Figures
Abstract
Myopia is the most common ocular disease worldwide. We investigated the association of high myopia with the common single nucleotide polymorphisms (SNPs) of five candidate genes – early growth response 1 (EGR1), v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS), jun oncogene (JUN), vasoactive intestinal peptide (VIP), and vasoactive intestinal peptide receptor 2 (VIPR2). We recruited 1200 unrelated Chinese subjects with 600 cases (spherical equivalent ≤−8.00 diopters) and 600 controls (spherical equivalent within ±1.00 diopter). A discovery sample set was formed from 300 cases and 300 controls, and a replication sample set from the remaining samples. Tag SNPs were genotyped for the discovery sample set, and the most significant haplotypes and their constituent SNPs were followed up with the replication sample set. The allele and haplotype frequencies in cases and controls were compared by logistic regression adjusted for sex and age to give Pa values, and multiple comparisons were corrected by permutation test to give Paemp values. Odd ratios (OR) were calculated accordingly. In the discovery phase, EGR1, JUN and VIP did not show any significant association while FOS and VIPR2 demonstrated significant haplotype association with high myopia. In the replication phase, the haplotype association for VIPR2 was successfully replicated, but not FOS. In analysis combining both sample sets, the most significant association signals of VIPR2 were the single marker rs2071625 (Pa = 0.0008, Paemp = 0.0046 and OR = 0.75) and the 4-SNP haplotype window rs2071623-rs2071625-rs2730220-rs885863 (omnibus test, Pa = 9.10e-10 and Paemp = 0.0001) with one protective haplotype (GGGG: Paemp = 0.0002 and OR = 0.52) and one high-risk haplotype (GAGA: Paemp = 0.0027 and OR = 4.68). This 4-SNP haplotype window was the most significant in all sample sets examined. This is the first study to suggest a role of VIPR2 in the genetic susceptibility to high myopia. EGR1, JUN, FOS and VIP are unlikely to be important in predisposing humans to high myopia.
Citation: Yiu WC, Yap MKH, Fung WY, Ng PW, Yip SP (2013) Genetic Susceptibility to Refractive Error: Association of Vasoactive Intestinal Peptide Receptor 2 (VIPR2) with High Myopia in Chinese. PLoS ONE 8(4): e61805. https://doi.org/10.1371/journal.pone.0061805
Editor: Yong-Gang Yao, Kunming Institute of Zoology, Chinese Academy of Sciences, China
Received: February 4, 2013; Accepted: March 14, 2013; Published: April 18, 2013
Copyright: © 2013 Yiu 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.
Funding: This work is a contribution from the Centre for Myopia Research of The Hong Kong Polytechnic University, and was supported by grants from The Hong Kong Polytechnic University (RP3C, JBB7P, G-U730, 87TP and 1.55.xx.99NE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Myopia is the most common eye disorder worldwide and has reached epidemic prevalence in East Asia [1]. Myopia can be classified into different categories based on the clinical entity [2]. High myopia is defined as a refractive error equal to or worse than −6.00 diopters (D). It is the most concerned type because of its association with irreversible visual impairment such as retinal detachment, glaucoma and, in severe cases, blindness [3]. Although both environmental and genetic factors play important roles in the development of myopia [4], [5], the principal factor is still under debate. Cross-sectional studies have shown that environmental factors such as educational level and near work are associated with the development of myopia [1], [6]. Evidence pointing to the role of genetic factors in the etiology of myopia comes mainly from the identification of myopia loci/genes in linkage and/or association studies [7]–[15]. Myopia is a complex disease, and genetic variations can increase the susceptibility to environmental factors and cause an early onset and/or aggressive progression. As the age of myopia onset is decreasing [16], [17], the chance of developing high myopia increases. In order to control the progression of myopia, the underlying pathways leading to this condition must be understood.
