Association between the Angiotensin-Converting Enzyme (ACE) Genetic Polymorphism and Diabetic Retinopathy—A Meta-Analysis Comprising 10,168 Subjects

Aims—to address the inconclusive findings of the association of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism on risk of diabetic retinopathy (DR), a meta-analysis was conducted. Methods—we conducted a meta-analysis on 4252 DR cases and 5916 controls from 40 published studies by searching electronic databases and reference lists of relevant articles. A random-effects or fixed-effects model was used to estimate the overall and stratification effect sizes on ACE I/D polymorphism on the risk of DR. Results—we found a significant association between the ACE I/D polymorphism and the risk of DR for all genetic model (ID vs. II: OR = 1.14, 95% CI: 1.00–1.30; DD vs. II: OR = 1.38, 95% CI: 1.11–1.71; Allele contrast: OR = 1.17, 95% CI: 1.05–1.30; recessive model: OR = 1.24, 95% CI: 1.02–1.51 and dominant model: OR = 1.21, 95% CI: 1.06–1.38, respectively). In stratified analysis by ethnicity and DM type, we further found that the Asian group with T2DM showed a significant association for all genetic models (ID vs. II: OR = 1.14, 95% CI: 1.01–1.30; DD vs. II: OR = 1.54, 95% CI: 1.14–2.08; Allele contrast: OR = 1.26, 95% CI: 1.09–1.47; recessive model: OR = 1.42, 95% CI: 1.07–1.88 and dominant model: OR = 1.26, 95% CI: 1.07–1.49, respectively). Conclusion—our study suggested that the ACE I/D polymorphism may contribute to DR development, especially in the Asian group with type 2 diabetes mellitus (T2DM). Prospective and more genome-wide association studies (GWAS) are needed to clarify the real role of the ACE gene in determining susceptibility to DR.


Introduction
Diabetic retinopathy(DR) is the premier cause of vision loss in adults aged 20-74 years [1]. From 1990 to 2010, DR ranked as the fifth most accpeted cause of preventable blindness and moderate to severe visual impairment [2]. Diabetic retinopathy (DR) is a microvascular complication occurring both in type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), and it was estimated that, of 285 million people worldwide with diabetes, over one-third had signs of DR in 2010 [3]. DR is a complex trait involving polygenic, metabolic, and environmental influences. Known risk factors, most notably the duration of diabetes and glycemic control, explain some, but not all, of the progression

Data Extraction and Conversion
Two investigators independently extracted data and reached a consensus on all of the items. Data extracted from these articles included the first author's name, year of publication, study design, ethnicity of the study population, type of DM, clinical characteristics, and the number of cases and controls for ACE I/D genotypes. The frequencies of the alleles and the genotypic distributions were extracted or calculated for both cases and controls. We defined that diabetic patients without retinopathy and/or matched healthy persons constituted the control group, and patients with DR were the case group. We merged the original data into the control group or case group if the study did not provide corresponding data. For some studies without sufficient information for extraction of data, we tried to contact the studies' authors by sending emails to request data missing from their articles.
In addition, it was tested whether the distribution of genotypes in the controls was consistent with the Hardy-Weinberg equilibrium (HWE) for each study, and calculated the frequency of the minor allele for ACE I/D polymorphism.

Quality Assessment and Study Stratification
The Newcastle-Ottawa scale (NOS) method was used to assess the observational included studies. The NOS is composed of three parts (8 entries): selection, comparability, and exposure. A quality item is given only one star for the study in selection and exposure, and a quality item is given, at most, two stars for the study in comparability. It is a semi-quantitative scale, and a score of 0-9 stars is assigned to each study. Studies whose scores were more than 6 stars were considered to be of relatively high quality [58]. The scores of included studies are shown in Table 1.

Meta-Analysis
The meta-analysis evaluated the relationship between the ACE I/D polymorphism and the risk of DR for each study by odds ratio (OR), with 95% confidence intervals (95% CI). For all studies, we calculated the ORs for the: (i) separate pairwise comparisons; (ii) allele contrast; (iii) recessive model; and (iv) dominant model. In addition, we conducted stratification analysis by ethnicity and DM type. A sensitivity analysis, which examines the effect of excluding specific studies, was also performed [59]. Our meta-analysis was subjected to sensitivity analysis for studies with the controls not in HWE (p < 0.05).
The χ 2 -based Q statistic test was used to assess the heterogeneity, and it was considered significant for p < 0.05. Heterogeneity was quantified with the I 2 metric, which is independent of the number of studies in the meta-analysis. I 2 takes values between 0% and 100%, with higher values denoting a greater degree of heterogeneity (I 2 > 50% was considered significant) [60]. We used the fixed-effects model and the random-effects model based on the Mantel-Haenszel method and the DerSimonian and Laird method, respectively, to combine values from each of the studies. When the effects were assumed to be homogenous, the fixed-effects model was then used; otherwise, the random-effects model was more appropriate [61]. In addition, we further conducted meta-regression analyses to estimate the source of heterogeneity. Publication bias was assessed according to the Egger regression asymmetry test and the Begg adjusted rank correlation test [62,63]. All analysis was done by using the Stata software (v.12.1) (StataCorp LP, College Station, TX, USA). All the p values were two-sided.

