Introduction

Diabetic retinopathy (DR), one of the most common chronic ocular complications of type 2 diabetes mellitus (T2DM), often causes blindness in diabetic patients worldwide. Hyperglycemia and prolonged duration of diabetics have been deemed to be the main causes of DR.1 However, some individuals develop DR in a shorter period despite having a tight metabolic control which indicates that other factors may have a role in the development of DR.2 In the past few years, many factors, including endothelial nitric oxide synthase, receptor for advanced glycation end products, vascular endothelial growth factor and angiotensin converting enzyme were demonstrated to be involved in the development of DR.3, 4, 5, 6

Angiotensin II (AngII), the main active peptide within the renin–angiotensin system (RAS), has been conformed to have a key role in the pathogenesis of T2DM by inhibiting the secretion of insulin, promoting the generation of inflammation and fibrosis, or producing insulin resistance.7, 8, 9, 10, 11 It is reported that AngII participates in the progress of retinopathy by inducing the synthesis of vascular endothelial growth factor, platelet-derived growth factor and connective tissue growth factor.12 Blockade of the RAS provides beneficial effects in terms of delaying the development and progression of diabetes and DR.13, 14

AngI converting enzyme 2 (ACE2), a novel component of RAS, as well as an ACE homolog, was reported to be able to degrade AngII to Ang-(I–VII) which exerts opposing actions to those of AngII.15, 16 ACE2 is highly expressed in normal kidney, heart, and retina.15, 17, 18 Previous studies on single nucleotide polymorphisms (SNPs, rs2074192 and rs714205) in ACE2 gene and diabetic nephropathy or hypertension were conducted;19, 20, 21 however, only SNP rs2074192 was reported to be associated with diabetic hypertension in male individuals, no association was found between the two SNPs in ACE2 gene and diabetic nephropathy in either men or women. It is not clear whether ACE2 gene variants influence the retinopathy progress in T2DM cohort, and whether gender is involved. Herein, we sought to clarify the association of polymorphisms rs2074192 and rs714205 in ACE2 gene with retinopathy in a Chinese T2DM cohort.

Materials and methods

Participants

A case-control study was conducted in 743 Chinese cases with T2DM from Qilu Hospital of Shandong University including 408 female cases and 335 male cases. Since ACE2 gene is located on the X chromosome, female and male cases were analyzed, respectively. Female cases were divided into two groups: (1) diabetes without retinopathy (DNR) group comprised 171 patients with DNR and (2) DR group consisted 237 patients with DR, which was further subclassified into nonproliferative DR (NPDR) group with 121 patients and proliferative DR (PDR) group with 116 patients. Male cases were also assigned to DNR group containing 153 patients and DR group involving 182 DR patients, which was further grouped into NPDR group with 86 patients and PDR group with 96 patients. The diagnosis of T2DM was according to clinical features and the guidelines of the Expert Committee Report of the American Diabetes Association.22 Retinopathy status was graded according to the Early Treatment Diabetic Retinopathy Study.23 All participants were carefully assessed to ensure meeting the included criteria. The study protocol was approved by the Institutional Review Boards of Qilu Hospital of Shandong University and adhered to the tenets of the Declaration of Helsinki. All subjects gave their informed consent before anonymously participating in the study.

Genotyping

Genomic DNA was extracted from peripheral venous blood using the DNA Purification kit (TIANamp Blood DNA Kit, Tiangen Biotech, Beijing, China). ACE2 genotyping was performed using PCR-ligase detection reaction technology. The primer pairs used was as follows: rs2074192, 5′-ACTTTGCCCTTAAACACAGC-3′, 5′-AAGTGTGGAAATGTATAAAT-3′; rs714205, 5′-ATCTTGTGGTCTGAGAATGC-3′, and 5′-TCCATGAGTTCTAGCCAGAC-3′. The PCR amplification was performed in the GeneAmp PCR system 9600 (Applied Biosystems, Foster City, CA, USA) in a 20-μl reaction system and the mixture were denatured at 94 °C for 15 min and then 35 cycles of 94 °C for 30 s, 56 °C for 1 min, and 72 °C for 1 min and a final extension at 72 °C for 7 min. LDR was performed in a total volume of 10 μl containing 1 μl PCR product, 0.05 μl Taq DNA ligase, 1 μl probe mix, 1 μl Taq DNA ligase buffer, and 6.95 μl deionized water. The LDR conditions were 2 min at 95 °C followed by 35 cycles of 30 s at 94 °C, 2 min at 50 °C. The fluorescent products of ligase detection reaction were differentiated by ABI sequencer 377 (Applied Biosystems).

