Study population
In this case-control study, a total of 336 subjects with T2DM and a defined ophthalmologic status were recruited from the Second People’s Hospital of Yunnan Province between June 2014 and October 2016. The diagnosis of diabetes was in accordance with the 1999 World Health Organization (WHO) criteria (fasting plasma glucose >7.0 mmol/ L, 2h plasma glucose >11.1 mmol/L, or both) (28). A complete ophthalmological examination (including corrected slit-lamp microscopic examination, funduscopic examination and fundus photography) was performed by senior ophthalmologists blinded to the T2DM status of the participants following a standardized protocol. DR was defined and staged according to the proposed Clinical International Classification Systems (29). The T2DM group consisted of 124 subjects with proliferative diabetic retinopathy (PDR), 70 subjects with non-proliferative diabetic retinopathy (NPDR) and 142 diabetic subjects without clinical signs of diabetic retinopathy (NDR). To avoid the confounding effect of other vascular diseases, patients with malignancy, type 1 diabetes, hypertension preceding T2DM, cardiac, primary renal and liver diseases were excluded from our study. Seventy-two age-matched healthy controls were also enrolled from the physical examination center of the Second People’s Hospital of Yunnan Province. In this study, the inclusion criteria for the control participants were: (1) no diabetes symptoms; (2) no history of dyslipidemia, coronary artery diseases, cerebral stroke, etc.; (3) no family history of diabetes, hypertension; (4) normal glucose tolerance assessed by a standard 75 g OGTT (fasting plasma glucose < 6.1 mmol/L, 2 h plasma glucose < 7.8 mmol/L).
Ethical principles
The study was approved by the institutional review board of the Second People’s Hospital of Yunnan Province and Yunnan University and was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was provided by each participant prior to the study.
Clinical evaluation and biochemical assays
All participants received an interviewer-administered questionnaire to collect general information regarding age, sex and history of diabetes and underwent a basic physical examination [e.g., weight, height, waist-to-hip ratio (WHR), systolic blood pressure and diastolic blood pressure] by trained nurses. Heparinized blood samples were collected after an overnight fast for at least 8 h. Lipid profiles [including total cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL)], blood urea nitrogen (BUN), creatinine (CRE) and uric acid (UA) were measured by an auto-analyzer (Hitachi 7600, Tokyo, Japan). Glycated hemoglobin (HbA1c) was estimated by high-performance liquid chromatography (HLC-73G7, Tokyo, Japan). All reagents and kits used for testing were purchased from Invitrogen (Carlsbad, CA, USA).
DNA extraction and GST genotyping analysis
DNA was extracted from the collected EDTA blood samples using a standard procedure of perchlorate/chloroform extraction (30). The quality of DNA extraction was done using spectrophotometric measurement (Nanodrop 2000, Wilmington, USA) (31). The frequency of the GSTT1 genotypes was determined by polymerase chain reaction (PCR) followed by agarose gel electrophoresis. PCR amplification was carried out on the GeneAmp PCR system 9700 (Applied Biosystems, CA, USA). The genotyping of GSTP1 was performed by providing the PCR products to Shanghai Sangon Biological Engineering Technology &Services Co, Ltd (Shanghai, China) for sequencing. The albumin locus was used as an internal control to distinguish the null genotype of GSTT1 from the aborted PCRs. The primers used in the present study were as follows: GSTT1, 5'-TTC CTT ACT GGT CCT CAC ATC TC-3' (forward) and 5'-TCA CCG GAT CAT GGC CAG CA-3' (reverse); GSTP1, 5'-ACC CCA GGG CTC TAT GGG AA-3' (forward) and 5'-TGA GGG CAC AAG AAG CCC CT-3' (reverse); and Albumin, 5'-GCC CTC TGC TAA CAA GTC CTA C-3' (forward) and 5'-GCCCTAAAAAGAAAATCGCCA ATC-3' (reverse) (21). PCR primers were offered by Dalian Bao Biological Engineering Technology &Services Co, Ltd (Dalian, China) and reagents were obtained from Sigma-Aldrich (St. Louis, USA).
Statistical analysis
Data were expressed as the mean ± standard deviation (SD) for normally distributed variables or as median (interquartile range 25-75%) for variable with a skewed distribution. The Kolmogorov-Smirnov test was used to determine data normality. The genotype frequencies were tested for the Hardy-Weinberg equilibrium (HWE) using Pearson’s chi-square test (32). For continuous variables, differences between the healthy control group and T2DM group were evaluated using Student’s t-test or Mann–Whitney U test. Comparisons among diabetic groups were evaluated by one-way ANOVA or the Kruskal–Wallis test as appropriate, with post hoc Tukey test for multiple comparisons (33). For categorical variables, data were presented as n (%), and comparisons between groups were analyzed by Chi-square test or Fisher's exact test as appropriate. The relationships between GSTT1 and GSTP1 genotypes and the risks of T2DM and DR were assessed by the means of the odds ratio (OR) with 95% confidence interval (CI) limits calculated by logistic regression analysis. All tests were two-tailed, and values of P < 0.05 were considered statistically significant. Statistics were performed with SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA).