| Objective: Diabetic retinopathy (DR) is one of the serious microvascular complications, and is a major cause of new-onset blindness among diabetic adults. Diabetic retinopathy is characterized by increased vascular permeability, tissue ischemia, and neovascularization. Vascular endothelial growth factor (VEGF) can stimulate angiogenesis and increase the permeability of the microvasculature. In diabetic retinopathy, VEGF plays a role in the neovascularization of proliferative retinopathy. PEDF, as a counterpart of VEGF, is an anti-inflammatory factor that may be actively involved in the pathogenesis of DR. It can postpone the development of DR and provide effective protection. The imbalance of VEGF and PEDF, that is VEGF increase and PEDF decrease, may play an important role in the development of DR. It is possible that polymorphisms in VEGF and PEDF SNPs, may contribute to the development of DR.This study aims to study the relationship between polymorphism of VEGF, PEDF and diabetic retinopathy Methods: According to the standards of diabetic diagnosis by WHO in 1999 and the standards of DR diagnosis in the third National Ophthalmic Conference put forward in 1985, a total of 160 unrelated patients with type 2 diabetes were randomly recruited in this study. These patients were further divided into proliferative diabetic retinopathy (PDR) and nonproliferative diabetic retinopathy (NPDR) groups according to their result of Fundus Fluorescein Angiography. At the same time 77 healthy controls were selected from population for regular physical examination in our hospital. 5ml blood was taken from the vein of each subject. Human genomic DNA was extracted by guanidine salt acid method. Genotyping for VEGF-460 C/T and PEDF Met72Thr(T/C) polymorphisms was first carried out by PCR-RFLP analysis (polymerase chain reaction- restriction fragment length polymorphism). Then the analysis to the gene polymorphism of more samples was carried out by HRM (High Resolution Melt). Clinical characteristics such as HbA1c, BMI, SBP, DBP, BUN, TC, TG etc were measured and calculated meanwhile. All data was tested the normality. Chi-square analysis was used to test whether VEGF and PEDF genotype distribution followed the Hardy-Weinberg equilibrium. Normally distributed data was demonstrated using means±SD ( x±s). Non-normally distributed data was demonstrated using median (quartile range). Measurement data was compared using t or t′-test. Numeration data was compared using Chi-square analysis. Risk factors for type 2 diabetic proliferative diabetic retinopathy were analyzed using multiple logistic regression analysis.Results: (1) Clinic experiment index indicated type 2 diabetic patients had higher HbA1c, BMI, SBP, DBP, BUN, TG and Cr levels than normal controls (p<0.05). The patients with PDR suffered with diabetes longered than those with NPDR (p<0.05). (2) Distribution of two allele and genotype of VEGF-460C/T:There were two allele gene (C,T)and three genotypes(CC,CT,TT) in the NPDR group,PDR group and Control group respectively. Test all actual observed values with the formulae of Hardy-Weinberg equilibrium. The result indicated that all of the values ofχ2 were less than 3.84(df=1,p=0.05;χ2=3.84),that was P>0.05. So there was no statistical difference between the actual genotype frequencies and the expected genotype frequencies. The distribution of allele and genotype of VEGF-460C/T was accordance with Hardy- Weinberg's Law of Equilibrium and exhibits the representative of population. The frequencies of CC genotype were 12.99%,17.07% and 27.94% in the Control group,NPDR group and PDR group respectively. The frequency of CC genotype in PDR group was significantly higher than those in Control group and NPDR group (χ2=11.095, 6.275, p<0.05). There was no statistical difference with the frequency of CC genotype between Control group and NPDR group (χ2=1.118, p>0.05). The frequencies of C allele were 37.01%,43.03%和55.88% in the Control group,NPDR group and PDR group respectively. The frequency of C allele in PDR group was significantly higher than those in Control group and NPDR group (χ2=7.13 and 5.70,p<0.05). There was no statistical difference with the the frequency