Correction
10 Oct 2019: Wong YL, Hysi P, Cheung G, Tedja M, Hoang QV, et al. (2019) Correction: Genetic variants linked to myopic macular degeneration in persons with high myopia: CREAM Consortium. PLOS ONE 14(10): e0223942. https://doi.org/10.1371/journal.pone.0223942 View correction
Figures
Abstract
Purpose
To evaluate the roles of known myopia-associated genetic variants for development of myopic macular degeneration (MMD) in individuals with high myopia (HM), using case-control studies from the Consortium of Refractive Error and Myopia (CREAM).
Methods
A candidate gene approach tested 50 myopia-associated loci for association with HM and MMD, using meta-analyses of case-control studies comprising subjects of European and Asian ancestry aged 30 to 80 years from 10 studies. Fifty loci with the strongest associations with myopia were chosen from a previous published GWAS study. Highly myopic (spherical equivalent [SE] ≤ -5.0 diopters [D]) cases with MMD (N = 348), and two sets of controls were enrolled: (1) the first set included 16,275 emmetropes (SE ≤ -0.5 D); and (2) second set included 898 highly myopic subjects (SE ≤ -5.0 D) without MMD. MMD was classified based on the International photographic classification for pathologic myopia (META-PM).
Results
In the first analysis, comprising highly myopic cases with MMD (N = 348) versus emmetropic controls without MMD (N = 16,275), two SNPs were significantly associated with high myopia in adults with HM and MMD: (1) rs10824518 (P = 6.20E-07) in KCNMA1, which is highly expressed in human retinal and scleral tissues; and (2) rs524952 (P = 2.32E-16) near GJD2. In the second analysis, comprising highly myopic cases with MMD (N = 348) versus highly myopic controls without MMD (N = 898), none of the SNPs studied reached Bonferroni-corrected significance.
Citation: Wong Y-L, Hysi P, Cheung G, Tedja M, Hoang QV, Tompson SWJ, et al. (2019) Genetic variants linked to myopic macular degeneration in persons with high myopia: CREAM Consortium. PLoS ONE 14(8): e0220143. https://doi.org/10.1371/journal.pone.0220143
Editor: Yong-Gang Yao, Kunming Institute of Zoology, Chinese Academy of Sciences, CHINA
Received: March 14, 2019; Accepted: June 20, 2019; Published: August 15, 2019
Copyright: © 2019 Wong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: YLW is an employee of Essilor International, Singapore. The funder provided support in the form of salaries for author YLW. Author KOM is a consultant for Bayer, Nevakah, and Santen. The funder provided support in the form of consultancy fees for author KOM. These funders do not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author are articulated in the ‘author contributions’ section.
Competing interests: YLW is an employee of Essilor International, Singapore and received funding in the form of salary. Author KOM is a consultant for Bayer, Nevakah, and Santen, and receiving support in the form of consultancy fees. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.
Introduction
Myopia is a refractive error condition that can usually be corrected with visual aids. It may however result in significant complications, as high myopia (HM) increases the risk of myopic macular degeneration (MMD). MMD, defined as the presence of myopia-specific retinal pathology from excessive axial elongation, is characterized by structural degeneration of the retina and associated with changes in the scleral wall [1]. MMD is one of the leading causes of irreversible loss of vision and blindness worldwide [2–5]. Numerous genome-wide association studies (GWAS) have identified multiple genetic variants associated with myopia or spherical equivalent (SE) in the general population [6–12]. Several association studies [13–19] also suggested overlapping genetic risk between myopia and HM that often correlate with blinding complications [20]. Currently, only a relatively small number of loci have been associated with HM [21–26].
Several single nucleotide polymorphisms (SNPs) associated with MMD have been identified in previous GWAS analyses in Japanese populations [27]. However, only one GWAS identified a locus specific to MMD at rs11873439 in CCDC102B, which compared high myopes with MMD (cases) with high myopes without MMD (controls) [28]. In addition, some studies had ambiguous definitions of MMD that did not refer to a single and formal classification system, which limits comparability of findings [29, 30]. Therefore, genetic determinants of MMD require validation using the recently established International META-PM classification system for MMD [28].
For two of the most highly significant SNPs first associated with refractive error (GJD2 and RASGRF1), no association with MMD was found in an ethnically-homogenous Chinese population [31]. Further international studies with ethnically diverse populations are needed to evaluate the roles of these variants in MMD.
In this study, we evaluated the roles of known myopia-associated genetic variants in HM and MMD, using case-control studies from the Consortium of Refractive Error and Myopia (CREAM).
Methods
Study population and design
Subjects of either European or Asian ancestry, with available genome-wide genotyping and MMD status information, from 10 different cohorts participating in the CREAM consortium (Table 1), were included in this study [7, 15]. Subjects were between 30 and 80 years of age. A previous history of cataract surgery or laser refractive procedures that could alter refraction, were criteria for exclusions from the analyses. A total of 17,521 subjects were included in this study.