Animal studies show that the development of myopia involves extracellular matrix (ECM) remodeling of the sclera [18], [19]. Therefore, candidate-gene association studies in myopia genetics have mainly focused on the genes expressed in the sclera [20]–[23]. However, it is well established that visual experience alters ocular growth and the changes are first mediated by local visual processing and signaling mechanisms [24]–[26]. We hypothesize that genes directly responsive to visual signals are the primary genes involved in the biological pathways. Activation of these genes may further activate other secondary genes in the pathways, and this in turn causes ECM remodeling of the sclera and ultimately leads to altered ocular growth. This study aims to investigate these primary genes for their potential role in the susceptibility to high myopia. Five functional candidate genes have been selected on the basis of this hypothesis: early growth response 1 (EGR1) located at chromosome 5q31.1, v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) at 14q24.3, jun oncogene (JUN) at 1p32-p31, vasoactive intestinal peptide (VIP) at 6q25, vasoactive intestinal peptide receptor 2 (VIPR2) at 7q36.3 (NCBI, http://www.ncbi.nlm.nih.gov/).
Consistent findings from different animal models demonstrate the participation of Egr-1 (also known as ZENK) in ocular growth [27]–[29]. The study of Egr-1 knockout mice has also provided convincing evidence for the involvement of Egr-1 in regulating ocular growth [30]. Egr-1 knockout mice had a myopic shift in refraction and tended to have eyes with a longer axial length. FOS encodes a protein that dimerizes with the protein encoded by JUN to form a transcription factor complex known as activating protein-1 (AP-1) [31]. Binding of AP-1 causes trans-activation of its target genes. AP-1 sites were found in the promoters of genes encoding matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitor of metalloproteinases or TIMPs) [32], [33]. In other words, AP-1 can regulate the expression of MMPs and TIMPs. MMPs are capable of degrading ECM proteins and are believed to be involved in ECM remodeling of the sclera. AP-1 can also repress the trans-activation of retinoid receptors [34], which have been shown to be critical for the regulation of eye growth [35]. The expression of VIP showed a positive correlation with the depth of the vitreous chamber [36] and this suggests that increased release of VIP may be responsible for ocular growth. VIPR2 is one of the VIP receptors and is located on chromosome 7q36, which is within the interval for a putative locus for autosomal dominant high-grade myopia (formerly called MYP4) [37], [38]. The expression of VIPR2 in the retina and the choroid was altered in chicks with form-deprivation myopia [39]. These findings suggest a potential role of VIPR2 in the development of myopia.
We conducted a case-control genetic association study to investigate the association between high myopia and the five selected candidate genes. Samples from a homogeneous population (southern Han Chinese) were used to minimize the possibility of false positive results due to population stratification. We also validated the initial positive findings with an independent sample set.
Materials and Methods
Recruitment of Subjects
We recruited unrelated Han Chinese aged between 18 and 45 years via the Optometry Clinic of The Hong Kong Polytechnic University as described previously [9]–[11]. Cases were subjects with refraction (spherical equivalent or SE) of −8.00 D or worse for both eyes while controls were subjects with SE within ±1.00 D for both eyes. Subjects with ocular disease or genetic disease associated with myopia were excluded from the study. The study was approved by the Human Subjects Ethics Sub-committee, The Hong Kong Polytechnic University, and adhered to the tenets of the Declaration of Helsinki. All participants gave written informed consent. Eye examination included retinoscopy, fundus examination, and measurement of refractive error, corneal power, lens thickness, anterior chamber depth, posterior chamber depth and axial length [9]–[11]. In total, we recruited 600 cases and 600 controls, and sequentially allocated them into two sample sets (the discovery sample set and the replication sample set). Each sample set consisted of 300 cases and 300 controls.
Selection and genotyping of single nucleotide polymorphisms (SNPs)
Genomic DNA was extracted from the subjects' blood samples [9]. Tag SNPs of the five candidate genes (Table S1) were identified from the HapMap database (release 23a/phase II March 08; http://hapmap.ncbi.nlm.nih.gov/) with the selection criteria of r2>0.8 and minor allele frequency (MAF)>0.10 for Han Chinese population by the Tagger software. The 3-kb regions upstream and downstream of the candidate genes were included for SNP selection [11], [23].