Literature Search
The study selection process is shown in Figure 1. A total of 660 articles (PubMed 572, CNKI 88) were identified from the databases, and 0 duplicates were excluded, using EndNote (X7) (Thomson ResearchSoft, Stamford, CT, USA). In addition, 581 articles were excluded, based on a review of the titles and abstracts, and 79 full-text articles were assessed for eligibility; 37 articles were excluded due to various reasons, such as being review articles or case reports, being written in languages other than English or Chinese, or could not provide each genotype frequency or other sufficient information for extraction of data. Finally, a total of 40 54,55] articles were included in this meta-analysis.

Literature Search
The study selection process is shown in Figure 1. A total of 660 articles (PubMed 572, CNKI 88) were identified from the databases, and 0 duplicates were excluded, using EndNote (X7) (Thomson ResearchSoft, Stamford, CT, USA). In addition, 581 articles were excluded, based on a review of the titles and abstracts, and 79 full-text articles were assessed for eligibility; 37 articles were excluded due to various reasons, such as being review articles or case reports, being written in languages other than English or Chinese, or could not provide each genotype frequency or other sufficient information for extraction of data. Finally, a total of 40 54,55] articles were included in this meta-analysis.

Eligible Studies and Study Characteristics
The selected study characteristics from the studies included in the meta-analysis are provided in Table 1, and the details on ACE I/D polymorphism allele/genotype prevalence are shown in Table  2. For 40 studies, 8 studies (7 Non-Asian, 1 Asian) involved cases with T1DM, 33 (9 Non-Asian, 24 Asian) with T2DM, and 1 study ( [21])with un-defined DM type (1 Asian study with 100 cases and 164 controls). It is worth emphasizing that 2 studies ( [17,44]) involved both T1DM and T2DM. The studies on T1DM Non-Asians contributed 599 cases and 614 control subjects, while the Asian studies included 33 cases and 104 control subjects. Among the T2DM studies, studies involving Non-Asians contributed 865 cases and 1541 control subjects, while the Asian studies included 2655 cases and 3659 control subjects. Thirty-three studies were case-control study design, 4 studies were cross-sectional study design, and 3 studies were cohort study design.

Eligible Studies and Study Characteristics
The selected study characteristics from the studies included in the meta-analysis are provided in Table 1, and the details on ACE I/D polymorphism allele/genotype prevalence are shown in Table 2. For 40 studies, 8 studies (7 Non-Asian, 1 Asian) involved cases with T1DM, 33 (9 Non-Asian, 24 Asian) with T2DM, and 1 study ( [21])with un-defined DM type (1 Asian study with 100 cases and 164 controls). It is worth emphasizing that 2 studies ( [17,44]) involved both T1DM and T2DM. The studies on T1DM Non-Asians contributed 599 cases and 614 control subjects, while the Asian studies included 33 cases and 104 control subjects. Among the T2DM studies, studies involving Non-Asians contributed 865 cases and 1541 control subjects, while the Asian studies included 2655 cases and 3659 control subjects. Thirty-three studies were case-control study design, 4 studies were cross-sectional study design, and 3 studies were cohort study design.

Summary Statistics
Data from 40 articles that investigated the association between the ACE I/D polymorphism and DR risk were included in the meta-analysis. The overall frequency (%) of minor D allele frequency (MAF) was 0.47/0.46 for cases and controls. The frequency of the MAF for each study polymorphism on controls is shown in Table 1. All studies suggested that the genotypes distribution in controls was consistent with the Hardy-Weinberg equilibrium except for 8 studies ( [22,28,30,35,38,43,48,51]), indicating genotyping errors and/or population stratification [59]; therefore, a sensitivity analysis was performed by excluding these studies.