The prevalence of MMD is higher in Asian cohorts, therefore most cases included in the analyses were of Asian ancestry. The Singapore Epidemiology of Eye Diseases (SEED) studies, consisting of the Singapore Chinese Eye Study (SCES), the Singapore Malay Eye Study (SiMES) and the Singapore Indian Eye Study (SINDI), contributed a total of 81 cases, 4,777 controls (set 1), and 245 controls (set 2) [32, 33]. Another study consisting of individuals of Asian ancestry, namely the Nagahama Study, contributed 130 cases, 1,537 controls (set 1), and 252 controls (set 2). The Rotterdam Studies (RS), comprising the RS1, RS2 and RS3 cohorts, contributed 106 cases, 7,267 controls (set 1), and 285 controls (set 2) [34–38]. Other studies with subjects of European ancestry contributed 22 cases, 1,480 controls (set 1) and 17 controls (set 2) from the Blue Mountains Eye Study (BMES); and a total of 9 cases, 1,214 controls (set 1) and 99 controls (set 2) from the Gutenberg Health Study (GHS) 1 and GHS2 [39]. All studies were performed with the approval of their local medical ethics committee, and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. The names of the ethics committees of the individual studies are listed in the Supporting Information (S2 Text).
Phenotypic assessment
Each subject underwent detailed ophthalmologic examination. Non-cycloplegic refraction status was determined by the use of an autorefractor and/or subjective refraction. SE of refractive error was defined as sphere plus half cylinder. Emmetropia and HM were defined as SE > -0.5 D and ≤ -5.0 D in the right eye, respectively [40].
Fundus photograph grading was performed by trained graders for all HM subjects with SE ≤ -5.0 D in the right eye. The graders from each participating study underwent training by experienced retinal specialist (K.O.M.), and all participating studies defined MMD based on the International META-PM Photographic Classification and Grading System for MMD [28]. The presence of MMD was defined and classified into Meta-PM categories. MMD was graded according to increasing severity: no macular lesions (category 0), tessellated fundus only (category 1), diffuse chorioretinal atrophy (category 2), patchy chorioretinal atrophy (category 3), and macular atrophy (category 4). Based on fundus photograph grading, the subject was considered to have MMD, if Meta-PM category 2, 3, or 4, was observed [1].
Evaluation of the role of myopia-associated genetic variants with MMD
We used a candidate gene approach that tested 50 genetic variants for association with MMD. The 50 selected genetic variants were reported to be associated with myopia from the largest GWAS study published to date [11]. Two case-control analyses were performed to evaluate the roles of known myopia-associated genetic variants with the development of MMD in highly myopic persons.
The first analysis aimed at identifying genetic variants associated with HM in highly myopic subjects with MMD. It compared 348 highly myopic cases with MMD (SE ≤ -5.0 D in the right eye; mean SE range between -6.9 and -13.6 D) with 16,275 emmetropic controls without HM or MMD (SE ≥ -0.5 D in the right eye; mean SE range between 0.6 and 2.5 D). Of the 348 cases included, 137 (39.4%) and 211 (60.6%) cases were of European and Asian ancestries, respectively. Of the 16,275 emmetropic controls, 9,961 (61.2%) and 6,314 (38.8%) were of European and Asian ancestries, respectively.
The second analysis aimed at identifying genetic variants specifically associated with MMD. We used the same group of cases (348 high myopes with MMD) and compared them with a control set different from the previous one, that comprised 898 high myopes without signs of MMD (mean SE range between -5.6 and -8.0 D). Of the 898 highly myopic controls, 401 (44.7%) and 497 (55.3%) were of European and Asian ancestries, respectively.
Genotyping and imputation were executed as previously described [41]. Stringent quality control (QC) procedures of genotyping before imputation were applied in each study. Briefly, duplicate DNA samples, subjects with low call rate (< 95%), gender mismatch, or ethnic outliers were excluded. SNPs were excluded if they had a low genotyping call rate (> 5% missingness), a minor allele frequency (MAF) of less than 1%, or were Hardy-Weinberg disequilibrium (P < 10−6). After QC filtering, genomic imputation was performed using the 1000 Genomes Project data as reference panel (build 37, phase 1 release, March 2012) with Minimac [42] or IMPUTE2 [43]. SNPs with MAF ≥ 5% and imputation quality of at least 0.5 (r2 for MACH or info score for IMPUTE) were included in further analyses.
Gene expression in human ocular tissues
Adult ocular samples were obtained from the normal eyes of an 82-year-old Caucasian female from the North Carolina Eye Bank, Winston-Salem, North Carolina, USA. Fetal ocular samples were obtained from 24-week fetal eyes by Advanced Bioscience Resources Inc., Alameda, California, USA. The adult ocular samples were stored in Qiagen RNA later within 6.5 hours of collection and shipped on ice overnight to the lab. Fetal eyes were preserved in RNA later within minutes of harvesting and shipped over night on ice. Whole globes were dissected on the arrival day. Isolated tissues were snap-frozen and stored at −80°C until RNA extraction. RNA was extracted from each tissue sample independently using the Ambion mirVana total RNA extraction kit. The tissue samples were homogenized in Ambion lysis buffer with an Omni Bead Ruptor Tissue Homogenizer per protocol. Reverse transcription reactions were performed with Invitrogen SuperScript III First-Strand Synthesis kit. The expression of the identified genes was assessed by running 10 μl reactions with QIAGEN’s PCR products consisting of 1.26 μl H2O, 1.0 μl 10× buffer, 1.0 μl dNTPs, 0.3 μl MgCl, 2.0 μl Q-Solution, 0.06 μl taq polymerase, 1.0 μl forward primer, 1.0 μl reverse primer, and 1.5.0 μl cDNA. The reactions were run on an Eppendorf Mastercycler Pro S thermocycler with touchdown PCR ramping down 1°C per cycle from 72°C to 55°C followed by 50 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s with a final elongation of 7 min at 72°C. All primer sets were designed by Primer3 [44]. Gel electrophoresis was run on a 2% agarose gel at 70 volts for 35 minutes. The primers were run on a custom tissue panel including the Clontech Human MTC Panel I, Fetal MTC Panel I, and an ocular tissue panel.