We genotyped the SNPs by restriction fragment length polymorphism, unlabelled probe melting curve analysis or primer extension reaction coupled with denaturing high-performance liquid chromatography [9]–[11], [23]. We used direct DNA sequencing of representative samples to confirm all observed genotypes. Details of the genotyping methods are described in Appendix S1. Tag SNPs and their genotyping methods are listed in Table S1 together with the respective restriction enzymes, primers, extension primer, and probes. Tag SNPs showing significant associations in single-marker or haplotype analyses using the discovery sample set were followed up with the replication sample set.
Statistical analysis
We used PLINK (ver. 1.07; http://pngu.mgh.harvard.edu/~purcell/plink/) as the software tool for association analysis [40]. Genotypes of the controls were tested for Hardy-Weinberg equilibrium (HWE) by exact test with a significance threshold set at P = 0.001 [41]. Single-marker (allelic test) and haplotype associations were tested using logistic regression adjusted for sex and age as covariates, and odds ratios (OR) and their 95% confidence intervals (CI) were also calculated accordingly. We performed haplotype analysis using an exhaustive sliding-window approach by testing all possible haplotypes made up of a variable number of constitutive SNPs. For each sliding window, we jointly assessed the significance of the haplotype effects by a single case-control omnibus test with (H – 1) degrees of freedom, where H is the number of haplotypes for the window concerned. For a particular window size with a given gene, we conducted the test for all possible windows of the same size, and shifted one SNP at a time to the 3′ end of the gene. Multiple testing was corrected by generating empirical P values based on 10 000 permutations across all SNPs or haplotype windows for a given sample set as appropriate. To increase the power of the study, the genotype data were also analyzed by combining the two sample sets with adjustment for sample set as another covariate in addition to sex and age. P values adjusted for the covariates are reported, and indicated as Pa if not corrected for multiple comparisons or as Paemp if corrected for multiple comparisons. A Paemp<0.05 indicates significant association. Note that the minimum Paemp value achievable by 10 000 permutations is 0.0001. Linkage disequilibrium (LD) maps were constructed by Haploview using solid spine of LD as the definition of haplotype blocks [42].
Results
Subject demographics
Measurements for all traits were highly correlated between the right and the left eyes, particularly for SE (r = 0.97) and axial length (r = 0.96). Hence, only measurements for the right eye were used for analysis. The characteristics of the subjects in the discovery and the replication sample sets are presented in Table 1. The average SE values were 0.03 D for controls and −10.56 D for cases of the discovery sample set, and 0.10 D for controls and −10.06 D for cases of the replication sample set. Most control subjects had SE between −0.10 D and 0.10 D with about 2.2% of controls in the range of 0.12 D and 0.40 D. Most case subjects had SE between −8.00 D and −15.00 D with 4.8% of cases in the range of less than −15.00 D and −24.00 D. This skewed distribution arose as a result of the criteria used for subject recruitment.
There were fewer females in the control group than in the case group for the discovery sample set (55.60% vs 73.33%; chi-squared test, P = 9.146×10−6) and for the replication sample set (59.33% vs 69.67%; chi-squared test, P = 0.0105). The subjects were younger in the control group than in the case group (mean age, 24.90 vs 27.75 years; unpaired t test, P = 3.02×10−7) for the discovery sample set. Although the mean age was similar in both the control and the case groups (33.34 vs 33.73 years; unpaired t test, P = 0.6118) of the replication sample set, we adjusted for sex and age in all subsequent association analyses for all sample sets to maintain consistency across the board.
Discovery sample set
In total, 26 tag SNPs were selected and genotyped for the discovery sample set: 1 from ERG1, 5 from FOS, 4 from JUN, 3 from VIP and 13 from VIPR2 (Table S1). Table 2 summarizes the genotype data. The control group was in HWE (P>0.001) for all 26 tag SNPs examined. Therefore, we included all SNPs for association analysis. In single-marker analysis, six SNPs showed significant nominal P values (Pa<0.05): rs4645874 (S04) of FOS, rs1407267 (S01) of VIP, and rs3828963 (S03), rs6973238 (S06), rs3793227 (S07) and rs2071623 (S10) of VIPR2. However, none of them remained significant (Paemp<0.05) after correction for multiple comparisons across 26 SNPs by permutation test.
