = 27)
p Value (versus control)
72.4 ± 8.8 104 (50.7%)
75.6 ± 8.4 114 (72.6%)
50.0001 50.0001
73.1 ± 8.2 253 (76.0%)
0.285 50.0001
82.7 ± 7.9 10 (37.0%)
50.0001 0.18
nAMD, neovascular age-related macular degeneration; PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatous proliferation
TABLE 2. Genetic characteristics of controls and patients with exudative age-related macular degeneration.
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Control (n = 205)
nAMD (n = 157)
rs800292 (CFH) Genotype GG 74 (36.1%) GA 89 (43.4%) AA 42 (20.5%) G allele frequencies 57.90%
85 (54.1%) 57 (36.3%) 15 (9.6%) 72.30%
rs10490924 (ARMS2) Genotype TT 26 (12.7%) TG 87 (42.4%) GG 92 (44.9%) T allele frequencies 33.90%
73 (46.5%) 60 (38.2%) 24 (15.3%) 65.6%
rs429608 (SKIV2L) Genotype AA 3 (1.5%) AG 51 (24.9%) GG 151 (73.6%) A allele frequencies 13.90%
0 (0%) 18 (11.5%) 139 (88.5%) 5.70%
p Value (versus control)
PCV (n = 333)
0.0007 50.0001
180 (54.1%) 122 (36.6%) 31 (9.3%) 72.30%
50.0001 50.0001
125 (37.5%) 150 (45.1%) 58 (17.4%) 60.00%
0.0014 0.0002
1 (0.3%) 46 (13.8%) 286 (85.9%) 7.20%
p Value (versus control)
RAP (n = 27)
p Value (versus control)
50.0001 50.0001
17 (63.0%) 10 (37.0%) 0 (0%) 81.50%
0.0063 0.0008
50.0001 50.0001
17 (63.0%) 9 (33.3%) 1 (3.7%) 79.60%
50.0001 50.0001
0.0013 0.0003
0 (0%) 2 (7.4%) 25 (92.6%) 3.70%
0.095 0.0345
nAMD, neovascular age-related macular degeneration; ARMS, age-related maculopathy susceptibility; CFH, complement factor H; PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatous proliferation; SKIV2L, super killer viralicidic activity 2-like
TABLE 3. Logistic regression analysis of risk factors associated with exudative age-related macular degeneration. nAMD versus control p Value Odds ratio Age Male gender CFH (G allele) ARMS2 (T allele) SKIV2L (A allele)
0.001 50.001 50.001 50.001 50.001
1.05 3.52 2.03 3.67 0.243
95% CI 1.02–1.08 2.06–6.04 1.39–2.95 2.58–5.22 0.122–0.484
PCV versus control p Value Odds ratio 0.26 50.001 50.001 50.001 0.001
1.01 3.06 1.93 2.96 0.429
95% CI 0.99–1.04 2.02–4.62 1.45–2.56 2.22–3.93 0.262–0.704
RAP versus control p Value Odds ratio 50.001 0.422 0.001 50.001 0.011
1.2 0.611 7.08 11.6 0.09
95% CI 1.1–1.31 0.18–2.04 2.27–22.1 4.05–33.0 0.014–0.581
nAMD, neovascular age-related macular degeneration; ARMS, age-related maculopathy susceptibility; CFH, complement factor H; CI, confidence interval; PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatous proliferation; SKILV2, super killer viralicidic activity 2-like
The distribution of genotypes and alleles in the three single-nucleotide polymorphisms (SNPs) are shown in Table 2. All three SNPs in the controls and cases were in Hardy-Weinberg equilibrium (all p40.05). All three SNPs were associated with both neovascular AMD and PCV. Rs10490924 (ARMS2 A69S) and rs800292 (CFH I62V) were associated with RAP (p = 0.0008, and p50.001, respectively). The T allele frequency of ARMS2 A69S was significantly higher in RAP than in AMD and PCV (p = 0.0418 and p = 0.0045, !
2014 Informa Healthcare USA, Inc.
respectively) though there was not a significant difference in minor allele frequencies in the CFH and the SKIV2L gene among three subtypes of exudative AMD. Table 3 showed the logistic regression analysis of risk factors associated with each subtype of exudative AMD. After adjusting for confounding factors, the A allele of rs429608 (SKIV2L) was significantly protective against each subtype of exudative AMD (AMD: odds ratio 0.24, p50.001, PCV: odds ratio 0.429, p = 0.001, RAP: odds ratio 0.09, p = 0.011).
154 S. Yoneyama et al.
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DISCUSSION In addition to established variants associated with exudative AMD including CFH and ARMS2, we investigated the genetic variant in the SKIV2L gene in the present study. We found that the A allele of rs429608 in the SKIV2L gene is protective against neovascular AMD, PCV and RAP. As was previously reported in Asians,23,24 rs800292 in CFH and rs10490924 in ARMS2 were strongly associated with neovascular AMD, PCV, and RAP in the current study, although the strength of the association with rs10490924 in ARMS2 was subtly different among three subtypes of exudative AMD. Hayashi and co-workers5 reported that the T allele frequencies at A69S of the ARMS2 gene are significantly higher in the order of RAP, neovascular AMD, and PCV. Tanaka and co-authors6 reported that there is no significant difference in the T allele frequency of ARMS2 A69S between neovascular AMD and PCV. In the present study, the T allele frequency of the ARMS2 gene was higher in RAP than other subtypes without a significant difference between neovascular AMD and PCV (p = 0.095, chi-square test). Inconsistency among the studies in terms of the strength of association with rs10490924 in ARMS2 among subtypes of exudative AMD may probably arise from the relatively small sample size especially of patients with RAP. SKIV2L is a putative RNA helicase and encodes proteins potentially involved in RNA splicing, translation, and turnover.25 It was first identified from an expression library derived from bovine retinal pigment epithelium (RPE) cells.26 Recently it has been reported that SKIV2L is expressed in human RPE.27 Since exudative AMD including neovascular AMD, PCV, and RAP are degenerative diseases at the level of RPE/choroid, SKIV2L might be involved in the integrity of RPE. A previous study reported by Lu and co-workers27 demonstrated that rs429608 in the SKIV2L gene is significantly associated with exudative AMD including neovascular AMD and PCV though they did not differentiate neovascular AMD from PCV. On the other hand, Liu and colleagues22 reported that rs429608 is associated with neovascular AMD, but not PCV. In the present study, A allele frequencies of rs429608 were significantly lower in neovascular AMD and PCV than in controls (p = 0.0002 and p = 0.0003, respectively). While the association between RAP and the variant of the SKIV2L was marginal (p = 0.0345, chi-square test), we confirmed the association using logistic regression analysis. As a result, the A allele of the SKIV2L gene was also protective against RAP (p = 0.011, logistic regression analysis). These results indicated that the A allele in the SKIV2L gene is significantly associated with protection against exudative AMD regardless of
subtype. This is the first report investigating the association between RAP and a variant of SKIV2L gene. There was no significant difference in the A allele frequency of rs429608 among three subtypes in exudative AMD, which might indicate that three subtypes of exudative AMD share an equivalent genetic background in terms of rs429608 in the SKIV2L gene. As rs429608 is located on the intronic region in the SKIV2L gene it does not change the amino acid sequence. Further studies are necessary to reveal how this variant affects the gene expression. The major limitation of the present study is that we examined only one SNP, which was previously reported to be associated with AMD, in the SKIV2L gene. Based on the present results, further studies would be needed by investigating the region spanning the C2-CFB-RDBP-SKIV2L gene. A second limitation of the study was the small sample size of RAP patients because it has been reported that prevalence of RAP is about 5% in exudative AMD. To confirm the present result, it would be necessary to perform a study including a larger number of RAP patients. In conclusion, rs429608 in the SKIV2L gene was significantly associated with protection against exudative AMD including neovascular AMD, PCV, and RAP.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported in part by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant Number 23791972.