Statistical analysis
Logistic regression models were performed for all studies with each SNP as predictors, and MMD as a binary outcome, with adjustments for age, gender, and principal components. To avert population stratification and inflation of the results in each cohort, the ancestry of all participants was checked via a Principal Component Analysis. Individuals who were not perfectly clustering with their respective ethnic groups were removed. Meta-analyses were performed to estimate the combined effects, using inverse-variance fixed-effect meta-analyses in METAL [45]. The meta-analyses were stratified by ancestry (European or Asian ancestry). Only SNPs that were available and polymorphic in at least 8 participating studies were considered. Of the 50 SNPs, 39 and 37 were included in the first analysis (highly myopic cases with MMD versus emmetropic controls without MMD) and second analysis (highly myopic cases with MMD versus highly myopic controls without MMD), respectively. Corrections for multiple testing were performed: PBonferroni = 0.05/39 = 1.28E-03 for the first analysis and PBonferroni = 0.05/37 = 1.35E-03 for the second analysis.
Results
The highly myopic cases with MMD (mean SE of -8.67 ± 3.3 D; N = 348) were more myopic than the highly myopic controls without MMD (mean SE of -6.89 ± 1.89 D; N = 898). The mean SE of the emmetropic controls without MMD was 1.43 ± 1.43 D (N = 16,275). Compared to the subjects of European ancestry, subjects of Asian ancestry were more myopic in the cases and two control groups (Table 1).
(1) Evaluation of genetic variants associated with HM in highly myopic subjects with MMD
In the first analysis (highly myopic cases with MMD versus emmetropic controls without MMD), two SNPs were significantly associated with HM in highly myopic subjects with MMD (Table 2). rs10824518 (P = 6.20E-07; Fig 1A) maps within the KCNMA1 gene genomic sequence and rs524952 (P = 2.32E-16; Fig 1B) about 38kbp downstream the GJD2 gene. A third SNP, rs13380104 (P = 1.73E-03; Fig 1C), located in the last intron of the RASGRF1 gene, was just short of our pre-defined Bonferroni corrected threshold of significance.
Plot of the effect on high myopia in highly myopic subjects with myopic macular degeneration for (A) rs10824518, (B) rs524952, and (C) rs13380104 in the population cohorts in first case-control study. For each cohort, the circle shows the β linear regression coefficient and the bars represent the standard error for the estimate. BMES, the Blue Mountains Eye Study, Australia; RS-I, the first Rotterdam Study cohort, Netherlands; RS-II, the second Rotterdam Study cohort, Netherlands; RS-III, the third Rotterdam Study cohort, Netherlands; GHS1, the first Gutenberg Health Study cohort, Germany; GHS2, the second Gutenberg Health Study cohort, Germany; SCES, the Singapore Chinese Eye Study, Singapore; SiMES, the Singapore Malay Eye Study, Singapore; SINDI, the Singapore Indian Eye Study, Singapore; Nagahama, the Nagahama Study cohort, Japan.
(2) Evaluation of genetic variants specifically associated with MMD
In the second analysis, (highly myopic cases with MMD versus highly myopic controls without MMD), none of the SNPs reached Bonferroni-corrected significance in this model (Table 3). The highest association was observed for rs479445 (P = 2.55E-02), located downstream of the NFIA gene.
To assess whether the SNPs associated with myopia had any role in HM and MMD predisposition, quantile-quantile plots of the P-values from each meta-analysis were examined (Fig 2). Associations of genetic variants for HM and MMD between highly myopic cases with MMD and emmetropic controls without MMD showed significance, beyond what would be expected under the assumption of a uniform distribution (Kolmogorov Smirnov for uniformity p = 3.87E-05), as the test statistic distribution deviated from expectations for the first analysis (Fig 2A). In contrast, after the influence of HM was removed in the second analysis, the associations of genetic variants for MMD between highly myopic cases with MMD and highly myopic controls without MMD were not significantly different from the null hypothesis of a uniform distribution (p = 0.64) (Fig 2B).
(A) Q-Q plot for association between analysed SNPs and HM in high myopes with MMD in first case-control study (highly myopic cases with MMD versus emmetropic controls without MMD); (B) Q-Q plot for association between analysed SNPs and MMD specifically in second case-control study (highly myopic cases with MMD versus highly myopic controls without MMD). Each dot represents an observed statistic (defined as–log10p) versus the corresponding expected statistic. The red line corresponds to the null distribution. The shaded areas represent the 95% confidence intervals.
The effect sizes of the genetic variants reported for myopia were strongly correlated with the effect sizes of the SNPs in the first analysis (Fig 3A), reflecting the correlation (Spearman’s ρ = 0.70, p = 1.15E-07) of myopia with HM and MMD. Several genetic loci (such as KCNMA1 and GJD2) displayed stronger effects over HM and MMD than SE. Alternatively, for some other loci previously associated with myopia [11], we observed weaker or no effect at all over HM and MMD (for example DLG and COL61A). There was a marginally weaker correlation of the effect sizes between the first and second case-control analysis (Spearman’s ρ = 0.59, p = 0.0001, Fig 3B), perhaps reflecting an overlap of genetic risks between HM and MMD.
