Study Of The Association Between Vascular Endothelial Growth Factor Gene Polymorphism And Type 2 Diabetic Retinopathy

Objective: Diabetes mellitus (DM) is a metabolic disorder symdrone diagnosed by increased sugar level in the blood and urine. It can lead to damages of many important organize and even nonfunction. Diabetic retinopathy (DR) is a serious appearance of diabetic microvascular, also it's a serious complication of diabete. As reported that DR is one of the four main diseases which leads to blind in the USA and other developed countries. With the economy and living standard in last decade, the prevalence of diabetes has risen to 3.2% in China. And DR accordingly become the chief disease which incidence rate rised rapdly in last decade.The pathogenesy of DR is still unclearly, some research indicated that DR is a diabetic microvasalar complication which course multiple factor. Long-term chronic hyperglycemia will lead to a series of metabolic disorders which are associated with the morbidity of DR. However, as the changes of morbidity time and serious levels of DR can't explain all by physiology biochemistry and environmental agents. The invasion of DR and DM almost at the same time in some patients while DR may happen ten or more years after invasion of DM in other patients; and it can delay the development of DR to control blood suger in some DM patients but it can not prerent them from DR, and others that have a poor blood suger control but can not have DR.Thus, genetic factor plays an important role in the invasion and development of DR. At present, scholars in and out china mainly proceed their study on the pathophysiology of the invasion and development of DR. they screened the possible candidate genes which may affect the invasion and development of DR, and analysised the hereditary susceptibility of DR by gene polymorplism and the regulation of gene expression. Presently these studies concentrate on the white people in Europe, Japanese and Indians in Asian. They have made some progress, but there are still no definite conclusions.Vascular endothelial growth factor (VEGF) can specially act on vascular endothelial cells, and it increases the permeability of micrvasculature and simulates neovacularization. It plays an important role on the invasion of DR especially proliferation diabetic retinopathy (PDR). The single nucleotide polymorphism(SNP)of VEGF gene may influence it's transcriptional level and protein expression,consequently it may influence the invasion of diseases. Study of relationship between VEGF gene polymorphism and the genetic susceptibility of DR have been reported in abroad, but there aren't uniform conclusion. Thus, the aim of our study is to approach the association between the -2578C/A polymorphism in promoter region and +936C/T polymorphism in the 3'-untranslated region (3'-UTR) of VEGF gene and DR in North Chinese han people.Methods:Our study adopts case-control studing method. All the patients were recruited from the department of ophthalmology and endocrinology in the Fourth Affiliated Hospital of Hebei Medical University and from DM screening in the city of Shi Jiazhuang and surrounding counties from March 2006 to December 2007, including 144 cases of diabetes mellitus without diabetic retinopathy(DM), 141cases of diabetic retinopathy (DR), and there are 105 cases of non proliferation diabetic retinopathy (NPDR) and 36 cases of proliferation diabetic retinopathy (PDR). All the patients were made a definite diagnosis by diabetes diagnosis standard of WHO and international diabetic retinopathy typing standard. 137 cases of healthy volunteers(Control)who had no clinical evidence of diabetes mellitus or any other diseases were randomly selected from Chinese blood donors as control subjects. All cases and controls were from North Chinese Han population. The informed consent was got from all the recruited subjects. Four milliter of venous blood from each subject was drawn in Vacutainer tubes containing 3.2% sodium citrate and stored at 4℃. The genomic DNA was extracted within one week after bleeding by using proteinase K digestion followed by a salting out procedure. Genotypes of the VEGF gene were analyzed by PCR-restriction fragment length