Main Results, Stratification, and Sensitivity Analyses
The estimation of the relationship of ACE I/D polymorphism with DR is presented in Table 3. Figure 2 shows the overall effect for the relationship between the polymorphism and the DR risk in dominant model.  As shown in Table 3, the overall analysis found a significant association between the ACE I/D polymorphism and the risk of DR for all genetic models (ID vs. II: OR = 1.14, 95% CI: In a stratified analysis by ethnicity and DM type, we further detected that the Asian group, T2DM group, and Asian group with T2DM all showed significant associations for all genetic models (ID vs. II: OR = 1.14, 95% CI: 1.01-1.29 for the Asian group, OR = 1.13, 95% CI: 1.00-1.24 for the T2DM group and OR = 1.14, 95% CI: 1.01-1.30 for the Asian group with T2DM, respectively; DD vs. II: OR = 1.54, 95% CI: 1.16-2.04 for the Asian group, OR = 1.39, 95% CI: 1.10-1.74 for the T2DM group and OR = 1.54, 95% CI: 1.14-2.08 for the Asian group with T2DM, respectively; Allele contrast: OR = 1.26, 95% CI: 1.10-1.45 for the Asian group, OR = 1.17, 95% CI: 1.04-1.32 for the T2DM group and OR = 1.26, 95% CI: 1.09-1.47 for the Asian group with T2DM, respectively; recessive model: OR = 1.42, 95% CI: 1.08-1.85 for the Asian group, OR = 1.24, 95% CI: 1.01-1.54 for the T2DM group and OR = 1.42, 95% CI: 1.07-1.88 for the Asian group with T2DM, respectively and dominant model: OR = 1.26, 95% CI: 1.08-1.47 for the Asian group, OR = 1.19, 95% CI: 1.05-1.36 for the T2DM group and OR = 1.26, 95% CI: 1.07-1.49 for the Asian group with T2DM, respectively). However, we did not find any significant effects for different genetic models in other subgroup. Further sensitivity analysis for HWE did not alter the pattern of results in both overall analysis and subgroup analysis. Health 2016, 13, 1142 12 of 18 # Sensitivity analysis for HWE; * test for heterogeneity; random-effects model was used when p value for heterogeneity test < 0.05 and I 2 > 50%; otherwise, fixed-effects model was used. In a stratified analysis by ethnicity and DM type, we further detected that the Asian group, T2DM group, and Asian group with T2DM all showed significant associations for all genetic models (ID vs. II: OR = 1.14, 95% CI: 1.01-1.29 for the Asian group, OR = 1.13, 95% CI: 1.00-1.24 for the T2DM group and OR = 1.14, 95% CI: 1.01-1.30 for the Asian group with T2DM, respectively; DD vs. II: OR = 1.54, 95% CI: 1.16-2.04 for the Asian group, OR = 1.39, 95% CI: 1.10-1.74 for the T2DM group and OR = 1.54, 95% CI: 1.14-2.08 for the Asian group with T2DM, respectively; Allele contrast: OR = 1.26, 95% CI: 1.10-1. 45

Source of Heterogeneity and Publication Bias
From Table 3, we found that the heterogeneity between studies was observed in overall comparisons as well as subgroup analyses. We estimated the source of heterogeneity in both dominant and recessive genetic models of the variant allele by ethnicity (Asian or Non-Asian), DM type (T1DM or T2DM), HWE (in HWE or not), and study design (case-control, cross-sectional, or cohort study design) by meta-regression analyses. It revealed that none of these four factors could influence significant between-study heterogeneity in genetic models for ACE I/D polymorphism: ethnicity (p = 0.78 for dominant model and p = 0.39 for recessive model), DM type (p = 0.59 for dominant model and p = 0.9 for recessive model), HWE (p = 0.26 for dominant model and p = 0.77 for recessive model), and study design (p = 0.06 for dominant model and p = 0.24 for recessive model).
The potential presence of publication bias was estimated by using a funnel plot of the evaluation of log-odds ratio for the genotype DD+ ID versus II against the reciprocal of its standard error ( Figure 3). As shown, we failed to find any significant funnel asymmetry to indicate publication bias. We further used the Egger regression asymmetry test and the Begg adjusted rank correlation test to estimate the publication bias of literatures included in the meta-analysis. As shown in Table 4, no publication bias was found for polymorphism and risk of DR in genetic models.
From Table 3, we found that the heterogeneity between studies was observed in overall comparisons as well as subgroup analyses. We estimated the source of heterogeneity in both dominant and recessive genetic models of the variant allele by ethnicity (Asian or Non-Asian), DM type (T1DM or T2DM), HWE (in HWE or not), and study design (case-control, cross-sectional, or cohort study design) by meta-regression analyses. It revealed that none of these four factors could influence significant between-study heterogeneity in genetic models for ACE I/D polymorphism: ethnicity (p = 0.78 for dominant model and p = 0.39 for recessive model), DM type (p = 0.59 for dominant model and p = 0.9 for recessive model), HWE (p = 0.26 for dominant model and p = 0.77 for recessive model), and study design (p = 0.06 for dominant model and p = 0.24 for recessive model).
The potential presence of publication bias was estimated by using a funnel plot of the evaluation of log-odds ratio for the genotype DD+ ID versus II against the reciprocal of its standard error ( Figure 3). As shown, we failed to find any significant funnel asymmetry to indicate publication bias. We further used the Egger regression asymmetry test and the Begg adjusted rank correlation test to estimate the publication bias of literatures included in the meta-analysis. As shown in Table 4, no publication bias was found for polymorphism and risk of DR in genetic models.