(A) Relation of effect sizes observed in original study for myopia (Pickrell et al 2016) versus that in the first case-control study (highly myopic cases with MMD versus emmetropic controls without MMD) for HM in high myopes with MMD; (B) Relation of effect sizes in first case-control study (highly myopic cases with MMD versus emmetropic controls without MMD) for development of MMD in those with HM versus that in the second case-control study (highly myopic cases with MMD versus highly myopic controls without MMD) for MMD specifically.
Gene expression in human ocular tissues
As the expression and role of GJD2 and RASGRF1 in eye and myopia development have been explored and reported previously [6–9, 15], we focused on the gene expression of KCNMA1 in human ocular tissues. KCNMA1 was expressed in most adult and fetal ocular tissues, including human retina, sclera, choroid or retinal pigment epithelium (RPE), and optic nerve (Table 4). In particular, KCNMA1 was highly expressed in human retina and sclera for both fetal and adult tissues.
Discussion
Using two case-control meta-analyses, this study evaluated genetic risk factors for the development of MMD in adults with HM who have MMD. We found that KCNMA1 is linked to HM in highly myopic individuals with MMD in CREAM, a locus that had been previously identified for myopia in CREAM and 23andMe [12]. Furthermore, we replicated previously reported association on GJD2 and RASGRF1 in highly myopic individuals with MMD compared to emmetropic controls without MMD. However, these results were not replicated in the second case-control study that compared highly myopic cases with MMD and highly myopic controls without MMD. Since these genetic variants were not tested positive in both case-control studies, we found no evidence that any of the variants that we analysed, confers risk specific to MMD, beyond risk mediated through HM. These genes might be linked to development of MMD only in these highly myopic subjects with MMD.
Several genetic variants associated with MMD have been reported in the literature [29, 30], but few have been consistently replicated [31]. A previous GWAS identified a genetic locus associated with MMD at rs11873439 in CCDC102B (N = 7739; P = 1.61E-10; odds ratio [OR] of 1.46; 95% confidence interval [CI], 1.30 to 1.64). The CCDC102B gene protein may be linked to weakened connective tissue in retinal and choroid layers, which predisposes the eye to MMD [27]. Another GWAS analysis (N = 2,741) found an association between rs577948 in BLID (OR of 1.37; 95% CI, 1.21 to 1.54; P = 2.22E-07) [29], which encodes an inducer of mitochondrial cell death and apoptosis and expressed in human retina [46]. We did not find similar results to previous studies, as this could potentially be due to small sample size or the greater complexity of MMD that may have multifactorial, polygenic and environmental influences.
We have confirmed at least the KCNMA1 locus (10q22) as a susceptibility locus for HM in persons with both HM and MMD. KCNMA1 was identified as a susceptibility locus for SE and myopia in the wider general population in two previous large GWAS [6, 11, 12], but we observed much stronger effects and association near the high myopic end of the refraction spectrum. Encoding a large potassium voltage-sensitive conductance calcium-activated channel (MaxiK+) [47], KCNMA1 is mainly involved in ion channel activity [48], control of smooth muscle and neuronal development [49], action potential repolarization of neurons [50], regulation of neurotransmitter release [51] and synaptic plasticity [47]. Notably, MaxiK+ channels control synaptic transmission exclusively in the rod pathway, a light-induced signalling pathway that contributes to myopia development [52]. KCNMA1 is expressed in neurons, retinal, and RPE tissues [47, 51, 53]. MaxiK+ channels in RPE control the changes in intracellular Ca2+, in turn regulating several cell functions including dark adaptation of photoreceptor activity, differentiation and vascular endothelial growth factor (VEGF) secretion [54, 55], thereby suggesting possible involvement of KCNMA1 in myopia-related pathologic changes, such as the initiation of choroidal neovascularization and changes in the blood-retinal barrier [56]. Validation of the role of KCNMA1 in myopia progression is needed, particularly in ion channel activity which is one of the major functional pathways implicated, with an existing pool of several associated genes (KCNQ5, KCNJ2, and CACNA1D) [7].
As the first two susceptibility loci found to be associated with myopia [6, 8, 9, 12, 18, 57, 58], GJD2 [59, 60] and RASGRF1 [13, 17, 61] were significantly associated with HM in those with HM and MMD in the current study and previous studies. However, similar to previous work, GJD2 [31] and RASGRF1 [17, 31] were not specifically associated with MMD. GJD2 [62–65] plays an essential role in synaptic transmission and processing of visual signals in photoreceptors and retinal cells [64, 66–69], and seems to be controlled by light exposure and dopamine [70], both of which have established roles in eye growth and myopia development [57, 71, 72]. RASGRF1 [66, 68, 73] is involved in neuronal signal transduction pathways for retinal maintenance and function, and synaptic transmission of the photoreceptor responses [68]. Downregulated RASGRF1 expression in mice models have resulted in impaired memory consolidation and learning [74], and deficiencies in photoreception and visual sensory processes [68].