Statistical analysis

Clinical characteristics were compared using the student’s t-test for continuous variables and Pearson’s χ2-test for categorical variables. All frequency and genotype distributions of studied SNPs were tested for Hardy–Weinberg equilibrium (HWE). Distribution of allele, genotype, and haplotype frequencies of each group were estimated using χ2-test. Statistical analyses were conducted using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Pair-wise linkage disequilibrium (LD) and haplotype frequency estimations were obtained using http://analysis.bio-x.cn, r2 and D′ measures LD for the two investigated SNPs, and those haplotypes that exhibited a frequency <0.03 were ignored in analysis. Logistic regression was used to adjust the risk of DR for age, gender, blood glucose, HbA1c level, and duration of disease. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of the association. P-values <0.05 were considered nominally significant. Data were shown as mean±SD.

Results

Table 1 shows that significant differences existed in the duration of diabetes, systolic blood pressure, the level of blood urea nitrogen, and total cholesterol in DR group than in DNR group in the Chinese T2DM cohort (P<0.05). However, no statistically significant differences were observed in age, body mass index, the level of fasting plasma glucose, hemoglobin A1c, and triacylglycero between the DR and DNR groups.

Table 1 Clinical characteristics of type 2 diabetic patients with retinopathy (DR) and without retinopathy (DNR)
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The ACE2 gene is X linked, herein the analyses in this study were performed on female and male subjects separately. In the female cases, the genotype distribution of the two SNPs rs2074192 and rs714205 among female cases are given in Table 2, they were in HWE in DNR and DR groups (P>0.05, respectively). The frequency of genotype TT in SNP rs2074192 was significantly increased in DR group (OR=1.65; 95% CI=1.01–2.71, P=0.046) and PDR group (OR=1.97; 95% CI=1.12–3.47, P=0.02) compared with DNR group, respectively. Moreover, genotype CC in rs2074192 was significantly lower in PDR group (OR=0.56; 95% CI=0.32–0.98, P=0.04) than in NPDR group. No significant difference was detected on the genotype distribution of SNP rs2074192 between NPDR and DNR groups. Compared with DNR group, the frequency of genotype CC in SNP rs714205 was demonstrated significantly higher in DR group (OR=1.61; 95% CI=1.004–2.59, P=0.047) and PDR group (OR=1.84; 95% CI=1.07–3.18, P=0.03), respectively. However, no positive results were shown on this genotype in the comparison between groups NPDR and DNR, PDR and NPDR, respectively.

Table 2 Frequencies of genotype and allele in female patients
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The frequencies of alleles at both loci in female T2DM cases are shown in Table 2. Compared with DNR group, the frequency of allele T in rs2074192 locus was significantly higher in DR group (OR=1.49; 95% CI=1.12–1.98, P=0.006) and PDR group (OR=1.81; 95% CI=1.29–2.87, P=0.0006), respectively. Moreover, allele T frequency in SNP rs2074192 was found to increase the risk of PDR compared with NPDR (OR=1.47; 95% CI=1.02–1.14, P=0.04). In rs714205 locus, the frequency of allele C was significantly higher in DR group (P=0.01) and PDR group (P=0.004) compared with DNR group respectively, the strength of their association with DR and PDR were indicated as OR=1.45, 95% CI=1.09–1.92 and OR=1.65, 95% CI=1.18–2.31, respectively.