REFERENCES 1. Friedman DS, O’Colmain BJ, Munoz B, et al. Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol 2004;122:564–572. 2. Spaide RF, Yannuzzi LA, Slakter JS, et al. Indocyanine green videoangiography of idiopathic polypoidal choroidal vasculopathy. Retina 1995;15:100–110. 3. Yannuzzi LA, Negrao S, Iida T, et al. Retinal angiomatous proliferation in age-related macular degeneration. Retina 2001;21:416–434. 4. Maruko I, Iida T, Saito M, et al. Clinical characteristics of exudative age-related macular degeneration in Japanese patients. Am J Ophthalmol 2007;144:15–22. 5. Hayashi H, Yamashiro K, Gotoh N, et al. CFH and ARMS2 variations in age-related macular degeneration, polypoidal choroidal vasculopathy, and retinal angiomatous proliferation. Invest Ophthalmol Vis Sci 2010;51:5914–5919. 6. Tanaka K, Nakayama T, Yuzawa M, et al. Analysis of candidate genes for age-related macular degeneration Ophthalmic Genetics
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subtypes in the Japanese population. Mol Vis 2011;17: 2751–2758. Koizumi H, Yamagishi T, Yamazaki T, et al. Subfoveal choroidal thickness in typical age-related macular degeneration and polypoidal choroidal vasculopathy. Graefes Arch Clin Exp Ophthalmol 2011;249:1123–1128. Sakurada Y, Yoneyama S, Imasawa M, Iijima H. Systemic risk factors associated with polypoidal choroidal vasculopathy and neovascular age-related macular degeneration. Retina 2013;33:841–845. Xu Y, Guan N, Xu J, et al. Association of CFH, LOC387715, and HTRA1 polymorphisms with exudative age-related macular degeneration in a northern Chinese population. Mol Vis 2008;14:1373–1381. Buentello-Volante B, Rodriguez-Ruiz G, Miranda-Duarte A, et al. Susceptibility to advanced age-related macular degeneration and alleles of complement factor H, complement factor B, complement component 2, complement component 3, and age-related maculopathy susceptibility 2 genes in a Mexican population. Mol Vis 2012;18:2518–2525. Abbas RO, Azzazy HM. Association of single nucleotide polymorphisms in CFH, ARMS2 and HTRA1 genes with risk of age-related macular degeneration in Egyptian Patients. Ophthalmic Genet 2013;34:209–216. Gold B, Merriam JE, Zernant J, et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet 2006;38: 458–462. Spencer KL, Hauser MA, Olson LM, et al. Protective effect of complement factor B and complement component 2 variants in age-related macular degeneration. Hum Mol Genet 2007;16:1986–1992. Francis PJ, Hamon SC, Ott J, et al. Polymorphisms in C2, CFB and C3 are associated with progression to advanced age related macular degeneration associated with visual loss. J Med Genet 2009;46:300–307. McKay GJ, Silvestri G, Patterson CC, et al. Further assessment of the complement component 2 and factor B region associated with age-related macular degeneration. Invest Ophthalmol Vis Sci 2009;50:533–539. Liu X, Zhao P, Tang S, et al. Association study of complement factor H, C2, CFB, and C3 and age-related macular degeneration in a Han Chinese population. Retina 2010;30:1177–1184.
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17. Nakata I, Yamashiro K, Yamada R, et al. Significance of C2/CFB variants in age-related macular degeneration and polypoidal choroidal vasculopathy in a Japanese population. Invest Ophthalmol Vis Sci 2012;53:794–798. 18. Tian J, Yu W, Qin X, et al. Association of genetic polymorphisms and age-related macular degeneration in Chinese population. Invest Ophthalmol Vis Sci 2012;53: 4262–4269. 19. Kim SJ, Lee SJ, Kim NR, Chin HS. Association of polymorphisms in C2, CFB and C3 with exudative agerelated macular degeneration in a Korean population. Exp Eye Res 2012;96:42–47. 20. Kopplin LJ, Igo Jr RP, Wang Y, et al. Genome-wide association identifies SKIV2L and MYRIP as protective factors for age-related macular degeneration. Genes Immun 2010;11:609–621. 21. Kondo N, Honda S, Kuno S, Negi A. Role of RDBP and SKIV2L variants in the major histocompatibility complex class III region in polypoidal choroidal vasculopathy etiology. Ophthalmology 2009;116:1502–1509. 22. Liu K, Chen LJ, Tam PO, et al. Associations of the C2-CFB-RDBP-SKIV2L locus with age-related macular degeneration and polypoidal choroidal vasculopathy. Ophthalmology 2013;120:837–843. 23. Mori K, Horie-Inoue K, Gehlbach PL, et al. Phenotype and genotype characteristics of age-related macular degeneration in a Japanese population. Ophthalmology 2010;117: 928–938. 24. Zhang X, Li M, Wen F, et al. Different impact of highdensity lipoprotein-related genetic variants on polypoidal choroidal vasculopathy and neovascular age-related macular degeneration in a Chinese Han population. Exp Eye Res 2013;108:16–22. 25. Schmid M, Jensen TH. The exosome: a multipurpose RNA-decay machine. Trends Biochem Sci 2008;33:501–510. 26. Lokki ML, Koskimies SA. Allelic differences in hemolytic activity and protein concentration of BF molecules are found in association with particular HLA haplotypes. Immunogenetics 1991;34:242–246. 27. Lu F, Shi Y, Qu C, et al. A genetic variant in the SKIV2L gene is significantly associated with age-related macular degeneration in a Han Chinese population. Invest Ophthalmol Vis Sci 2013;54:2911–2917.