Discussion
Why some diabetics develop retinopathy, whereas others do not, despite having long-term hyperglycemia, remains an undetermined question. Because known environmental factors do not fully explain this, researchers have sought the answer in the genetic background of the host [32]. The rennin-angiotensin-aldosterone system (RAAS) has been strongly implicated in the pathogenesis of progressive diabetes [64]. The RAAS is a critical regulator of sodium balance, extracellular fluid volume, vascular resistance, and, ultimately, arterial blood pressure by angiotensin II [61,65,66]. Thus, the RAAS serves as one of the most powerful regulators of arterial blood pressure and atherosclerosis and could be considered candidate genes involved in the pathogenesis of diabetic complications, including DR [67,68]. As the gene-encoding components of the RAAS, the ACE gene plays an important role in the RAAS, which is a complicated regulatory network with intrinsic like extrinsic agonistic and antagonistic hormones. It has been increasingly recognized that ACE inhibition demonstrates function and tissue protection of considered organs, to improve eye function of patients with diabetes mellitus and reduce the development and progression of DR [69,70]. In 1990, Rigat et al. described the polymorphism of the ACE gene based on the presence (insertion I) or absence (deletion D) of a 287 base pair element in intron 16 [11]. In plasma ACE levels, this genotype accounts for 47% of the total phenotypic variance in healthy individuals in a way that individuals with D alleles have an increased activity [11]. In addition, Danser et al. showed that the ACE I/D polymorphism also influences ACE tissue concentrations [9]. Numerous investigations into the potential role of ACE as a susceptibility gene for DR have been conducted over the past decades, with controversial results. Early meta-analyses attempted to reconcile these findings, but attempts to draw definite conclusions have been hindered by limited data, particularly when examining specific patient subgroups and increased relative studies [56,57].
It is worth emphasizing that our current meta-analysis obtained several critically different conclusions from the previous reports [56,57]. In Zhou's [56] report, they conducted a separate analysis of only the T2DM and T1DM groups, which showed that the ACE genotype has a non-significant association with DR, regardless of diabetic type. Lu et al. [57] performed the meta-analysis on only the Chinese population, without any subgroup analysis on DM type and ethnicity. However, from the present meta-analysis of 40 studies reported from 1994 to 2016 and comprising 10,168 subjects, we not only found the main effects of ACE I/D polymorphism on DR risk, but also found a significant relationship in the T2DM group. From the stratification analysis by ethnicity and DM type, we found that the ACE I/D polymorphism was significantly associated with DR risk in the T2DM and Asian groups, especially in the Asian group with T2DM. These findings may indicate that genetic factors may have more impact on the Asian population with T2DM, rather than on other subgroups like the T1DM and Non-Asian population.
We conducted a comprehensive meta-analysis on 40 published studies with 4252 diabetic retinopathy cases and 5916 controls relating the variant of the ACE I/D to the risk of DR, which can provide better ability to detect smaller effect sizes. Its strength was based on the accumulation of published data, giving greater information to detect significant differences. In order to estimate the power of the study, we used the Power and Precision 4 software to conduct the power calculation by respectively accumulating the frequency of ACE D allele in case and control groups from all studies, and the result showed the power of our study is 80.2%.
In this study, the effect of separate pairwise comparisons, allele contrast, and the dominant and recessive genetic models were evaluated. Substratification analysis by DM type andethnicity, and sensitivity analysis for studies not in HWE, was performed. In addition, we further evaluated the source of heterogeneity and the publication bias of included literatures.
Despite this, we still have some limits. In the meta-analysis, non-English/Chinese, non-indexed, and non-published studies literature was not reviewed, thus, some bias might be introduced [71]; only the unadjusted pooled ORs were calculated, since data for probable confounding factors that influence the estimates of associations (e.g., age, sex, BMI) were not provided; sampling variability and stratification in genetic association studies could be a possible confounding factor in the role of genetic markers. In addition, the risk effect may depend on the interaction with other risk factors: diabetes duration, HbA 1c , blood pressure, total serum cholesterol, control of diabetes, and body mass index, all of which modulate the development of DR [3]. Furthermore, small numbers of individuals and inadequate information of lifestyle factors and dietary intake by the published studies limited our statistic power to fully investigate the gene-environment interactions [61]. Therefore, further well-designed large studies, particularly referring to GWAS and gene-environment interactions are warranted to determinate the real contribution of these polymorphisms to DR risk susceptibility and might further indicate the genetics of DR.

Conclusions
In conclusion, the present meta-analysis finds an association between DR and ACE I/D polymorphism, especially in the Asian group with T2DM. Prospective and more genome-wide association studies (GWAS) are needed to clarify the real role of the ACE gene in determining susceptibility to DR.