We acknowledge that there may be limitations to our study. Our study is likely to be underpowered to detect associations between the candidate genetic variants and HM and MMD, due to the small sample size of MMD cases. The direction of effect in our study were similar to that in the previously reported GWAS for KCNMA1 and GJD2,[11] thus indicating the lack of sufficient power in this study. As the prevalence of MMD in the population is low at 1–3% [2], it is logistically difficult to collect a sufficient number of cases with both MMD and genotyping information. It is unclear if the genetic variants reported in our findings are associated with HM, as the control group used in the first analysis should ideally be low and moderate myopes without MMD, instead of emmetropes without MMD. Therefore, these genetic variants are associated with HM in a specific population of those with both MMD and HM. We do not have individual data from each participating study on other factors that might be associated with MMD, for instance myopia duration. Although our study population has ethnic diversity, comprising individuals of European and Asian ancestries, the nonsignificant associations may be due to genetic heterogeneity across populations of varying ethnicity. It may be due to the multifactorial influences on this complex ocular disease as well. In addition, due to the nature of our candidate study, we focused on SNPs with strong prior evidence of association with myopia. In that respect, it may be unsurprising that these loci did not confer any significant effect over MMD, independent of their effect over HM. We did not conduct a GWAS and this study evaluated only the top SNPs for myopia, thus genetic variants that specifically affect MMD but have weak or no associations with myopia were not examined in this study. As only the top SNPs with stronger association with myopia were tested, we may have missed significant associations of untested SNPs with weaker associations with myopia present in the same locus as the top SNPs that were tested. Further verification and replication of our findings are required.
Conclusions
In our study, we did not find any myopia-associated variant that was specifically associated with MMD. However, we report a significant association between HM in highly myopic subjects with MMD and the rs10824518 SNP in the KCNMA1 locus in an international and multi-ethnic study. We also replicated and verified associations between HM in highly myopic subjects with MMD and the first gene associated with SE (GJD2). Further studies of larger sample sizes are required to elucidate susceptibility loci exclusive to MMD.
Supporting information
S2 Text. Study populations and acknowledgments.
https://doi.org/10.1371/journal.pone.0220143.s002
(DOCX)
S1 Appendix. Meta-analysis on genetic association studies checklist.
https://doi.org/10.1371/journal.pone.0220143.s003
(DOCX)
S2 Appendix. Plot of the effect on high myopia in highly myopic subjects with myopic macular degeneration for all 39 tested SNPs in the population cohorts in first case-control study.
https://doi.org/10.1371/journal.pone.0220143.s004
(DOCX)
S3 Appendix. Plot of the effect on myopic macular degeneration in highly myopic subjects with myopic macular degeneration for all 37 tested SNPs in the population cohorts in second case-control study.
https://doi.org/10.1371/journal.pone.0220143.s005
(DOCX)
Acknowledgments
We sincerely thank all study participants, their relatives and the staff at the recruitment centers for their contributions. We thank all contributors to the CREAM Consortium for their generosity in sharing data and help in the production of this publication. Funding agencies that facilitated the execution of the individual studies are acknowledged in the Supporting Information (S2 Text).
References
- 1. Ohno-Matsui K. What is the fundamental nature of pathologic myopia? Retina. 2017;37(6):1043–8. pmid:27755375
- 2. Wong TY, Ferreira A, Hughes R, Carter G, Mitchell P. Epidemiology and disease burden of pathologic myopia and myopic choroidal neovascularization: an evidence-based systematic review. American journal of ophthalmology. 2014;157(1):9–25. e12. pmid:24099276
- 3. Xu L, Cui T, Yang H, Hu A, Ma K, Zheng Y, et al. Prevalence of visual impairment among adults in China: the Beijing Eye Study. American journal of ophthalmology. 2006;141(3):591–3. pmid:16490524
- 4. Iwase A, Araie M, Tomidokoro A, Yamamoto T, Shimizu H, Kitazawa Y, et al. Prevalence and causes of low vision and blindness in a Japanese adult population: the Tajimi Study. Ophthalmology. 2006;113(8):1354–62. e1. pmid:16877074
- 5. Wong YL, Saw SM. Epidemiology of Pathologic Myopia in Asia and Worldwide. Asia-Pacific journal of ophthalmology. 2016;5(6):394–402. pmid:27898442
- 6. Kiefer AK, Tung JY, Do CB, Hinds DA, Mountain JL, Francke U, et al. Genome-wide analysis points to roles for extracellular matrix remodeling, the visual cycle, and neuronal development in myopia. PLoS genetics. 2013;9(2):e1003299. pmid:23468642
- 7. Verhoeven VJ, Hysi PG, Wojciechowski R, Fan Q, Guggenheim JA, Höhn R, et al. Genome-wide meta-analyses of multiancestry cohorts identify multiple new susceptibility loci for refractive error and myopia. Nature genetics. 2013;45(3):314–8. pmid:23396134
- 8. Hysi PG, Young TL, Mackey DA, Andrew T, Fernandez-Medarde A, Solouki AM, et al. A genome-wide association study for myopia and refractive error identifies a susceptibility locus at 15q25. Nat Genet. 2010;42(10):902–5. pmid:20835236
- 9. Solouki AM, Verhoeven VJ, van Duijn CM, Verkerk AJ, Ikram MK, Hysi PG, et al. A genome-wide association study identifies a susceptibility locus for refractive errors and myopia at 15q14. Nat Genet. 2010;42(10):897–901. pmid:20835239
- 10. Stambolian D, Wojciechowski R, Oexle K, Pirastu M, Li X, Raffel LJ, et al. Meta-analysis of genome-wide association studies in five cohorts reveals common variants in RBFOX1, a regulator of tissue-specific splicing, associated with refractive error. Human molecular genetics. 2013;22(13):2754–64. pmid:23474815
- 11. Pickrell JK, Berisa T, Liu JZ, Ségurel L, Tung JY, Hinds DA. Detection and interpretation of shared genetic influences on 42 human traits. Nature genetics. 2016;48(7):709–17. pmid:27182965
- 12. Tedja MS, Wojciechowski R, Hysi PG, Eriksson N, Furlotte NA, Verhoeven VJ, et al. Genome-wide association meta-analysis highlights light-induced signaling as a driver for refractive error. Nature genetics. 2018;50(6):834–48. pmid:29808027
- 13. Qiang Y, Li W, Wang Q, He K, Li Z, Chen J, et al. Association study of 15q14 and 15q25 with high myopia in the Han Chinese population. BMC genetics. 2014;15(1):51.