RESEARCH REPORT
Genetic Variants in the SKIV2L Gene in Exudative Age-related Macular Degeneration in the Japanese Population
Ophthalmic Genet Downloaded from informahealthcare.com by Mcgill University on 01/26/15 For personal use only.
Seigo Yoneyama1, Yoichi Sakurada1, Fumihiko Mabuchi1, Atsushi Sugiyama1, Takeo Kubota2 and Hiroyuki Iijima1 1
Departments of Ophthalmology and 2Departments of Epigenetics, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan
ABSTRACT Background: To investigate whether genetic variant in superkiller viralicidic activity 2-like (SKIV2L) gene is associated with exudative age-related macular degeneration (AMD) including neovascular AMD, polypoidal choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP). Materials and Methods: A total of 517 patients with exudative AMD comprised of 157patients with neovascular AMD, 333 patients with PCV, and 27patients with RAP, and 205 controls were enrolled in this study. Rs429608 inSKIV2L, rs800292 in complement factor H (CFH), rs10490924 in age-related maculopathy susceptibility2 (ARMS2) gene was genotyped using TaqMan technology. Logistic regression analysis was performed to correlate the risk for exudative AMD with demographic and genetic factors. Results: The A allele frequency of rs429608 in the SKIV2L gene was significantly higher in controls (13.9%) than in those with neovascular AMD (5.7%, p = 0.002), PCV (7.2%, p = 0.003) and RAP (3.7%, p = 0.0345). After adjusting for age, gender, ARMS2 A69S, and CFHI62V, the A allele of rs429608 was significantly protective against neovascular AMD (odds ratio [OR] 0.24, 95% confidence interval [CI] 0.122–0.484, p50.001), PCV (OR 0.43, 95% CI 0.262–0.704, p = 0.001), RAP (OR 0.09, 95% CI 0.014–0.581, p = 0.011). Conclusions: A SKIV2L variant was associated with protection against exudative AMD regardless of subtypes in the Japanese population. Keywords: Age-related macular degeneration; polypoidal choroidal vasculopathy; retinal angiomatous proliferation; superkiller viralicidic activity 2-like gene
INTRODUCTION
neovascularization (CNV). PCV is characterized by a branching vascular network that terminates polypoidal lesions on indocyanine green angiography (ICGA).2 RAP is characterized by proliferation of retinal capillaries and downward progression from subretinal space to CNV.3 In a Japanese clinic-based study, PCV accounts for more than half of presumed exudative AMD.4 Previous reports have described the many similarities and differences between neovascular AMD, PCV and RAP including their manifestations, genetic and clinical background.5–8
Age-related macular degeneration (AMD) is a leading cause of severe vision loss in older people in developed countries.1 The advanced stage of AMD is classified into dry or exudative types. Exudative AMD is subdivided into neovascular AMD, polypoidal choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP). Neovascular AMD is characterized by progressive breakdown of the macula caused by choroidal
Received 12 November 2013; revised 14 March 2014; accepted 11 April 2014; published online 23 May 2014 Correspondence: Yoichi Sakurada, Department of Ophthalmology, Faculty of Medicine, University of Yamanashi, Shimokato 1110, Chuo, Yamanashi, 409-3898, Japan. Tel: +81 55 273 9657. Fax: +81 55 273 6757. E-mail: [email protected]
151
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152 S. Yoneyama et al. Two major genetic loci were found which increase the risk of AMD in different races.9–11 These are located in the complement factor H (CFH) gene at 1q32 and age-related maculopathy susceptibility 2(ARMS2) at 10q26. It has been demonstrated that these genetic loci are also associated with PCV and RAP in the Japanese population.5,6 In addition to these loci in genetic susceptibility to exudative AMD, complement component 2 (C2) and factor B (CFB), which encode the regulation of the complement pathway, were found to be