For candidate genes with two or more tag SNPs examined, we constructed LD patterns with Haploview and defined LD blocks by solid spine of LD. In general, LD between SNPs was very weak with no LD block identified for any of the four genes (FOS, JUN, VIP and VIPR2) examined for LD patterns (Figures S1A, S1D and S1E; and Figure 1A). Therefore, we performed haplotype analysis using an exhaustive sliding-window approach (Table 3). Of all the 123 possible sliding windows, 2 windows of the FOS gene and 13 windows of the VIPR2 gene displayed significant association (Paemp<0.05) with high myopia even after correction for multiple comparisons (n = 123) by permutation test. For the FOS gene, the most significant haplotype window was the 3-SNP window S03…S05 consisting of rs4645869, rs4645874 and rs17103109 (Pa = 6.43×10−5 and Paemp = 0.0016). For the VIPR2 gene, the most significant haplotype window was the 4-SNP window S10…S14 made up of rs2071623, rs2071625, rs2730220 and rs885863 (Pa = 8.47×10−8 and Paemp = 0.0001). We, therefore, followed up these 7 SNPs by genotyping an independent sample set – the replication sample set (Table 1).
LD measures are indicated as r2 values for cases and controls together for (a) the discovery sample set, (B) the replication sample, and (C) the combined sample set. Note that, as defined by solid spine of LD, no LD bock is identified in any of the sample sets.
Replication sample set
We examined seven SNPs with the replication sample set (Table 1) in light of the significant haplotype association obtained with the discovery sample set. We summarize the genotype data of the replication sample set in Table 2. The control group was in HWE for all seven SNPs.
We did not find any significant association in single-marker analysis of FOS SNPs, not even when we jointly analyzed both sample sets (hereafter called the combined sample set) (Table 2). However, single-marker analysis of the replication sample set revealed significant association of rs2730220 (S12) of VIPR2 with high myopia even after correction for multiple testing across seven SNPs (Pa = 0.0017 and Paemp = 0.0110). The OR of its minor allele A was 0.51 (95% CI, 0.34–0.78) with reference to its major allele G. Interestingly, instead of rs2730220 (S12), rs2071625 (S11) of VIPR2 demonstrated significant association with high myopia for the combined sample sets (Pa = 0.0008 and Paemp = 0.0046) with the OR of its minor allele G being 0.75 (95% CI, 0.63–0.89).
LD between SNPs was also very weak for FOS and VIPR2 in both the replication sample set and the combined sample set (Figures S1B and S1C; and Figures 1B and 1C). We also did not identify any LD block for these sample sets. Exhaustive sliding-window haplotype analysis failed to demonstrate any significant association for FOS for both sample sets (Table 3). However, we were able to replicate significant haplotype association (Paemp<0.05) of VIPR2 with high myopia: five significant haplotype windows for the replication sample set and six significant haplotype windows for the combined sample set (Table 3). Most importantly, the same most significant VIPR2 haplotype window was identified as in the discovery sample set: S10…S13 consisting of rs2071623, rs2071625, rs2730220 and rs885863 (Pa = 1.15×10−5 and Paemp = 0.0002 for the replication sample set; and Pa = 9.10×10−10 and Paemp = 0.0001 for the combined sample set).
The most significant VIPR2 haplotype window
The 4-SNP haplotype window S10…S13 (rs2071623-rs2071625-rs2730220-rs885863) of VIPR2 was the most significant haplotype sliding window in the discovery sample set, the replication sample set and the combined sample set (Table 3). The constituent haplotypes of this sliding window are shown in Table 4. We did not observe any haplotypes displaying opposite directions of association in the discovery and the replication sample sets although the GAGA haplotype was found in the replication sample set, but not in the discovery sample set. Therefore, the directions of association of these 4-SNP haplotypes were compatible between the discovery and the replication sample sets.
