Regulatory SNPs Alter the Gene Expression of Diabetic Retinopathy Associated Secretary Factors

Abstract

Objectives: Diabetic retinopathy (DR) is a common microvascular complication in both type I and type II diabetes. Several previous reports indicated the serum centration of some secretary factors were highly associated with DR. Therefore, we hypothesis regulatory SNPs (rSNPs) genotype in secretary factors may alter these gene expression and lead to DR.

Methods: At first, pyrosequencing were applying to screen the SNPs which present allele frequency different in DR and DNR. Then individual genotyping was processed by Taqman assays in Taiwanese DR and DNR patients. To evaluate the effect of SNP allele on transcriptional activity, we measured promoter activity using luciferase reporter constructs.

Results: We found the frequencies of the CC, CG, and GG genotype of the rs2010963 polymorphism were 15.09%, 47.14%, and 37.74% in DR and 12.90%, 19.35%, and 67.74% in DNR, respectively (p = 0.0205). The prevalence of DR was higher (p = 0.00793) in patients with the CC or CG genotype (62.26% and 32.26% for DR and DNR, respectively) compared with the patients with the GG genotype. To evaluate the effect of rs2010963-C allele on transcriptional activity, we measured promoter activity using luciferase reporter constructs. The rs2010963-C reporter showed 1.6 to 2-fold higher luciferase activity than rs2010963-G in 3 cell lines.

Conclusion: Our data proposed rs2010963-C altered the expression level of VEGFA in different tissues. We suggested small increase but long term exposure to VEGFA may lead to DR finally.

Keywords: DR; SNP; VEGFA..

Figure 1

Allele frequency determine in DNA pool by pyrosequencing. A) The workflow of allele frequency determination in DNA pools by pyrosequencing. B) Minor allele frequency for 29 SNPs in DR and DNR pools.

Figure 2

Effects of the rs2010963 variants on luciferase reporter expression. A) The construction map of pGL4 luciferase reporter containing VEGF promoter sequence with the allele C or G allele at SNP rs2010963. B) The reporter vector with the allele C or G allele at SNP rs2010963 were transfected into A549, sw480 and SNU398 cells.