Association Of Polymorphisms Of ACE2with Diabetic Retinopathy And A Meta-analysis On Color Doppler Imaging For Retrobulbar Blood Flow Velocities

OBJECTIVE Diabetic retinopathy (DR), one of the most common chronic ocular complications of Type2diabetes mellitus (T2DM), often causes blindness in diabetes patients around the world. Hyperglycemia and prolonged duration of diabetics have been deemed to be the main causes of DR. However, some individuals develop DR in a shorter period despite having a tight metabolic control; which indicates that other factors may play a role in the development of DR。In recent years, studies about single nucleotide polymorphism(SNP) in DR get more and more attention. Moreover, change of eyeball blood supply in patients with DR got the attention of scholars both at home and abroad. Therefore, we explore the related factors of DR from the following two aspects. First, a case-control study. was performed to survey the relation of SNP rs2074192and rs714205in ACE2gene and DR in Chinese T2DM risk, providing a molecular basis for the early detection and treatment of DR. Second, a meta-analysis was conducted on related literatures to assess the condition of eyeball blood supply in DR patients.METHODS Case-control study:A case-control study was carry out to detect two SNPs (rs2074192and rs714205) in ACE2gene for the association with retinopathy in the Chinese T2DM cohort.743T2DM participants were involved in this study including408female cases and335male cases which were analyzed respectively since ACE2gene is positioned in the X-chromosome. Female cases were assigned into two groups:(1) Diabetes without retinopathy (NDR) group comprised171patients with NDR;(2) Diabetic retinopathy (DR) group consisted237patients with DR, which was additionally subgrouped into nonproliferative DR (NPDR) group with121patients and proliferative DR (PDR) group with116patients. Male cases were also assigned to NDR group containing153patients and DR group involving182DR patients, which was further grouped into NPDR group with86patients and PDR group with96patients. The genotypes were identified by polymerase chain reaction-ligase detection reaction (PCR-LDR) techniques.SPSS software was used for statistical analyses (version17.0, SPSS Inc, Chicago, IL).Clinical data were analyzed using the student’s t-test for continuous variables and Pearson’s chi-square test for classified variables. The distributions of genotype of control group were detected for Hardy-Weinberg equilibrium (HWE). The distribution of allele, genotype, and haplotype frequencies of each group were assessed using chi-square test. Pair-wised linkage disequilibrium (LD) and haplotype distribution were obtained utilizing SHEsis, r2and D’measures LD for the two investigated SNPs. Odds ratios (ORs) and95%confidence intervals (CIs) were counted to estimate the size of relation, p-values under0.05were identified to have statistical significance. Data were shown as mean±SD.Meta analysis:Two researchers independently performed a computerized search in four database-PubMed, ISI Web of Knowledge (Version4.5), the Cochrane Central Register of Controlled Trials, Chinese national knowledge infrastructure (CNKI), and Wanfang (Chinese). RevMan software5.0was used for this meta-analysis. For each participant study, we calculated the MD of the continuous outcomes (PSV, EDV or RI) along with95%CIs. The between-study heterogeneity was tested by the chi-square-based Cochran’s statistics and the I2. Statistically significant heterogeneity was considered present with Pheterogeneity<0.05and I2>50%. In the presence of substantial heterogeneity (P>50%), the random effect model (REM) was adopted as the pooling method; otherwise, the fixed effect model (FEM) was used. The sensitivity analysis was performed to find the key studies with a substantial impact on between-study heterogeneity. A funnel plot was conducted to look for evidence of publication bias.RESULTS1、The results of case control study(1) Allele distribution①rs2074192In female T2DM cases, The allele distribution analyses showed that alleles T in SNP rs2074192was considered as the risk alleles of DR group (OR:1.49,95%CI:1.12-1.98,p=0.006) or PDR group (OR:1.81,95%CI:1.29-2.87, p=0.0006) compared with NDR group。Allele T in SNP rs2074192was also likely the risk allele of PDR group (p=0.04) compared with NPDR group. No posotive realtion was seen between rs2074192mutation and DR in male T2DM individuals.②rs714205The allele distribution analyses showed that alleles C in SNP rs714205were considered as the risk alleles of DR group (OR:1.45,95%CI:1.09-1.92,p=0.01) or PDR group (OR:1.65,95%CI:1.18-2.31,p=0.004) compared with DNR group. No positive results in other comparisons. No significant relation was detected between rs714205mutation and DR in male T2DM individuals.(2) Genotype distribution①rs2074192In female T2DM cases, the frequency of genotypes TT in rs2074192was suggested to be associated with the increased risk of DR group (OR:1.65,95%CI:1.01-2.71,p0.046) and PDR group (OR:1.97,95%CI:1.12-3.47, p=0.02) compared with NDR group. Moreover, the distribution of genotype CC in SNP rs2074192was lower in PDR group (OR:0.56,95%CI:0.32-0.98, p=0.04) than in NPDR group.②rs714205the frequency of genotypes CC in rs714205was suggested to be associated with the increased risk of 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) compared with DNR group.(3) LD and Haplotype distributionThe two investigated SNPs were in LD in female T2DM participants (D’-1.00). Haplotype distribution analyses indicated that haplotype TC was remarkable higher in DR (OR:1.49,95%CI:1.12-1.98,p=0.006) and PDR group (OR:1.81,95%CI:1.29-2.54,p=0.0006); however, 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).2%The results of Meta-analysisIn group1, NDR eyes have statistically significant increase in PSV of OA (p=0.002); however, significant reduction of PSV and EDV were shown in CRA (p=0.002,p=0.007; respectively). A significant increase in RI-OA (p=0.02) was found inNDR eyes. In group2, PSV and EDV of CRA in DR eyes were shown significant decrease (p<0.00001, respectively). Similar results appeared in EDV-OA (p=0.0003) and in SPCA (p<0.00001). RI-OA was significant higher in DR eye (p=0.0008) than controls. In group3, PSV in OA was lower in DR eyes (p=0.04) than in NDR eyes, and PSV and EDV of CRA in DR eyes were shown significant decrease (p=0.004, p<0.00001, respectively) compared with NDR eyes. Meanwhile, RI in OA and CRA were found in DR eyes (p=0.05, p<0.00001, respectively).CONCLUSIONS (1) Our study reported the association of ACE2gene polymorphisms and DR in Chinese T2DM individuals for the first time. SNPs rs2074192and rs714205in ACE2gene were associated with the susceptibility to DR and PDR in Chinese female T2DM patients. Moreover, haplotypes rs2074192T-rs714205C and rs2074192C-rs714205G were found to be the risk and protective haplotypes respectively in DR group in Chinese female T2DM patients. The SNP loci rs2074192and rs714205in ACE2gene can be used as an index forecasting the occurrence and development of DR and PDR for clinical application (2) The results of meta-analysis showed that the condition of eyball blood supply in NDR and DR patients had certain degree changes and retrobulbar blood flow parameters can be used as an objective indicators monitoring the progress of DR. Moreover, because of the advantage of CDI, it can be perfored as a routine inspection in patients with DR.