- 14. Lu B, Jiang D, Wang P, Gao Y, Sun W, Xiao X, et al. Replication study supports CTNND2 as a susceptibility gene for high myopia. Investigative ophthalmology & visual science. 2011;52(11):8258–61.
- 15. Verhoeven VJ, Hysi PG, Saw SM, Vitart V, Mirshahi A, Guggenheim JA, et al. Large scale international replication and meta-analysis study confirms association of the 15q14 locus with myopia. The CREAM consortium. Human genetics. 2012;131(9):1467–80. pmid:22665138
- 16. Schache M, Richardson AJ, Mitchell P, Wang JJ, Rochtchina E, Viswanathan AC, et al. Genetic association of refractive error and axial length with 15q14 but not 15q25 in the Blue Mountains Eye Study cohort. Ophthalmology. 2013;120(2):292–7. pmid:23131718
- 17. Oishi M, Yamashiro K, Miyake M, Akagi-Kurashige Y, Kumagai K, Nakata I, et al. Association between ZIC2, RASGRF1, and SHISA6 genes and high myopia in Japanese subjects. Investigative ophthalmology & visual science. 2013;54(12):7492–7.
- 18. Yoshikawa M, Yamashiro K, Miyake M, Oishi M, Akagi-Kurashige Y, Kumagai K, et al. Comprehensive Replication of the Relationship Between Myopia-Related Genes and Refractive Errors in a Large Japanese CohortMyopia-Related Genes in Japanese. Investigative ophthalmology & visual science. 2014;55(11):7343–54.
- 19. Gao Y, Wang P, Li S, Xiao X, Jia X, Guo X, et al. Common variants in chromosome 4q25 are associated with myopia in Chinese adults. Ophthalmic & physiological optics: the journal of the British College of Ophthalmic Opticians. 2012;32(1):68–73.
- 20. Ohno-Matsui K, Lai TY, Lai CC, Cheung CM. Updates of pathologic myopia. Progress in retinal and eye research. 2016;52:156–87. pmid:26769165
- 21. Li YJ, Goh L, Khor CC, Fan Q, Yu M, Han S, et al. Genome-wide association studies reveal genetic variants in CTNND2 for high myopia in Singapore Chinese. Ophthalmology. 2011;118(2):368–75. pmid:21095009
- 22. Li Z, Qu J, Xu X, Zhou X, Zou H, Wang N, et al. A genome-wide association study reveals association between common variants in an intergenic region of 4q25 and high-grade myopia in the Chinese Han population. Human molecular genetics. 2011;20(14):2861–8. pmid:21505071
- 23. Fan Q, Barathi VA, Cheng CY, Zhou X, Meguro A, Nakata I, et al. Genetic variants on chromosome 1q41 influence ocular axial length and high myopia. PLoS genetics. 2012;8(6):e1002753. pmid:22685421
- 24. Meng W, Butterworth J, Bradley DT, Hughes AE, Soler V, Calvas P, et al. A Genome-Wide Association Study Provides Evidence for Association of Chromosome 8p23 (MYP10) and 10q21. 1 (MYP15) with High Myopia in the French PopulationA Genome-Wide Association Study in the French Population. Investigative ophthalmology & visual science. 2012;53(13):7983–8.