polymorphism analysis (RFLP). Statistical analysis was performed using SPSS13.0 software package. P<α=0.05 was considered significant for all statistical analyses and in the method of Chi-square division test P<α'=0.0125 was considered significant. Comparison of the VEGF genotype, allelotype distribution in patients and healthy controls was performed by means of two-sided contingency tables using Chi-square test. The odds ratio (OR) and 95% confidence Interval (CI) were calculated using an unconditional logistic regression model.Results:1. The allelotype frequencies and the genotype frequencies of VEGF in the group of healthy controls were found in Hardy-weinberg equilibrium.2. Association between the VEGF -2578C/A SNP in promoter region and diabetic retinopathy: The C and A allele frequencies in Control, DM, DR groups are 74.8%, 25.2%; 75%, 25%; 83.7%, 16.3%, respectively. And the results have significant deviation (χ~2=8.390,P=0.015). There's no significant deviation of C and A allele frequency between the Control group and the DM group(χ~2=0.002,P>0.0125). While there are significant deviation when compare the N Control group and DR group(χ~2=6.665,P<0.0125) or the DM group and DR group(χ~2=6.551,P<0.0125). The C/C, C/A,A/A genotypic frequencies of cases in the three groups are 57.7%,34.7%,8%;55.6%,38.9%,5.6%;69.5%,28.4%,2.1%, respectively. The results also have significant deviation (χ~2=9.653,P=0.047). There's no significant deviation of C/C,C/A,A/A genotypic grequency between the Control group and DM group(χ~2=1.093,P>0.0125), while there are significant deviation between the Control group and DR group(χ~2=7.118,P<0.0125) or the DM group and DR group(χ~2=6.729,P<0.0125). Compared with the C/A+A/A genotype, the C/C genotype may increase the onset risk of diabetic retinopathy. The odds ratio (OR) between the Control group and DR group is 1.673(95%CI =1.022~2.740); between the DM group and DR group is 1.823(95%CI=1.121~2.965).3. Association between the VEGF 3'UTR+936C/T SNP and diabetic retinopathy: The C and T allele frequencies in Control, DM, DR groups are 86.4%,15.6%; 83.7%,16.3%; 83.7%,16.3%, respectively. And there's no significant deviation between them(χ~2=1.123,P=0.570).The C/C, C/T, T/T genotypic frequencies of cases in the three groups are 75.9%,21.2%,2.9%;70.1%,27.1%,2.8%;68.8%,29.8%,1.4%,respectively. Also, there's no significant deviation between them(χ~2=1.948 ,P=0.378).Compared with the C/T+T/T genotype, the C/C genotype won't increase the onset risk of diabetic retinopathy. The odds ratio (OR) between the Control group and DR group is 1.430 (95%CI =0.842 ~2.427) ; Between the DM group and DR group is 1.065(95%CI=0.643~1.764).4. Stratified by international diabetic retinopathy typing: The C/C, C/A, A/A genotypic frequencies of VEGF-2578C/A site in NPDR group are 72.4%,26.7%,1%,respectively, and there's no significant deviation when compared with the PDR group (61.1%,33.3%,5.6%) (χ~2=1.606 , P=0.205). Compared with the C/A+A/A genotype, the C/C genotype won't increase the onset risk of PDR, OR=0.600 (95%CI=0.271~1.328).The C and A allele frequencies in the two groups are 85.5%, 14.2%; 77.8%, 22.2%, respectively, and there's no significant deviation between them(χ~2=2.474,P=0.116).The C/C, C/T, T/T genotypic frequencies of VEGF+936C/T site in NPDR group are 67.6%,31.4%,1%,respectively, and there's no significant deviation when compared with the PDR group (72.2%,25.0%,2.8%)(χ~2=0.265,P=0.607). Compared with the C/T+T/T genotype, the C/C genotype won't increase the onset risk of PDR,OR=0.803(95%CI=0.348~ 1.853).The C and T allele frequencies in the two groups are 83.3%,16.2%; 84.2%,15.3%, respectively, and there's no significant deviation between them(χ~2=0.676,P=0.783).Conclusions:1. The VEGF -2578C/A SNP may play a role in the genetic susceptibility to the invasion of diabetic retinopathy. Compared with the -2578C/A, A/A genotype, the VEGF-2578C/C genotype did significantly increase the risk of the development of the diseases.2. The VEGF +936C/T SNP is not associated with the susceptibility of DR in North Chinese han population.3. The VEGF -2578C/A SNP and +936C/T SNP is not associated with the development of NPDF to PDF of DR in North Chinese han population.