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Abstract
Purpose The endothelial and cell-specific angiopoietin-Tie pathway plays an important regulatory role in angiogenesis. In this study, we investigated the associations of the TIE2 (tyrosine kinase, endothelial, TEK) gene with neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV), using haplotype-tagging single-nucleotide polymorphisms (SNPs) analysis.
Methods This study involved totally 2343 subjects, including a Hong Kong Chinese cohort (214 nAMD patients, 236 PCV patients and 433 control subjects), a Shantou Chinese cohort (189 nAMD patients, 187 PCV patients and 531 control subjects) and an Osaka Japanese cohort (192 nAMD patients, 204 PCV patients and 157 control subjects). Thirty haplotype-tagging SNPs in TIE2 were genotyped in the Hong Kong cohort using TaqMan technology. Two SNPs (rs625767 and rs2273717) showing association in the Hong Kong cohort were genotyped in the Shantou and Osaka cohorts. The SNP-disease association of individual and pooled cohorts were analysed.
Results Two SNPs (rs625767 and rs2273717) showed suggestive association with both nAMD and PCV in the Hong Kong cohort. In the meta-analysis involving all the three cohorts, rs625767 showed significant associations with nAMD (p=0.01; OR=0.82, 95% CI 0.70 to 0.96; I2=0%), PCV (p=0.02; OR=0.83, 95% CI 0.71 to 0.97; I2=27%) and pooled nAMD and PCV (p=0.002; OR=0.82, 95% CI 0.72 to 0.93; I2=0%), with low inter-cohort heterogeneities.
Conclusion This study revealed TIE2 as a novel susceptibility gene for nAMD and PCV in Japanese and Chinese. Further studies in other populations are warranted to confirm its role.
- genetics
- macula
- experimental–laboratory
- neovascularisation
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information. Data are available upon reasonable request.
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Introduction
Age-related macular degeneration (AMD) is a common degenerative disease of the macula among elderly individuals. It is a leading cause of irreversible central vision impairment worldwide. Advanced AMD can be further classified into two forms, neovascular age-related macular degeneration (nAMD) and geographic atrophy. The nAMD, which is featured by choroidal neovascularisation (CNV), accounts for the majority of cases with severe visual loss due to AMD. Polypoidal choroidal vasculopathy (PCV), which is characterised by polyp-like choroidal vascular abnormalities,1 is usually considered as a subtype of nAMD. Similar to nAMD, PCV can also cause submacular haemorrhage, exudation and scarring, resulting in severe impairment of central vision. However, nAMD and PCV have differences in some aspects. The prevalence of nAMD is approximately 0.46% in both Asian and Caucasian populations, respectively.2 In contrast, the prevalence of PCV is higher in Asians than in Caucasians, ranging from 22.3% to 61.6% among Asians and 8%–13% among Caucasians.3 Therefore, PCV represents an important subtype of exudative AMD in Asian populations. Antivascular endothelial growth factor (anti-VEGF) therapy is the mainstay therapeutic approach for nAMD and PCV, but the treatment responses are different. While nAMD responses well to anti-VEGF monotherapy, PCV usually requires combined anti-VEGF and photodynamic therapy.4 Such similarities and differences suggest there could be shared and specific components in the mechanisms underlying nAMD and PCV.
Both AMD and PCV are complex diseases resulted from the interaction of multiple genetic and environmental risk factors. Previous genome-wide association studies had identified single-nucleotide polymorphisms (SNPs) in over 30 genetic loci associated with nAMD.5 Among them, the complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) were the major genes that have been confirmed in different populations.5–7 PCV shares some genetic components with nAMD, such as the CFH, ARMS2-HTRA1, ABCG1 and CETP.8 9 In a study in East Asians, PCV was found genetically highly correlated with nAMD, with AMD known loci accounting for up to 36% variation.10 However, the effect sizes of certain gene variants, such as those at the ARMS2-HTRA1 locus, varied between PCV and nAMD, with weaker association for PCV.10–12 Additionally, a whole-exome sequencing study found FGD6 to be associated with PCV rather than nAMD,13 while other genes, such as PGF 14 and SKIV2L,15 were associated with nAMD but not with PCV. These findings suggested nAMD and PCV have shared and distinct genetic components.