associated with protection against AMD in Caucasians although several studies in East Asian showed inconsistent results.12–19 The superkiller viralicidic activity 2-like (SKIV2L) and RD RNA-binding protein (RDBP) genes are located adjacent to the C2/CFB region at 6p21. A recent genome-wide association study revealed that this region is protective against AMD in Caucasians.20 Kondo and co-authors21 reported that rs2075702 in the SKIV2L gene is significantly associated with protection against PCV in the Japanese cohort. Moreover, Liu and colleagues22 investigated the C2-CFB-RDBP-SKIV2L region and reported that rs429608 and rs453821in the SKIV2L gene are associated with neovascular AMD and they also reported that rs429608 is a responsible variant in the C2-CFB-RDBP-SKIV2L region for neovascular AMD in the Chinese cohort though this variant is not associated with PCV. In the present study, we investigated rs429608 in the SKIV2L gene and the association between this variant and neovascular AMD, PCV, and RAP in the Japanese population.
MATERIALS AND METHODS Consecutive patients with exudative AMD including 333 patients with PCV, 157 patients with neovascular AMD and 27 patients with RAP were recruited from the Macular Clinic, Department of Ophthalmology, University of Yamanashi Hospital. A total of 205 patients who had no retinal or choroidal vascular pathology were recruited from the Department of Ophthalmology, University of Yamanashi Hospital and served as controls. This study was reviewed and approved by the Ethics and Gene Analysis Committee of the Faculty of Medicine, University of Yamanashi and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from each subject. All patients with exudative AMD including neovascular AMD, PCV, and RAP underwent comprehensive ophthalmic examination including best-corrected visual acuity, slit-lamp biomicroscopy with a +78D lens, color fundus photography, fluorescein angiography (FA), indocyanine angiography (ICGA) using a fundus camera
(TRC-50LX/IMAGENET2000, Topcon, Tokyo, Japan) or a confocal laser scanning ophthalmoscope HRA2 (Heidelberg Engineering, Dossenheim, Germany) and optical coherence tomography (OCT) using SD-OCT Spectralis (Heidelberg Engineering) or Cirrus HD-OCT system (Carl Zeiss Meditec, Dublin, California, USA). The diagnosis of PCV was mainly based on the ICGA findings, which indicated clusters of polypoidal dilation of the vessels with or without abnormal vascular networks in the superficial choroid in eyes with PCV. Eyes with neovascular AMD exhibited classic CNV or occult CNV in FA without polypoidal lesions in ICGA. The diagnosis of RAP is based on OCT and angiography including FA and ICGA, which showed retinal–retinal anastomosis and retinal–choroidal anastomosis. Peripheral blood was collected from the study participants. Genomic DNA was purified using a PUREGENE DNA Isolation Kit (Gentra Systems, Minneapolis, MN). We genotyped three single nucleotide polymorphisms (SNPs) including rs429608 in the SKIV2L gene, rs800292 in the CFH gene, and rs10490924 in the ARMS2 gene. Genotyping was performed using TaqMan genotyping assays with a 7300/7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) in accordance with the manufacturer’s recommendations. Statistical analyses were performed using DR SPSS for Windows (SPSS Inc., Tokyo, Japan). Differences of categorical variables including gender distribution, genotypic and allelic frequencies between cases and control subjects were estimated by chi-square test. Differences of continuous variables including age between cases and controls were compared using the Mann-Whitney U test. A chi-square test of the Hardy-Weinberg equilibrium for each variant was performed for cases and controls. A p value less than 0.05 was statistically significant. Logistic regression analysis was also performed to reveal the demographic and genetic risk factors for each subtype of exudative AMD.