In the combined sample set, GGGG (1211) was a protective haplotype with an OR of 0.52 (Pa = 3.37×10−6 and Paemp = 0.0002) and GAGA (1112) a high-risk haplotype with an OR of 4.68 (Pa = 0.0001 and Paemp = 0.0027). Note that the haplotypes are indicated in both the ACGT and the 1–2 (major-minor) formats. The GGGG (1211) haplotype had a frequency of 9.82% in cases and 16.08% in controls. The GAGA (1112) haplotype was much less common with a frequency of 3.68% in cases and 1.45% in controls.
Discussion
Previous studies indicate that genes responsive to visual signals are involved in the early part of the biological pathways concerned with altered ocular growth while genes responsible for ECM remodeling of the sclera participate in the later part of the pathways [29]–[31]. Therefore, we selected EGR1, FOS, JUN, VIP and VIPR2 as functional candidates and investigated their potential association with high myopia. We assumed a “common disease common variants” model [43] in this study and hence selected SNPs, the most abundant sequence variation in the human genome, as the genetic markers for the present genetic association study.
We performed haplotype analysis in addition to single-marker analysis. In the absence of LD block defined for the genes under study (Figures S1 and 1), we used the variable-sized sliding-window strategy to further explore possible association by comprehensively examining haplotype windows of all possible sizes. This strategy has been shown to be more powerful in detecting genetic association than single-marker analysis and LD-block-based haplotype analysis [44]. This is particularly true for genomic regions of low LD such as candidate loci evaluated in this study (Figures S1 and 1).
We did not find any evidence to support the role of ERG1, FOS, JUN and VIP in the genetic susceptibility to high myopia (Tables 2 and 3). We failed to confirm with the replication sample set the initial significant association of FOS haploypes with high myopia in the discovery sample set. Our EGR1 data complement a recent study that assumed a “common disease rare variants” model and did not find any pathological mutation in the EGR1 coding regions by DNA sequencing of 96 Chinese subjects with high myopia [45].
On the contrary, we first found the significant association of VIPR2 haplotypes with high myopia in the discovery sample set and then successfully replicated the significant association in the replication sample set (Tables 2 and 3). We found it reassuring that the haplotype window S10…S13 (rs2071623-rs2071625-rs2730220-rs885863) was the most significant sliding window among all possible haplotype windows examined in the discovery, the replication and the combined sample sets (Table 3). Although the haplotype GAGA (1112) of the S10…S13 window was significant in the replication sample set, it was not found in the discovery sample set (Table 4). Other than this, the directions of association were consistent for the haplotypes identified in the discovery and the replication sample sets. In the combined sample set, we identified one protective haplotype (GGGG or 1211, OR = 0.52) and one high-risk haplotype (GAGA or 1112, OR = 4.68). The high-risk haplotype GAGA was much less common particularly in the controls: 1.64% (∼20 chromosomes out of 1200 chromosomes) in the discovery sample set and 1.45% (∼17 chromosomes out of 1200 chromosomes) in the replication sample set. Note that analysis for this rare haplotype might be subject to random variation.
In the combined sample set, the four constituent SNPs of the VIPR2 S10…S13 window each contributed independent effects to the significant haplotype association as shown by conditional logistic regression: P = 5.37×10−8 for S10 (rs2071623), P = 4.31×10−7 for S11 (rs2071625), P = 0.0181 for S12 (rs2730220) and P = 1.09×10−8 for S13 (rs885863). We note that these four SNPs are located at the 3′ end of the VIPR2 gene (Figure S2). This region harbors a few sequence features that may be important in regulating the expression of VIPR2.