The VEGF pathway is important for angiogenesis, and it is currently the major therapeutic target against angiogenesis-related diseases. The VEGFA gene has been associated with both nAMD and PCV.10 The angiopoietin-TIE pathway is another essential endothelial and cell-specific pathway in regulating angiogenesis.16 It may provide excellent candidate genes for nAMD and/or PCV. Of note, the ANGPT2 gene in this pathway has been demonstrated to be associated with nAMD and PCV.17 Angiopoietin (Ang) ligands (Ang1, Ang2 and Ang4) and the TIE (Tie1 and Tie2) receptor tyrosine kinases form the main part of the angiopoietin-TIE pathway. The Tie2 protein is a tyrosine kinase receptor in angiogenic endothelial cells, and could bind the Ang ligands. Tie2 plays a vital role during the developing and remodelling of the vasculatures, whose activity is regulated by two secretory ligands Ang1 and Ang2.18 19 Ang1 could stimulate Tie2 to stabilise retinal and choroidal vessels, reducing their ability to sprout new vessels. In contrast, Ang2 could stimulate dephosphorylation of Tie2, leading to destabilisation of blood vessels and stimulation of neovascularisation.20 Therefore, the TIE2 gene (also known as TEK, tyrosine kinase, endothelial) could represent a potential genetic risk factor for nAMD and PCV. However, while the ANGPT2 gene has been implicated in nAMD and PCV,17 the role of the TIE2 gene in these diseases remained unknown. Of note, rare mutations in TIE2 had been found to cause primary congenital glaucoma (PCG), an important cause of childhood blindness.21 Therefore, it would be interesting to investigate whether common variants in TIE2 are implicated in AMD and PCV, a group of late-onset disease. In this study, we performed a haplotype-tagging SNP association analysis to evaluate the role of TIE2 in nAMD and PCV.
Materials and methods
Subjects and samples
Totally 2343 unrelated participants from three independent East Asian cohorts were enrolled in this study: a Hong Kong Chinese cohort of 214 nAMD patients, 236 PCV patients and 433 controls recruited from the eye clinics of the Prince of Wales Hospital and the Hong Kong Eye Hospital, Hong Kong; a Shantou Chinese cohort of 189 nAMD patients, 187 PCV patients and 531 controls recruited from the Joint Shantou International Eye Center, Shantou, China; an Osaka Japanese cohort of 192 nAMD patients, 204 PCV patients and 157 controls recruited from the Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan. Written informed consents were obtained from all subjects. All procedures in this study were performed in accordance with the tenets of the Declaration of Helsinki.
All patients had undergone detailed ophthalmic examinations, including best-corrected visual acuity measurement, ocular tonometry, slit-lamp biomicroscopy, fundus photography, fluorescein angiography (FA) and indocyanine green angiography (ICGA). Diagnosis and classification of AMD were made according to the Age-Related Eye Disease Study criteria.22 PCV was diagnosed according to the presence of nodular polypoidal lesions shown in ICGA.8 15 Diagnosis of nAMD and PCV was differentiated by FA and ICGA. All patients had nAMD or PCV in at least one eye. Other forms of CNV, such as myopic CNV, were excluded. Control subjects were recruited from attendants to the clinics for unrelated eye conditions and were also given detailed ophthalmic examinations; they were ≥50 years old and had no macular degeneration of any causes. All patients and controls in this study had no PCG or family history of PCG.
SNPs selection and genotyping
Totally 31 haplotype-tagging SNPs of the TIE2 gene were selected from HapMap Han Chinese (CHB), using the HapMap Genome Browser release #27 dataset (http://hapmap.ncbi.nlm.nih.gov). The tagger-pairwise programme was applied, with an R square (r2) cut-off of 0.8 and a minor allele frequency (MAF) cut-off of 0.1. However, rs10967724 failed the assay design and was removed from further analysis.
Genomic DNA from peripheral blood was extracted with a QIAamp Blood Kit (Qiagen, Hilden, Germany) referring to the standard protocol. All of the 30 SNPs were tested in all of the Hong Kong subjects, using TaqMan genotyping technology (Applied Biosystems, Foster City, California, USA) in a Roche LightCycle 480 Real-Time PCR machine (Roche Diagnostics; Basel, Switzerland), following the manufacturer’s instructions. Two SNPs (rs625767 and rs2273717) showing association in the Hong Kong samples were further investigated in the Shantou and Osaka cohorts.