RESULTS Table 1 showed the age and gender distribution in the study participants. Mean age was significantly younger in the control subjects than in neovascular AMD and RAP patients (p50.001) though the mean age of the PCV patients was not significantly different from that of PCV (p = 0.285). The proportion of females is significantly higher in control subjects than in PCV patients and neovascular AMD patients (both p50.001) though there was not a significant difference in gender distribution between controls and patients with RAP (p = 0.18). Ophthalmic Genetics
SKIV2L Gene in Exudative AMD 153 TABLE 1. Demographic characteristics of controls and patients with exudative age-related macular degeneration.
Age Gender male
Controls (n = 205)
nAMD (n = 157)
p Value (versus control)
PCV (n = 333)
p Value (versus control)
RAP (n = 27)
p Value (versus control)
72.4 ± 8.8 104 (50.7%)
75.6 ± 8.4 114 (72.6%)
50.0001 50.0001
73.1 ± 8.2 253 (76.0%)
0.285 50.0001
82.7 ± 7.9 10 (37.0%)
50.0001 0.18
nAMD, neovascular age-related macular degeneration; PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatous proliferation
TABLE 2. Genetic characteristics of controls and patients with exudative age-related macular degeneration.
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Control (n = 205)
nAMD (n = 157)
rs800292 (CFH) Genotype GG 74 (36.1%) GA 89 (43.4%) AA 42 (20.5%) G allele frequencies 57.90%
85 (54.1%) 57 (36.3%) 15 (9.6%) 72.30%
rs10490924 (ARMS2) Genotype TT 26 (12.7%) TG 87 (42.4%) GG 92 (44.9%) T allele frequencies 33.90%
73 (46.5%) 60 (38.2%) 24 (15.3%) 65.6%
rs429608 (SKIV2L) Genotype AA 3 (1.5%) AG 51 (24.9%) GG 151 (73.6%) A allele frequencies 13.90%
0 (0%) 18 (11.5%) 139 (88.5%) 5.70%
p Value (versus control)
PCV (n = 333)
0.0007 50.0001
180 (54.1%) 122 (36.6%) 31 (9.3%) 72.30%
50.0001 50.0001
125 (37.5%) 150 (45.1%) 58 (17.4%) 60.00%
0.0014 0.0002
1 (0.3%) 46 (13.8%) 286 (85.9%) 7.20%
p Value (versus control)
RAP (n = 27)
p Value (versus control)
50.0001 50.0001
17 (63.0%) 10 (37.0%) 0 (0%) 81.50%
0.0063 0.0008
50.0001 50.0001
17 (63.0%) 9 (33.3%) 1 (3.7%) 79.60%
50.0001 50.0001
0.0013 0.0003
0 (0%) 2 (7.4%) 25 (92.6%) 3.70%
0.095 0.0345
nAMD, neovascular age-related macular degeneration; ARMS, age-related maculopathy susceptibility; CFH, complement factor H; PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatous proliferation; SKIV2L, super killer viralicidic activity 2-like
TABLE 3. Logistic regression analysis of risk factors associated with exudative age-related macular degeneration. nAMD versus control p Value Odds ratio Age Male gender CFH (G allele) ARMS2 (T allele) SKIV2L (A allele)
0.001 50.001 50.001 50.001 50.001
1.05 3.52 2.03 3.67 0.243
95% CI 1.02–1.08 2.06–6.04 1.39–2.95 2.58–5.22 0.122–0.484
PCV versus control p Value Odds ratio 0.26 50.001 50.001 50.001 0.001
1.01 3.06 1.93 2.96 0.429
95% CI 0.99–1.04 2.02–4.62 1.45–2.56 2.22–3.93 0.262–0.704
RAP versus control p Value Odds ratio 50.001 0.422 0.001 50.001 0.011
1.2 0.611 7.08 11.6 0.09
95% CI 1.1–1.31 0.18–2.04 2.27–22.1 4.05–33.0 0.014–0.581
nAMD, neovascular age-related macular degeneration; ARMS, age-related maculopathy susceptibility; CFH, complement factor H; CI, confidence interval; PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatous proliferation; SKILV2, super killer viralicidic activity 2-like
The distribution of genotypes and alleles in the three single-nucleotide polymorphisms (SNPs) are shown in Table 2. All three SNPs in the controls and cases were in Hardy-Weinberg equilibrium (all p40.05). All three SNPs were associated with both neovascular AMD and PCV. Rs10490924 (ARMS2 A69S) and rs800292 (CFH I62V) were associated with RAP (p = 0.0008, and p50.001, respectively). The T allele frequency of ARMS2 A69S was significantly higher in RAP than in AMD and PCV (p = 0.0418 and p = 0.0045, !