of C allele between Control group and NPDR group (χ2=1.01,p>0.05). (3) Distribution of allele and genotype of PEDF Met72 Thr:There were two allele gene (T,C) and three genotypes(TT,TC,CC) in the NPDR group,PDR group and Control group respectively. Test all actual observed values with the formulae of Hardy-Weinberg equilibrium. The result indicated that all of the values ofχ2 were less than 3.84 (df=1, p=0.05;χ2=3.84), that was p>0.05. So there was no statistical difference between the actual genotype frequencies and the expected genotype frequencies. The distribution of two allele gene and genotype of PEDF was accordance with Hardy-Weinberg's Law of Equilibrium and exhibited the representative of population. The frequencies of TT genotype were 32.46%,46.74% and 48.53% in the Control group,NPDR group and PDR group respectively. The frequency of TT genotype in PDR group was higher than those in Control group (χ2=3.882, p<0.05). There was no statistical difference with the frequency of TT genotype between PDR group and NPDR group (χ2=0.50, p>0.05). There was no statistical difference with the frequency of TT genotype between NPDR group and Control group (χ2=3.550, p>0.05). The frequencies of T allele were 48.05%,57.61% and 60.29% in the Control group,NPDR group and PDR group respectively. The frequency of T allele in PDR group was higher than those in Control group (χ2=4.355, p<0.05). There was no statistical difference with the frequency of T allele between PDR group and NPDR group (χ2=0.233, p>0.05); There was no statistical difference with the frequency of T allele between NPDR group and Control group (χ2=3.076,p>0.05). (4) Compared between different genotype of VEGF and the incidence of proliferative retinopathy: the rate of TT genotypes in patients with proliferative retinopathy patients was 27.5%, while the rate of the C allele carrying patients suffering from proliferative retinopathy was 47.5%. The rate of C allele carrying in patients with proliferative retinopathy was significantly higher than that of patients with TT genotype (χ2=4.91,p<0.05). The risk of C allele carrying in patients with proliferative retinopathy was 2.39 times than that of patients with TT genotype (OR = 2.39 , 95 % CI: 1.09-5.21). (5) Compared between different genotype of PEDF and the incidence of proliferative retinopathy: the rate of CC genotypes in patients with proliferative retinopathy patients was 39.58%, while the rate of the T allele carrying patients suffering from proliferative retinopathy was 43.75%. There was no significant diference between the rate of CC genotypes suffering from proliferative retinopathy and that of the T allele carrying patients (χ2=0.239,p=0.625).(6) Risk factors analysis of Type 2 DM complicated with proliferative retinopathy: It is PDR as the dependent variable and there are VEGF genotype, PEDF genotype, gender, age, course of disease, body mass index, systolic blood pressure, diastolic blood pressure, cholesterol, triglycerides, blood urea nitrogen, creatinine, glycated hemoglobin as independent variables, researching many factors affect the incidence of PDR by making non-conditional Logistic regression analysis. The results showed that VEGF gene polymorphisms (OR=2.12,95%CI: 1.20–3.73) and duarition were risk factors of type 2 DM complicated with proliferative retinopathy. by interaction analysis between VEGF genotype and duarition, the results showed that the odds ratio was 2.04 after interaction, and it was between the OR of VEGF genotype and duarition, so their interaction was not obvious. By interaction analysis of Genotype of VEGF and PEDF, the results showed that the odds ratio was 2.26 after interaction, and it was higher than the OR value of Genotype of VEGF and PEDF, showing that the genotype of VEGF and PEDF have synergy role.Conclusions: (1) VEGF-460 C/T genetic polymorphism was associated with PDR.C allele is probably a susceptible gene of PDR. (2) There is no correlation between the PEDF Met72Thr T/C genetic polymorphism with PDR. (3) These results demonstrate that the VEGF gene, in cooperation with the PEDF gene, may contribute to the development of DR. |
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