The importance of S11 (rs2071625) was highlighted by its significant association in single-marker analysis (Table 2) and its being a constituent SNP in all significant haplotype windows (Table 3) in the combined sample set. In the discovery sample set, S11 (rs2071625) was the only SNP included in all 13 significant haplotype windows (Table 3) even though it was not associated with high myopia as a single marker (Table 2). On the other hand, S12 (rs2730220) stood out in the replication sample set because of its significant association as a single marker. In both scenarios, association was statistically more significant with haplotype windows than with single markers. Therefore, we speculate that these SNPs or their haplotypes are more likely tagging some untyped causal variants that drive the genuine association with high myopia. Nevertheless, some databases (e.g., Patrocles, http://www.patrocles.org/Patrocles_targets.htm; and FuncPred, http://manticore.niehs.nih.gov/snpinfo/snpfunc.htm) predict that S12 (rs2730220) and S13 (rs885863) may affect the binding of certain microRNAs and hence influence the expression of VIPR2 accordingly. Despite these predictions, we doubt the involvement of these SNPs as causal variants in the genetic susceptibility to high myopia. Therefore, we recommend that future studies be aimed at identifying the causal variants. Since this is also the first study that has identified VIPR2 as a myopia susceptibility gene, our positive results should be replicated using samples from other populations, particularly those of different ethnicities.
VIPR2, also known as VPAC2, is located on chromosome 7q36 and lies within a putative locus for autosomal dominant high myopia (once called MYP4) [37], [38]. As a G-protein coupled receptor, VIPR2 is in fact a receptor for VIP. The expression of VIPR2 in the retina and the choroid was up-regulated in the treated eyes with reference to the fellow control eyes, but down-regulated with increasing axial length in chicks with form-deprivation myopia [39]. An unselective antagonist of VIP receptors (including VIPR2) could also suppress the development of form-deprivation myopia in chicks in a dose-dependent manner [46]. VIPR2 is also an input gene involved in circadian networks. Intriguingly, some studies have shown that transgenic mice over-expressing or lacking VIPR2 show deranged circadian rhythms [47]–[49]. Interestingly, growing eyes of chicks and monkeys display a diurnal rhythm in axial length, which is in anti-phase with the rhythm in choroidal thickness [50]. These phases are distorted in eyes that grow too fast or too slowly. Therefore, we speculate that VIPR2 may influence genetic susceptibility to myopia through its involvement in circadian rhythms. In light of the significant association between VIPR2 gene polymorphisms with high myopia, this hypothesis is worth exploring in future studies.
In summary, EGR1, JUN, FOS and VIP were not associated with high myopia. However, we identified VIPR2 as a novel myopia susceptibility gene. We obtained consistent results with sliding-window haplotype analysis and the S10…S13 haplotype window (rs2071623-rs2071625-rs2730220-rs885863) was the most significant sliding window in all sample sets. In combined sample set, S11 (rs2071625) also showed significant association with high myopia as a single marker.
Supporting Information
Figure S1.
Linkage disequilibrium (LD) patterns for single nucleotide polymorphisms of FOS, JUN and VIP.
https://doi.org/10.1371/journal.pone.0061805.s001
(PDF)
Figure S2.
Sequence features at the 3′ end of the VIPR2 locus.
https://doi.org/10.1371/journal.pone.0061805.s002
(PDF)
Table S1.
SNP genotyping: restriction enzymes, and sequences of primers, extension primer, and probes used.
https://doi.org/10.1371/journal.pone.0061805.s003
(DOC)
Appendix S1.
Genotyping of single nucleotide polymorphisms (SNPs).
https://doi.org/10.1371/journal.pone.0061805.s004
(DOC)
Acknowledgments
This work is a contribution from the Centre for Myopia Research of The Hong Kong Polytechnic University. We thank all the subjects participating in our myopia genetics study. The discovery study of this work was presented in the 13th International Myopia Conference in Tubingen, Germany, in 26–29 July 2010.
Author Contributions
Conceived and designed the experiments: WCY SPY MHY. Performed the experiments: WCY WYF PWN. Analyzed the data: WCY SPY. Contributed reagents/materials/analysis tools: WCY WYF PWN SPY. Wrote the paper: WCY SPY. Obtained funding: MKY SPY.
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