Statistical analysis
Hardy-Weinberg equilibrium (HWE) of all SNPs in controls was assessed using χ2 test in PLINK (v1.07; http://zzz.bwh.harvard.edu/plink/).23 SNP-disease associations were assessed by comparing the allelic distributions between cases and controls using the χ2 test, with OR and 95% CIs estimated. Also, the effects of SNPs were assessed by logistic regression analysis adjusted for age and gender. In this study, the SNP-disease associations were initially assessed in nAMD and PCV individually, and then in a combined disease group involving all nAMD and PCV patients.
Pairwise gene-gene interaction was analysed in the Hong Kong sample using the epistasis option in PLINK. Those tested were between individual TIE2 SNPs and other AMD/PCV-associated SNPs reported in our previous studies, including CFH rs800292,24 HTRA1-ARMS2 rs11200638,12 SKIV2L rs42960815, CETP rs3764261,8 ABCG1 rs57137919,8 C3 rs17030,25 PGF rs226861514 and ANGPT2 rs4455855 and rs13269021.17 Genotype data of these genes were extracted from our previous studies.8 12 14 17 23 25
For data from the three study cohorts, a meta-analysis was performed to generate the pooled-ORs and 95% CIs for the two SNPs (rs625767 and rs2273717), using the fixed-effect (I2≤50%) or random-effect (I2>50%) model according to heterogeneity test results. The meta-analyses were conducted using Review Manager (RevMan, V.5.2, The Cochrane Collaboration, Copenhagen, Denmark). P value of <0.05 was considered statistically significant among individual association analysis and the meta-analysis. The p values were adjusted by the Bonferroni correction in multiple testing. A final p value of <0.0017 (0.05/30, where 30 was the number of SNPs involved in data analysis) was considered study-wide significant.
Results
Individual SNP association and SNP*SNP interaction in the Hong Kong cohort
Table 1 showed the characteristics of the study subjects in the three cohorts. In the Hong Kong cohort, the genotype call rates were 99.5% for all SNPs. None of the SNPs was deviated from HWE. In the association analysis, SNP rs625767 was associated with both nAMD and PCV, while rs2273717 was associated with PCV only (table 2). Other tagging SNPs in TIE2 did not show statistical significance (p>0.05; table 2). Since the mean ages and gender proportions were significantly different among the patient and control groups in the three cohorts (p<0.05), age and gender were adjusted in the association analyses by logistic regression. The association results remained largely unchanged after controlling for age and gender. SNP rs625767 (nAMD: p=0.027; OR=0.76, 95% CI 0.60 to 0.97; table 3) remained nominally associated with nAMD, while rs2273717 remained associated with PCV (p=0.029; OR=1.37, 95% CI 1.03 to 1.81; table 3). Moreover, these two SNPs were associated with combined nAMD and PCV (rs625767: p=0.013; OR=0.78, 95% CI 0.65 to 0.95; rs2273717: p=0.03; OR=1.29, 95% CI 1.03 to 1.62; table 3).
Characteristics of study subjects
Allelic association of SNPs in the TIE2 gene with nAMD and PCV in the Hong Kong cohort
Associated TIE2 SNPs in nAMD and PCV adjusted for age and gender
In epistatic analysis, no significant epistasis was detected between individual TIE2 SNPs and the SNPs in other genes: CFH, HTRA1-ARMS2, SKIV2L, CETP, ABCG1, C3, PGF and ANGPT2 (all p values were >0.05 for the interaction terms).
Replication study and meta-analysis
The two SNPs, rs625767 and rs2273717, followed HWE in controls of the Shantou and Osaka cohorts. In the Shantou cohort, rs625767 was associated with PCV after age and gender adjustment (p=0.024; OR=0.74, 95% CI 0.58 to 0.96), while the ORs for nAMD (OR=0.93, 95% CI 0.73 to 1.18) and pooled nAMD and PCV (OR=0.83, 95% CI 0.68 to 1.02) followed the same directions with those in the Hong Kong cohort (table 3). In the Osaka cohort, the ORs of rs625767 for nAMD (OR=0.70, 95% CI 0.46 to 1.06) and pooled nAMD and PCV (OR=0.91, 95% CI 0.64 to 1.28) followed the same trends as those in the Hong Kong and Shantou cohorts, but the OR for PCV was to the opposite trend (OR=1.09, 95% CI 0.75 to 1.58; table 3).