- 25. Khor CC, Miyake M, Chen LJ, Shi Y, Barathi VA, Qiao F, et al. Genome-wide association study identifies ZFHX1B as a susceptibility locus for severe myopia. Human molecular genetics. 2013;22(25):5288–94. pmid:23933737
- 26. Shi Y, Gong B, Chen L, Zuo X, Liu X, Tam PO, et al. A genome-wide meta-analysis identifies two novel loci associated with high myopia in the Han Chinese population. Human molecular genetics. 2013;22(11):2325–33. pmid:23406873
- 27. Hosoda Y, Yoshikawa M, Miyake M, Tabara Y, Shimada N, Zhao W, et al. CCDC102B confers risk of low vision and blindness in high myopia. Nature communications. 2018;9(1):1782. pmid:29725004
- 28. Ohno-Matsui K, Kawasaki R, Jonas JB, Cheung CM, Saw SM, Verhoeven VJ, et al. International photographic classification and grading system for myopic maculopathy. Am J Ophthalmol. 2015;159(5):877–83 e7. pmid:25634530
- 29. Nakanishi H, Yamada R, Gotoh N, Hayashi H, Yamashiro K, Shimada N, et al. A genome-wide association analysis identified a novel susceptible locus for pathological myopia at 11q24.1. PLoS genetics. 2009;5(9):e1000660. pmid:19779542
- 30. Yu Z, Zhou J, Chen X, Zhou X, Sun X, Chu R. Polymorphisms in the CTNND2 gene and 11q24. 1 genomic region are associated with pathological myopia in a Chinese population. Ophthalmologica. 2012;228(2):123–9. pmid:22759899
- 31. Chen C-d, Yu Z-q, Chen X-l, Zhou J-q, Zhou X-t, Sun X-h, et al. Evaluating the association between pathological myopia and SNPs in RASGRF1. ACTC1 and GJD2 genes at chromosome 15q14 and 15q25 in a Chinese population. Ophthalmic genetics. 2015;36(1):1–7. pmid:23834555
- 32. Lavanya R, Jeganathan VSE, Zheng Y, Raju P, Cheung N, Tai ES, et al. Methodology of the Singapore Indian Chinese Cohort (SICC) eye study: quantifying ethnic variations in the epidemiology of eye diseases in Asians. Ophthalmic epidemiology. 2009;16(6):325–36. pmid:19995197
- 33. Foong AW, Saw S-M, Loo J-L, Shen S, Loon S-C, Rosman M, et al. Rationale and methodology for a population-based study of eye diseases in Malay people: The Singapore Malay eye study (SiMES). Ophthalmic epidemiology. 2007;14(1):25–35. pmid:17365815
- 34. Hofman A, Breteler MM, van Duijn CM, Krestin GP, Pols HA, Stricker BHC, et al. The Rotterdam Study: objectives and design update. European journal of epidemiology. 2007;22(11):819–29. pmid:17955331
- 35. Hofman A, Breteler MM, van Duijn CM, Janssen HL, Krestin GP, Kuipers EJ, et al. The Rotterdam Study: 2010 objectives and design update. European journal of epidemiology. 2009;24(9):553–72. pmid:19728115
- 36. Hofman A, van Duijn CM, Franco OH, Ikram MA, Janssen HL, Klaver CC, et al. The Rotterdam Study: 2012 objectives and design update. European journal of epidemiology. 2011;26(8):657. pmid:21877163
- 37. Hofman A, Murad SD, van Duijn CM, Franco OH, Goedegebure A, Ikram MA, et al. The Rotterdam Study: 2014 objectives and design update. European journal of epidemiology. 2013;28(11):889–926. pmid:24258680
- 38. Hofman A, Brusselle GG, Murad SD, van Duijn CM, Franco OH, Goedegebure A, et al. The Rotterdam Study: 2016 objectives and design update. European journal of epidemiology. 2015;30(8):661–708. pmid:26386597
- 39. Höhn R, Kottler U, Peto T, Blettner M, Münzel T, Blankenberg S, et al. The ophthalmic branch of the Gutenberg Health Study: study design, cohort profile and self-reported diseases. PloS one. 2015;10(3):e0120476. pmid:25775251
- 40. Holden BA, Fricke TR, Wilson DA, Jong M, Naidoo KS, Sankaridurg P, et al. Global prevalence of myopia and high myopia and temporal trends from 2000 through 2050. Ophthalmology. 2016;123(5):1036–42. pmid:26875007
- 41. Fan Q, Verhoeven VJ, Wojciechowski R, Barathi VA, Hysi PG, Guggenheim JA, et al. Meta-analysis of gene-environment-wide association scans accounting for education level identifies additional loci for refractive error. Nature communications. 2016;7.
- 42. Howie B, Fuchsberger C, Stephens M, Marchini J, Abecasis GR. Fast and accurate genotype imputation in genome-wide association studies through pre-phasing. Nature genetics. 2012;44(8):955–9. pmid:22820512
- 43. Howie BN, Donnelly P, Marchini J. A flexible and accurate genotype imputation method for the next generation of genome-wide association studies. PLoS genetics. 2009;5(6):e1000529. pmid:19543373
- 44. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Bioinformatics methods and protocols: Springer; 2000. p. 365–86.
- 45. Willer CJ, Li Y, Abecasis GR. METAL: fast and efficient meta-analysis of genomewide association scans. Bioinformatics. 2010;26(17):2190–1. pmid:20616382
- 46. Broustas CG, Gokhale PC, Rahman A, Dritschilo A, Ahmad I, Kasid U. BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner. Journal of Biological Chemistry. 2004;279(25):26780–8. pmid:15069058
- 47. Turner SC, Shieh CC. Medicinal Chemistry of Ca2+‐activated K+ Channel Modulators. Voltage-Gated Ion Channels as Drug Targets, Volume 29. 2006:310–34.
- 48. Hysi PG, Wojciechowski R, Rahi JS, Hammond CJ. Genome-Wide Association Studies of Refractive Error and Myopia, Lessons Learned, and Implications for the FutureGWAS of Refractive Error and Myopia. Investigative ophthalmology & visual science. 2014;55(5):3344–51.