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respectively) though there was not a significant difference in minor allele frequencies in the CFH and the SKIV2L gene among three subtypes of exudative AMD. Table 3 showed the logistic regression analysis of risk factors associated with each subtype of exudative AMD. After adjusting for confounding factors, the A allele of rs429608 (SKIV2L) was significantly protective against each subtype of exudative AMD (AMD: odds ratio 0.24, p50.001, PCV: odds ratio 0.429, p = 0.001, RAP: odds ratio 0.09, p = 0.011).
154 S. Yoneyama et al.
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DISCUSSION In addition to established variants associated with exudative AMD including CFH and ARMS2, we investigated the genetic variant in the SKIV2L gene in the present study. We found that the A allele of rs429608 in the SKIV2L gene is protective against neovascular AMD, PCV and RAP. As was previously reported in Asians,23,24 rs800292 in CFH and rs10490924 in ARMS2 were strongly associated with neovascular AMD, PCV, and RAP in the current study, although the strength of the association with rs10490924 in ARMS2 was subtly different among three subtypes of exudative AMD. Hayashi and co-workers5 reported that the T allele frequencies at A69S of the ARMS2 gene are significantly higher in the order of RAP, neovascular AMD, and PCV. Tanaka and co-authors6 reported that there is no significant difference in the T allele frequency of ARMS2 A69S between neovascular AMD and PCV. In the present study, the T allele frequency of the ARMS2 gene was higher in RAP than other subtypes without a significant difference between neovascular AMD and PCV (p = 0.095, chi-square test). Inconsistency among the studies in terms of the strength of association with rs10490924 in ARMS2 among subtypes of exudative AMD may probably arise from the relatively small sample size especially of patients with RAP. SKIV2L is a putative RNA helicase and encodes proteins potentially involved in RNA splicing, translation, and turnover.25 It was first identified from an expression library derived from bovine retinal pigment epithelium (RPE) cells.26 Recently it has been reported that SKIV2L is expressed in human RPE.27 Since exudative AMD including neovascular AMD, PCV, and RAP are degenerative diseases at the level of RPE/choroid, SKIV2L might be involved in the integrity of RPE. A previous study reported by Lu and co-workers27 demonstrated that rs429608 in the SKIV2L gene is significantly associated with exudative AMD including neovascular AMD and PCV though they did not differentiate neovascular AMD from PCV. On the other hand, Liu and colleagues22 reported that rs429608 is associated with neovascular AMD, but not PCV. In the present study, A allele frequencies of rs429608 were significantly lower in neovascular AMD and PCV than in controls (p = 0.0002 and p = 0.0003, respectively). While the association between RAP and the variant of the SKIV2L was marginal (p = 0.0345, chi-square test), we confirmed the association using logistic regression analysis. As a result, the A allele of the SKIV2L gene was also protective against RAP (p = 0.011, logistic regression analysis). These results indicated that the A allele in the SKIV2L gene is significantly associated with protection against exudative AMD regardless of
subtype. This is the first report investigating the association between RAP and a variant of SKIV2L gene. There was no significant difference in the A allele frequency of rs429608 among three subtypes in exudative AMD, which might indicate that three subtypes of exudative AMD share an equivalent genetic background in terms of rs429608 in the SKIV2L gene. As rs429608 is located on the intronic region in the SKIV2L gene it does not change the amino acid sequence. Further studies are necessary to reveal how this variant affects the gene expression. The major limitation of the present study is that we examined only one SNP, which was previously reported to be associated with AMD, in the SKIV2L gene. Based on the present results, further studies would be needed by investigating the region spanning the C2-CFB-RDBP-SKIV2L gene. A second limitation of the study was the small sample size of RAP patients because it has been reported that prevalence of RAP is about 5% in exudative AMD. To confirm the present result, it would be necessary to perform a study including a larger number of RAP patients. In conclusion, rs429608 in the SKIV2L gene was significantly associated with protection against exudative AMD including neovascular AMD, PCV, and RAP.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was supported in part by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant Number 23791972.
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