By pooling the genotype data of all study subjects from the Hong Kong, Shantou and Osaka cohorts using meta-analysis, SNP rs625767 showed a significant association with nAMD (p=0.01; OR=0.82, 95% CI 0.70 to 0.96; I2=0%), PCV (p=0.02; OR=0.83, 95% CI 0.71 to 0.97; I2=27%) and combined nAMD and PCV (p=0.002; OR=0.82, 95% CI 0.72 to 0.93; I2=0%), with low inter-cohort heterogeneities (figure 1).
The forest plots of meta-analysis for rs625767. Upper panel: nAMD; middle panel: PCV; lower panel: pooled nAMD and PCV. Individual and pooled OR were estimated for the A-allele frequencies. Squares indicate the study-specific OR. The size of the box is proportional to the per cent weight that each cohort contributed in the pooled OR. Horizontal lines indicate 95% CI. A diamond indicates the pooled or with its corresponding 95% CI. nAMD, neovascular age-related macular degeneration; PCV, polypoidal choroidal vasculopathy.
Discussion
In this work, we have identified the association of a haplotype-tagging SNP rs625767 in the TIE2 gene with nAMD and PCV in Chinese and Japanese groups, suggesting that TIE2 is an associated gene for nAMD and PCV. Further replication studies in more ethnic cohorts are warranted to confirm the role of TIE2 in nAMD and PCV in view of potential ethnic variations.
The TIE2 gene, spanning a 1 21 038 bp region on chromosome 9, encodes the Tie2 protein, which plays an important role in angiogenesis. In a previous study, variants rs80338908, rs80338909 and rs387906745 in the exons of TIE2 were associated multiple cutaneous and mucosal venous malformations.26 Another two TIE2 SNPs, rs638203 and rs639225, were found to be associated with vascular malformations.27 Regarding eye diseases, the TIE2 SNP rs581724 was associated with allergic conjunctivitis in asthmatic children.28 Also, rare mutations in TIE2 was found to cause PCG.21 These suggested TIE2 could have play a role in multiple ocular pathologies. In the present study, we adopted a haplotype-tagging SNP association analysis to explore the association of common SNPs in TIE2 with nAMD and PCV in three Asian cohorts. This is a relatively unbiased method to assess the association between a disease and a gene with good coverage. Interestingly, the SNP rs639225, which was associated vascular malformations,27 was among the TIE2 tagging SNPs in our present study. However, this SNP was not associated with nAMD, PCV or pooled nAMD and PCV (table 2). Instead, another tagging SNP rs625767, located in the intron 1 of TIE2, conferred increased risk for both nAMD and PCV. Currently, the role of this intronic SNP in TIE2 is not well known. By speculation, it could be correlated with the expression level of Tie2, playing a regulatory role or in linkage disequilibrium (LD) with other causal exonic variants. Further functional characterisation should be warranted. Of note, rs625767 is not in LD with rs80338908, rs80338909, rs387906745, rs638203 or rs639225 (r2<0.1), suggesting that TIE2 is a susceptibility gene for different diseases with allelic diversities, and that the association of TIE2 rs625767 with nAMD and PCV is a novel and independent association signal. Our findings enriched the growing understanding of TIE2 in ocular diseases.