- 49. Knaus H-G, Schwarzer C, Koch R, Eberhart A, Kaczorowski GJ, Glossmann H, et al. Distribution of high-conductance Ca (2+)-activated K+ channels in rat brain: targeting to axons and nerve terminals. Journal of Neuroscience. 1996;16(3):955–63. pmid:8558264
- 50. Sun X, Gu XQ, Haddad GG. Calcium influx via L-and N-type calcium channels activates a transient large-conductance Ca2+-activated K+ current in mouse neocortical pyramidal neurons. Journal of Neuroscience. 2003;23(9):3639–48. pmid:12736335
- 51. Misonou H, Menegola M, Buchwalder L, Park EW, Meredith A, Rhodes KJ, et al. Immunolocalization of the Ca2+‐activated K+ channel Slo1 in axons and nerve terminals of mammalian brain and cultured neurons. Journal of Comparative Neurology. 2006;496(3):289–302. pmid:16566008
- 52. Grimes WN, Li W, Chávez AE, Diamond JS. BK channels modulate pre-and postsynaptic signaling at reciprocal synapses in retina. Nature neuroscience. 2009;12(5):585. pmid:19363492
- 53. Wimmers S, Halsband C, Seyler S, Milenkovic V, Strauß O. Voltage-dependent Ca2+ channels, not ryanodine receptors, activate Ca2+-dependent BK potassium channels in human retinal pigment epithelial cells. Molecular vision. 2008;14:2340. pmid:19096717
- 54. Wimmers S, Karl MO, Strauss O. Ion channels in the RPE. Progress in retinal and eye research. 2007;26(3):263–301. pmid:17258931
- 55. Rosenthal R, Heimann H, Agostini H, Martin G, Hansen LL, Strauss O. Ca2+ channels in retinal pigment epithelial cells regulate vascular endothelial growth factor secretion rates in health and disease. Molecular vision. 2007;13:443. pmid:17417605
- 56. Seko Y, Seko Y, Fujikura H, Pang J, Tokoro T, Shimokawa H. Induction of vascular endothelial growth factor after application of mechanical stress to retinal pigment epithelium of the rat in vitro. Investigative ophthalmology & visual science. 1999;40(13):3287–91.
- 57. Tideman JWL, Fan Q, Polling JR, Guo X, Khawaja A, Höhn R, et al. When do myopia genes have their effect? Comparison of genetic risks between children and adults. Genetic epidemiology. 2016;40(8):756–66. pmid:27611182
- 58. Miyake M, Yamashiro K, Tabara Y, Suda K, Morooka S, Nakanishi H, et al. Identification of myopia-associated WNT7B polymorphisms provides insights into the mechanism underlying the development of myopia. Nature communications. 2015;6:6689. pmid:25823570
- 59. Jiao X, Wang P, Li S, Li A, Guo X, Zhang Q, et al. Association of markers at chromosome 15q14 in Chinese patients with moderate to high myopia. Molecular vision. 2012;18:2633. pmid:23170057
- 60. Li Y-T, Xie M-K, Wu J. Association between ocular axial length-related genes and high myopia in a Han Chinese population. Ophthalmologica. 2016;235(1):57–60. pmid:26485405
- 61. Hayashi H, Yamashiro K, Nakanishi H, Nakata I, Kurashige Y, Tsujikawa A, et al. Association of 15q14 and 15q25 with high myopia in Japanese. Investigative ophthalmology & visual science. 2011;52(7):4853–8.
- 62. Söhl G, Joussen A, Kociok N, Willecke K. Expression of connexin genes in the human retina. BMC ophthalmology. 2010;10(1):27.
- 63. Lin B, Jakobs TC, Masland RH. Different functional types of bipolar cells use different gap-junctional proteins. Journal of Neuroscience. 2005;25(28):6696–701. pmid:16014731
- 64. Deans MR, Volgyi B, Goodenough DA, Bloomfield SA, Paul DL. Connexin36 is essential for transmission of rod-mediated visual signals in the mammalian retina. Neuron. 2002;36(4):703–12. pmid:12441058
- 65. Kihara A, Paschon V, Cardoso C, Higa G, Castro L, Hamassaki D, et al. Connexin36, an essential element in the rod pathway, is highly expressed in the essentially rodless retina of Gallus gallus. Journal of Comparative Neurology. 2009;512(5):651–63. pmid:19051319
- 66.
Sangwan SK, Sharma A, Sharma N, Tandon R. Genetics of Myopia. Advances in Vision Research, Volume I: Springer; 2017. p. 403–11.
- 67. Seeliger MW, Brombas A, Weiler R, Humphries P, Knop G, Tanimoto N, et al. Modulation of rod photoreceptor output by HCN1 channels is essential for regular mesopic cone vision. Nature communications. 2011;2:532. pmid:22068599
- 68. Fernández‐Medarde A, Barhoum R, Riquelme R, Porteros A, Núñez A, De Luis A, et al. RasGRF1 disruption causes retinal photoreception defects and associated transcriptomic alterations. Journal of neurochemistry. 2009;110(2):641–52. pmid:19457086
- 69. Güldenagel M, Ammermüller J, Feigenspan A, Teubner B, Degen J, Söhl G, et al. Visual transmission deficits in mice with targeted disruption of the gap junction gene connexin36. Journal of Neuroscience. 2001;21(16):6036–44. pmid:11487627
- 70. Bloomfield SA, Völgyi B. The diverse functional roles and regulation of neuronal gap junctions in the retina. Nature Reviews Neuroscience. 2009;10(7):495. pmid:19491906
- 71. Feldkaemper M, Schaeffel F. An updated view on the role of dopamine in myopia. Experimental eye research. 2013;114:106–19. pmid:23434455
- 72. Witkovsky P. Dopamine and retinal function. Documenta ophthalmologica. 2004;108(1):17–39. pmid:15104164
- 73. Wolfman A, Macara IG. A cytosolic protein catalyzes the release of GDP from p21ras. Science. 1990;248(4951):67–9. pmid:2181667
- 74. Brambilla R, Gnesutta N, Minichiello L, White G, Roylance AJ, Herron CE, et al. A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature. 1997;390(6657):281. pmid:9384379