The two investigated SNPs were in LD with each other (DR vs DNR: r2=0.83, D′=1.00; PDR vs DNR: r2=0.80, D′=1.00; NPDR vs DNR: r2=0.85, D′=1.00). Haplotype frequencies of the two loci among female T2DM subjects are summarized in Table 3. Of the haplotypes constructed, three were found to be common, and were included in subsequent analysis. Significantly increased frequency of haplotype TC was seen in DR (OR=1.49; 95% CI=1.12–1.98, P=0.006) and PDR groups (OR=1.81; 95% CI=1.29–2.54, P=0.0006) compared with DNR, respectively. Meanwhile, reduced frequency of haplotype CG were found in DR (OR=0.68; 95% CI=0.52–0.90, P=0.008) and PDR groups (OR=0.60; 95% CI=0.43–0.84, P=0.003). No significant differences were found in NPDR compared with DNR.

Table 3 Analysis of haplotype frequency for rs2074192 and rs714205 in female patients
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No significance was observed on the frequency distribution analyses of genotypes and alleles at both loci among male T2DM subjects (Table 4).

Table 4 Frequencies of allele in male patients
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Discussion

Some studies have been conducted to report the association between polymorphisms (SNPs, rs2074192 and rs714205) in ACE2 gene and diabetic nephropathy or hypertension.19, 20, 21 It is not clear whether ACE2 gene variants influence the retinopathy progress in T2DM cohort. In this study, we analyzed these two SNPs in the ACE2 gene in T2DM patients.

This study showed a strong correlation of SNPs rs2074192 and rs714205 polymorphism in ACE2 gene with DR and PDR in Chinese female T2DM patients, not in male T2DM patients. The ACE2 gene is located on chromosome Xp22 and consists of 18 exons and 17 introns, which infers its gender characteristics.24 Therefore, we analyzed the two SNPs in female and male, respectively. In the female group, the genotypes TT or CC and alleles T or C in SNPs rs2074192 or rs714205 were demonstrated as risk genotypes or alleles in DR and PDR; however, minor homozygous genotype CC in SNP rs2074192 was shown protective on PDR individuals. Moreover, strong LD was detected between the two loci. The haplotype TC comprised by the risk alleles of the two SNPs was significantly higher in DR group, especially in PDR group, whereas the protective haplotype CG was significantly over represented in DNR group. It is a crucial finding that SNPs rs2074192 and rs714205 in the ACE2 gene were associated with the susceptibility to DR and PDR in the Chinese female T2DM cohort. The minor homozygous genotype in SNP rs2074192 can reduce the risk of PDR. We also found that TC or CG may be the risk or protective haplotypes of DR subjects, especially PDR subjects.

To the best of our knowledge, this is the first study to investigate the association of ACE2 gene polymorphism with DR in T2DM. It is demonstrated that ACE2 cleaves AngII to form Ang-(I–VII) with a high catalytic efficiency; whereafter, Ang-(I–VII) exerts opposing actions to those of AngII by inhibiting vasoconstriction and cell proliferation and consequently protect the retina.16, 25 The reduction of serum level of Ang-(I–VII) may reduce the protective function on retina and cause the occurrence and development of DR. In this study, we found that ACE2 gene variants were probably involved in the pathogenesis of DR and PDR in the Chinese female T2DM cohort. The possible reason may be that the variants rs2074192 and rs714205 located on the intron area of ACE2 gene which may affect the expression of ACE2 gene by regulating the splicing of mRNA and eventually results in the decrease of serum level of Ang-(I–VII).

As we know, gender and sex hormones can probably affect the components of the RAS,26 ACE2 is one of the components of RAS gene that is located on X chromosome.27 The escapes of X inactivation are reported on many genes may contribute to the sexual differences of ACE2 gene in this Chinese T2DM cohort.27

In conclusion, the present study demonstrates that the ACE2 gene polymorphisms rs2074192 and rs714205 are correlated with DR in the Chinese female T2DM cohort. The two SNPs can increase the susceptibility of DR and PDR. The minor homozygous genotype at SNP rs2074192 can reduce the risk of PDR. Moreover, the risk and protective haplotypes comprised by the two SNPs were found in DR. Further researches focusing on the exact biological mechanism and other possible factors underlying the association of the ACE2 gene with DR in T2DM individuals are needed to be confirmed in the future.