Tie2 is a ubiquitous tyrosine kinase receptor in the vascular endothelium. Tie2 is constitutively phosphorylated in quiescent vasculature,29 suggesting a role in vascular maintenance. The loss of Tie2 signalling may cause vessel destabilisation, which facilitates VEGF-dependent angiogenesis.16 Tie2 is mainly regulated by Ang1 and Ang2, which are secreted by endothelial cells. Ang1, an important vascular stabiliser, activates Tie2 to protect vascular endothelial cells. Contrarily, Ang2 is an antagonist of Tie2. When the balance among Ang1, Ang2 and Tie2 is broken, there could be pathogenic consequences in blood vessels.30 Like Ang2, the vascular endothelial-protein tyrosine phosphatase (VE-PTP) also participates in suppressing the Tie2 pathway.31 Thus, the Tie2 axis should play a role in the development and stabilisation of blood vessels. Also, Tie2 is likely a crucial factor in systemic inflammation and a target of controlling host vascular response.32 In our previous study, we found that the ANGPT2 gene, which encodes the Ang2 protein, was associated with nAMD and PCV.17 In the present study, we further identified the association of the TIE2 gene with nAMD and PCV. These findings altogether suggested that the Tie2 protein and axis could be involved in the pathogenesis of the age-related choroidal vascular diseases, although further functional studies of the Tie2 axis in nAMD and PCV are warranted. The biological interaction between Ang1, Ang2 and Tie2 in angiogenesis arouse the interest to investigate whether there is also gene-gene interaction between the ANGPT2 and TIE2 genes in the genetic architecture of nAMD and/or PCV. However, epistasis analyses in this study revealed that there was no significant SNP-SNP interaction between TIE2 and ANGPT2 or other AMD-associated genes, namely CFH, HTRA1-ARMS2, SKIV2L, CETP, ABCG1, C3 and PGF. Therefore, TIE2 may not play an interactive role with these genes in the genetic architecture of nAMD and PCV. However, the lack of significant epistasis could be due to our limited sample size, therefore further interaction analysis in larger samples should be warranted.
Currently, anti-VEGF therapy is the mainstay of treatment for nAMD and PCV. However, there are patients who showed fair response to anti-VEGF therapy. Therefore, new therapies, including new drugs, are needed for broadening our capacity to treat nAMD and PCV. Targeted regulation of the secretion of Ang1 and/or Ang2, or the introduction of agonist and/or inhibitor to modify the Tie2 axis is a new therapeutic option for treating angiogenesis. For example, a small molecule AKB-9778, which can activate the Tie2 through competitively suppressing VE-PTP, could inhibit neovascularisation and VEGF-dependent angiogenesis.33 Notably, AKB-9778 had been shown effective in treating diabetic macular oedema.34 Whether it is also effective in treating nAMD and PCV remained to be evaluated.
There were some limitations in the present study. First, the statistical significance for the association of TIE2 rs625767 with nAMD and PCV were borderline in individual cohorts and also the pooled subjects from all cohorts. This could be due to the relatively small sample sizes in each cohort. Nevertheless, the association of TIE2 with nAMD and PCV is still convincing, as the pooled p values in the meta-analysis were significant with low inter-cohort heterogeneities. However, since we only included one Japanese cohort from Osaka, in which the TIE2 SNPs did not show statistical significance, further replication studies in other Japanese cohorts are needed to confirm the role of TIE2 in nAMD and PCV in Japanese. Second, this study involved only SNPs with MAF >10%, it is therefore underpowered to detect rare variants in TIE2 that could be associated with the disease with greater effect sizes. Third, smoking and endophenotypes data35 of most patients were incomplete, rendering difficulties in analysing the gene-smoking interaction and genotype-endophenotypes correlation in this study. Further studies in well-characterised cohorts of nAMD and PCV are warranted.
In summary, the present study identified a SNP rs625767 in the TIE2 gene to be associated with nAMD and PCV in Japanese and Chinese subjects, suggesting TIE2 as a susceptibility gene for nAMD and PCV. These findings enriched the growing understanding of the genetic architecture of nAMD and PCV, and provided suggestion of a novel treatment target for the diseases.
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information. Data are available upon reasonable request.
Ethics statements
Ethics approval
The study protocol was approved by the respective institutional Ethics Committee at the collaborating institutions.
Acknowledgments
The authors thank all the participants in this study.
References
Footnotes
ZJC and LM contributed equally.
Contributors LJC and CPP conceived the idea and designed the study. MB, HC, MT, TYYL, MH, KS, CH, NH, ALY, KN, CCT and LJC recruited the study subjects and performed clinical examinations. ZJC, LM and POST performed the experiments and collected data. ZJC, LM and LJC analysed the data and wrote the manuscript. LJC, CCT, ALY and CPP revised the paper. All authors approved the submitted version.
Funding This study was supported in part by the General Research Fund, Hong Kong (14120516 (LJC)), Direct Grant of Chinese University of Hong Kong Medical Panel, Hong Kong (4054281 (LJC)), National Natural Science Foundation of China (NSFC, 81500764 (LJC)), and the Endowment Fund for Lim Por-Yen Eye Genetics Research Centre, Hong Kong.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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